LIPIDS AND PHOSPHOLIPASE ACTIVITY OF VIBRIO CHOLERAE

DISSERTATION

Presented to the Graduate Council of the

North Texas State University in Partial

Fulfillment of the Requirements

For the Degree of

DOCTOR OF PHILOSOPHY

BY

Buford L^Brian, M. A. \ \\

Denton, Texas

August, 1972

« < '

Brian, Buford L., Lipids and Phospholipase Activity

of Vibrio choleras. Doctor of Philosophy (Biological

Sciences}, August, 1972, 137 pp., 27 tables, 14 figures,

bibliography, 170 titles.

One purpose of this investigation is to determine the

fatty acid and lipid content of typical Vibrio cholerae cells.

The comparison of cholera lipid constituents with those of

closely-related bacteria might be of taxonomic value.

Furthermore, chemical characterization of the cholera vibrio

could provide useful criteria for identification of these

disease-producing microorganisms.

In an early V. cholerae lipid study, large amounts of

free fatty acid and phospholipid were observed when cholera

lipids were separated by thin-layer chromatography, and the

existence of a powerful cholera lipase was thus suggested.

Therefore, another purpose of this study is to determine

whether cholera cells contain phospholipase activity capable

of rapid hydrolysis of fatty acids from phospholipids.

Companion studies were conducted for the purpose of

development and extension of gas chromatographic techniques.

The esterification reagents boron trichloride and boron

trifluoride in methanol were tested for quantitative con-

version of cyclopropane fatty acids to their respective

methylesters. Hydrogenation and bromination procedures were

applied to the detection of cyclopropane fatty acids in

Pseudomonas aeruginosa. Fatty acids of lymphosarcoma and

liver of tumor-bearing DBA/1J mice were studied during tumor

development. Fatty acids of carcass, liver, and fat bodies

of Cnemidophorus ticrris prior to hibernation were investi-

gated. A new phenylthiohydantoin amino acid derivative, the

. acetate, was introduced and compared to the trifluoroacetate

with respect to gas-chromatographic applicability.

V. cholerae strain 569 3 (Inaba) contains 5.4-6.6%

lipid. Cholera lipids consist of ca 75% phospholipid and

ca 25% free fatty acid. Major phospholipids separated by

thin-layer chromatography are tentatively identified as

phosphatidyl ethanoiamine, phosphatidyl glycerol, and

cardiolipin.

Gas-liquid chromatography of fatty acids from thirty

cholera strains was conducted. Major acids found are myristate

(2-9%), palmitate (21-39%), hexadecenoate (34-46%), stearate

(2-5%), and octadecenoate (12-26%). Cyclopropane-ring-

containing compounds are not observed. The feasibility of

utilizing such chemical analyses for identification of

cholera vibrios is suggested.

Pnospho1ipase activity of the acyl hydrolase type

is j.ound in V. cno^srae strain 569 B {, Inaba) cell sonicates

32 1A

using P and C radioactive phospholipid substrates.

Results indicate that both fatty acids are removed from

phosphatidyl ethanoiamine, and that no detectable amounts

of lys©phosphatidyl ethanolamine are present in reaction

products. The phospholipase appears to be membrane-bound.

Reaction in the presence of EDTA 910 mM) and 3-hydroxyqu ino-

line (1 mM) shows that no divalent cation is required. One

and 10 mM concentrations of the chloride salts of calcium,

barium, magnesium, manganese(ous), zinc, iron (ferrous), and

mercury cause inhibition of the cholera phospholipase. No

inhibition is observed with potassium or sodium chloride

(1 and 10 mM). Triton X 100 optimum concentration is 1 mg/ml.

The of the cholera phospholipase for Escherichia coli -^P

4 -

phospholipid is 63 (~6) uM using sonicate (1.6 mg/ml protein)

in 25 mM borate buffer, pH 7.5 with 1 mg/ml Triton X 100.

An enzyme capable of selectively hydrolyzing membrane

phospholipids could contribute to the outpouring of fluids

and ions into the intestinal lumen, causing the extreme

diarrhea observed in cholera victims. A major contribution

to the understanding of the processes by which intestinal

pathogens cause diarrheal symptoms in disease victims might

be made by elucidation of the effect of bacterial phospho-

lipase activity on intestinal membrane constituents.

LIPIDS AND PHOSPHOLIPASE ACTIVITY OF VIBRIO CHOLERAE

DISSERTATION

Presented to the Graduate Council of the

North Texas State University in Partial

Fulfillment of the Requirements

For the Degree of

DOCTOR OF PHILOSOPHY

BY

Buford L^Brian, M. A. \ \\

Denton, Texas

August, 1972

TABLE OF CONTENTS

Page

LIST OF TABLES v

LIST OF ILLUSTRATIONS

Chapter

I. LIPIDS OF VIBRIO CHOLERAE 1

Introduction Materials and Methods Results and Discussion

II. PHOSPHOLIPASE ACTIVITY OF VIBRIO CHOLERAE . . . 42

Introduction Materials and Methods Results and Discussion

III. GAS CHROMATOGRAPHY OF CYCLOPROPANE FATTY ACID METHYLESTERS PREPARED WITH METHANOLIC BORON TRICHLORIDE AND BORON TRIFLUORIDE . . . . 84

Introduction Materials and Methods Results and Discussion

IV. CYCLOPROPANE FATTY ACIDS OF PSEUDOMONAS AERUGINOSA 92

Introduction Materials and Methods Results and Discussion

V. TUMOR AND LIVER FATTY ACIDS OF DBA/1J MICE DURING LYMPHOSARCOMA DEVELOPMENT 100

Introduct i on Materials and Methods Results and Discussion

xxi

Chapter Page

VI. FATTY ACID DISTRIBUTION OF LIPIDS FROM CARCASS, LIVER AND FAT BODIES OF THE LIZARD, CNEMIDOPHORUS TIGRIS, PRIOR TO HIBERNATION 112

Introduction Materials and Methods Results and Discussion

VII. ANALYSIS OF ACETYLATED AND TRIFLUORO-ACETYLATED PHENYLTHIOHYDANTOIN AMINO ACIDS BY GAS CHROMATOGRAPHY 118

Introduction Materials and Methods Results and Discussion

LITERATURE CITED 123

IV

LIST OF TABLES

Table Page

Vibrio Cholerae Strains 6

II. Non-Cholera Vibrios 7

III. Phosphorus Analysis of Thin-layer Chromatography-Separated Phospholipids of Vibrio Cholerae Strain 569 B (Inaba) and Comparison with Escherichia Coli . . . . 17

IV. Percent Yield of the Simmons-Smith Reaction as Determined by Gas Chromatography . . . . 20

V. Gas Chromatographic Reproducibility Using One Sample of Fatty Acid Methylesters (NIH 41) Analyzed Ten Times 22

VI. Effects of Hydrogenation on Percent

Fatty Acid Methylesters 24

VII. Fatty Acid Distribution of Vibrio Cholerae . . 25

VIII. Fatty Acid Distribution of Non-Cholera Vibrios 27

IX. Effects of Incubation Temperature on Fatty Acid Distribution (Percentages) of NIH 41 29

X. Percent Fatty Acids in Lipid Fractions . . . . 30

XI. Phospholipase D Assay 60

XII. Assay of Vibrio Cholerae Sonicate for Phospholipase Activity Using 32P Phosphatidyl Glycerol as Substrate 63

XIII. Effects of Tris-HCl (pH 8.0), EDTA, 8-Hydroxyquinoline and Dialysis on Reaction of Sonicate 68

XIV. Percent Inhibition by Chloride Salts (One and Ten mM) on Phospholipase Activity of V. Cholerae Sonicate 69

v

Table

XV.

XVI.

XVII.

XVIII.

XIX.

XX.

XXI.

XXII.

XXIII.

XXIV.

XXV.

XXVI.

Effects of Triton X 100 Concentration on Phospholipase Activity of Sonicate . .

Phospholipase Activity of XM-100A, PM-30, and UM-10 Membrane Filter Retentates of V. Cholerae Sonicate . . .

Page

71

75

Ratio of the Peak Areas of Cis-9,10-Methylene Octadecanoate (Cyc C-̂ g) to Heptadecanoate (^7) Following Esterification (1 mg of Each Acid) . . . .

Effects of Incubation Temperature on Pseudomonas Aeruginosa Fatty Acids . . . .

Fatty Acids (%) of Pseudomonas Aeruginosa Strains Incubated 40 C

Results of Hydrogenation and Bromination on Pseudomonas Aeruginosa Fatty Acids . .

Mean Net Weight of Tumors and Livers from Tumor-Bearing Mice at Various Stages of Tumor Development

Percent of Lipid in Tumors and Livers from Tumor-bearing Mice at Various Stages of Tumor Development ,

Percentage of Fatty Acids Occurring in Liver Lipids of Tumor-bearing Mice at Various Stages after Implanation . . . . ,

Percentage of Fatty Acids Occurring in Tumor Lipids at Various Stages after Implanation ,

Male and Female C. Tigris Body Measurements (mm), Tissue Dry Weights (mg), and Lipid Content Expressed as Percentages of the Dry Weight of Each Tissue

88

94

95

96

104

106

107

108

115

Male and Female C. Tigris Fatty Acids from Carcasses, Livers, and Fat Bodies Expressed as Percentages (Mean Values) of the Total Fatty Acid Content 116

VI

Table Page

XXVII. Relative Retention Times of Amino Acids Phenylthiohydantoin Acetates and Trifluoroacetates 120

LIST OF ILLUSTRATIONS

Figure Page

1. Growth Curve of Vibrio Cholerae Strain 569 B (Inaba) by Optical Density (O.D.) Readings (540 nm) 9

2. Thin-layer Chromatogram of V. Cholerae (569 B) Lipids with a Standard Mixture of Phospholipids 15

3. Thin-layer Chromatogram of E. Coli (ATCC 11775) Lipids and V. Cholerae (560 B) Lipids 16

4. Gas-liquid Chromatogram of Fatty Acid Methylesters of V. Cholerae Strain NIH 41 21

5. Thin-layer Chromatogram of Major Egg Yolk Phospholipids and Purified Phosphatidyl Choline . 53

6. Assay of Crotalus atrox Venum (2.5 ug) Phospholipase A? Activity on Phospha-tidyl Choline (10 umoles) with Time (22 C) 5 5

7. Assay of Crotalux atrox venom (2.5 ug) Phospholipase A? Activity on 5-50 umoles of Phosphatidyl Choline (10 min, 27 C.) 5 5

8. Thin-layer Chromatogram of Bacterial Phosphatidyl Ethanolamine (PE) and Lysophosphatidyl Ethanolamine (LPE) Prepared by Reaction of PE with Crotalux atrox venom 57

9. Optical Density (o. D.) Readings (526 nm) Following Reaction of 4-20 umoles of Choline Hydrochloride with Ammonium Reineckate (Average of Two Determinations) . . 59

Vlll

Figure Page

10. Thin-layer Chromatogram of E. coli (ATCC 11775) 32p phospholipids 62

11. Thin-layer Chromatograms of Cholera Phospholipase Products Following Reaction with Phosphophatidyl Ethanolamine (21 nmoles) for 1 hr, 37 C 65

12. Assay of Vibrio Cholerae Strain 569 B (Inaba) "Roux Flaslc Supernatant" (6 mg/ml Protein) for Phospholipase Activity Using 32p phosphatidyl Ethanolamine (11 nmoles) as Substrate 66

13. Effect of Enzyme (Sonicate) Concentration on Reaction Rates of V. Cholerae Phospholipase Activity Using -^P (16.6 nmoles) as Substrate 72

14. Initial Velocities (nmoles/hr) of "Roux Flask Supernatant," 6 mg/ml Protein, and Sonicate, 1.6 mg/ml Protein, with Lineweaver-Burk plots 73

xx

CHAPTER I

LIPIDS OF VIBRIO CHOLERAE

Introduction

The introduction of gas chromatography to microbial

lipid chemists (55, 56) began a surge of investigations on

the fatty acid content of bacteria. Reports of fatty acid

and lipid content of many bacterial species have been exten-

sively reviewed (3, 29, 49, 53, 61, 62, 68, 72, 85, 87).

Bacterial fatty acids consist chiefly of straight-chain or

branched-chain saturated compounds, monoenes, hydroxyl- or

cyclopropane-ring-containing acids (85, 87).

Hydroxy fatty acids were found as major constituents

of 1ipopolysaccharides in Escherichia coli (26) and Proteus

(82). Branched-chain (iso, anteiso) compounds were reported

to be in large amounts in Sarcina lutea (52, 100, 102),

Bacillus species (57, 58, 66), and Staphylococcus aureus

(107). Fatty acids containing cyclopropane rings were

reported in Serratia marcescens (6, 7, 64, 65), E. coli

(28, 33, 36, 59, 60, 70, 78, 94, 106), Pseudomonas

fluorescens (16, 34), Pseudomonas aeruginosa (23, 47, 103),

Lactobacillus species (49, 50), Aqrobacterium tumefaciens

( 6 0 )' Streptococcus species (75, 76), Rhodomicrobium

vannielii (88), Klebsiella pneumoniae (37), rugose variants

1

2

of Vibrio cholerae (17, 19), and a strain of Vibrio fetus

(101). The most commonly reported cyclopropane fatty acids

are cis-9,10-methylene hexadecanoate (59) and cis-11,12-

methylene octadecanoate (lactobacillic acid, 50).

The mechanisms involved in the synthesis of cyclopro-

panes were the subject of several papers (30, 31, 35, 48, 71,

84, 86, 90, 109) and were shown to involve construction of a

methylene bridge across the double bond of palmitoleic or

cis-vaccenic acids with S-adenosylmethionine as the carbon

donor to produce the two cyclic compounds described. Bacteria

do not synthesize polyunsaturated acids (85). The chief

monounsaturates reported were cis-9,10-hexadecenoate

(palmitoleate) and cis-11,12-octadecenoate (cis-vaccenate).

Oleic acid (cis-9,10-octadecenoate) was found in the tubercle

baccilus (27). Hexadecenoate and octadecenoate appear to be

major fatty acids of V. cholerae (18).

Differences found in fatty acid content of bacterial

species have prompted some investigators to suggest that

such gas chromatographic data might prove valuable for pur-

poses of identification of bacteria (2, 11, 98). Patty acid

profile patterns could only be useful for characterization

if cultural and analytical techniques were standardized,

since age of culture, incubation temperature, and media

constituents are known to have drastic effects on fatty acid

distribution (65, 66, 70, 78, 94). Furthermore, unreliability

3

of analytical techniques or strain differences introduce

further variables and might have accounted for failure by some

-workers to report cyclopropane acids in P. aeruginosa (9, 38,

91), since others (23, 47, 103) have indicated their presence

in this species. However, use of gas chromatographic data

for characterization of bacteria deserves attention by those

equipped for such analyses.

Thin-layer, gas-liquid, and silicic acid column

chromatography have replaced earlier separation methods such

as paper chromatography, distillation, and electrophoresis

for lipid purification. However, many cases of technical

incompetency cloud the literature in this area, and it

should be emphasized that complete utilization of the

methodology requires a highly-trained technologist.

The most widely-studied species for lipid content is

c o l i (60, 83), which was reported to contain phosphatidyl

ethanolamine, phosphatidyl glycerol, and cardiolipin as major

phospholipids. It seemed feasible to use E. coli for com-

parative purposes in lipid studies of V. cholerae.

Phospholipid chemistry and metabolism of bacteria.

Plants, and animals have been reviewed by many workers (1,

3, 28, 45, 46, 61, 62, 68, 72, 87). Lipids are used for

energy storage as well as for structural elements in cell

membrane construction (92). Attempts have been made to dis-

cern correlations between lipid composition and pathogenecity

4

(18, 81) of microorganisms, but no clear-cut relationship has

been found.

Lipid is known to comprise an integral part of bac-

terial endotoxins (26). Investigation of purified Type 2

toxin (choleragen) of V. cholerae strain 569 B (25, 32) by

Kaur, et al. (69) showed that the exo-entero-toxin contained

30% lipid. Fatty acids of the toxin (90) were similar to

those previously reported in whole-cell vibrios (18).

Further purification of the choleragen (40-43, 73) resulted

in an active toxin with a molecular weight of 84,000 and

with little lipid remaining. Pierce and Greenough (89)

reported that the Type 2 toxin stimulated glycerol produc-

tion in isolated fat cells. The means by which such lipolytic

activity occurs is not known.

A paucity of knowledge is available on the lipids of

¥• cholerae. One investigation (4) of cholera lipids

utilized cholera vaccine. Fatty acids were analyzed by paper

chromatography (4) and bear little resemblance to those

determined by gas—liquid chromatography by Brian and Gardner

(13, 14—19). Blass (8) investigated the nitrogen—containing

phospholipids of the cholera vibrio. Electrophoretic

techniques were used to separate nitrogenous lipid components.

Ethanolamine, ornithine, and several amino acids were found.

In an early V. cholerae lipid study (18), large

amounts of free fatty acid and phospholipid were observed

5

when cholera lipids were separated by thin-layer chromatography,

and the existence of a powerful cholera lipase was thus sug-

gested. Discovery of the presence of lipolytic activity

in the vibrio might provide ample incentive for rapid

extraction of cells with lipid solvents. Cells which are

allowed to stand at room temperature for some appreciable

time would surely be a poor source for lipids (a possibility

given little consideration by some workers).

The purpose of this investigation was to determine

the fatty acid and lipid content of typical V. cholerae

cells. The comparison of cholera lipid constituents with

those of closely-related bacteria might be of taxonomic

value. Furthermore, chemical characterization of the

cholera vibrio could provide useful criteria for identifi-

cation of these disease-producing microorganisms.

Materials and Methods

Bacterial Species. Vibrio cholerae strains investi-

gated are given in Table I. The three major antigenic types

(AB - Ogawa, AC - Inaba, and R - Rough) were determined by

slide agglutination with monospecific rabbit antisera (22).

V. cholerae rugose (108) strains studied have been previously

described (10, 11, 17, 19, 22). Non-cholera vibrios investi-

gated are shown in Table II. Strains which demonstrated

typical vibrio morphology and monotrichous polar flagella-

tion but which failed to agglutinate in anti-cholera sera

6

TABLE I

VIBRIO CHOLERAE STRAINS

Strain Antigenic Components Source

NIH 41 AB (Ogawa) National Institutes of Health

NIH 35 A3 AC (Inaba) II

ATCC 14035 AB American Type Culture Collection

569 B AC R. A. Finkelstein Ca 72 AB, Rough (R) W. Burrows, and C. E.

Lankford, isolated in Calcutta, India, 1953

Ca 113 AB II

Ca 323 R II

Ca 324 R II

Ca 325 R II

Ca 385 R II

Ca 412 AB II

Ca 424 AB II

IRAN 46 AB J. C. Feeley, National Institutes of Health .

Iraq 230 AB 11 B 29112 AB 11 B 1307 AB H "7 "

J 8001 AB II

ROK 350 AB II

M 3735 AB 28/62 AB II

VC 12 Rxl AB II

El 36 AB II

CH-1 AB II

Ubon 13 AB II

0-4 AB II

VN-1 AB II

CRC 31/64 • AB II

1222 AB HK-1 AB II

V 86 AC II

TABLE II

NON-CHOLERA VIBRIOS3

Strain Source Geographic Location

8032 Diarrhea Philippines

485 II Thailand

942 11 India

8288 Freshwater Philippines

8305 II II

6471 II East Pakistan

9682 Sewage United States

aObtained from H. L. College.

Smith and K. Goodner, Jefferson Medical

8

were designated as non-cholera vibrios (96). Morphological

characteristics of vibrios were determined by T. 0. McDonald

(Alcon Laboratories, Port Worth, Texas) using an electron

microscope. Escherichia coli (ATCC 11775) was also studied

for purposes of comparison with cholera lipids. Bacteria

were maintained in the lyophilized form or on slants (2 C)

of Trypticase Soy Broth (BBL) with 2 percent agar (Difco).

Cultural Conditions. Bacterial species were grown

in Trypticase Soy Broth (TSB), previously shown to be essen-

tially lipid-free (18, 75, 76), or in Rome flasks containing

100 ml of TSB with one-two percent agar (Difco). One ml of

a sixteen hour (37 C) broth culture was used to inoculate

each liter of broth or each Roux flask. Broth cultures

(one to ten liters) were incubated (37 C)until the late

stationary growth phase as determined by optical density

(Fig. 1) using a Spectronic 20 colorimeter (540 nm).

Bacteria were harvested in 250 ml bottles by centrifugation

(5000 x g, 15 min, 4 C) using a Sorvall RC-2B refrigerated

centrifuge equipped with a GSA rotor. Roux flasks were

incubated 37 C, 24 hours. Cells were harvested by washing

the agar surface with distilled water followed by centrifu-

gation. Wet cell paste was extracted for lipid, but in

some cases bacteria were lyophilized to determine dry

weight. Bacteria were streaked for isolation onto Petri

plates containing agar media both before and after initial

.6

.5'

£ A c.4 O

2.3 o

.2

.1

"T" 8

—r-11

—i 13 10

Hours

12

Fig. 1—Growth curve of Vibrio cholerae strain 569 B (Inaba) by optical density (0. D.) readings (540 nm).

10

inoculation as well as after "harvest to determine presence of

contaminating microorganisms. If no contamination was appar-

ent, lipid extraction was begun.

Lipid Extraction. Lipids were extracted from known

weight of tissues or bacteria by the method of Folch, et al.

(44) as previously described (11-21, 23). A minimum of

twenty volumes of chloroform:methanol (2:1, volume/volume)

was used per gram of tissue or cells. Both chloroform and

methanol were distilled before use. Lipid was concentrated

either by a stream of nitrogen or by rotary evaporation

under partial vacuum and then diluted to known volumes with

chloroform:methanol (2:1, volume/volume). Portions of lipid

materials were dried (70 C) on pre-weighed aluminum foil for

dry-weight determination. Phosphorus was quantitated by

the Bartlet procedure (5, 74) using a Cary 14 spectrophoto-

meter at 830 nm.

Thin-layer Chromatography. Thin-layer chromatography

(TLC) was performed using 20 x 20 cm, 5 x 20 cm, and 1 x 3

inch glass plates. Silica gel G (Curtin Chemical Co.)

aqueous slurries were spread onto plates with a thickness

of 0.25-0.5 mm and dried overnight at 26 C. Ammonium sul-

fate (1%, weight/volume) was added to the aqueous slurry

when sample charring, following TLC separation, was desired

(79, 104).

11

TLC-separated lipids were visualized by iodine vapors

or by heating TLC plates (220 C) which contained ammonium

sulfate. Sulfuric acid which results from this treatment

effectively decomposed lipids to visible carbon spots.

Phosphatidyl ethanolamine (PE) was detected by spraying TLC

plates with 0.3% ninhydrin in acetone. Phosphatidyl glycerol

(PG) was detected with periodate-schiff reagent (63), and

Dragendorff reagent (97) was used to detect phosphatidyl

choline (PC). Standards of PC and PE were purchased from

Applied Science Laboratories and cardiolipin (CL) from

Supelco, Inc.; PG was produced by reaction of PC with cabbage

transphosphatidylase in the presence of 20% glycerol (70).

Oleic acid, triolein, methyl oleate, and cholesterol oleate

were from Applied Science Laboratories.

TLC solvent systems used were:

Solvent A (105)—Petroleum ether: diethyl ether:

glacial acetic acid (90:10:1, volume/volume).

Solvent B (77)—Chloroform: methanol: water

(60:30:5, volume/volume).

Solvent C (74)—Chloroform: methanol: glacial

acetic acid: water (80:13:8:0.3, volume/volume).

Gas-liquid Chromatography (GLC). GLC (15, 20, 24,

54, 56, 99) was performed using methylesters of fatty acids

hydrolyzed from lipid constituents. Lipids were hydrolyzed—

esterified by boiling one to ten mg lipid with 0.5 N

12

methanolic sodium "hydroxide (2 ml) followed by 3 ml of 10%

boron trichloride in methanol (Applied Science Laboratories)

as detailed earlier (24, 80). Standard fatty acids and

methyl esters were purchased from Applied science Laboratories

unless otherwise noted.

Fatty acid ester mixtures were hydrogenated (18, 39)

using a platinum catalyst followed by bromination (16) to

detect unsaturated and cyclopropane fatty acids. Cyclopro-

pane methylesters (cis-9,10-methylene hexadecanoate, cis-9,

10-methylene octadecanoate, and cis-11,12-methylene octade-

canoate) were synthesized from palmitoleate, oleate, and

cis-vaccenate, respectively, using the simplified zinc-

copper couple of Shank and Schecter (93) and the Simmon-

Smith reaction (95). Synthesized compounds were purified

using a Varian Aerograph (Varian Associates, Palo Alto,

California) gas chromatograph equipped with a hydrogen-

flame detector, column-effluent splitter, and a column

(6 feet x 0.25 inch, O.D.) packed with 15% diethylene

glycol succinate polyester on Chromosorb W (60/80 mesh)

operated at 180 C.

Quantitative GLC of fatty acid methylesters of

lipid extracts was accomplished using a Varian Aerograph

gas chromatograph Model 204 series equipped with dual flame

hydrogen detectors and a i m volt (full scale) recorder.

One-ten ug of fatty acid methylesters was injected with a

13

10 ul syringe with a range setting of lO--^ and attenuation

of 4-16. Columns (10 feet and 5 feet x 0.125 inch, O.D.)

containing 15-20% diethylene glycol succinate polyester

(DEGS) on Chromosorb W, 60/80 mesh (Applied Sciences Lab-

oratories), were operated isothermally at 180 C. Carrier

flow rate (helium or nitrogen) was 20-25 ml/min, hydrogen

was 30 ml/min, and air was 200-300 ml/min. Peaks obtained

were quantitated by a disc integrator (Disc Instruments,

Inc.) or by multiplication of peak height x width at 1/2 peak

height. Quantitative standards (51) were used to test

linearity of detector response over a wide range of

methylesters.

Results and Discussion

Lipids of Vibrio cholerae. Percent lipid on a dry

cell basis of typical cholera vibrios was reported by Brian

and Gardner (18). Average amount of weighed lipid ranged

from 5.8-7.6% (18). Strain 569 B (Inaba) was found to con-

tain 5.4-6.6% lipid. Phosphorus analysis of the lipid

extracts indicated phosphorus levels of ca 3% of weighed

lipid and ca 0.17% of dry cell weight. Since phosphorus

comprises 4-4.4% of a phospholipid, it was calculated that

cholera lipid investigated consisted of 70-75% phospholipid.

The remaining lipid (25-30%) appeared to be free fatty acids.

The observation of large amounts of free fatty acids in

cholera strains by TLC using solvent A suggested that

14

phospholipase activity had occurred.

A standard mixture containing PE, PG, and cardiolipin

(bacterial) was spotted on TLC plates beside cholera (569 B)

lipids and separated by solvent C (Pig. 2). Migration of

569 B lipid components suggested presence of PE, PG, CL, \

neutral lipid (fatty acids), and ninhydrin-positive material

at the origin. PE was ninhydrin-positive, and the PG spot

reacted positively with periodate-schiff reagent. Tentative

identification of cardiolipin was by Rf only (solvents B and

C). Solvent B did not separate PE and PG. Therefore, solvent

C was chosen for subsequent phospholipid separation by TLC.

The separation of E. coli and V. cholerae lipids

indicated no qualitative differences in phospholipid content

(Fig. 3). This finding was not surprising since the cultural

conditions, media, and extraction techniques used with these

two Gram-negative species were similar. Differences in lipid

content of bacteria which appear in the literature often

have been due to inconsistencies in growth and lipid tech-

niques (85). It seemed feasible to use the most vigorously

studied bacterium (E. coli) for comparative investigation

of cholera lipids.

Strain 569 B lipids containing 0.082 umoles of

phosphorus were spotted on TLC plates and separated by sol-

vent C. Spots were visualized with iodine, scraped into

tubes, and analyzed for phosphorus content (5). Table III

15

- S o l v e n t

o N e u t r a l l i p id

O o CL

0 0 PG

0 0 PE

0 • O r i g i n

Fig. 2—Thin-layer chromatogram of V. cholerae (569 B) lipids (left) with a standard mixture of phospholipids (right) Neutral lipid = fatty acid. Phosphatidyl glycerol (PG) was periodate-schiff-positive, phosphatidyl ethanolamine (PE) was ninhydrin-positive. Origin contained ninhydrin-positive material. Solvent was chloroform: methanol: acetic acid: water (80:13:8:0.3, volume/volume). Cardiolipin = CL.

16

Solvent Neutral lipid CL

00 Origin

Fig. 3—Thin-layer chromatogram of E. coli (ATCC 11775) lipids (left) and V. cholerae (569 B) lipids (right). Solvent chloroform: methanol: acetic acid: water (80:13* 8:0.3, volume/volume).

17

TABLE III

PHOSPHORUS ANALYSIS OF THIN-LAYER CHROMATOGRAPHY-SEPARATED PHOSPHOLIPIDS OF VIBRIO CHOLERAE STRAIN 569 B (INABA)

AND COMPARISON WITH ESCHERICHIA COLI

Tentative Identification Per cent Phosphorus V. cholerae E. colia

Origin 8

Phosphatidyl ethanolamine 71 78

Phosphatidyl glycerol 17 16

Cardiolipin 4 6

Neutral lipid (fatty acids) 0

a Calculated from published data (83).

18

shows relative percent of phosphorus found in each spot.

Values obtained from an area of silica gel containing no

visable lipid were subtracted from visable spots. Results

suggest that PE is by far the major component (71%) with

PG second (17%). Cardiolipin (4%) and a spot at the origin

(ninhydrin-positive) appear as minor constituents. The

origin may contain small amounts of phosphatidyl glycerol-

O-aminoacyl ester as reported in Serratia marcescens (64).

However, it is not uncommon in lipid extractions to trap free

amino acids as well as other water-soluble compounds by their

mutual attraction to phospholipid. Table III also shows the

phospholipid composition of E. coli which was calculated from

published data (83). A close relationship between phospho-

lipids of V. cholerae and E. coli was demonstrated. Ikawa

(53) has shown that closely-related bacterial species con-

tain similar lipids. Chemical characterization, in order to

be valuable for taxonomic purposes, must be conducted with

a large number of bacterial species. This report is an attempt

to provide such information of V. cholerae lipids.

Gas-liquid Chromatography (GLC). GLC techniques have

been discussed previously (11-12, 23). National Institutes

of Health fatty acid standards (51) were used to determine

linearity of detector (hydrogen flame) response over a wide

range of carbon numbers. An attempt was made to set up GLC

conditions that would provide less than 10% error on minor

19

components (less than 10% of total fatty acids) and less than

5% error on major acids (more than 10% of total).

Cyclopropane fatty acids were synthesized using a

simple zinc-copper couple (93) and the Simmons-Smith reaction

(95). The reaction mixture was analyzed toy GLC for percent

yield (Tatole IV) and then purified toy preparatory GLC (24).

Purified cyclopropane fatty acid methylesters (99% pure toy

GLC) were used as reference standards. Cyclopropanes were

resistant to mild hydrogenation tout reacted with bromine

(16, 17).

Esterification of fatty acids to their corresponding

methylesters using bacterial lipids containing cyclopropanes

has been reported (20). Boron-trichloride (BCI3) in methanol

(10%) was found to be a satisfactory esterification reagent.

Fatty Acids of Vibrio cholerae. The fatty acids of

typical V. cholerae (strain NIH 41) as revealed by GLC is

shown in Fig. 4. Major fatty acids were myristic (^4)/,

palmitic (C16), hexadecenoic (c16_)/ stearic (C1Q), and

octadecenoic (C2g=) acids. No cyclopropanes were found.

NIH 41 fatty acids were injected into the gas chromatograph

ten consecutive times and analyses made in order to deter-

mine GLC reproducibility (Table V). Average percent seemed

less significant than range obtained. Fatty acids comprising

over 30% of the total deviated less than 5% from the average

while low values deviated considerably. Subsequent reports

20

TABLE IV

PERCENT YIELD OP THE SIMMONS-SMITH REACTION AS DETERMINED BY GAS CHROMATOGRAPHY

Reactant Product Percent Yield

methyl palmitoleate methyl cis-9,10-methylene hexade-canoate 69

methyl oleate methyl cis-9,10-methylene octa-decanoate 53

methyl cis-vaccenate

methyl cis-11,12-methylene octa-decanoate 51

21

So I v e n t

Fig, 4—Gas-liquid chromatogram of fatty acid methyl-esters of V. cholerae strain NIH 41. Column (10 ft. x 0.125 in.) was operated at 180 C (isothermally).

22

TABLE V

GAS CHROMATOGRAPHIC REPRODUCIBILITY USING ONE SAMPLE OF FATTY ACID METHYLESTERS (NIH 41)

ANALYZED TEN TIMES

Fatty acida Per cent found (average) Range

14:0 3.5 3.4 - 3.8

iso 16:0 1.0 0.5 - 1.1

16:0 32.6 31.1 - 33.6

16:1 39.8 38.2 - 40.8

17:0 1.1 0.7 - 1.4

iso 18:0 i. 4 0.4 - 2.5

18:0 4.0 3.5 - 4.7

18:1 15.8 14.8 - 16.8

aNumber preceding colon indicates number of carbons, and number following designates degree of unsaturation.

23

of cholera lipids did not include fatty acids found to be

less than 1% of the total.

Brian and Gardner reported on the use of hydrogenation

followed by bromination for the detection of cyclopropane-

ring-containing fatty acids in bacterial lipids (16). Table

VI shows the effects of hydrogenation of methylesters of

fatty acids of E. coli and V. cholerae. Unsaturated com-

pounds were converted to the corresponding saturates.

Cyclopropane fatty acids (eye 17:0 and eye 19:0) were not

affected by hydrogenation but were eliminated from gas

chromatograms following bromination (16). Brominated fatty

acid methylesters were retained by the gas chromatographic

column, and their elution was not observed. The cholera

vibrio did not appear to contain cyclopropanes as major

lipid constituents, thus indicating the absence of the

cyclopropane fatty acid synthetase enzyme (71). Fatty

acids of E. coli (Table VI) were comparable to those found

by other workers (28, 29, 33, 36, 59, 60, 70, 78, 94, 106).

The small amounts of branched-chain compounds (iso 16:0 and

iso 18:0) found in V. cholerae lipids (16) appeared to be

absent in E. coli.

Tables VII and VIII give fatty acids of a wide range

of cholera and non-cholera vibrios. Qualitatively, the

distributions of acids are identical. Quantitatively, little

differences exist. Fatty acids reported here are similar to

24

TABLE VI

EFFECTS OF HYDROGENATION ON PER CENT FATTY ACID METHYLESTERS

Fatty acida Rtb E. coli V. cholerae Rtb Before After Before After

14:0 0.56 3.8 4.2 3.9 4.6

15:0 0.74 1.2 1.3 t t

iso 16:0 0.87 _d 2.0 2.3

16:0 1.00 43.8 46.2 29.5 62.8

16:1 1.18 2.1 - 34.2 -

17:0 1.34 1.4 t 1.2 1.9

eye 17:0C 1.54 20.9 22.0 - -

iso 18:0 1.56 - - 1.8 t

18:0 1.82 te 7.6 3.1 26.8

18:1 2.08 7.2 - 22.9 -

eye 19:0C 2.82 18.8 17.8 - -

3 Number preceding colon indicates number of carbons, and number following designates degree of unsaturation.

•^Retention time relative to 16:0 (palmitic acid).

cCyclopropane fatty acids (eye 17:0 and eye 19:0) were resistant to hydrogenation but reacted with bromine.

^No peak detected.

et = trace (less than

25

TABLE VII

FATTY ACID DISTRIBUTION OF VIBRIO CHOLERAEa

Strain 14: 0 iso 16:C I 16: 0 16: Fatty Acid*3

1 17:0 iso 18:< D 18 i:0 18: 1

NIH 41 3. 9 2. O 29. 5 34. 2 1.2 1. 8 3. 1 22. 9

NIH 35 A3 4. .4 1. 0 37. 7 36. 9 t c 1. 1 3. 1 14. 7

ATCC 14035 3. ,3 2. 7 28. ,3 42. , 3 1.0 1. 5 2. 6 17. 0

569 B 1. ,7 2. 6 24. ,8 40. ,2 t 2. 5 3. ,1 24. 6

Ca 72 2. ,2 t 38. ,0 39. ,5 1.1 1. 1 2. 18 13. 2

Ca 113 4. ,7 1. 6 37. ,2 36. ,2 t t 2. ,5 17. ,5

Ca 323 3. ,3 1. 8 30. .1 42. ,0 1.1 1. 8 3. ,3 15. ,0

Ca 324 6. ,4 t 34. ,5 41. ,0 t t 3. ,3 12. ,7

Ca 325 9. .0 t 38. ,6 37. .5 t t 2. ,6 12. .0

Ca 385 1. .3 t 26. .0 43. .4 t t 3. .7 23. .9

Ca 412 4. .0 1. 5 33. .0 39. .0 1.2 1. 5 3. .7 15. .0

Ca 424 4. .1 1. 7 34. A 38. .2 1.2 1. 5 2. .9 14. .8

Iran 46 1. .8 2. 0 24, .3 41. .0 t 2. 0 2. .8 25. .0

Iraq 230 2. .0 2. 2 23, .3 40, .0 t 2. 1 3. .5 25, .6

B 29112 2. .0 2. 0 25, .0 41, .0 t 1. 0 3, .0 25, .0

B 1307 3, .0 3. 0 24, .0 44, .0 1.0 2. 0 2, .0 20, .0

J 8001 3, .7 1. 0 28, .0 40, .9 t 1. 0 2, .4 21, .6

ROK 350 2, .1 3. 4 22, .0 39, .7 1.0 2. 6 3, .6 24, .6

M 3735 2, .6 2. 0 22, .6 42, .0 t 2. 1 2, .9 24, .0

28/62 1, .4 2. 0 21, .4 39, .2 1.0 2. 5 5, .0 26. .4

VC 12 Rxl 2, .7 2. 5 23, .6 46, .0 1.1 2. 5 2, .5 18, .0

26

TABLE VII—Continued

Strain 14:0 iso 16:0 16:0 16:1 17:0 iso 18:0 18:0 18:1

El 36 1.7 2.5 24.6 40.3 t 1.7 3.5 25.0

CH-1 2.8 1.8 27.5 41.0 1.1 1.7 3.9 18.8

Ubon 13 1.5 2.7 23.2 41.8 t 2.2 2.8 24.6

0-4 2.9 2.2 27.6 44.6 t 1.7 1.7 17.7

VN-1 1.5 2.1 22.9 39.6 t 2.3 4.0 26.1

CRC 31/64 4.3 1.4 29.3 44.8 t 1.0 2.6 15.4

1222 1.8 1.8 24.2 38.1 t 1.8 4.2 26.5

HK-1 1.8 1.8 22.4 44.3 t 1.3 2.8 24.6

V 86 2.1 2.7 28.4 40.5 1.0 2.0 4.2 18.0

<3.

Percent of each fatty acid.

ID Number preceding colon indicates number of carbons, and number following colon designates degree of unsaturation. c t = trace (less than 1% of total).

21

TABLE VIII

FATTY ACID DISTRIBUTION OF NON-CHOLERA VIBRIOS9

Strain 14:0 iso 16:0 16:0 Fatty Acid13

16:1 17:0 iso 18:0 18:0 18:1

8032 2.4 1.2 26.0 40.0 1.0 1.6 4.8 22.8

485 2.0 3.6 25.2 36.8 1.0 2.1 4.0 24.0

942 2.0 1.0 23.3 35.6 1.2 2.3 9.8 24.2

8288 2.5 2.3 28.8 37.0 1.2 2.2 3.9 21.2

8305 1.6 1.4 26.1 38.8 1.0 1.6 4.4 24.4

6471 4.1 1.3 33.2 41.4 t c t 2.8 14.4

9682 1.6 2.8 25.6 35.8 1.0 2.8 4.9 24.3

aPer cent of each fatty acid.

^Number preceding colon indicates number of carbons, and number following designates degree of unsaturation.

°t = trace (less than 1% of total).

28

those reported for Vibrio metchnikovii (28), Vibrio fetus

(101), and the halophile Vibrio costicolus (67).

Although composition of the culture media is known

to affect bacterial lipids (33, 38, 66, 70, 100), no factor

affects lipid and fatty acid more profoundly than incubation

temperature (7, 23, 65, 70, 78, 83, 94). Table IX gives

changes in fatty acid content of V. cholerae strain NIH 41

grown at 25, 32, and 37 C. As temperature of incubation

increases, unsaturation decreases. Hexadecenoic acid (16:1)

is the major fatty acid at incubation temperatures of 37 C

and below. No other bacterial species has been reported to

contain such a large amount of 16:1 under these growth con-

ditions. It has been suggested that fatty acid profiles may

be used for bacterial identification (2, 61, 85, 87, 98).

In order to utilize effectively GLC data for identification

purposes, incubation temperature, as well as other cultural

conditions, must be rigorously controlled.

Of all the cholera vibrios studied to date, only the

rugose morphological mutant (10, 22, 108) has been found to

contain cyclopropane fatty acids (17, 19). It was suggested

that cyclopropanes may play a role in the survival mechanism

of these resistant variants (17).

Distribution of fatty acids in polar (phospholipid)

and neutral (fatty acid) lipids separated by TLC (Table X)

was investigated (18). The major acids of the phospholipid

fraction were palmitate (47-50%), hexadecenoate (22-24%),

29

TABLE IX

EFFECTS OF INCUBATION TEMPERATURE ON FATTY ACID DISTRIBUTION (PERCENTAGES) OF NIH 41

Fatty Acida

25 C Incubation Temperature

32 C 37 C

14:0 1.7 2.1 3.3

iso 16:0 t b 1.0 1.0

16:0 16.4 21.8 31.2

16:1 51.0 41.5 40.3

17:0 t t 1.1

iso 18:0 t 1.9 1.2

18:0 t 3.1 3.9

18:1 29.5 26.7 16.9

3 Number preceding colon indicates number of carbons, and number following designates degree of unsaturation.

= trace (less than 1% of total).

30

TABLE X

PER CENT FATTY ACIDS IN LIPID FRACTIONS

Extractable lipids Polar lipids Neutral lipids Fatty acida (phospholipids)

NIH 41 Ca 324 NIH 41 Ca 324 NIH 41 Ca 324

14:0 3.3 6.4 3.5 7.4 2.9, 4.5

iso 16:0 1.0 t b 1.0 t t t

16:0 31.2 34.5 46.8 50.2 13.9 13.3

16:1 40.3 41.1 22.3 23.6 64.8 66.7

17:0 1.1 t 1.5 t t 1.5

iso 18:0 1.2 t 1.3 1.0 t t

18:0 3.9 3.3 6.0 5.1 1.6 1.7

18:1 16.9 12.7 16.5 11.8 13.8 10.9

aNumber preceding colon indicates number of carbons? and number following designates degree of unsaturation.

b t = trace (less than 1% of total).

31

octadecenoate (12-17%), sterate (5-6%), and myristate (4-7%).

The major free fatty acid was hexadecanoate (65-67%). If a

phospholipase was responsible for the large amount of free

fatty acid found, the enzyme activity appeared to favor

hydrolysis of hexadecenoic acid.

This report has shown that the fatty acid distribu-

tions from a brpad spectrum of cholera vibrios were similar

but were different from those reported in other bacterial

species. The feasibility of utilizing such data in the

chemical-taxonomic characterization and identification of

these pathogenic microorganisms is thus suggested.

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78. Marr, A. G., and J. L. Ingraham. 1962. Effect of temperature on the composition of fatty acids of E. coli. J. Bacterid. 84:1260-1267.

79. Marsh, J. B., and D. B. Weinstein. 1966. Simple charring method for determination of lipids. J. Lipid Res. 7:574.

80. Metcalfe, L. D., A. A. Schmitz, and J. R. Pelka. 1966. Rapid preparation of fatty acid esters from lipids for gas chromatographic analysis. Anal. Chem. 38:514-515.

39

81. Neilsen, H. S., Jr. 1966. Variation in lipid content of strains of Histoplasma capsulatum exhibiting dif-ferent virulence properties for mice. J. Bacterid. 91:273-277.

82. Nesbitt, J. A., Ill, and W. J. Lennarz. 1965. Com-parison of lipids and lipopolysaccharide from the bacillary and L forms of Proteus P18. J. Bacterid. 89:1020-1025.

83. Okuyama, H. 1969. Phospholipid metabolism in Escherichia coli after a shift in temperature. Biochim. Biophys. Acta 176:125-134.

84. O'Leary, W. M. 1959. Involvement of methionine in bacterial lipid synthesis. J.Bacteriol. 78:709-713.

85. O'Leary, W. M. 1962. The fatty acids of bacteria. Bacterid. Rev. 26:421-447.

86. O'Leary, W. M. 1962. S-adenosylmethionine in the biosynthesis of bacterial fatty acids. J. Bacterid. 84:967-972.

87. O'Leary, W. M. 1967. The chemistry and metabolism of microbial lipids. The World Publishing Co., New York.

88. Park, E. E., and L. R. Berger. 1967. Fatty acids of extractable and bound lipids of Rhodomicrobium vannielii. J. Bacterid. 93:230-236.

89. Pierce, N. F., and W. B. Greenough, III. 1970. Stimu-lation of glycerol production in fat cells by cholera toxin. Nature 226:658-659.

90. Pohl, S., J. H. Law, and R. Ryhage. 1963. The path of hydrogen in the formation of cyclopropane fatty acids. Biochim. Biophys. Acta 70:583-585.

91. Romera, E. M., and R. R. Brenner. 1966. Fatty acids synthesized from hexadecane by Pseudomonas aeruginosa. J. Bacteriol. 91:183-188.

92. Selkirk, J. K., J. C. Elwood, and H. P. Morris. 1971. Study on the proposed role of phospholipid in tumor cell membrane. Cancer Res. 31:27-31.

93. Shank, R. S., and H. Schecter. 1959. Simplified zinc-copper couple for use in preparing cyclopropanes from methylene iodide olefins. J. Org. Chem. 24:1825-1826.

40

94. Shaw, M. K., and J. L. Ingraham. 1965. Fatty acid composition of Escherichia coli as a possible controlling factor of the minimal growth temperature. J. Bacterid. 90:141-146.

95. Simmons, H. E., and R. D. Smith. 1959. A new synthesis of cyclopropanes. J. Am. Chem. Soc. 81:4256-5264.

96. Smith, H. L., Jr., and K. Goodner. 1965. On the classification of vibrios. Cholera Research Symposium, Honolulu, Hawaii, U. S. Government Printing Office, Washington, D. C., pp. 4-8.

97. Stahl, E. 1969. Thin-layer chromatography. (A laboratory handbook.) Springer-Verlag, New York.

98. Steinhauer, J. E., R. L. Flentge, and R. V. Lechowich. 1967. Lipid patterns of selected microorganisms as determined by gas-liquid chromatography. Appl. Microbiol. 15:826-829.

99. Supina, W. R. 1964. Analysis of fatty acids and derivatives by gas chromatography, pp. 271-305. In H. A. Szymanski, Biomedical applications of gas chromatography. Plenum Press, New York.

100. Tornabene, T. G., E. 0. Bennett, and J. Oro. 1967. Fatty acid and aliphatic hydrocarbon composition of Sarcina lutea grown in three different media.

101. Tornabene, T. G., and J. E. Ogg. 1971. Chromatographic studies of the lipid components of Vibrio fetus. Biochim. Biophys. Acta 239:133-141.

102. Tornabene, T. G., and J. Oro. 1967. 14C incorporation into the fatty acids and aliphatic hydrocarbons of Sarcina lutea,, J. Bacterid. 94:349-358.

103. Vaczi, L., J. K. Makleit, A. Rethy, and I. Redai. 1964. Studies on lipids in Pseudomonas pyocyanea. Acta Microbiol. 11:383-390.

104. Walder, B. L. 1971. A novel charring technique for detection of lipids on thin-layer chromatograms. J. Chromatog. 56:320-323.

105. Walsh, D. E., 0. J. Banasid, and K. A. Gilles. 1965. Thin-layer chromatographic separation and colori-metric analysis of barley or malt lipid classes and their fatty acids. J. Chromatog. 17:278-287.

41

106. Weinbaum, G., and C. Panos. 1966. Fatty acid distribu-tion in normal and filamentous Escherichia coli. J. Bacteriol. 92:1576-1577.

107. White, D. C., and F. E. Frerman. 1968. Fatty acid composition of the complex lipids of Staphylococcus aureus during the formation of the membrane-bound electron transport system. J. Bacteriol. 95:2198-2209,

108. White, P. B. 1938. The rugose variant of vibrios. J. Pathol. Bacteriol. 46:1-6.

109. Zalkin, H., J. H. Tav, and H. Goldfine. 1963. Enzymatic synthesis of cyclopropane fatty acids catalyzed by bacterial extracts. J. Biol. Chem. 238:1242-1248.

CHAPTER II

PHOSPHOLIPASE ACTIVITY OP VIBRIO CHOLERAE

Introduction

Lipid extracts of Vibrio cholerae have been shown to

contain large amounts of free fatty.acids and phospholipids

(5). The possibility that phospholipase activity had

occurred prior to lipid extraction was suggested by the high

free fatty acid content (5) since bacterial lipids are known

to consist chiefly of complex lipid molecules.

The purpose of this study was to determine if

phospholipase activity exists in typical V. cholerae which

is capable of cleaving fatty acids from cholera phospholipids.

The existence of such phospholipase activity might be ample

reason for rapid extraction of cells with lipid solvents.

Phospholipids of bacteria would be rapidly hydrolyzed by an

active phospholipase if cells were allowed to remain for

long periods of time in aqueous solution.

Phospholipases have been reviewed by several workers

(1, 20, 23, 27). Enzymes which remove one fatty acid from

a phospholipid are designated phospholipase A (EC 3.1.1.4).

Phospholipase A (phosphatide acyl-hydrolase) is of two types:

phospholipase A.̂ which cleaves fatty acid from the 1-

position of a phosphoglyceride, has been studied in human

42

4 3

and beef pancreas (27) as well as in rat and calf brain (18);

the enzyme specifically designated phospholipase A2, found in

Crotalus atrox (Western Diamondback rattlesnake)' and Crotalus

adamanteus (Eastern Diamondback rattlesnake) venoms, cleaves

the fatty acyl group from the 2-position of a phosphoglyceride

(10, 19, 21, 40, 43, 47-49) to give a lysophosphoglyceride.

This enzyme is also found in bee, wasp, and scorpion venoms

(23) .

Phospholipase B (EC 3.1.1.5) or lysophospholipase (9,

25, 38) cleaves either both fatty acids from a phosphoglycer-

ide (in some cases, in conjunction with phospholipase A) or

the remaining fatty acid from a lysophosphoglyceride.

Lysophosphoglycerides are known to be powerful hemolytic

agents (20).

Phospholipase C (EC 3.1.4.3, phosphatidyl choline t

cholinephosphohydrolase), found in culture supernatants of

Pseudomonas fluorescens (11), Bacillus cereus (50), and

Clostridia (27), hydrolyzes phosphatidyl choline to

digylceride and phosphorylcholine. The substrate specificity

of this enzyme differs with the enzyme source. This enzyme

(Clostridium perfrinqens) was said to be hemolytic and lethal

(27), and its action on artificial membranes (22) resulted

in decreased membranes resistance.

Phospholipase D (EC 3.1.4.4, phosphatidyl choline

phosphatidohydrolase), found in cabbage, spinach, and sugar

44

beet plastids, cleaves choline from phosphatidyl choline to

give phosphatidic acid and free choline (27). Several

phosphoglycerides act as substrate. An unusual phospholipase

D was found (32, 33) in Haemophilus parainfluenzae which

hydrolyzed cardiolipin to phosphatidic acid and phosphatidyl

glycerol.

Phospholipases found in bacteria (other than those

mentioned above) include phospholipase A in Escherichia

coli (3/ 12, 17, 31, 36, 41), Salmonella typhimurium (3),

and Bacillus megaterium (37), and lysophospholipase in E.

coli (12, 35) and Mycoplasma laidlawii (44). These enzymes

were reported to be membrane-bound.

Materials and Methods

Cultural Conditions. Vibrio cholerae strain 569 B

(Inaba), received from R. A. Finkelstein (The University of

Texas Southwestern Medical School, Dallas, Texas), was used

in this study. A large number of cholera workers (8, 13-16,

26, 34) have utilized strain 569 B in studies of V. cholerae

Type 2 (exo—entero) toxin (6). Bacteria were maintained in

the lyophilized form or on slants (2 C) of Trypticase Soy

Broth (BBL) with 2% agar (Difco).

Cells were grown in Trypticase Soy Broth (TSB) or in

Roux flasks containing 100 ml of TSB with 1% agar. One ml

of a sixteen hour (37 C) broth culture was used to inoculate

each liter of broth or each Roux flask. Broth cultures (one

45

to ten liters) were incubated (37 C) until the late station-

ary growth phase as determined by optical density using a

Spectronic 20 colorimeter (540 nm). Bacteria were

harvested in 250 ml bottles by centrifugation (5000 x g,

15 min, 4 C) using a Sorvall RC-2B refrigerated centrifuge

equipped with a GSA rotor. Roux flasks were incubated 37 C,

24 hr. Cells were harvested by washing the agar surface

with distilled water followed by centrifugation. Bacteria

were streaked for isolation onto Petri plates containing

agar media both before and after initial inoculation as well

as after harvest to determine presence of contaminating

microorganisms. If no contamination was apparent, cholera

enzyme solutions were prepared.

Cholera Phospholipase Preparations. V. cholerae cells

harvested by centrifugation were diluted in distilled water

(3.2-6.0 mg/ml protein) and sonicated (6-9 amps, D. C.) for

5 min while in an ice bath. Temperature was not allowed to

exceed 10 C. The sonicate was diluted 1:1 with 0.05 M

borate buffer, pH 7.5. "Roux flash supernatant," pH 6.0,

was prepared by freezing Roux media (-12 C) after most cells

had been removed by washing. Thawed media yielded ca 50 ml

of liquid per Roux flask (6-8 mg/ml protein) and was assayed

for phospholipase activity directly. Protein determinations

were made by the Lowry procedure (28) using bovine serum

albumin as the standard.

46

Thin-layer Chromatography. Thin-layer chromatography

(TLC) was performed using 20 x 20 cm, 5 x 20 cm, and 1 x 3

inch glass plates. Silica gel G (Curtin Chemical Company)

aqueous slurries were spread onto plates with a thickness

of 0.25-0.5 mm and dried overnight at 26 C. Ammonium sul-

fate (1%, weight/volume) was added to the aqueous slurry

when sample charring, following the TLC separation, was

desired (30, 45).

TLC-separated lipids were visualized by iodine vapors

or by heating TLC plates (220 C) which contained ammonium

sulfate. Sulfuric acid which results from this treatment

effectively decomposes lipids to visible carbon spots.

Phosphatidyl ethanolamine (PE) was detected by spraying TLC

plates with 0.3% ninhydrin in acetone. Phosphatidyl glycerol

(PG) was detected with periodate-schiff reagent (24), and

Dragendorff reagent (42) was used to detect phosphatidyl

choline (PC). Standards of PC and PE were purchased from

Applied Science Laboratories and cardiolipin (CL) from

Supelco, Inc.; PG was produced by reaction of PC with

cabbage transphosphatidylase in the presence of 20% glycerol

(27). Oleic acid, triolein, methyl oleate, and cholesterol

oleate were from Applied Science Laboratories.

TLC solvent systems used were:

Solvent A (46)—Petroleum ether: diethyl ether:

glacial acetic acid (90:10:1, volume/volume),

47

Solvent B (29)—Chloroform: methanol: water

(60:30:5, volume/volume), and

Solvent C (27)—Chloroform: methanol: glacial

acetic acid: water (80:13:8:0.3, volume/volume).

Crotalus atrox Phospholipase A^. C. atrox (Western

Diamondback rattlesnake) venom was obtained from J. Smith

(N. T. S. U.) and lyophilized. Ten mg was placed in 100 ml

of buffer (20, 21) containing (per liter) 0.372 g EDTA, 2.22

g calcium chloride, and 12.8 g sodium chloride. The pH was

adjusted to 7.5 with 0.1 N potassium hydroxide.

The enzyme assay (20) consisted of titration of fatty

acids released after incubation (room temperature) of known

quantities of PC or PE (in 2 ml diethyl ether) with 25 ul of

enzyme solution using phenol red as indicator. A 100 ul

syringe was used to deliver known quantities of 0.02 N sodium

hydroxide. Standard lysophosphatidyl ethanolamine (LPE) was

synthesized by this enzyme system from bacterial PE (Calbiochem)

LPE purity was determined by TLC (solvent B).

Cabbage Phospholipase D. Inner cabbage leaves were

washed with distilled water, and plastids were extracted from

200 g (wet leaves) using methods described by Lowenstein (27).

Plastid preparations were lyophilized. Total yield was 450

mg dry weight.

The assay system consisted of 10 mg plastids, 0.25 ml

48

of 1 M calcium chloride in 1.25 ml of 0.2 M acetate buffer,

pH 5.6, and 1 ml of diethyl ether solution with 15 umoles

PC. Following incubation at 25 C, the aqueous phase was

reacted with ammonium reineckate (27) and optical density

determined at 526 nm with a Spectronic 20 colorimeter.

Cabbage plastid transphosphatidylase activity was utilized

to synthesize PG using the above reaction (15 umoles PC)

with 20% glycerol in the aqueous phase (pH 5.6). PG was

purified by elution through heat-activated (110 C, 1 hr)

silicic acid (39) with acetone. Purity was established by

TLC (solvents B and C), and the presence of PG was determined

by periodate-schiff reagent (83).

Preparation of Purified Egg Yolk Phosphatidyl Choline

(PC). Six chicken eggs were used to prepare purified PC.

Yolks (100 ml) were placed in a 500 ml glass—stoppered

graduated cylinder, and lipid was extracted (27) with

chloroform: methanol (2:1, volume/volume). The lipid extract

was dissolved in chloroform, and phospholipids were precipi-

tated with ten volumes of acetone (repeated several times) at

2 C. The acetone-insoluble egg yolk phospholipid was dis-

solved in 10 ml chloroform: methanol (1:1, volume/volume).

A 2.5 x 30 cm glass chromatography column (teflon stopcock)

packed with 10 g aluminum oxide G (Merck) was used to purify

PC. The phospholipid mixture was placed on the top of the

49

alumina bed and elution of PC effected by addition of chloro-

form-methano1 (10 p.s.i. nitrogen pressure) until Dragendorff-

positive material ceased to elute. PE (a major egg yolk

phospholipid) failed to elute from alumina. PC purity was

determined by TLC in solvent B. Purified PC was quantitated

by phosphorus analysis (2). PC was also purified by silicic

acid column chromatography (39), but this method was more

difficult and time-consuming.

Preparation of Radioactive Substrates. Phosphorus-32

(32P)-labeled phospholipids were prepared by growing E. coli

(ATCC 11775) in the presence of 32P disodium hydrogen

32

phosphate (Na2H P04) purchased from International Chemical

and Nuclear Corporation. One liter (distilled water) con-

taining 10 g Bacto peptone (Difco) and 0.5 mM Na2HP0^ was

inoculated with bacteria and incubated at 37 C. After 32

visible turbidity was apparent, 5 mC of Na2H PO^ (0.5 ml)

was added and incubation continued for two hours. Radio-

active cells were harvested as previously described by

centrifugation. 14

Carbon-14 ( C)-labeled phospholipids were prepared

as above using ^ C sodium acetate (500 uC) purchased from

New England Nuclear. 32 14

Phospholipids ( P and C) were extracted from cells

in 16 x 150 mm test tubes with teflon-lined screw-caps by

the Bligh and Dyer method (4). Chloroform extracts were

50

evaporated (vacuum rotary evaporator), taken up in chloro-

form, and analyzed for phosphorus content (2).

32? radioactivity was assayed by drying samples on

planchets and reading directly with a Geiger-Muller tube and

scaler. 14C was assayed with a Beckman liquid scintillation

counter in scintillation vials containing 10 ml of the

following fluid: toluene (1 liter), 5 g PPO, and 130 mg

POPOP (27) .

Purified E. coli "^PE and "^PG as well as "^C PE

were obtained by separating radioactive phospholipids by TLC

in solvent C. Separated components were eluted into 10 ml

chloroform: methanol (1:2, volume/volume); solvent was

evaporated; and the isolated compounds were analyzed for

phosphorus content.

Total "^P phospholipids were separated in solvent C

and visualized by iodine vapors; spots were scraped onto

planchets and read directly with a geiger counter to deter-

mine percentage of label in "^PE, "^PG, and "^P CL.

Cholera Phospholipase Assay. Phospholipid ("^P)

preparations (0.084-4.81 umoles P) were placed into 16 x 150

mm screw-cap test tubes, and solvent was evaporated with a

stream of nitrogen (30 C). One ml of a 2% solution of Triton

X 100 in water was added to each tube and vortexed for 5 min.

Radioactivity in solution was assayed and compared to chloro-

form solutions. Not less than 95% of the radioactive label

51

was rendered water-soluble in the presence of Triton X 100.

The cholera phospholipase assay system consisted of

1 ml of enzyme preparation ("Roux flask supernatant" or 09 14.

sonicate in 0.025 M borate) plus 50 ul of P or C

phospholipid in Triton X 100 solution. After addition of

phospholipid, tubes were vortexed for 1 min and incubated

at 37 C. Following incubation, lipids were partitioned into

a chloroform phase by the Bligh and Dyer method (4) as

modified by Scandella and Kornberg (41). To the 1 ml of

enzyme reaction mixture was added 2.25 ml methanol and 1 ml

chloroform. Vortexing resulted in a single phase. After

standing (26 C) for 15 min, 1 ml chloroform and 1 ml water

were added and mixed. The resulting two-phase system was

centrifuged 5 min (International Clinical Centrifuge) at 32

maximum speed. Water-soluble P was determined by evaporat-

ing 1 ml of aqueous phase on planchets and counting with a

Geiger-Muller tube and scaler. Tubes containing buffer (no

enzyme), but with 50 ul of labeled phospholipid and incubated

under identical conditions as enzyme solutions, served as

controls. Control readings were subtracted from those

obtained with enzymes since controls indicated background

radioactivity plus ubiquitous water-soluble label. Counts

per min (CPM) thus obtained were compared with CPM obtained

by reading 50 ul (100%) of phospholipid (32P) utilized in

each test.

52

The 14C assay of chloroform-soluble products was

more difficult and was used primarily to identify products.

14 After reaction of cholera phospholipase with C phospholipid

*

and Bligh and Dyer lipid extraction (4), the chloroform phase

was evaporated and taken up in 100 ul chloroform. Ten ul of

lipids were spotted on TLC plates and separated in solvent A.

Free fatty acid and phospholipid were assayed for radio-

activity. Solvent B was used to separate (TLC) both -^C and

32

P chloroform-soluble compounds in an effort to determine

presence of LPE as a reaction product. Standards (PE and LPE)

were spotted with samples to be separated and visualized by

ninhydrin spray.

Results and Discussion

Egg Yolk Phosphatidyl Choline (PC). Separation of

egg yolk phospholipids by TLC in solvent B is shown in Fig. 5

(left). Major components were PE and PC. Also shown is the

PC which was purified by aluminum oxide column chromatography

(right). No PE eluted from the alumina column, but a small

amount of neutral lipid was present. PC was used as substrate

f o r atrox phospholipase A2 and cabbage phospholipase D

assays without further purification.

PC as well as other phospholipid substrates were

quantitated by phosphorus (P) analyses (2). Optical density

(830 nm) readings using a Cary 14 spectrophotometer were

made with 0.025-0.15 umoles of KH2P04. Optical density

53

Solvent.

0 PE

0 0 PC

O r i g i n

Fig. 5-7 Thin-layer chromatogram of major egg yolk phospholipids (left) and purified phoaphatidyl choline (right) Solvent was chloroform: methanol: water (60:30:5, volume/ volume).

5 4

(0. D.) was a linear function of P concentration. When the

Cary 14 instrument was not available, readings were made with

a Beckman spectrophotometer at 735 nm where 0. D. values were

1/2 of those at 830 nm. Duplicate samples of phospholipids

were analyzed by the Bartlet procedure (2) and quantitated

by comparison to similar standard curves.

Crotalus atrox Phospholipase A2. Results of incuba-

tion (22 C) of 10 umoles of.PC with C. atrox venom is shown

in Fig. 6. The reaction appears to proceed in a linear

fashion for at least 20 min. The products of the reaction

were lysophosphatidyl choline (LPC) and free fatty acids.

Fatty acids were released at an initial velocity of 50 umoles/

min/mg of venom under these conditions.

Substrate (PC) was increased from 5-50 umoles and the

venom enzyme allowed to react 10 min at 27 C. Hydrolysis

rate increases were linear until at least 20 umoles of PC

were present (Fig. 7) . Enzyme satiation with substrate was

never reached, but initial velocities began to decrease

before 30 umoles PC.

The purpose of investigating the snake venom phospho-

lipase A2 was to develop techniques for production of LPE

using PE as substrate. Bacterial PE (20 umoles) was reacted

with 2.5 ug of venom (1 hr, 25 C), and LPE was purified by

precipitation from cold ether (27). Fig. 3 shows a TLC

separation of PE and LPE (solvent B). Ether-insoluble LPE,

55

40 Min u f e s

Fig. 6—Assay of Crotalus atrox venom (2.5 ;ug) phospho-lipase A2 activity on phosphatidyl choline (10 jumoles) with time (22 C).

56

20 30 40 Phospha t idy l c h o l i n e (Mmoles)

Fig. 7—Assay of Crotalus atrox venom (2.5 jig) phospho-lipase A2 activity on 5-50 jumoles of phosphatidyl choline (10 min, 27 C) .

57

S o l v e n t

Origin

Fig. 8—Thin-layer chromatogram of bacterial phospha-tidyl ethanolamine (PE) and lysophosphatidyl ethanolamine (LPE) prepared by reaction of PE with Crotalus atrox venom. Solvent was chloroform: methanol: water (60:30:5, volume/ volume). Similar separations were used in attempts to detect 14C or 32P LPE following cholera phospholipase reactions.

58

after several ether washings, gave only one ninhydrin-

positive spot by TLC.

Cabbage Phospholipase D. 0. D. readings (526 nm)

following reaction of ammonium reineckate with 4-20 umoles

of choline hydrochloride are shown in Fig. 9. Linear 0. D.

responses were achieved over the determined range of choline.

Similar standard curves were used to quantitate the cabbage

plastid (10 mg) phospholipase D after reaction with 15 umoles

of PC (Table XI). Initial velocity under the conditions

employed appeared to be 2.8 unmoles/min/10 mg plastids.

Products of the reaction were phosphatidic acid (purified

by methanol precipitation, 2 C) and free choline.

The purpose of investigating phospholipase D was to

develop techniques for the production of LPC from PC by

utilizing the transphosphatidylase (27) activity of the

cabbage enzyme. If a large concentration (20%) of an alcohol

is placed in the water phase, choline is liberated from PC,

and the alcohol is substituted. PG was synthesized using

this system by reacting 10 mg of plastid preparation with

15 umoles PC in a 20% solution of glycerol (pH 5.6) for 2

hr, 25 C. Choline analysis indicated 12 umoles of choline

were released. Separation of reaction products by TLC

(solvent B) revealed a new spot which was periodate-schiff

positive (24), thus indicating presence of phosphatidyl

59

.5

.4"

i c .3 <o cs •o

D O*

..2

6 8 10 12 14 C h o l i n e ( M m o l e s )

16 "20

fr,l ,„Fl9' 9 Optical density (0. D.) readings (526 mil) following reaction of 4-20 Mmoles of choline hydrochloride with ammonium reineckate (average of two determinations).

60

TABLE XI

PHOSPHOLIPASE D ASSAY

Time (min) 0. D. (526 rati) jumoles Choline Released

10 .060 2.8

20 .114 5.4

30 .137 ' 6.4

40 .155

CN

I •

A

50 .187 00 t

61

glycerol. Silicic acid column chromatography (39) was used

to purify PG.> The only TLC spot Which eluted from silicic

acid with acetone was PG. No ninhydrin-positive (PE) or

Dragendorff-positive (PC) material was present.

Radioactive Substrates. E. coli 32P- ancj ^c-labeled

lipid was spotted on TLC plates and separated in solvent C

(Fig. 10).. Geiger counter readings of spots obtained by Op

iodine vapors suggested that 95% of P label was in PE, 5%

in PC, and less than 1% in CL. Similar separations were used

32 3 o 14 to prepare purified PG, °^PE, and C PE. Since the vast

majority of label was in 32PE, crude phospholipid extracts

were utilized in some enzyme assays, and results were similar

to those using purified 32PE.

Cholera Phospholipase. Initial cholera phospholipase

studies were performed using 32PG (4.2 nmoles) and sonicate

with 3 mg/ml protein (Table XII). Since products of the

32

reaction were unknown, PG in the assay system would have

detected phospholipase A (lysophosphatidylglycerol is water-

soluble), lysophospholipase, and phospholipase C activities.

All would have given water-soluble 32P but different

chloroform-soluble products. Product was formed at a linear

rate until ca 25% of 32PG was utilized. An overnight reaction

(21 hr) resulted in 43% of 32P in the water phase.

Chi or of orrn- sol ubl e products wore detected using

purified C PE (21 nmoles) as substrate followed by TLC

62

0

SoIvenf Neutral lipid

€1

SPG

P E

• i O r i g i n

32 Pig. 10—Thin-layer chromatogram of E. coli (ATCC 11775) P phospholipids. Solvent was chloroform: methanol: acetic

acid: water (80:13:8:0.3, volume/volume). Similar separations were used to determine per cent 32p in separated phospholipids as well as preparation of purified 32p phosphatidyl glycerol (PG), phosphatidyl ethanolamine (PE), and carbon-14-labeled phosphatidyl ethanolamine. Cardiolipin = CL.

63

TABLE XII

ASSAY OF VIBRIO CHOLERAE SONICATE FOR PHOSPHOLIPASE ACTIVITY

USING 32P PHOSPHATIDYL GLYCEROL AS SUBSTRATE

Hours (37 C) Product (nmoles)3

1 0.45

2 0.87

3 1.05

4 1.24

21 1.76 (43%)

^Average of duplicate tests.

r

64

separation (solvent A). Fig. 11 shows that chloroform-

soluble material corresponded to free fatty acid and phospho-

lipid. Scintillation counting of separated spots indicated

that 4.2 nmoles (ca 20% of CPM) was accounted for by free O p

fatty acid (a reaction rate comparable to P assay with

18.5 nmoles 32P). Phospholipids of both 32P and reaction

mixtures using purified PE were separated by TLC in solvent B

with standards to detect LPE. No LPE was found in 1 hr or

21 hr reactions, and the only radioactive phospholipid

detected was unreacted substrate (32PE and 14C PE). Thus it

appeared that both fatty acids of a phospholipid were removed

by the cholera phospholipase with no appreciable amount of

chloroform-soluble intermediate compound (LPE). The 32P

assay system thus became valid for use with 32PE as sub-

strate, since water-soluble 32P was essential for detection

of activity. Activity was achieved with "Roux flask supernatant"

material using purified 32PE (11 nmoles) as substrate. Water-32

soluble P was released from purified 3^pe a t j_nj_tial

rate of 2.3 nmoles/hr until 30% of substrate was utilized

(Fig. 12). Overnight reactions averaged 50-56% water-

soluble 32P.

Dilution of sonicate (3.2 mg/ml protein) with 0.5 M

borate buffer (pH 7.5) gave a 1.6 mg/ml enzyme solution which

was used to test effects of various parameters on enzyme

65

0

Solvent

C h o l e s t e r y 1 o l e a t e

Methyl o l e a t e

T r i o l e i n

O l e a t e

Origin ( P h o s p h o l i p i d )

Fig. 11—Thin-layer chromatograms of cholera phospho-lipase products (left) following reaction with -^c phospho-phatidyl ethanolamine (21 nmoles) for 1 hr, 37 C. Released fatty acid was 4.2 nmoles based on per cent "^C in unreacted substrate versus product. A standard lipid mixture (right) was used to determine products of the reaction. Solvent was petroleum ether: diethyl ether: acetic acid (90:10:1, volume/volume).

66

Hou rs

Fig. 12—Assay of Vibrio cholerae strain 569 B (Inaba) "Roux flask supernatant" (6 mg/ml protein) for phospholipase activity using substrate.

32 P phosphatidyl ethanolamine (11 nmoles) as

67

activity. Table XIII shows the effects of Tris-HCl, EDTA,

8-hydroxyquinoline, and dialysis of enzyme on activity using

8.3 nmoles of 32P substrate. EDTA and 8-hydroxyquinoline

had no effect, thus suggesting no divalent cation require-

ment. Dialysis of 2 ml sonicate (one liter borate buffer)

had no effect, which suggested the absence of loosely-bound

activators or inhibitors. Use of 0.1 M Tris-HCl buffer

(pH 8.0) also did not increase or inhibit phospholipase

activity. These results were surprising, since many phospho-

lipases require the divalent cation calcium (41). Calcium may

act by sequestering hydrolyzed fatty acids. Other divalent

cations were shown to be inadequate substitutes for calcium.

After observing that divalent cations were not neces-

sary for phospholipase activity by the cholera sonicate,

experiments were conducted to determine effects of such

cations on the enzyme. Table XIV shows the level of inhibition

of the cholera phospholipase by chloride salts of barium,

magnesium, manganese, zinc,•calcium, iron, and mercury at 1

and 10 mM concentrations using 25 mM borate buffer, pH 7.5.

No effect was observed, with KC1 or NaCl. Prior to this

investigation, mercuric chloride was shown to inhibit

phospholipase activity (41). Less than 15% inhibition of E.

coli phospholipase A^ was effected by 0.1 or 1 mM mercuric

chloride, while 0.08 M sodium chloride decreased activity

by 50% (41) . Increased ionic strength did not appear to

68

TABLE XIII

EFFECTS OF TRIS-HCl (pH 8.0), EDTA, 8-HYDROXYQUINOLINE

AND DIALYSIS ON REACTION OF SONICATE

Conditions of assay Product (nmoles)/hr

Borate buffer (0.025 M, pH 7.5) 2.1

Variables " 1 r - -- -1-1 *

EDTA (10 mM) 2.1

8-OH quinoline (1 mM) 1.8

Dialysis 1.9

Tris-HCl buffer (0.01 M, pH 8.0) 2.0

a Reaction time = 1 hr, 37 C.

69

TABLE XIV

PER CENT INHIBITION BY CHLORIDE SALTS (ONE AND TEN mM)

ON PHOSPHOLIPASE ACTIVITY OF V. CHOLERAE SONICATE

Cation Per cent inhibition

1 mM 10 mM

Potassium 0 0

Sodium 0 0

Barium 7 37

Magnesium 12 35

Manganese(ous) 44 68

Zinc 56 95

Calcium 80 95

Iron (ferrous) 37 100

Mercury 100 100

70

affect the cholera phospholipase (Table XIV). Further, buffer

concentration (6-50 mM) did not affect cholera phospho-

lipase significantly. Attempts at pH studies were hampered

by the large buffering capacity of the sonicate and must

await enzyme purification.

32 Since Triton X 100 was essential in making P and

14

C phospholipids water-soluble, the concentration of the

detergent for maximum enzyme activity was determined (Table

XV) using 8.3 nmoles substrate. Maximum activity was

achieved at 1 mg/ml detergent. Lower activity was evident

both below and above the 1 mg/ml level. Triton X 100 con-

centrations below the optimum level may not have been suffi-

cient to allow formation of phospholipid micelles necessary

for enzymatic activity. Above the optimum concentration,

Triton X 100 may have interfered with the activity by com-

peting with the substrate for hydrophobic binding sites on

the enzyme (41). Incubation of the cholera phospho1ipase

with 2-4 mg/ml Triton X 100 for 1 hr, 37 C had no inhibiting

effect, thus suggesting that the detergent did not cause

enzyme denaturation.

A linear relationship was shown between enzyme con-

centrations and initial velocities of the cholera phospho-

lipase (Pig. 13). Substrate (^P) concentration was increased

from 18.5—240.5 nmoles/ml in the presence of the sonicate and

"Roux flask supernatant" as shown in Fig. 14. Reaction rates

71

TABLE XV

EFFECTS OF TRITON X 100 CONCENTRATION ON PHOSPHOLIPASE

ACTIVITY OF SONICATE9

Triton X 100 (mg/ml) Product (nmoles)/hr

0.25 1.27

0.50 2.04

1.00 2.18

2.00 1.34

4.00 0.50

'Reaction time = 1 hr, 37 C.

72

.4 0.6 0.8 1.0 1.2 1A

S o n i c a t e ( m g / m l p r o t e i n )

Fig. 13—Effect of enzyme (sonicate) concentration on reaction rates of V. cholerae phospholipase activity using 32 P phospholipid (16.6 nmoles) as substrate.

73

l/V XIO9 P r o d u c t ( n rno les /hour )

Fig. 14—Initial velocities (nmoles/hr) of "Roux flask supernatant" (A), 6 mg/ml protein, and sonicate (B), 1.6 mg/ml protein, with Lineweaver-Burk plots (below).

74

increased in a hyperbolic fashion with substrate concentra-

tion, but the enzyme was not saturated at the highest sub-

strate level. Lineweaver-Burk plots (Fig. 14) of reciprocal

values suggested a of 60 (SE + 7) uM for "Roux flask

supernatant" and 63 (SE + 6) uM for the sonicate. These

data suggest that both enzyme preparations contained the

same phospholipase activity. The "Roux flask supernatant"

solution was centrifuged (5000 x g, 15 min), and the

particulate fraction was assayed for activity. Activity was

higher in the pellet than in the supernatant. A membrane-

bound phospholipase was thus suggested.

The cholera sonicate was fractionated using an

Amicon Ultrafiltration Cell model 52 and Diaflo membranes

as shown in Table XVI. The substrate was 16.6 nmoles 32P

phospholipid. One-half of the activity was retained on the

XM-100A filter, which was probably due to its membrane-bound

characteristic. The PM-30 and UM-10 filters demonstrated no

apparent phospholipase. Combination of retentates of the

three fractions resulted in recovery of most of the phospho— I

lipase. Since the assay system employed was applicable only

to phospholipase activity, which released both fatty acids

from a phospholipase, the possibility that phospholipases A^

and/or A^ were retained on the PM-30 and UM-10 filters was

investigated. However, no 32P LPE could be found in the

chloroform phase by TLC in solvent B. However, optimum

75

TABLE XVI

PHOSPHOLIPASE ACTIVITY OF XM-100A, PM-30, AND UM-10

MEMBRANE FILTER RETENTATES OF V. CHOLERAE SONICATE

Fraction Product (nmoles/hr)a

Sonicate (100% activity) 4.6 - 4.9

Retentates

XM-100Ab 2.3 - 2.7

PM-30c 0.0 - 0.3

UM-10d o • o - 0.3

XM-100A + PM-30 2.5

00 • CN

1

XM-100A + UM-10 2.9 - 3.2

PM-30 + UM-10

o • o - 0.5

XM-100A + PM-30 + UM-10 4.3 - 4.7

Range of three determinations.

^Retains globular proteins with molecular weights of 100,000 and above.

cRetains globular proteins with molecular weights of 30,000 and above.

^Retains globular proteins with molecular weights of 10,000 and above.

76

conditions for phospholipase A activity, should it exist in

cholera cells, have not been determined.

Scandella and Kornberg (41) isolated the phospho-

lipase activity of E. coli B. The Km for PG using Triton

X 100 (0.5 mg/ml) was 15 uM and in the absence of detergent

was 0.34 uM. No effect on V__„ was noted with different lUcLX

detergent concentrations. A Lineweaver-Burk plot might

measure the affinity of the enzyme for phospholipid vesicles

or miscelles (41). If is a measure of enzyme affinity

for phospholipid, the cholera phospholipase (K^ of 60-63 uM)

compares favorably with the E. coli enzyme in that respect.

Such kinetic data published to date on bacterial phospho-

lipases have come only from Kornberg and co-workers (37, 41).

Isolation and purification of the E. coli enzyme required

1 pound (41) of starting material (bacteria), and the

purified enzyme resulting was 2 mg. It is not surprising

that other reports on bacterial phospholipases discussed

only a few parameters of the enzyme reaction (17, 31-33,

36, 36) .

Bacterial phospholipases (acyl hydrolases) were

found to be localized in the cell envelope (3) and were

tightly bound to their structural elements. The phospho-

lipase A^ was located in the outer membrane (3). This fact

presented a difficult problem in the isolation of the enzyme.

Scandella and Kornberg (41) and Raybin, et al. (37) were able

77

to remove phospholipase A-^rom the cell envelope by the

detergent sodium dodecyl sulfate (SDS). Removal of enzyme

from the cell envelope was a prerequisite to subsequent

purification. The cholera phospholipase activity was com-

pletely destroyed by SDS. Attempts to partially purify the

cholera enzyme by ultrafiltration using the Amicon Dyaflo

membrane (SM-100A) have resulted in partial retention of

activity in every case, thus suggesting that the enzyme

was structurally bound or aggregated in a form which exceeded

molecular weight of 100,000. Prior to future purification

attempts, some means of removal of enzyme from the envelope

which does not destroy activity must be determined.

The possible role of cholera lipase in the disease

mechanism can be hypothesized. An enzyme capable of selec-

tively hydrolyzing membrane constituents (phospholipids)

could contribute to the outpouring of fluids and ions into

the intestinal lumen, causing the extreme diarrhea observed

in cholera victims. Pierce and Greenough (34) have recently

found that the Type 2 (choleragen) cholera toxin (6, 8, 13-16,

26), said to be the fluid transport active factor, stimulated

lipolysis in isolated fat cells. Antisera prepared against

purified toxin destroyed lipolytic activity. Chiappe and

Van Den Bosch (7) recently reported on the lipolysis in

isolated fat cells by phospholipase A and lysophospholipase.

Lipase activity was stimulated by cyclic 3', 5'-AMP. Any

78

relationship between lipolysis of fat cells by choleragen

(34) and phospholipase activity of V. cholerae is not known.

Study of cholera lipases with respect to disease mechanisms

seems important and long overdue. A major contribution to

the understanding of the processes by which intestinal

pathogens cause diarrheal symptoms in disease victims might

be made by elucidation of the effect of bacterial phospho-

lipase activity on intestinal membrane constituents.

>- ;!:I Ij >_J : i v.i.v ' .'-J

1. Boudham, A.D. 1963. Physical structure a^d behavior of lipids ai!-l lipid ixizyuies. Adv. Lipid Res. 1: t' 3-104 .

2. Barfclet, G.H. 1^59. phosphorus in coluain ciiromato-gxaphy. J . Biol. Char,. 2 34 ; 4 b6-4 68 .

3. Bellf 'R.M. , R.D. Mavis, M.J. Osborrx, and P.R. Vagelos. ly?!, Enzymes of phospholipid metabolism; Localisat.1 on in the cytoplasmic ancl outer membrane of the cell envelope of Escharichxa coli and Sq.lT'ionelIa typhiirvur iii-nri. Biochim- Biophys• Acta 249; G2 8~63i>V"~

4. Bixyh, K.C4. f a;»U W.J• Dyer.. 3,959. v, rapid method of total lip,;d or';raci.iOi' find purification. Can. J. B1 o c i i c m. p 11 y s i o 1. 3 7: S i 1 - 917 .

5. Brian, a.P., ana S.w. Gardner. J.it.ds,, Fatty acids from Xt£££:"l Ixp-ds. J. Infect. Diseases i18:47-53- *

6. Burrows, w. 1958. Cholera toxins. Ann. Rev, Microbiol. 22:245-268. '

7. Chiappa Do Cxngoxani» G.E., H. Van Der; Bosch, and L.L.M. Ja.ii , i9 72 . j?no s p ft o i xpa.ae A and iysophospho— xipa^e a.cbi.v3 txes in isolated fat cells; Effect of cyclic 3' , 5 ' -I.ilP. Biochim. Biophys. Acta 2 60:3 o 7-332 .

8. Co ie it u*n f Vv'.d./ J. Katix", M.E, Ivert, G.J. nasai,. and V>. Barrows. 1360. Cholera toxins: Purification and pial.Litij.aa/y ch-iinctcii^atioa oC ileal loop reactive 'i'ypo 2 to:;in. J. ;>acrt:.©r.iol. 96 :1137»i:i.4 3 ,

9. T;aW;;oa, R. M. r. _ ̂ 5S. Phe i,V.atiil'ica tion of tv:o li^id component;; in .liver vhich enable .?.>rj.c:U Ixum

^X!:^cr*3 co ̂ ycirolyse lecithin, aiocben>.

80

10. Deenen, L. L. M., and G. H. DeHaas. 1963. The sub-strate specificity of phospholipase A. Biochim. Biophys. Acta 70:538-553.

11. Doi, 0., and S. Nojima. 1971. Phospholipase C from Pseudomonas fluorescens. Biochim, Biophys. Acta 248:234-244."

12. Doi, 0., and S. Nojima. 1972. Two kinds of phospho-lipase A and lysophospholipase in Escherichia coli. Biochim. Biophys. Acta 260:244-258.

13. Finkelstein, R. A., P. Atthasampunna, M. Chulasamaya, and P. Charunmethee. 1966. Pathogenesis of experimental cholerae: Biological activities of purified procholeragen A. J. Immunol. 96:440-449.

14. Finkelstein, R. A., and J. J. LoSpalluto. 1969. Pathogenesis of experimental cholera. Preparation and isolation of choleragen and choleragenoid. J. Exp. Med. 13:185-202.

15. Finkelstein, R. A., and J. J. LoSpalluto. 1970. Production of highly purified choleragen and cholera-genoid. J. Infec. Diseases 121:563-574.

16. Finkelstein, R. A., and J. J. LoSpalluto. 1972. Crystalline cholera toxin and toxoid. Science 75:529-530.

17. Fung, C. K. and P. Proulx. 1969. Metabolism of phosphoglycerides in E. coli. III. The presence of phospholipase A. Can. J. Biochem. 47:371-373.

18. Gatt, S. 1968. Purification and properties of phospholipase A, from rat and calf brain. Biochim. Biophys. Acta*156:304-316.

19. Hachimori, Y., M. A. Wells, and D. J. Hanahan. 1971. Observations on the phospholipase of Crotalus atrox. Molecular weight and other properties. Biochemistry 10:4084-4089.

20. Hanahan, D. J. 1960. Lipid chemistry. John Wiley and Sons, Inc., New York.

21. Hanahan, D. J., M. Rodbell, and L. D. Turner. 1954. Enzymatic formation of monopalmitoleyl- and monopalmitoyllecithin (lysolecithins). J. Biol. Cherrt. 206:431-441.

81

22. Hendrickson, H. S., and E. M. Scattergood. 1972. The action of phospholipase C on black film bilayer membranes. Biochem. Biophys. Res. Commun. 46: 1961-1969.

23. Kates, M. 1960. Lipolytic enzymes, pp. 165-237. In K. Block (ed.), Lipid metabolism. John Wiley and Sons, Inc., New York.

24. Kates, M. 1967. Paper chromatography of phosphatides and glycolipids on silicic-acid-impregnated filter paper, pp. 1-39. In V. Marinetti (ed.), V. 1. Lipid chromatographic analysis. Marcel Dekker, Inc., New York.

25. Kates, M., J. R. Madeley, and J. L. Beare. 1965. Action of phospholipase B on ultrasonically dispersed lecithin. Biophys. Acta 106:630-634.

26. LoSpalluto, J. J., and R. A. Finkelstein. 1972. Chem-ical and physical properties of cholera exo-enterotoxin (choleragen) and its spontaneously formed toxoid (choleragenoid). Biochim. Biophys. Acta 257:158-166.

27. Lowenstein, J. M. (ed.). 1969. Methods in enzymology. V. 14. Lipids. Academic Press, New York.

28. Lowry, 0. H., N. J. Rosenbrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193:265-275.

29. Mangold, H. K. 1969. Aliphatic lipids, pp. 363-421. In E. Stahl (ed.), Thin-layer chromatography. (A laboratory handbook.) Springer-Verlag, New York.

30. Marsh, J. B., and D. B. Weinstein. 1966. Simple charring method for determination of lipids. J. Lipid Res. 7:574.

31. Okuyama, H., and N. Shoshichi. 1969. The presence of phospholipase A in Escherichia coli. Biochim. Biophys. Acta 176:120-124.

32. Ono, Y., and D. C. White. 1970. Cardiolipin-specific phospholipase D activity in Haemophilus para-influenzae. J. Bacteriol. 103:111-115.

33. Ono, Y., and D. C. White. 1971. Consequences of the inhibition of cardiolipin metabolism in Haemophilus parainfluenzae. J. Bacteriol. 108:1065-1071.

82

34. Pierce, N. F., and W. B. Greenough, III. 1970. Stimu-lation of glycerol production in fat cells by cholera toxin. Nature 226:658-659.

35. Proulx, P., and L. L. M. Van Deenen. 1966. Acylation of lysophosphoglycerides by Escherichia coli. Biochim. Biophys. Acta 125:591-593.

36. Proulx, P., and L. L. M. Van Deenen. 1967. Phospho-lipase activities of Escherichia coli. Biochim. Biophys. Acta 144:171-174.

37. Raybin, D. M., L. L. Bertsch, and A. Kornberg. 1972. A phospholipase in Bacillus meqaterium unique to spores and sporangia. Biochemistry 11:1754-1760.

38. Roelofsen, B., R. F. A. Zwaal, P. Comfurious, C. B. Woodward, and L. L. M. Van Deenen. 1971. Action of pure phospholipase A2 and phospholipase C on human erythrocytes and ghosts. Biochim. Biophys. Acta 241:925-929.

39. Rouser, G. G. Kritchevsky, and A. Yamomoto. 1967. Column chromatographic and associated procedures for separation and determination of phosphatides and glycolipids, pp. 99-162. In V. Marinetti (ed.), V. 1. Lipid chromatographic analysis. Marcell Dekker, Inc., New York.

40. Saito, K., and D. J. Hanahan. 1962. A study of the purification and properties of the phospholipase A of Crotalus adamanteus venom. Biochemistry 1:521-532.

41. Scandella, C. J., and A. Kornberg. 1971. A membrane-bound phospholipase A-, purified from Escherichia coli. Biochemistry 10:4447-4456.

42. Stahl, E. 1969. Thin-layer chromatography. (A laboratory handbook.) Springer-Verlag, New York.

43. Uthe, J. F., and W. L. Magee. 1971. Phospholipase Ao: Action on purified phospholipids as affected by deoxycholate and divalent cations. Can. J. Biochem. 49:776-784.

44. Van Golde, L. M. G., R. N. McElhaney, and L. L.' M. Van Deenen. 1971. A membrane bound lysoohospho-lipase from Mycoplasma laidlawii strain B". Biochim. Biophys. Acta 231:245-249.

83

45. Walker, B. L. 1971. A novel charring technique for detection of lipids on thin-layer chromatograms. J. Chromatog. 56:320-323.

46. Walsh, D. E., 0. J. Banasid, and K. A. Gilles. 1965. Thin-layer chromatographic separation and color-imetric analysis of barley or malt lipid classes and their fatty acids. J. Chromatog. 17:278-287.

47. Wells, M. A. 1971. Evidence that the phospholipases A2 °f Crotalus adamanteus venom are dimers'. Bio-chemistry 10:4074-4078.

48. Wells, M. A. 1971. Spectral peculiarities of the monomer-dimer transition of the phospholipases A~ °f Crotalus adamanteus venom. Biochemistry 10:4078-4083'.

49. Wells, M. A., and D. J. Hanahan. 1969. Studies on phospholipase A. I. Isolation and characteriza-tion of two enzymes from Crotalus adamanteus venom. Biochemistry 8:414-424.

50. Zwall, R. F. A., B. Roelofsen, P. Comfurius, and L. L. M. Van Deenen. 1971. Complete purification and some properties of phospholipase C from Bacillus cereus. Biochim. Biophys. Acta 233:474-479.

CHAPTER III

GAS CHROMATOGRAPHY" OP CYCLOPROPANE FATTY ACID

METHYLESTERS PREPARED WITH METHANOLIC BORON

TRICHLORIDE AND BORON TRIFLUORIDE

Introduction

Christie (4) has suggested that the use of boron

trichloride in methanol (BC1^-CH^OH) by Brian and Gardner

(1-3) for esterification of bacterial fatty acids may have

resulted in unreliable gas chromatographic (GC) data.

Minnikin and Polgar (10) showed that methanolic boron

trifluoride (BF̂ -CH-̂ OH) reacted with disubstituted cyclo-

propanes to give methoxyesters and the corresponding olefins,

The possibility that a similar and unnoticed side-

reaction may have occurred when BCl^-CH^OH was used to

esterify cyclopropane fatty acids of bacterial origin (2,

3) was implied (4).

BF^ has been shown to be superior to BCl^ as a

catalyst for fatty acid transesterification (11). Detailed

studies using BF^ or "̂ or esterification (8, 9, 11) did

not include lipids containing cyclopropane fatty acids. A

large number of papers have been published in which BF3 or

BCl^ was used to catalyze the reaction of CH^OH with

bacterial fatty acids. Few authors who used BF^ or BCl^

84

85

in CH^OH "have indicated that another procedure was employed

to check recovery of cyclopropane acids from bacterial

lipids (3).

The purpose of this investigation was to determine

the reliability of GC data following use of commercially

available BF3~CH3OH and BC13-CH OH in the esterification

of bacterial fatty acids containing cyclopropane rings.

Materials and Methods

Esterification of Fatty Acid Standards. A standard

solution was prepared containing 1 mg/ml each of cis-9,10-

methylene octadecanoic (eye C^g) acid (Supelco, Inc.,

Bellefonte, Pa.) and heptadecanoic (C17) acid (Applied

Science Laboratories, State College, Pa.) in chloroform

(CHCI3).

One-ml aliquots were transferred to 15 x 150 mm

screw-cap tubes, and CHC13 was evaporated with a stream of

N 2 (30 C). Two ml of 14% BF3 in CH OH (w/v) or 10% BC13 in

CH30H (w/v), both from Applied Science Laboratories, were

added, and the open tube was placed in boiling water for 2

min (8). The tube was cooled, and the contents were trans-

ferred to a 30-ml separatory funnel. The tube was washed

with 4 ml of CHC13, the CHC13 and 1 ml of water were added to

the separatory funnel, and the contents were shaken and allowed

to separate. The CHCl^ phase containing methylesters was

evaporated with N2 in a screw-cap tube.

86

The fatty acid standards were also esterified in an

open tube for 0.5 rain (100 C), and for 5 min (100 C) while

tightly closed by a teflon-lined screw-cap.

Gas Chromatography of Fatty Acid Methylesters. Dried

methylesters were dissolved in 1 ml of CHCl^, and 1 ul was

injected into an Aerograph (Varian Associates, Palo Alto,

Calif.; Model 204-1C) gas chromatograph with flame ioniza-

tion detectors. Columns (5 ft. x 0.125 in.) containing 15%

diethylene glycol succinate polyester on Chromosorb W (60-80

mesh) were operated at 180 C. Detector and injector tempera-

tures were 220 C, and the flow was 25 ml/min. Range was

10-10, an<^ attenuation was 8. Areas of peaks were calculated

by multiplication of peak height by peak width at 1/2 height.

Esterification of Bacterial Fatty Acids. Escherichia

coli (ATCC 11775) was incubated for 16 hr at 40 C in Trypti-

case Soy Broth (BBL). Higher incubation temperature is

known to favor cyclopropane fatty acid production (7). Lipids

were extracted with CHCl^-CH^OIi (2:1) (5). A solution con-

taining 1 mg/ml C17 and 1.2 mg/ml E. coli lipid in CHC13 was

esterified with BC1 -CH_OH and BF -CHo0H by the method of o J 3 J

Metcalfe, Schmitz, and Pelka (9). Fatty acids were iden-

tified by hydrogenation, bromination, and by comparison with

authentic standards. Cyclopropane methylesters (cis-9,10-

methylene hexadecanoate, eye C17, and cis-11,12-methylene

87

octadecanoate, eye Cn g) were synthesized from palmitoleate

and cis-vaccenate using a simplified zinc-copper couple

(12) and the Simmons-Smith reaction (13).

Results and Discussion

Results of BF^-CH^OH and BCl^-CH^OH were compared

with the reaction of 2 -ml of freshly distilled diazomethane

at 0 C for 30 min (Table XVII). Diazomethane gave quantita-

tive recovery of eye C (99-101%). Recovery of eye

(retention time relative to C-̂-j = 2.03) using BCl^-CH^OH

at 100 C for 2 min (open tube) or 5 min (closed tube) was

similar to that obtained with diazomethane (93-100%).

BFo-CH^OH gave poor recovery of eye C (10-50%) depending o 29

on conditions of the reaction (see Table XVII). Similar

results were obtained with a 10% solution of BF^ in CH^OH

prepared from a BF^-ether complex, BF̂ O(C2H,_)2 (Eastman

Chemical Co.).

Five additional peaks (retention times relative to

c17 = 1.40, 1.64, 1.83, 3.12, and 4.23) were obtained when

BF -CH OH was used (Table XVII). J Results of BC1 -CH OH for esterification of E.

3 3 ~ coli fatty acids indicated that eye C comprised 29% and

eye C^g 16% of the total acids. Chromatograms obtained,

following use of BF CH OH, revealed considerable loss of •3 3

cyclopropane esters as well as additional small peaks.

Therefore, BF^-CH OH appears to be an undesirable esterifi-J 3 cation reagent for fatty acid mixtures containing cyclopropanes,

38

TABLE XVII

RATIO OF THE PEAK AREAS OF CI S - 9,10-METHYLENE OCTADECANOATE (CYC C19) TO HEPTADECANOATE (C17) FOLLOWING

ESTERIFI CATION (1 MG OF EACH ACID)9

"K n

Esterification method eye C-̂ g Other peaks

1. Diazomethane 0.99-1.01

2. Open tube, 0.5 min, 100 C BF3-CH3OH 0.45-0.59 0.14-0.15

3. Open tube, 2 min, 100 C BCI3-CH3OH 0.93-1.00 BF3CH3OH 0.12-0.13 0.31-0.32

4. Closed tube, 5 min, 100 C BCI3CH3OH 0.96-0.98 BF3CH3OH 0.10-0.11 0.46-0.50

Range of three determinations is given,

17 ^Retention time relative to C-j-y = 2.03.

cPeaks other than eye C19 with retention times relative to Cj_7 of 1.40, 1.64, 1.83, 3.12, and 4.23.

89

BCl^-CH^OH, on the other hand, is suitable for this purpose

and appears to quantitatively esterify cyclopropane fatty

acids which are prevalent in the lipids of many bacterial

species.

CHAPTER BIBLIOGRAPHY

1. Brian, B. L., and E. W. Gardner. 1967. Preparation of bacterial fatty acid methyl esters for rapid char-acterization by gas-liquid chromatography. Appl. Microbiol. 15:1499-1500.

2. Brian, B. L., and E. W. Gardner. 1968. A simple pro-cedure for detecting the presence of cyclopropane fatty acids in bacterial lipids. Appl. Microbiol. 16:549-552.

3. Brian, B. L., and E. W. Gardner. 1968. Cyclopropane fatty acids of rugose Vibrio cholerae. J. Bacteriol. 96:2181-2182.

4. Christie, W. W. 1970. Cyclopropane and cyclopropene fatty acids. Topics Lipid Chem. 1:1-49.

5. Folch, J., M. Lees, and G. H. Sloane-Stanley. 1957. A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. 226:497-509.

6. Goldfine, H., and C. Panos. 1971. Phospholipids of Clostridium butyricum. IV. Analysis of the positional isomers of monounsaturated and cyclopro-pane fatty acids and alk-l'-enyl ethers by capillary column chromatography. J. Lipid Res. 12:214—220.

7,. Marr, A. G., and J. L. Ingraham. 1962. Effect of temperature on the composition of fatty acids in E. coli. J. Bacteriol. 84:1260-1267.

8. Metcalfe, L. D., and A. A. Schmitz. 1961. The rapid preparation of fatty acid esters for gas chroma-tographic analysis. Anal. Chem. 33:363-364.

9. Metcalfe, L. D., A. A. Schmitz, and J. R. Pelka. 1966. Rapid preparation of fatty acid esters from lipids for gas chromatographic analysis. Anal. Chem. 38:514-515.

10. Minnikin, D. E., and N. Polgar. 1967. Structural studies on the mycolic acids. Chem. Communic., pp. 312-314.

90

91

11. Morrison, W. R., and L. M. Smith. 1964. Preparation of fatty acid methyl esters and dimethyl acetals from lipids with BF -CH OH. J. Lipid Res. 5:600-608. 6 6

12. Shank, R. S., and H. Schecter. 1959. Simplified zinc-copper couple for use in preparing cyclopropanes from methylene iodide and olefins. J. Org. Chem. 24:1825-1326.

13. Simmons, H. E., and R. D. Smith. 1959. A new synthesis of cyclopropanes. J. Am. Chem. Soc. 81:4256-4264.

CHAPTER IV

CYCLOPROPANE FATTY ACIDS OF

PSEUDOMONAS AERUGINOSA

Introduction

Pseudomonas species have been shown to contain cyclo-

propane fatty acids as major constituents of cellular lipids

(2, 4, 7, 15). Several investigations of Pseudomonas

aeruginosa fatty acids (1, 5, 8, 12) failed to demonstrate

cyclopropane acids in lipid extracts. Increased time and

temperature of incubation of some bacteria have resulted

in higher amounts of cyclopropane acids (9, 10, 15). This

investigation was conducted to determine the level of

cyclopropane fatty acid synthetase activity in typical P.

aeruginosa strains.

Materials and Methods

Two serologically distinct strains of P. aeruginosa

Verder and Evans (16) strains 2108 (serogroup III) and 1369

(serogroup II), obtained from Joe A. Bass (N.T.S.U.), were

used in this study. The organisms were grown on Trypticase

Soy Broth (BBL) with 2% agar (2). Incubation time and

temperature are indicated in Tables XVIII, XIX, and XX.

Wet cells were extracted with chloroform-methanol (2:1,

volume/volume) (6), fatty acids were esterified with

92

93

BC13-CH30H (10), and the methyl esters were analyzed by gas-

liquid chromatography (2). Cyclopropane fatty acid methyl-

esters (C-j.7 an<^ 9) were synthesized from methyl palmitole-

ate and methyl cis-vaccenate using an easily-prepared zinc-

copper couple (13) and the Simmons-Smith reaction (14).

Fatty acids were identified by comparison of retention times

with authentic standards on a diethylene glycol succinate

polyester column at 180 C (isothermal) and by results of

hydrogenation (3) and bromination (2). Fatty acids which

were detected in amounts of 0.5% or more in any one sample

were considered as major components.

Results and Discussion ' *

Increasing incubation temperature from 37 C to 40 C

resulted in higher amounts of methylene hexadecanoic and

methylene octadecanoic acids (Table XVIII). Strain 2108

grown at 37 C for 24 hr contained less than 1% total cyclo-

propane acids. Both strains, when incubated at 40 C for 16,

24, and 48 hours, demonstrated and cyclopropane

fatty acids as major lipid constituents (Table XIX).

Increased incubation time resulted in slightly larger amounts

of cyclic acids. Table XX presents effects of hydrogenation

and bromination (2) on P. aeruginosa fatty acids.

Strains 2108 and 1369 of P. aeruginosa demonstrate

cyclopropane fatty acid synthetase activity. This activity

94

TABLE XVIII

EFFECTS OF INCUBATION TEMPERATURE AERUGINOSA FATTY ACIDS

ON PSEUDOMONAS (°/o)

Fatty Acida Strain 2108 Fatty Acida

37 C, 24 hr 40 C, 24 hr

14:0 0.9 . tb

16:0 35.6 40.7

16:1 14.4 6.6

eye 17:0 0.8 1.3

18:0 1.4 2.1

18:1 46.8 45.5

eye 19:0 t 3.7

aNumber preceding colon indicates number of carbons; number following colon designates degree of unsaturation (eye = cyclopropane ring).

ht = trace (less than 0.5% of total).

95

TABLE XIX

FATTY ACIDS (%) OF PSEUDOMONAS AERUGINOSA STRAINS INCUBATED 40 C

Fatty acid Strain 2108 Strain 1369 16 hr 24 hr 48 hr 16 hr 24 hr 48 hr

16:0 44.2 40.7 42.4 40.0 40.2 41.8

16:1 5.6 6.6 5.6 5.9 6.4 4.9

eye 17:0 0.8 1.3 1.4 1.5 1.6 1.9

18:0 2.9 2.1 2.3 2.1 2.0 3.0

18:1 43.1 45.5 44.0 44.7 44.2 41.6

eye 19:0 3.3 3.7 4.2 5.8 5.5 6.7

aNumber preceding colon indicates number of carbons; number following colon designates degree of unsaturation (eye = cyclopropane ring).

96

TABLE XX

RESULTS OF HYDROGENATION AND BROMINATION ON PSEUDOMONAS AERUGINOSA FATTY ACIDS (%)

Fatty acida Retention time

Strain 1369, 40 C, 48 hr Fatty acida Retention

time Hydrogenation results Before After

Bromination results0

16:0 1.00 41.8 46.4

16:1 1.18 4.9 -

eye 17:0 1.54 1.9 1.7 +

18:0 1.82 3.0 45.0

18:1 2.08 41.6 -

eye 19:0 2.82 6.7 6.9 +

Number preceding colon indicates number of carbons: number following colon designates degree of unsaturation (eye -cyclopropane ring).

^Retention time relative to 16:0 (Palmitic acid).

:Plus (+) = peak eliminated.

97

appears to be low in relation to some bacterial species.

Incubation of Escherichia coli (ATCC 11775) 24 hr, 40 C

gave the following fatty acids: C14:0 (6.5%), C16:0 (53.3%),

C16:1 (trace), eye C17:0 (27.7%), CIS:1 (1.6%), and eye

C19:0 (10.3%). The low level of production of cyclopropane

fatty acids by P. aeruginosa could explain their absence in

fatty acid data of several investigators (1, 5, 7, 11).

However, the unknown acids presented by Edmonds and Cooney

(5) might have been cyclopropane acids. In addition to gas

chromatographic retention data, other chemical techniques

should be employed to detect presence of cyclopropane fatty

acids. Hydrogenation and bromination (2) are simple, rapid,

and sensitive procedures for detection of cyclopropane acids

which occur at low levels, as in P. aeruginosa.

CHAPTER BIBLIOGRAPHY

1. Bobo, R. A., and R. G. Eagon. 1968. Lipids of cell walls of Pseudomonas aeruginosa and Brucella abortus. Can. J. Microbiol. 14:503-513.

2. Brian, B. L., and E. W. Gardner. 1968. A simple pro-cedure for detecting the presence of cyclopropane fatty acids in bacterial lipids. Appl. Microbiol. 16:549-552.

3. Brian, B. L., and E. W. Gardner, 1968. Fatty acids from Vibrio cholerae lipids. J. Infect. Diseases 118:47-53,

4. Crowfoot, P. D., and A. L. Hunt. 1970. The effect of oxygen tension of methylene hexadecanoic acid forma-tion in Pseudomonas fluorescens and Escherichia coli. Biochim. Biophys. Acta 202:550-552.

5. Edmonds, P., and J. J. Cooney. 1969. Lipids of Pseudomonas aeruginosa cells grown on hydrocarbons and on Trypticase Soy Broth. J. Bacterid. 98:16-22.

6. Folch, J., M. Lees, and G. H. Sloane-Stanley. 1957. A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. 226:497-509.

7. Hancock, I. C., and P. M. Meadow. 1969. The extractable lipids of Pseudomonas aeruginosa. Biochim. Biophys. Acta 187:366-379.

8. James, A. T., and A. J. P. Martin. 1956. Gas-liquid chromatography. The separation and identification of the methyl esters of saturated and unsaturated acids from formic acid to n-octadecanoic acid. Biochem. J. 63:144-152.

9. Kates, M., and P. O. Hagen. 1964. Influence of temperature on fatty acid composition of psychro-philic and mesophilic Serratia species. Can. J. Biochem. 42:481-488.

10. Marr, A. G., and J. L. Ingraham. 1962. Effect of temperature on the composition of fatty acids in

coli. J. Bacteriol. 84:1260-1267.

98

99

11. Metcalfe, L. D., A. A. Schmitz, and J. R. Pelka. 1966. Rapid preparation of fatty acid esters from lipids for gas chromatographic analysis. Anal. Chem. 33:514-515.

12. Romero, S. M., and R. R. Brenner. 1966. Fatty acids synthesized from hexadecane by Pseudomonas aeruginosa. J. Bacteriol. 91:183-188.

13. Shank, R. S., and H. Schecter. 1959. Simplified zinc-copper couple for use in preparing cyclopropanes from methylene iodide and olefins. J. Org. Chem. 24:1825-1826.

14. Simmons, H. E., and R. D. Smith. 1959. A new synthesis of cyclopropanes. J. Am. Chem. Soc. 81:4256-4264.

15. Vaczi, L., J. K. Makleit, A. Rethy, and I. Redai. 1964. Studies on lipids in Pseudomonas Pyocyanea. Acta Microbiol. 11:383-390.

16. Verder, E., and J. Evans. 1961. A proposed antigenic schema for the identification of strains of Pseudomonas aeruginosa. J. Infect. Diseases 109:183-193.

CHAPTER V

TUMOR AND LIVER FATTY ACIDS OF DBA/lJ MICE

DURING LYMPHOSARCOMA DEVELOPMENT

Introduction

Fatty acid composition of tumor lipids have been

studied in a number of animal systems (1, 3, 4, 7, 9-12).

A major difference between tumor and liver fatty acids was

found to be the ratio between stearic and oleic acids (9,

11). Selkirk, et al. (9) found a greater percentage of

unsaturated fatty acids in phospholipids of a fast-

developing Morris 3924A hepatoma than on those of slower-

growing Reuber H-35 hepatoma. The ratio of stearic acid

(18:0) to oleic (18:1) was higher in normal liver than in

either hepatoma. Carruthers (3) found that the amount of

18:1 decreased, while that of linoleic acid (18:2) increased

following methylcholanthrene treatment of mouse skin.

Newland, et al. (7) have shown that 18:2 was the predominant

fatty acid in phosphatides of mice infected with mammary

tumor virus.

Lipid levels were shown to be lower in organs of

tumor-bearing mice. The fatty acids 18:0 and 18:2 increased,

while 18:1 decreased in organs during tumor growth (10).

Wood and Harlow (12) found a C24 dienoic acid in

100

101

sphingomyelin and ceramide fractions of Ehrlich ascites

carcinoma cells which did not appear in normal tissues.

Fatty acid studies from one investigation to another

have shown little relationship. This may have been because

of varying time periods the tumors were growing in the

animals being studied. Scholes (8) has pointed out the

importance of determining a time-activity relationship in

studying malignancy-associated changes.

The purpose of this study was to determine what

changes, if any, occurred in fatty acids of tumors and

livers of mice bearing tumors for varying periods of time.

Materials and Methods

Tissue Isolation. Male DBA/lJ mice, twelve weeks

old, were obtained from Jackson Memorial Laboratory, Bar

Harbor, Maine. The tumor line (8) has been maintained in

our laboratory for 135 passages. Transplantation of tumor

(lymphosarcoma) was performed by surgically implanting a

small mass (5-10 mg) of freshly removed, minced tissue

subcutaneously in the region of the anterior axial lymph

node.

Tumor-bearing mice were sacrificed by cervical dis-

location on the 4th, 6th, 8th, 10th, and 12th days following

tumor implant. Mice with tumors die, on an average, 12.5

days following tumor implant (8). DBA/lJ mice which did

102

not receive tumor implants were used as liver controls. Two

separate experiments were conducted in which tumors and livers

from 3 mice were pooled on each day of tissue isolation.

Livers and tumors were removed, pooled, immediately chilled

on ice, and total wet weight determined.

Lipid Extraction. All solvents were distilled before

use. Immediately after weighing, tumors and livers were

homogenized at 5 C for 5 min with a Vertis Blender at high

speed in 20 tissue-volumes of chloroform: methanol (2:1,

volume/volume). Homogenates were filtered through solvent-

washed Whatmann #3 paper and were extracted twice with addi-

tional amounts of chloroform: methanol. Lipid extracts were

washed by the method of Folch, et al. (5). NaCl (0.73%) in

distilled water (0.2 volume) was used to wash extracts. The

lower organic phase was reduced to approximately 5 ml under

partial vacuum (rotary evaporator), then taken to dryness

with a stream of nitrogen. Lipid residue was dissolved in

10 ml of chloroform and stored under nitrogen at -10 C.

Two ml aliquots were used to obtain lipid weights. Aliquots

were placed on pre-weighed aluminum foil cups and dried in

vacuo over CaCl2 ^ hrs at which time constant weight

was obtained.

Fatty .Acid .Analysis. Lipid (10 mg) was dried with a

stream of nitrogen and saponified-esterified by the method

103

of Metcalfe, et al. (6) using 0.5 N KOH in methanol followed

by boron trichloride: methanol (.Applied Science Laboratories,

College Station, Pa.). Fatty acid methyl esters were stored

under nitrogen at -10 C until analyzed by gas-liquid

chromatography.

Methyl esters were injected into a Varian Aerograph

Model 204 -1C gas chromatograph equipped with dual hydrogen

flame detectors and temperature programmer. Columns were 5

feet in length, 0.25 inch in diameter, packed with 15%

diethylene glycol succinate on Chromosorb W (60-80 mesh).

Detectors and injectors were 210 C. Columns were programmed

10 C/min between 150 C and 195 C and held at the upper limit,

or were operated isothermally at 180 C. Helium carrier was

20 ml/min. Methyl esters were tentatively identified by

comparison with authentic standards (Applied Science

Laboratories) and confirmed by hydrogenation (2).

Results and Discussion

Table XXI presents average wet weights (g) of livers

and tumors of each duplicate group of mice studied. The

fact that wet weight was determined could account for some

variation. However, an increase in liver weight is apparent

on the 10th and 12th days following tumor implant. Livers

became noticeably enlarged and proceeded from a normal

brown color to a characteristic gray as the terminal stage

of tumor development was approached. Tumor weight also

104

TABLE XXI

MEAN WET WEIGHT3 OP TUMORS AND LIVERS FROM TUMOR-BEARING MICE AT VARIOUS STAGES OF TUMOR DEVELOPMENT

Day post-implant Liver (g) Tumor (g)

Control*3 2.92 ± 0.16 -

4 00 •

CM 9 + 0.05 -

6 2.79 + 0.21 0.88 + 0.35

8 2.19 4- 0.04 2.42 + 0.25

10 4.59 -f* 0.51 4.75 + 0.20

12 5.16 0.56 4.07 + 0.49

aEach weight presented is the average of livers or tumors from two groups consisting of 3 mice per group + range.

^Control = no tumor implanted.

105

increased rapidly between 8 and 10 days post-implant. Prior

to the 3th day, it was difficult to separate tumor growth

from surrounding fatty material.

Table XXII lists the average percent lipid extracted

from wet livers and tumors. Livers increased in lipid con-

tent on the 8th day, while tumor lipid continued to decrease

from day 6 through day 10. Difficulty in separating fatty

tissues from early tumors could account for some increase

observed in lipid percentage.

Table XXIII shows the distribution of various fatty

acids in liver lipids. Only those acids present in amounts

of 0.5% or more in any one sample are presented. Changes

occurred as tumor development progressed. Palmitic acid

(16:0), stearic acid (18:0), arachidonic acid (20:4), and

a polyunsaturated acid decreased considerably, while

18:1 (oleate) and 18:2 (linoleate) increased significantly.

Total unsaturation increased slightly. Hydrogenation (2)

of selected samples resulted in saturated C14, C^, C1Q,

^20' an<^ ^22 acids. The C unsaturated acid has not been

identified but contains more than one unsaturated bond.

Table XXIV shows the distribution of fatty acids

from tumor lipids. Fatty acids 16:1, 18:1, and 18:2

decreased while 18:0, 20:4, and the C22 unsaturated acid

increased as tumors developed. Total unsaturation did not

appear to change appreciably. Fewer changes are evident in

106

TABLE XXII

PER CENT OF LIPID IN TUMORS AND LIVERS FROM TUMOR-BEARING MICE AT VARIOUS STAGES OF TUMOR DEVELOPMENT3

Days post-implant Livers Tumors

Control*3 5.5 + 0.1 -

4 4.8 + 0.1 -

6 4.5 + 0.3 9.3 + 2.6

8 8.2 + 0.2 6.8 + 0.1

10 8.5 ± 1.4 4.2 + 0.6

12 9.3 2.4 5.0 + 0.2

a Each percentage presented is the average per cent lipid of livers or tumors from two groups consisting of 3 mice per group + range.

^Control = no tumor implanted.

107

TABLE XXIII

PERCENTAGE OF FATTY ACIDS OCCURRING IN LIVER LIPIDS OF TUMOR-BEARING MICE AT VARIOUS STAGES

AFTER IMPLANTATION3

Fatty acid b

0 4 Days post-implant

6 8 10 12

14:0 trace trace trace trace trace trace

16:0 30.4 +2:8

28.3 +1.9

26.7 +0.4

26.2 +0.1

23.5 +0.3

24.6 ±0.5

16:1 1.7 +0.2

1.4 +0.2

1.5 +0.2

2.0 +0.1

2.6 +0.7

2.3 ±0.5

18:0 12.7 +0.' 2

13.3 +0.8

12.4 +0.1

8.4 +0.6

7.7 ±1.4

7.7 ±1.1

18:1 10.9 +0.8

11.6 +0.4

13.1 +0.9

18.0 +0.1

24.3 +0.8

23.3 ±1.7

18:2 20.3 +1.6

17.2 +4.0

23.6 +0.2

29.7 +0.5

29.3 ±1.4

31.2 ±0.4

20:4 10.7 +0.2

12.8 ±0.7

10.4 +0.4

6.6 +0.2

6.4 ±0.1

5.1 ±0.5

22:Unc 13.5 +0.7

15.2 ±1.2

12.3 +1.3

9.2 +0.5

6.8 ±1.7

5.9 +0.4

Average per cent fatty acids from 2 groups consisting of livers from 3 mice per group ± range.

^Number preceding colon indicates number of carbons, and number after colon designates degree of unsaturation.

C ^ Fatty acid with 22 carbons and more than one unsaturated bond,

108

TABLE XXIV

PERCENTAGE OF FATTY ACIDS OCCURRING IN TUMOR LIPIDS AT VARIOUS STAGES AFTER IMPLANTATION3

Days post-implant Fatty acid*3 6 8 10 12

1 4 : 0 2 . 0 + 0 . 2 1 . 9 + 0 . 2 1 . 8 + 0 . 1 1 . 7 ± 0 . 3

1 6 : 0 2 5 . 7 + 0 . 4 2 5 . 9 -r 0 . 4 2 2 . 7 + 0 . 8 2 5 . 1 + 1 . 7

1 6 : 1 4 . 7 + 1 . 0 4 . 0 + 0 . 3 3 . 3 + 0 . 2 3 . 1 + 0 . 1

1 8 : 0 6 . 7 + 1 . 1 7 . 6 + 0 . 2 1 0 . 1 + 0 . 4 9 . 8 + 0 . 5

1 8 : 1 3 2 . 5 + 1 . 2 3 2 . 4 -f 0 . 4 2 9 . 8 i 0 . 5 2 8 . 7 + 0 . 6

1 8 : 2 2 8 . 3 + 1 . 0 2 6 . 6 ± °-5 2 4 . 2 +_ 0 . 1 2 4 . 1 + 0 . 9

2 0 : 4 trace 2 . 2 ± o.i 4 . 2 + 0 . 1 3 . 8 + 0 . 5

22:UnC trace trace 3 . 9 + 0 . 2 3 . 0 + 0 . 4

a Average percentage fatty acids from 2 groups consisting of tumors from 3 mice per group _+ range.

ID Number preceding colon indicates number of carbons, and number after colon designates degree of unsaturation.

cFatty acid with 22 carbons and more than one unsaturated bond.

109

tumor lipids than in liver lipids. Both the C20 and C ^

unsaturated acids decreased in liver and increased in tumor.

It would be presumptive to hypothesize mechanisms

of tumor development or relationships between observed fatty

acids and carcinogenesis based upon the data presented here.

However, apparent changes in fatty acid synthesis during

tumor development suggest feasibility of using this model

animal system for future tumor lipid studies. Future

experiments should include investigation of fatty acid dis-

tribution changes in neutral lipids, phospholipids, and

specific lipid classes in developing tumor as well as in

various organ systems during tumor growth.

CHAPTER BIBLIOGRAPHY

1. Bergelson, L. D., E. V.' Daytlovitskaya, T. I. Torkhovskaya, I. B. Sorokina, and N. P. Gorkova. 1970. Phospholipid composition of membranes in the tumor cell. Biochim. Biophys. Acta 210:287-298.

2. Brian, B. L., and E. W. Gardner. 1968. A simple procedure for detecting the presence of cyclopro-pane fatty acids in bacterial lipids. Appl. Microbiol. 16:549-552.

3. Carruthers, C. 1962. The fatty acid composition of dermal and epidermal triglycerides and phosphatides in mouse skin during normal and abnormal growth. Cancer Res. 22:294-298.

4. Cartuthers, C. 1967. The fatty acid composition of the phosphatides of normal and malignant epidermis. Cancer Res. 27:1-6.

5. Folch, J., M. R. Lees, and G. H. Sloane-Stanley. 1957. A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. 226:497-509.

6. Metcalfe, L. D., A. A. Schmitz, and J. R. Pelka. 1966. Rapid preparation of fatty acid esters from lipids for gas chromatographic analysis. Anal. Chem. 38:514-515.

7. Newland, J., R. F. McGregor, and W. E. Carnatzer. 1965. Phosphorus metabolism in viral-induced neoplasia.

• hk vivo and in vitro studies of the milk and mammary gland lipides of strains of mice susceptible to the mammary tumor virus. Can. J. Biochem. 43:297-307.

8. Scholes, V. E. 1969. Skin nucleic acid phosphorus metabolism of DBA/lJ mice during implanted tumor development and methycholanthrene carcinogenesis. Cancer Res. 29:1416-1419.

110

Ill

9. Selkirk, J.K. , J.C. Elwood, and H.P. Morris. 1971. Study on the proposed role of phospholipid in tumor cell membrane. Cancer Res. 31:27-31.

10. Uezumi, N., H. Shinichi, and K. Kasama. 1969. Lipids of neoplastic tissues. II. Lipid contents and fatty acid compositions of the lipids of several organs of tumor (NF-Sarcoma)-bearing mice. Mie Med.J. 19:141-147.

11. Verrkamp, J.H., I. Mulder, and L.L.M. Van Deenen. 1962. Comparison of the fatty acid composition of lipids from different animal tissues including some tumors. Biochim. Biophys. Acta 57:299-309.

12. Wood, R., and R.D. Harlow. 1970. Tumor lipids: structural analyses of the phospholipids. Arch. Biochem. Biophys. 141:183-189.

CHAPTER VI

FATTY ACID DISTRIBUTION OP LIPIDS FROM CARCASS,

LIVER AND FAT BODIES OF THE LIZARD,

CNEMIDOPHORUS TIGRIS, PRIOR

TO HIBERNATION

Introduction

Temperate-zone lizards are known to store lipids in

their carcass, liver, and abdominal fat bodies (1, 3, 6, 9).

Lipids in several lizard species have been associated with

vitellogenesis (6) and energy storage for winter hibernation

(1, 4). However, no information comparable to that on the

salamander, Ambystoma tigrinum (7), is available on the

exact roles of carcass, liver, and fat body lipids in

vitellogenesis and energy metabolism in lizards. Studies

producing such information must await chemical analyses of

these lipid reserves. The objectives of this study were to

analyze the distribution and fatty acid composition of lipids

in the carcass, liver, and fat bodies in male and female

Chemidophorus tigris lizards.

Materials and Methods

Lizards, collected in late August 1970, in El Paso

County, Texas, by F. G. Gaffney (N.T.S.U.), were frozen and

112

113

stored at 0 C until lipid determinations were made. Five

sexually mature males and females (snout-vent length greater

than 80 mm) were studied. Gastrointestinal tracts, post-

coelomic fat bodies, and livers were removed from carcasses

prior to homogenization in a blender. Lipids were extracted

with chloroform-methanol (2:1, volume/volume) and purified

by the method of Folch, et al. (5). Solvent was evaporated

with a stream of N2 (40 C). Total dry weights of livers, fat

bodies, and carcasses were determined as the weight of dried

lipid-extracted residues plus weight of extracted lipids.

Fatty acids were hydrolyzed and esterified by boiling

lipids in 0.5 N KOH in methanol followed by boron trichloride

(10%) in methanol (8). A known amount of internal standard,

heptadecanoic acid, was added to lipid samples prior to the

hydrolysis step. No heptadecanoate was found in control

samples. Fatty acid methylesters were analyzed using a

Varian Aerograph Model 204-1C gas chromatograph equipped

with hydrogen flarae detectors. Columns were 5 ft. x 0.125 in.

packed with 15% diethylene glycol succinate polyester on

60/80 Chromosorb W. The columns were programmed from

150-200 C, 6 C/min and held at the upper temperature. The

helium carrier flow rate was 20 ml/min. Detector and

injector temperatures were 220 C. Fatty acid methylesters

were identified by comparison with retention times of

authentic standards and by hydrogenation (2). Areas of

114

the chromatograph peaks were determined as peak height x

width at half peak height.

Resul-ts and Discussion

Table XXV shows body measurements, tissue dry weights,

and lipid content of male and female carcasses, livers, and

fat bodies. Females had a larger mean fat body weight. Fat

bodies in both males and females contained the highest mean

percentage of lipids (77 and 88 percent, respectively).

Data (Table XXV) for range of male and female fat body lipid

percentages (66-97 percent) agree with those of Rose and

Lewis (10), who found 96.6-99.7 percent in A. tiqrinum,

a salamander. Table XXVI gives the fatty acid content of

carcasses, livers, and fat bodies of males and females.

Major components were myristic, palmitic, palmitoleic,

stearic, oleic, linoleic, linolenic, and arachidonic

acids. Arachidonic acid was highest in liver lipids (8.2-

14.4 percent), lower in carcasses (4.2-4.5 percent), but was

barely detectable (trace) in any fat body lipid extract.

Qualitatively, the data in Table XXVI are comparable to

those of Rose and Lewis (10) for fat body fatty acids.

Total fatty acid content (mg of fatty acid per 100

mg lipid) in male carcasses, liver, and fat bodies was

determined to be 58-60 mg, 34-46 mg, and 66-89 mg, respec-

tively. Both males and females showed the highest percent-

age of total fatty acid content in fat body lipids (66-94

mg fatty acid per 100 mg of lipids).

115

TABLE XXV

MALE AND FEMALE C. TIGRIS BODY MEASUREMENTS (mm), TISSUE DRY WEIGHTS (mg), AND LIPID CONTENT EXPRESSED AS PERCENTAGES OF THE DRY

WEIGHT OF EACH TISSUE

Males Mean Range

Females Mean Range

Body lengths (mm)

Snout-vent 84 8 1 - 8 9 85 81-88

Tail 209 1 6 3 - 2 4 5 196 1 7 0 - 2 1 2

Tissue dry weights (mg)

Carcass 4 0 8 1 3 4 2 6 - 5 1 6 4 4385 3 8 9 0 - 4 7 8 1

Liver

Fat bodies

Lipid percentages

41 2 6 - 6 7

57 1 4 - 1 7 3

58

142

4 4 - 7 9

5 6 - 3 1 2

Carcass

Liver

Fat bodies

4

28

77

2 - 1 0

1 0 - 4 6

6 6 - 9 3

8

36

88

3 - 1 4

2 0 - 4 8

7 6 - 9 7

116

TABLE XXVI

MALE AND FEMALE C. TIGRIS FATTY ACIDS FROM CARCASSES, LIVERS, AND FAT BODIES EXPRESSED AS PERCENTAGES

(MEAN VALUES) OF THE TOTAL FATTY ACID CONTENT

Males Females Fatty acid Carcass Liver

Fat bodies Carcass Liver

Fat bodies

iViyristic 1.2 Trace 1.5 1.1 Trace 1.4

Palmitic 18.0 11.5 21.4 18.3 16.3 21.6

PaIraitoleic 2.9 1.0 4.2 2.3 2.7 2.7

Stearic 9.1 13.8 6.7 8.6 8.2 6.5

Oleic ' 47.3 25.2 50.5 43.7 37.7 47.1

Linoleic 14.0 29.7 11.3 16.8 23.3 15.5

Linolenic 3.0 4.4 4.4 5.0 3.6 5.2

Arachidonic 4.5 14.4 Trace 4.2 8.2 Trace

CHAPTER BIBLIOGRAPHY

1. Avery, R. A. 1970. Utilization of caudal fat by hiber-nating common lizards, Lacerta vivipara. Comp. Biochem. Physiol. 37:119-121.

2. Brian, B. L., and E. W. Gardner. 1968. Fatty acids from Vibrio cholerae. J. Infect. Diseases 118:47-53.

3. Dessauer, H. C. 1953. Hibernation of the lizard, Anolis carolinensis. Proc. Soc. Exp. Biol. Med. 82:351-353.

4. Dessauer, H. C. 1955. Seasonal changes in the gross organ composition of the lizard, Anolis carolinensis. J. Exp. Zool. 128:1-12.

5. Polch, J., M. Lees, and G. H. Sloane-Stanley. 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497-509.

6. Hahn, W. E., and D. W. Tinkle. 1965. Fat body cycling and experimental evidence for its adaptive significance to ovarian follical development in the lizard Uta stansburiana. J. Exp. Zool. 158:79-86.

7. Lewis, H. E., ana F. L. Rose. 1969. Effects of fat body fatty acids on ovarian and liver metabolism of Ambystoma tiqrinum. Comp. Biochem. Physiol. 30:1055-1060.

8. Metcalf, L. D., A. A. Schmitz, and J. R. Pelka. 1966. Rapid preparation of fatty acid esters from lipids for gas chromatographic analysis. Analyt. Chem. 38:514-515.

9. Moberly, W. R. 1963. Hibernation in the desert iguana Dipsosaurus dorsalis. Physiol. Zool. 36:152-160.

10. Rose, F. L., and H. L. Lewis. 1968. Changes in weight and free fatty acid concentration of fat bodies of paedogenic Ambystoma tiqrinum during vitello-genesis. Comp. Biochem. Physiol. 26:149-154.

117

CHAPTER VII

ANALYSIS OF ACETYLATED AND TRIFLUORACETYLATED

PHENYLTHIOHYDANTOIN AMINO ACIDS

BY GAS CHROMATOGRAPHY

Introduction

Reaction of phenylisothiocyanate with the N-terminal

amino acid of a peptide or protein followed by cleavage in

acid results in formation of a 3-phenyl-2-thiohydantoin

(PTH) amino acid derivative (2, 3). Protein sequence

studies have been facilitated by analysis of the isolated

PTH using thin-layer or paper chromatography (7, 8).

Several PTH amino acids have been successfully separated

and analyzed by gas chromatography as trimethylsilyl

derivatives (5, 6). Roda and Zamorani (9) separated six

trifluoracetylated PTH's by gas chromatography using a

stainless steel column containing 5% SE-30 stationary

phase. Prior to this investigation, glass columns alone

had been used to analyze PTH's (5-8).

This report (1) compares the gas chromatographic

behavior of trifluoroacetylated and acetylated PTH amino

acids using a stainless steel column.

118

119

Materials and Methods

N-acetylation and N-trifluoroacetylation of PTH Amino

Acids. Alanine, glycine, valine, proline, leucine, iso-

leucine, methionine, and phenylalanine PTH's were purchased

from Mann Research Laboratories. Acetates and trifluoro-

acetates were made by a procedure similar to that of Roda

and Zamorani (9). To 2 mg of each PTH in 2 ml of chloroform

was added 0.2 ml acetic anhydride or trifluoroacetic anhydride

(TFAA). Reaction mixtures were allowed to stand at least 30

min at room temperature before gas chromatography.

Gas Chromatography. A Varian Aerograph Model 204-1C

gas chromatography with dual flame ionization detectors was

used. Injector and detector temperatures were 220 C. A

5 ft. x 0.125 in. (O.D.) stainless steel column containing

1% SE-30 on Chromosorb G (acid washed and silanized) was

operated at 180 C isothermally to obtain the data in Table

XXVII or programmed from 150-200 C, 10 C/min then held at

200 C. Helium carrier gas flow rate was 25 ml/min. Two ul

samples were injected with a Range of 10"11 and Attentuation

of 16.

Results and Discussion

Table XXVII gives retention times of acetates and

trifluoroacetates relative to unreacted proline PTH.

Since proline PTH was not N-acetylated, the retention time

120

TABLE XXVII

RELATIVE RETENTION TIMES OF AMINO ACID PHENYLTHIOHYDANTOIN ACETATES AND TRIFLUOROACETATES a

Acetates Trifluoroacetates

Alanine 0. .51 0. .26

Glycine 0. .60 0. .33

Valine 0. .71 0. .37

Leucine, Isoleucine 0. . 96 0. .49

Methionine 2. .36

Phenylalanine 2. .88 1, .51

aProline PTH, 5.75 min = 1.00.

121

of the derivative was not affected by acetic anhydride or

TFAA treatment. Proline was the only compound which could

be successfully chromatographed as the free PTH on the

stainless stell column, and therefore it was chosen as a

marker compound for derivatives. All PTH acetates separated

except leucine and isoleucine. Roda and Zamorani (9) also

were unable to resolve these two as the PTH trifluoroacetates.

Methionine PTH trifluoroacetate gave no peak, and the

solution turned dark brown upon standing for several hours

at room temperature. Chromatography of methionine or

phenylalanine PTH1s was not attempted by Roda and Zamorani

(9). The PTH acetates of these two amino acids were readily

separated (Table XXVII). The order of appearance of alanine

and glycine derivatives appears to conflict with that

reported previously (9).

Thus, from a variety of considerations, acetic

anhydride appears to b'e superior to TFAA for derivation of

simple amino acid PTH's.

CHAPTER BIBLIOGRAPHY

1. Brian, B. L., R. W. Gracy, and V. E. Scholes. 1971. Analysis of acetylated and trifluoroacetylated phenylthiohydantoin amino acids by gas chroma-tography. J. Chromatog. 63:386-388.

2. Edman, P. 1950. Preparation of phenyl thiohydantoins from some natural amino acids. Acta Chem. Scand. 4:277-282.

3. Edman, P. 1950. Method for determination of the amino acid sequence in peptides. Acta Chem. Scand. 4:283-293.

4. Eriksson, S., and J. Sjoquist. 1960. Quantitative determination of N-terminal amino acids in some serum proteins. Biochim. Biophys. Acta 45:290-296.

5. Guerin, M. R., and W. D. Shults. 1969. Gas chromatography of silylated phenylthiohydantoin amino acids. Utility of a sulfur-specific detection. J. Chromatog. Sci. 7:701-803.

6. Karman, R. E., J. L. Patterson, and W. J. A. Vanden-Heuvel. 1968. Gas chromatographic behavior of trimethylsilated phenylthiohydantoin amino acids. Anal. Biochem. 25:452-458.

7. Pisano, J. J., and T. J. Bronsert. 1969. Analysis of amino acid phenylthiohydantoins by gas chromatography. J. Biol. Chem. 244:5597-5607.

8. Pisano, J. J., and W. J. A. Vandenheuvel, and E. C. Horning. 1962. Gas chromatography of phenylthio-hydantoin and dinitrophenyl derivatives of amino acids. Biophys. Res. Commun. 7:82-86.

9. Roda, G., and A. Zamorani. 1970. Gas chromatographic analysis of amino acids as trifluoroacetylated phenylthiohydantoins. J. Chromatog. 46:315-316.

10. Sjoquist, J. 1960. Determination of amino acids as phenyl thiohydantoin derivatives. III. Quantita-tive determination of 3-phenyl-2-thiodydantoins from paper chromatograms. Biochim. Biophys. Acta 41:20-30.

122

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