THE JOURNAL OF BIOLOQICAL CHEMISTRY Vol. 248, No. 3, Issue of February 10, pp. 1098-1105, 1973

Printed in U.S.A.

Function of Cytidine Diphosphate-Diglyceride and Deoxycytidine Diphosphate-Diglyceride in the Bio- genesis of Membrane Lipids in Ewherichia coZi*

(Received for publication, September 28, 1972)

CHRISTIAN R. H. RAETZ$. AND EUGENE P. KENNEDY

From the Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts 0.2116

SUMMARY

CDP-diglyceride synthesized by chemical procedures is an efficient donor of phosphatidyl residues for the enzymatic synthesis of phospholipids in cell-free extracts of Escherichia coli. However, prior to the present work, CDP-diglyceride had never been isolated from living cells of E. coli or any other organism. We have, therefore, isolated and characterized the cytosine-containing liponucleotide fraction of E. coli and find it to be a mixture of almost equal amounts of CDP- diglyceride and dCDP-diglyceride. In these experiments, cells growing in the log phase were simultaneously labeled with tritiated cytosine and sn-glycero-3-[32P]phosphate. Under the conditions employed sn-glycero-3-[32P]phosphate was taken up without prior hydrolysis and was rapidly con- verted to labeled lipids. Since the intracellular pools of phosphatidic acid and of cytosine liponucleotides are very small and turn over very rapidly, the amounts of 32P in these fractions may be assumed to reflect their relative intracel- lular concentrations. The 32P radioactivity of the phospha- tidic acid fraction was about 20 times higher than that of the cytosine liponucleotide fraction. Since phosphatidic acid is itself less than 1% of the lipids of E. coli, the steady state levels of the cytosine liponucleotides must be exceed- ingly low, supporting the hypothesis that the conversion of phosphatidic acid to cytosine liponucleotides may be rate- making for the biosynthesis of the membrane phospholipids in this organism.

The relative activity of CDP-diglyceride and of dCDP- diglyceride for the enzymatic synthesis of phosphatidylserine and of phosphatidylglycerophosphate was found to vary considerably with the concentration of liponucleotide used in the assay system. At concentrations greater than 0.1 mrvr, dCDP-diglyceride is relatively much more active in the synthesis of phosphatidylglycerophosphate than in the syn- thesis of phosphatidylserine. Below this concentration,

* This research was supported in part by Grant NB-02946 from the National Institute of Neurological Diseases and Stroke, and Grant GM-13952 from the National Institute of General Medical Sciences.

$ Life Insurance Foundation Medical Scientist Fellow. Some of the work described here forms part of a dissertation to be sub- mitted to the Faculty of Arts and Sciences of Harvard University in partial fulfillment of the requirements for the Ph.D. degree.

however, dCDP-diglyceride is a more effective substrate in both reactions.

The specificity of these enzymes for other nucleotides was studied with synthetic UDP-diglyceride, ADP-diglyceride, and GDP-diglyceride. The non-cytosine derivatives were found to have low, but measurable, activity as phosphatidyl donors for the enzymatic synthesis of phospholipids in ex- tracts of E. coli.

Earlier studies in this laboratory (l-3) demonstrated that chemically synthesized GDP-diglyceride serves as a donor of phosphatidyl residues in the enzymic synthesis of phosphatidyl- serine and of phosphatidylglycerophosphate in cell-free extracts of Es&e&&a coli (Scheme l), in a role essentially similar to that previously discovered in enzyme preparations from animal tissues (4). The synthesis of CDP-diglyceride from CTP and phosphatidic acid has also been found to be catalyzed by bac- terial enzymes (5-7) in a reaction like that previously found in extracts of mammalian tissues (8).

The evidence from study of cell-free enzymes strongly sup- ports the central role (Scheme 1) of CDP-diglyceride in the bio- synthesis of membrane phospholipids in E. coli and other bac- teria. However, as pointed out by Cronan and Vagelos (9), there has as yet been no real proof of the occurrence of CDP- diglyceride in E. coli or, for that matter, elsewhere in nature. The present paper reports the isolation and characterization of the cytosine-containing liponucleotides of E. coli, which have been found to be a mixture of CDP-diglyceride and dCDP-di- glyceride. The activity of these liponucleotides and of synthetic UDP-diglyceride, ADP-diglyceride and GDP-diglyceride in the synthesis of phosphatidylserine and of phosphatidylglycerophos- phate catalyzed by E. coli enzymes will also be described.

The levels of cytosine-containing liponucleotides in growing cells of E. coli appear to be only about &th those of phos- phatidic acid, based on estimates from studies with isotopes. The cytosine liponucleotides, like phosphatidic acid (9), also turn over rapidly in vivo. These findings support the hypothesis that the conversion of phosphatidic acid to liponucleotides may

1098

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~HtOOCR CH,OOCR

RCOOCH 0 RCOObH 0 0 CH,-0-;-0-CH,CH(OH)CH,-O-:-OH

OH NH, AH AH

(3) (5) 4 4

RCOObH’ CH200CR 0 RCOOLH CH200CR 0

&H,-0-%-0-CH,CH,NH,

6H

+ co, ~H,-~-~-~-~H,CH(~H)CH~~H + Pi

6H

J (6)

CH200CR RCOOCH rH,OOCR 0

ORCOOCH

~H~-~-~~-~-CH,CHCH,-O%-O-~H,

6H 6H 6H

PATHWAYS OF PHOSPHOLIPID METABOLISM IN ESCHERICHIA COLI -

%!HEME 1

be a rate-making step for the biogenesis of membrane lipids in E. coli.

MATERIALS AND METHODS

nn-[3-i4C]Serine, [5, 6-aH]cytosine, and ATP labeled with 32P in the y position were products of the New England Nuclear Company, Boston. The procedure of Chang and Kennedy was used to prepare sn-glycero-3-[32P]phosphate and sn[2-aH]glycero- 3-P (3). Nucleoside monophosphomorpholidatesl were pur- chased as dicyclohexylammonium salts from the Sigma Chemical Co., St. Louis. Dipalmitoyl-L-a-glycerophosphate was syn- thesized chemically by a method based on that of Baer (10). Triton X-100 (octylphenoxy polyethoxy ethanol) was obtained from Rohm and Haas, Philadelphia.

Synthesis and Purijication of Liponucleotides-A procedure adapted from that of Agranoff and Suomi was used (II). The Tris salt of phosphatidic acid (33 Mmoles) and a nucleoside mono- phosphomorpholidate (51 pmoles) were dissolved in 2 ml of anhydrous pyridine. This solution was incubated at 37” for 3 days in a tightly stoppered glass vessel. The pyridine was re- moved in a rotary evaporator. The residue was redissolved in 12 ml of chloroform-methanol-water (2:3: 1, v/v). After the addition of 20 ml of 0.05 N HCI, the resulting two phases were thoroughly mixed and then separated by a IO-min centrifugation at 2000 x g. The aqueous methanol layer was removed without disturbing the interface (at which a substantial portion of the liponucleotides may be concentrated), and the chloroform layer was washed twice again with 20 ml of water in similar manner. The final chloroform phase, along with material at the interface, was mixed with 10 ml of methanol to form a homogeneous solu- tion, which was adjusted to about pH 7 with 1 M Tris (free base).

This solution was applied to a column (1 x 30 cm) of DEAE-

1 The nucleoside diphosphate diglycerides of the following nucleosides were synthesized: cytidine, 2’-deoxycytidine, uridine, guanosine, and adenosine.

cellulose (Whatman DE-52) in the acetate form, suspended in and washed with chloroform-methanol-water (2:3:1, v/v). The liponucleotides were eluted at 25” in the same solvent with a 500-ml linear gradient (0 to 0.2 of ammonium acetate, pH 7.4), and 5-ml fractions were collected. The liponucleotides were detected in the effluent by their absorbance in the ultraviolet region and were cleanly separated from unreacted phosphatidic acid, which emerged from the column in a peak just preceding the liponucleotides.

Fractions containing the desired nucleoside diphosphate di- glycerides were pooled, partitioned after acidification between chloroform and aqueous methanol, as described above, and taken to dryness in a rotary evaporator. The residue was redissolved in water and neutralized with 2 eq of Tris. Any remaining insoluble debris was removed by centrifugation. Final liponu- cleotide stock solutions of about 5 mM were stable when stored for several months at -10”.

The yield of liponucleotides, based on phosphatidic acid, was about 40%, if care was taken to ensure anhydrous conditions during the coupling reaction in pyridine.

CDP-diglyceride synthesized in this manner had the correct ratio of ester-phosphate-cytosine and was quantitatively con- verted to phosphatidylserine by phosphatidylserine synthetase preparations from E. coli. Treatment with the specific hydrolase described by Raetz et al. (12) led to the cleavage of the synthetic CDP-diglyceride with the production of CMP and phosphatidic acid in stoichiometric amounts. All liponucleotides were also analyzed for purity by thin layer chromatography on Silica Gel F plates (E. Merck, Darmstadt) in the solvent chloroform-meth- anol-water-acetic acid (25 : 15 : 4 : 2, v/v). Every compound migrated as a single spot of ultraviolet-absorbing material, which also contained phosphate and fatty acids, as judged by spray reagents. Each compound had the expected A26,,:A280 ratio when dissolved in chloroform-methanol (2:1, v/v) containing 0.01 M

HCl. Growth Conditions for E. coli-In most of these experiments,

we used cells of strain 205, a derivative of E. coli K-12 (13) gen- erously provided by E. C. C. Lin.z This strain lacks both aerobic and anaerobic glycerophosphate dehydrogenases as well as alka- line phosphatase. It is constitutive for the uptake of glycero- phosphate and converts exogenous sn-glycero-3-P to lipid with high efficiency. Cells were grown in a rotary shaker at 37” on mineral medium 63 (14) supplemented with 1 y0 casein hydrolyzate, 0.3% glucose, and thiamine (2 pg per ml). Some experiments were also carried out with E. coli K-12, A-324.

Enzyme Assays-CDP-diglyceride-L-serine phosphatidyltrans- ferase (phosphatidylserine synthetase) and CDP-diglyceride-sn- glycero-3-P phosphatidyltransferase (phosphatidylglycerophos- phate synthetase) were assayed essentially as described previously (15). Incubations were carried out at 37”. The final concentration of the liponucleotides to be tested was usually 0.3 mM.

Enzyme Preparations-Phosphatidylserine synthetase was prepared by streptomycin precipitation as described previously (15). Phosphatidylserine decarboxylase was pursed about 600-fold3 by the same general methods which we recently re- ported for the CDP-diglyceride hydrolase of E. coli (12). Phos- phatidylglycerophosphate synthetase was obtained in a par- ticulate, membrane-bound form from sonicated preparations of

2 Strain 205 was derived from strain 202 by Lin and co-workers (13).

3 W. T. Wickner, W. Dowhan, and E. P. Kennedy, manuscript in preparation.

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frozen E. coli B (Grain Processing Co., Muscatine, Iowa). Membrane pellets were prepared by centrifugation for 2 hours at 40,000 X g and were washed with 0.01 M Tris-HCl buffer (pH 7.5) containing 1 mM MgC12, 30 mM ammonium chloride, and 6 mM 2-mercaptoethanol.

RESULTS

Double Labeling of E. coli Lipids-If living cells synthesize CDP-diglyceride as shown in Scheme 1, the CDP-diglyceride, formed during growth in medium containing tritiated cytosine and sn-glycero-3-[32P]phosphate should contain tritium in the cytosine portion of the molecule and 32P in the phosphatidyl moiety. A culture (75 ml) of the strain 205 was grown to a cell density of about 7 x 16* per ml, at which time tritiated cytosine (log cpm per pmole) was added to give a final concentration of 3 PM. Shaking was continued for 20 min and sn-glycero-3- [a2P]phosphate (5 X lo7 cpm per pmole) was quickly added to a level of 40 PM. The total time of exposure of the cells to glyc- erophosphate was 10 s.

The labeling was terminated by pouring the culture directly into 46 ml of chloroform-methanol-water (30:15:0.7, v/v), ti- trated to pH 1 with HCl in a 200-ml glass centrifuge tube. Car- rier CDP-dipalmitin (40 mg) was dissolved in this mixture, just prior to the addition of the labeled culture. To extract the la- beled lipids, the tube was tightly stoppered, mixed, and cen- trifuged for 10 min at 2000 x g to separate the phases. Addi- tional acid was added during the partitioning, if the pH of the aqueous methanol layer was greater than 2. Protein, nucleic acid, and cell debris were layered at the interface after centrif- ugation, whereas phospholipids were largely dissolved in the lower, chloroform layer or were at the interface.

The aqueous methanol layer was carefully aspirated without disturbing the interface, as described above for the synthesis of liponucleotides, and the chloroform layer was washed twice with 150-ml portions of distilled water. The final chloroform layer and material at the interface were mixed with 40 ml of methanol to make a single solvent phase. The remaining precipitate was dispersed to maximize the extraction of the phospholipids. This precipitate was then removed by centrifugation at 2000 x g for 20 min.

DEAE-Cellulose Chromatography at pH 7.4-The doubly la- beled phospholipids (containing about 0.1% of the tritium and 1% of the 32P added to the medium) were initially chromato- graphed on DEAE-cellulose at pH 7.4 with a linear salt gradient, as shown in Fig. 1. Carrier CDP-dipalmitin, added as a marker during extraction of the lipids, emerged at a sodium acetate con- centration of 0.06 M, as judged by the absorbance of the fractions at 271 nm. It was recovered with a yield of 60%. The major peak of tritiated lipid, which represents 0.005% of the total tritiated cytosine added to the culture, overlapped with the optical density peak of the carrier CDP-dipalmitin. However, the leading edge of the relatively broader tritium peak emerged significantly ahead of the CDP-dipalmitin carrier. The com- position of the second, smaller tritium peak in Fig. 1 is not known.

Re-chromatography of Cytosine Liponucleotides on DEAE-cel- lulose-As shown in Fig. 1, only small amounts of 32P were re- covered in the peak containing the carrier CDP-diglyceride, in comparison to the phosphatidic acid peak at Fraction 32. Fur- thermore, about 80% of this radioactivity was removed during further purification of the cytosine liponucleotide fraction. Fractions 40 to 47 were pooled, acidified, and partitioned be- tween chloroform and aqueous methanol to remove salts. The

-2 500- ; 08

2

a c

k

$ s

e 250- g 0.4

s 9

OL 0 20 40 60

FRACJ/ONS 13mlJ

FIG. 1. DEAE-cellulose chromatography of labeled lipids. The labeled lipid extract, which also contained nonradioactive carrier CDP-dipalmitin, was chromatographed on a column (1 X 30 cm) of DEAE-cellulose in chloroform-methanol-water (2:3: 1, v/v). After the sample was applied, the column was washed with 2 bed volumes of the solvent. The lipids were eluted at 4” with a 500.ml linear salt gradient (0 to 0.2 in) of ammonium acetate of pH 7.4 prepared in the same solvent; 3-ml fractions were collected and analyzed for absorbance at 271 (l-cm path length) and for radio- activity as indicated.

0.6 c

FRACTIOIVS /3.8m//

FIG. 2. Rechromatography of the cytosine liponucleotide frac- tion on DEAE-cellulose. Tubes from Fig. 1, containing carrier CDP-dipalmitin, were pooled and rechromatographed on an identical column, except that the pH of the ammonium acetate buffer was 5.7.

lipids were redissolved in chloroform-methanol-water (2 : 3 : 1, v/v), applied to a second DEAE-cellulose column, and eluted as in the experiment of Fig. 1, except that the pH of the ammo- nium acetate buffer was 5.7. The results are shown in Fig. 2.

Purijication on Silicic Acid-Final purification of the doubly labeled lipid required further fractionation on silicic acid (Bio- Sil HA, Bio-Rad Laboratories, Richmond, California). Frac- tions 21 to 26 of Fig. 2 were pooled and applied to a small silica gel column (0.2 x 3 cm) in the solvent chloroform-pyridine- formic acid (50:30:7, v/v). Both the carrier and the tritium but only 20% of the azP .were retained by the column, whereas the bulk of 3sP emerged in the run-through. The carrier CDP- diglyceride, together with all of the applied tritium and 20% of the 32P, was eluted with chloroform-methanol (3:2, v/v).

Enzymic Assays of Isolated, Labeled Cytosine Liponucleotides- CDP-diglyceride (or dCDP-diglyceride) synthesized according to Scheme 1 during growth in a medium containing sn-glycero-3-

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“,Jgi, s ‘O I2 I4 t I6 Solvent Front

cm

FIG. 3. Enzymatic conversion of the 32P of the purified cytosine liponucleotides to [32P]phosphatidylethanolamine. The purified liponucleotide was incubated for l-hour at 37” in the presence of 100 UM carrier CDP-dinalmitin. 3 mu n-serine. 0.1% Triton X-100. 0.1 & potassium phosphate of pH 7.4, 50 rg of phosphatidylserine synthetase and 20 pg of phosphatidylserine decarboxylase in a final volume of 0.3 ml. A similar reaction mixture was incubated in parallel without enzyme. The reaction was terminated by the addition of 3 ml of chloroform-methanol (2:1, v/v), 0.7 ml of 0.1 M HCl. and 1 ml of water. The chases were mixed and then separated by a 5-min centrifugation at 2000 X 9. The radio- ac^tivity in -the aqueous methanol and chloroform layers was determined. The chloroform layer contained all of the 3V, and the aqueous layer contained over 90% of the 3H. The chloroform layer was dried under a stream of Nf, and a portion of the material was spotted on a Silica Gel F plate (E. Merck, Darmstadt). This was developed with chloroform-methanol-water-acetic acid (25:15:4:2, v/v)-at room temperature. Phospholipid standards were located on the plate either by their ultraviolet absorbance or by reaction with ninhgdrin. Strips (1 cm) were then scraped into-scintillation vials, aid the 32P content of each segment &s determined by liquid scintillation counting in 10 ml of Patterson- Greene fluid and 1 ml of water (23). The upper panel shows that incubation of the lipid in the absence of enzymes had no effect on the mobilitv of the 32P. which still chromatoaravhed with carrier CDP-dipalmitin. Incubation with the phogph‘atidylserine syn- thetase and the phosphatidylserine decarboxylase, shown in the lower panel, resulted in quantitative conversion of the a2P to phosphatidylethanolamine. Positions of standards (C and PE) are schematically indicated by circles.

[zP]phosphate should contain 32P exclusively in the phosphatidyl moiety. Treatment of such labeled liponucleotide(s) with phosphatidylserine synthetase in the presence of L-serine and phosphatidylserine decarboxylase should lead to the formation of a2P-labeled phosphatidylethanolamine via Reactions 2 and 3 of Scheme 1. These enzymic reactions offered a sensitive assay for the identity and purity of the isolated liponucleotide frac- tion.

Such an experiment is shown in Fig. 3. The 32P of the iso- lated liponucleotide fraction was quantitatively converted to phosphatidylethanolamine, as revealed by thin layer chromatog- raphy of the lipid products of the enzymic reactions. No water- soluble s2P was liberated during the reaction. The identification of the labeled product as phosphatidylethanolamine was con- firmed by deacylation with mild alkali (16), with the formation of glycerophosphorylethanolamine, identified by chromatography on Whatman No. 1 paper in the solvent propanol-ammonia- water (6 : 3 : 1, v/v). Glycerophosphorylethanolamine has an RF of 0.5 in this system.

Q. :: 20

With Hydrolose

J I6

t + Origin sdmt

Front

cm FIG. 4. Enzymatic conversion of the 32P in the purified, doubly

labeled lipid to [32P]phosphatidic acid. This was accomplished by incubation under the conditions described in Fig. 3, except that n-serine was omitted, and 2 pg of purified CDP-diglyceride hydro- lase was employed as enzyme (12). After the reaction was com- pleted, lipids were extracted as in Fig. 3, but thin layer chromato- grams were developed with chloroform-pyridine-formic acid (50:30:7, v/v). In this system the untreated lipid remained on the origin with the CDP-dipalmitin standard, as demonstrated in the upper panel. The lower panel shows that the 32P of the lipid was quantitatively converted to [32P]phosphatidic acid by the CDP-diglyceride hydrolase. Positions of standards (C and PA) are schematically indicated by circles.

A highly specific hydrolase that cleaves CDP-diglyceride with formation of phosphatidic acid and CMP was recently discovered in this laboratory (12). When the isolated 32P-labeled liponu- cleotide fraction was treated with purified hydrolase, labeled phosphatidic acid was formed in almost quantitative yield (Fig. 4).

Identification of 3H-Labeled CMP and dCMP Released by Treat- ment with Phosphatidylserine Synthetase-Treatment of the iso- lated liponucleotide fraction in an experiment such as that of Fig. 3 led to the conversion of essentially all of the tritium to water-soluble products in a reaction completely dependent upon added phosphatidylserine synthetase and L-serine. The water- soluble fraction, obtained as described in Fig. 3, was mixed with carrier CMP and dCMP, which were added in quantities suffi- cient for detection by optical methods, and subjected to chro- matography on Dowex l-X2 (Fig. 5). All of the radioactivity and the carrier were retained by the resin. The nucleotides were eluted with a gradient of ammonium formate in the presence of sodium borate (17). Carrier CMP and dCMP were located in the effluent by their absorbance at 271 nm. Almost equal amounts of tritium were recovered with CMP and dCMP, The over-all recovery of tritium was 85%. Thus the labeled cytosine liponucleotide fraction of E. coli is a mixture of CDP-diglyceride and dCDP-diglyceride.

The results of Fig. 5 were confirmed by paper chromatography. The solvent employed in Fig. 6 resolved two peaks of radioac- tivity from the water-soluble fraction which coincided with car- rier CMP and dCMP.

Relative Amounts of CDP-diglyceride and dCDP-diglyceride- As noted above, a considerable fraction of the major tritium peak of Fig. 1 emerged slightly ahead of the CDP-dipalmitin carrier. When tested with the phosphatidylserine synthetase, the tritiated substance comprising the leading edge of the main tritium peak of Fig. 1 was also found to be enzymically active. Chromatography of the water-soluble product so obtained in

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dCMP CMP TABLE I

Speci$city of the phosphatidylserine synthetase of E. coli for nucleoside diphosphate &glycerides

Initial rates were measured by the standard phosphatidylserine synthetase assay (15). The liponucleotide concentration was 0.3 lll~ in each instance. The incubation mixtures contained 22 pg of partially purified phosphatidylserine synthetase in a final vol- ume of 60 ~1; this amount of enzyme converted about 6 nmoles (out of 20) of CDP-diglyceride to phosphatidylserine in 10 min at 37”. This rate was arbitrarily defined as 100%.

Substrates Relative initial rates L

0 50 60 70 SO 90 100

fRACT/ONS /I m/l

FIG. 5. Dowex l-X2 chromatography of tritiated, water-soluble products. The aqueous methanol layer, obtained by partitioning the incubation mixture described in Fig. 3, contained the tritium which was released enzymically by the phosphatidylserine synthe- tase. The aqueous layers of several experiments were pooled to increase the total counts available for analysis, and the solution was titrated to pH 9 with 1 M Tris (free base). Nonradioactive CMP and dCMP (2 rmoles of each) were added as carrier. The mixture was applied to a column (0.8 X 25 cm) of Dowex l-X2 formate, prepared in 5 mM sodium borate of pH 9.2. After wash- ing with 1 column volume’of the same borate solution, the CMP and dCMP were eluted with a 120 ml (0.2 to 0.6 M), linear am- monium formate gradient of pH 9.2, which contained 5 mM borate throughout. Fractions (1 ml) were analyzed for optical density of the carrier at 271 nm (l-cm path length). CMP emerges after dCMP, and the tritium in each fraction was determined by liquid scintillation counting.

5 IO 15 20 25

t Origin

t SOlVeilt Front

cm

FIG. 6. Paper chromatography of tritiated water-soluble prod- ucts. A sample (1 ml) of the same water-soluble fraction em- ployed in Fig. 5 (containing about 100 cpm) was dried down to a volume of 20 ~1 under a stream of NP. Carrier CMP and dCMP (100 nmoles each) were added in a volume of 20 ~1, and the mixture was spotted on Whatman No. 1 paper. This was developed for 26 cm in the ascending direction at 25” in the solvent propanol-con- centrated HCl-water (85:20:20, v/v). Carrier CMP and dCk?P were located under ultraviolet light, as indicated by the circles. Radioactivity was determined after cutting the paper into l-cm strips.

the paper system of Fig. 6 revealed that it was primarily dCMP. Thus dCDP-diglyceride is eluted slightly ahead of CDP-diglyc- eride under the conditions of Fig. 1. This accounts for the dis- placement of the tritium peak relative to the CDP-dipahnitin carrier. The pooled fractions used for the purification of the liponucleotide had thus not included some of the fractions en- riched in dCDP-diglyceride. When a correction was made to allow for this, it was calculated that the ratio of dCDP-diglyc-

CDP-diglyceride ........................ (100) dCDP-diglyceride ........................ 19 UDP-diglyceride ......................... 4 ADP-diglyceride ........................ 3 GDP-diglyceride ......................... <l

eride to CDP-diglyceride in strain 205 must be about 1.1. For this calculation, it is assumed that after 20 min of growth on labeled cytosine, the specific activities of the cytosine nucleoside and deoxynucleoside pools are the same.

Since it was important to confirm this finding, and because it was also essential to determine whether the presence of a rel- atively high proportion of dCDP-diglyceride was an anomalous property of strain 205, the labeling, isolation, and assay of the liponucleotide fractions as described above was repeated with cells of E. coli A324, a K-12 derivative that is the wild type for the utilization of glycerol. The ratio of dCDP-diglyceride to CDP-diglyceride in this strain was 0.8. Thus, on the basis of cytosine labeling, the ratio of dCDP-diglyceride to CDP-diglyc- eride in both strains is considerably higher than accepted values (0.1 to 0.2) for the ratio of dCTP to CTP in E. coli (18, 19).

Specificity of E. coli Phosphatidyltransjerases for Liponucleo- tides--The relative activity of dCDP-diglyceride and CDP-di- glyceride, as well as of other chemically synthesized nucleoside diphosphatide diglycerides, in the enzymic synthesis of phos- phatidylserine is shown in Table I. The cytosine liponucleotides were far more active than the others. However, with the pos- sible exception of GDP-diglyceride, the slow rates of conversion of the analogs to phosphatidylserine do not reflect trace contam- ination with CDP-diglyceride, since nearly complete conversion of the analogs to phosphatidylserine was achieved by prolonged incubation with a large amount of enzyme.

The rate of phosphatidylserine synthesis with CDP-diglyc- eride was about 5 times faster than with dCDP-diglyceride under the conditions of the standard test system. In contrast, dCDP- diglyceride is more active in the synthesis of phosphatidylglyc- erophosphate (Table II) than is CDP-diglyceride.

The results of the experiments in Tables I and II raise the possibility that CDP-diglyceride might be preferentially utilized for the pathway of Scheme 1 leading to the formation of phos- phatidylethanolamine, whereas dCDP-diglyceride might be preferred as a precursor of the polyglycerophosphatides. How- ever, the relative activity of the cytosine liponucleotides was found to vary greatly with experimental conditions of the assay. When the concentrations of the liponucleotides were varied over a considerable range during the enzymic synthesis of phospha- tidylserine, for example, the ratio of activities was found to be dependent on concentration (Fig. 7). The non-cytosine analogs, however, were much less active throughout the range of con- centrations shown in Fig. 7, but dCDP-diglyceride was pref-

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TABLE II Specijcity of the phosphatidylglycerophosphak synthetase of E. coli

for nucleoside &phosphate &glycerides Initial rate was measured by the standard phosphatidylglyc-

erophosphate synthetase assay previously described (15), except that the liponucleotide concentration in each instance was 0.3 IXIM. The final incubation mixtures contained 14 pg of enzyme protein, which w&s prepared as described under “Materials and Methods.” This amount of enzyme converted about 3 nmoles of CDP-diglyceride (out of 20) to phosphatidylglycerophosphate in 20 min at 37”, which was defined as 100% activity.

Substrates Relative initial rates

CDP-diglyceride. . dCDP-diglyceride. . . UDP-diglyceride. ADP-diglyceride . . . . GDP-diglyceride . . . .

- -

. . . .

.......

.......

.......

.

(100) 170

8 9

<2

O 0.2 0.4 0.6

80,000 j A -j 0.8

FRAC T/ON

FIG. 8. DEAE-cellulose chromatography of phospholipids after labeling with sn-glycero-3-[32Plphosphate and after pulse labeling followed by chase with nonradioactive glycerophosphate. Condi- tions of chromatography were identical with those described in Fig. 1, except that 20 mg of CDP-dipalmitin was used as carrier. Panel A (3.6-ml fractions) shows the results of the pulse labeling, whereas Panel B (3.9-ml fractions) shows the results of the chase. Phosphatidylglycerol, phosphatidic acid, and CDP-dipalmitin eluted around fractions 10,22, and 31, respectively. The identity of the a%P-labeled substances eluting at Fractions 14 and 33 in A has not been established.

L IPOWCL EOTDE /m MJ

FIG. 7. Activity of phosphatidylserine synthetase as a function of cytosine liponucleotide concentration. The initial rate of phos- phatidylserine formation was measured under standard assay conditions (15) with 3 pg of phosphatidylserine synthetase at con- centrations of liponucleotides ranging from 0.04 to 0.6 mM.

erentially utilized in phosphatidylglycerophosphate synthesis at all concentrations tested (data not shown).

Relative Levels of Phosphatidic Acid and of Cytosine Liponu- cleotides-In the experiment of Fig. 1, the principal peak of 32P emerged from the column about 10 fractions earlier than the carrier CDP-dipalmitin, in a region of the chromatogram ex- pected to contain phosphatidic acid. Thin layer chromatog- raphy of the material recorded in this peak revealed that more than 90% of the radioactivity chromatographed with a phos- phatidic acid standard. Deacylation by mild alkaline hydrolysis (16) yielded labeled glycerophosphate, which was identified by paper chromatography, confirming the identity of the principal azP-labeled lipid as phosphatidic acid.

On the assumption that the specific radioactivity of the phos- phatidyl moiety of CDP-diglyceride and dCDP-diglyceride is the same as that of phosphatidic acid, the relative amounts of phos- phatidic acid and cytosine liponucleotides in growing cells of E. coli can be estimated from comparison of the amounts of radio- activity recovered in phosphatidic acid and in the purified lipo- nucleotide fraction. In the experiment of Fig. 1, the radio- activity of the purified liponucleotide was about 5% of that of the phosphatidic acid.

The determination of the relative amounts of phosphatidic

acid and of cytosine liponucleotide was carried out, in a second experiment in which cells were labeled with sn-[2-3H]glycero-3-P for 2 min, rather than 10 s. The amount, of liponucleotide in this experiment was also estimated to be about 5 To of the amount of phosphatidic acid, although phosphatidylethanolamine and phosphatidylglycerol were the predominant radioactive lipids recovered under these conditions. The good agreement of this result, with that obtained with much shorter time of labeling argues that phosphatidic acid and cytosine liponucleotide do indeed attain rapid isotopic equilibrium.

Pulse Labeling of Lipids with sn-Glycero-S-[32P]phosphate fol- lowed by Chase with Nonradioactive sn-Glycero-S-P-If cytosine liponucleotides are rapidly metabolized intermediates, as is suggested by their low levels in E. coli, the a2P radioactivity derived from sn-glycero-3-[32P]phosphate in the cytosine lipo- nucleotide fraction should be lost when pulse labeling of cells is followed by a chase with excess, nonradioactive sn-glycero-3-P. Exponentially growing cells of strain 205 (10 ml at a cell density of 7 x lo* per ml) were divided into two equal portions, both of which were labeled with sn-glycero-3-3zP (4 x log cpm per pmole) added to give a final concentration of 16 PM. After 30 s, the lipids from one culture were rapidly extracted by the methods described above. Unlabeled m-ar-glycerophosphate (final con- centration 2 mM) was added to the other culture to reduce the specific activity of sn-glyceroi3-P by at least 60-fold, and the incubation was continued for a further 30 s. The lipids of both cultures were worked up in parallel in the presence of chemically synthesized CDP-dipalmitin carrier, as described above, and the extracts were chromatographed on DEAE-cellulose columns, shown in Fig. 8. The total incorporation of 32P radioactivity into phospholipids was about the same in both cultures.

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Fig. 8A shows the pattern of radioactive lipids recovered after a 30-s period of pulse labeling. The profile of 32P radioactivity is similar to that of the 10-s labeling shown in Fig. 1, except that the phosphatidylglycerol peak at Fraction 9 in the experiment of Fig. 8A is relatively more prominent than the phosphatidic acid peak at Fraction 22. Fig. 8A also demonstrates the large excess of phosphatidic acid over cytosine liponucleotides pre- viously noted.

Fig. 8B shows the pattern of radioactive lipids obtained after 30 s of “chase.” Over 900jo of the radioactivity in the phos- phatidic acid peak (Fraction 23) was lost, although the phos- phatidylglycerol peak was relatively unchanged. The radio- activity recovered in the cytosine liponucleotide fractions was also greatly reduced after chase with m-a-glycerophosphate.

To determine quantitatively the extent of chase in the cytosine liponucleotide fractions, these were further purified on silicic acid as described above. The identity and purity of the endog- enous 32P-labeled cytosine liponucleotides obtained after the silicic acid step was established by enzymic conversion to phos- phatidylethanolamine under the conditions of Fig. 3. Over 95yo of the 32P radioactivity had been chased from the cytosine liponucleotide pool.

DISCUSSION

Cytosine-containing coenzymes appear to play an essential and highly specific role in the de novo biogenesis of the phos- phodiester bond in phospholipids throughout nature. Thus, CDP-choline, the first of these coenzymes to be discovered, can- not be replaced in the ensymic synthesis of phosphatidylcholine by synthetic UDP-choline, GDP-choline, or ADP-choline (20). In the present study, a similar specificity of phosphatidylserine synthetase and of phosphatidylglycerophosphate synthetase of E. coli for the cytosine coenzymes is apparent. The activity of the non-cytosine nucleoside diphosphate diglycerides, although measurable, was much lower than that of the cytosine liponucleo- tides. Thus, a significant involvement of the non-cytosine ana- logs in Qhospholipid biosynthesis is unlikely. In this regard the intracellular level of ADP-diglyceride, if it is present at all, is lower than that of CDP-diglyceride, as judged by labeling of cells with tritiated adenine under conditions similar to those described in Fig. 1. Nonetheless, the possible role of the non- cytosine derivatives as phosphatidyl donors in some circum- stances is not completely excluded by these experiments.

Phosphatidic acid is present in very low amounts in E. COG, representing less than 1% of the total lipids (9). It is very rapidly renewed in growing cells (16), as is the cytosine lipo- nucleotide fraction. For these reasons, it is probably safe to assume that the specific radioactivity of the phosphatidyl moiety of CDP-diglyceride and of dCDP-diglyceride is the same as that of phosphatidic acid. With this assumption, the relative amounts of phosphatidic acid and of cytosine liponucleotides in growing cells of E. coli can be estimated from the radioactivity in these fractions after labeling with sn-glycero-3-[32P]phosphate. In one such experiment, in which the cells were labeled for 10 s, it appeared that the level of cytosine liponucleotides was about soth that of phosphatidic acid, or less than 0.05% of the total lipids. When the experiment was repeated, but with a period of labeling of 2 min, essentially the same result was obtained, strengthening the assumption discussed above.

The very low levels of cytosine liponucleotide relative to phos- phatidic acid lead us to suggest that the conversion of phospha- tidic acid to liponucleotide may be the rate-making step in the

over-all process of the biogenesis of membrane phospholipids in E. coli.

The discovery that the cytosine liponucleotides of E. coli con- sist of a mixture of the ribonucleoside and deoxyribonucleoside derivatives is not altogether unexpected, in view of the fact that deoxyribonucleoside forms of CDP-choline and CDP-ethanol- amine have been isolated from a variety of natural sources (17). In a study of the possible function of dCDP-choline and of dCDP-ethanolamine in enzyme systems derived from the livers of various species, Kennedy et al. (17) found little difference between CDP-choline and dCDP-choline. However, dCDP- ethanolamine appeared to be less active than CDP-ethanolamine as a precursor of phosphatidylethanolamine in the systems studied.

Kennedy et al. (17) suggested that the ratio of CDP-choline to dCDP-choline found in various tissues might simply reflect the ratio of CTP to dCTP in the same tissues, since there ap- peared to be little difference between the activity of CTP and dCTP in the enzymic synthesis of the nucleotide forms of choline. However, it has been reported that levels of dCTP in E. coli are much lower than those of CTP (18). I f this is the case, it would appear that dCTP may be utilized more efficiently than CTP in the formation of the cytosine liponucleotides, since dCDP-diglyceride and CDP-diglyceride are about equally abundant in viva under our conditions. Steady state levels of liponucleotides, however, may not accurately reflect their re- spective rates of synthesis. The results of the experiment shown in Fig. 8B make it clear that both CDP-diglyceride and dCDP-diglyceride turn over very rapidly in living cells of E. COli.

Ter Schegget et al. (21) have demonstrated the enzymatic synthesis of dCDP-diglyceride from dCTP by a mitochondrial fraction from liver. These interesting results, however, leave open the question of what liponucleotides actually are present in liver.

The ratio of phosphatidylethanolamine to polyglycerophospha- tides in E. coli may be altered under various conditions as re- viewed by Lusk and Kennedy (22). The branchpoint of the pathways leading to these end products involves the transfer of phosphatidyl units from CDP-diglyceride or dCDP-diglyceride either to L-serine or to sn-glycero-3-P (Scheme 1). In the ex- periments outlined in Tables I and II, the striking difference in the ratios of activities of CDP-diglyceride and dCDP-diglycer- ides, as precursors of phosphatidylserine and phosphatidyl- glycerophosphate, makes it necessary to consider the possibility that the specificity for ribonucleotide or deoxyribonucleotide coenzyme may be an import,ant regulatory mechanism deter- mining the relative proportions of phospholipids synthesized. However, the results observed are strongly influenced by the conditions of the assay. Since it is not possible to determine which set of conditions most closely represents those of the living cell, it is difficult to assess the possible physiological sig- nificance of the differences not.ed. Since there is no doubt, however, that there is a considerable specificity under some conditions at least, the possibility deserves further consideration that dCDP-diglyceride is utilized largely as a precursor of phos- phatidylglycerol and cardiolipin, whereas CDP-diglyceride is preferentially used for the synthesis of phosphatidylethanola- mine via phosphatidylserine.

The rate of turnover of the pools of phosphatidic acid and of cytosine liponucleotides was too fast for accurate measurement in the experiment of Fig. 8. The time required for the synthesis and utilization of half the pools appears to be about 5 s or less.

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This result offers striking evidence in support of the postulated 12. central role of phosphatidic acid and of cytosine liponucleotides in the biogenesis of membrane phospholipids in E. coli (Scheme 13 1).

1.

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Christian R. H. Raetz and Eugene P. KennedyEscherichia coliDiphosphate-Diglyceride in the Biogenesis of Membrane Lipids in

Function of Cytidine Diphosphate-Diglyceride and Deoxycytidine

1973, 248:1098-1105.J. Biol. Chem. 

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