JOURNAL OF BACTERIOLOGY, Nov. 1970, p. 712-718 Vol. 104, No. 2Copyright © 1970 American Society for Microbiology Printed In U.S.A.

Cardiolipin-Specific Phospholipase D ofHaemophilus parainfluenzae

II. Characteristics and Possible SignificanceYOSHIE ONO AND DAVID C. WHITE

Department ofBiochemistry, Albert B. Chandler Medical Center,University ofKentucky, Lexington, Kentucky 40506

Received for publication 6 August 1970

A phospholipase specific for cardiolipin (CL) was found in the membrane ofHaemophilus parainfluenzae. The enzyme hydrolyzed CL to phosphatidic acid (PA)and phosphatidylglycerol (PG), indicating that it was a phospholipase D (an enzymeactivity believed to be confined to higher plants). In addition to its substrate spec-ificity, this enzyme was unusual in its requirement for Mg,+ (Km of 1.3 mM) formaximal activity and its inhibition by chelating agents, heavy metals, some deter-gents, and organic solvents. When inhibitors of phospholipase activity were addedto the growth medium, CL accumulated and PG disappeared in the membrane, sug-gesting that the phospholipase D was active in vivo. The activity of phospholipaseD in cell-free homogenates was greater than expected from earlier studies of CLmetabolism and greater than the other phospholipase activities detected in thehomogenate. The high activity of the CL-specific phospholipase D suggests theremight be a very active degradation of CL to PG and PA and an active resynthesisof CL from the hydrolysis products.

In an earlier study (8), an unusual phospholi-pase which hydrolyzed cardiolipin (CL) to phos-phatidic acid (PA) and phosphatidylglycerol (PG)was detected in Haemophilus parainfluenzae. Theenzyme is unusual in its specificity for CL and thefact that it is a phospholipase D (8). Phospholi-pase D has previously been thought to be con-fined to higher plants. A surprising feature of theenzyme proved to be its very high activity, con-sidering that CL accounts for only 3 to 5% of thetotal phospholipid and that CL has a relativelyslow metabolism in pulse-chase experiments.Previous studies have shown that the CL loseshalf of the 32p and 14C from the middle glycerol infour bacterial doublings in exponentially growingcells (14). The high enzymatic activity found incell-free homogenates suggested that the rapidhydrolysis of CL was coupled with a rapid resyn-thesis from the hydrolysis products without greatdilution from other pools.

MATERIALS AND METHODSMaterials. Triton X-100 (t-octylphenoxy polyethoxy

ethanol) was supplied by Rohm and Haas, Philadel-phia, Pa.; Sarcosyl NL-30 (sodium lauryl sarcosinate)was supplied by Geigy Industrial Chemicals, Ardsley,N.Y., through the kindness of H. J. McElhone;Triton N-101 (nonylphenoxy polyethoxy ethanol),Tween 20 (polyoxyethylene sorbitan monolaurate),

sodium dodecyl sulfate (SDS), hexadecylpyridiniumchloride, sodium deoxycholate, bis-p-nitrophenyl-phosphate (B-PNPP), and ethylenediaminetetraace-tate (EDTA) were supplied by Sigma Chemical Co.,St. Louis, Mo. Other materials were supplied as de-scribed (8, 10, 14).

Growth of bacteria. The growth of H. parainfluenzaeas a source of phospholipases and Escherichia coliK-12 as a source ofCL has been described (8).Enzyme preparation and assay. H. parainfluenzae

was harvested and ruptured by ultrasonic vibration toform the cell-free homogenate used in most of the ex-periments reported (8). The substrates were emulsifiedin 2% (w/v) Triton X-100 after their isolation fromE. coli grown with 32p or 14C (8). The chromatographicmethods for the separation and analysis of the lipidshave been described (8, 12). Phospholipase D activitywas based on the hydrolysis of CL to PA and PG. Atypical reaction mixture contained: 100 ug of enzymeprotein, 10 mM MgCl2, 0.07% (w/v) Triton X-100, 20nmoles of CL (20,000 counts/min of 32P) in 50 mmpotassium phosphate buffer (pH 7.5) in 0.5 ml. Themixture was incubated at 37 C for 30 min, and thelipids were extracted and separated by chromatog-raphy on silica gel-loaded paper, the lipids were lo-cated by autoradiography, and the radioactivity wasdetermined (8). The activity of phospholipases A andC in H. parainfluenzae were assayed after ammoniumsulfate precipitation by a modification of a proceduredeveloped previously (7). Phospholipase A and C ac-tivities assayed after ammonium sulfate precipitationrepresented about 90% of the activity present in the

712

CARDIOLIPIN-SPECIFIC PHOSPHOLIPASE D

total homogenate. The reaction in 1 ml was stoppedby adding 0.5 ml of 20% (w/v) trichloroacetic acidand 0.5 ml of 20% (w/v) sodium deoxycholate. Aftermixing with a Vortex mixer (Scientific Industries, Inc.,Springfield, Mass.), the suspension was extracted with5 ml of n-hexane. The mixing was then repeated andfollowed by centrifugation at 4,000 X g for 3 min. Thehexane layer was recovered, and the 14C and 8P weredetermined. Reconstruction experiments indicatedthat 100% of added fatty acids or diglycerides andless than 2% of the phospholipids (also lysophos-pholipids) were recovered in the hexane in the presenceof a final concentration of 5% sodium deoxycholate.The n-hexane-extractable products of phospholipidhydrolysis were identified as diglycerides and fattyacids by Silica Gel G thin-layer chromatography in asolvent system of petroleum ether (boiling point, 30 to80 C)-diethyl ether-acetic acid (60:40:1, v/v) orchloroform-methanol-water (65:35:4, v/v). Reactionproducts formed after incubation with Bacillus cereusphospholipase C (diglyceride) and venom phospholi-pase A (fatty acids) were used as standards.

Phosphatidylserine (PS) decarboxylase activity wasassayed by comparing the loss of PS to the increase inphosphatidylethanolamine (PE). The lipid extract waschromatographed in the two-dimensional, silica gel-impregnated paper system described (8). The sub-strate consisted of PS-3-14C (Tracerlab, Waltham,Mass.) diluted with unlabeled PS (Applied ScienceLabs., State College, Pa.) suspended in 2% (w/v)Triton X-100. In the incubation mixture, 100 nmolesof PS (50,000 counts/min of 14C) was added to thehomogenate as in the phospholipase D assay. Phos-phodiesterase was assayed with B-PNPP in the pres-ence of 10 mm MgCl2 (2). Protein and phosphate weredetermined colorimetrically (3, 13).

RESULTS

Phospholipase D activity. Previously, the prod-ucts of the CL-specific phospholipase D activityin H. parainfluenzae were shown to be PA andPG (8). The rate of the reaction was linear withprotein concentrations up to 100 to 150 ,ug ofprotein (Fig. 1). At higher protein concentration,PA was degraded in 30 min of incubation. Theenzyme activity was linear with time for 50 min inthis assay system (Fig. 2). The enzyme had a pHoptimum between pH 7.5 and 8.0 (Fig. 3). Therate of hydrolysis of CL depended on the sub-strate concentration between 20 and 200 nmolesper ml with an apparent Km value of 2 X 104 to4 X 10-4 M and a maximal velocity (Vmax) of 20nmoles per min per mg of protein (Fig. 4). Highersubstrate concentrations were impossible to testas emulsions formed. The enzymatic activity de-creased by 98% when the preparation was heatedto 60 2 C for 10 min in the assay mixture con-taining 100 Mg of protein per 0.5 ml. The crudeenzyme can be stored at -20 C for several monthswithout loss of activity.

Activity and inhibition of phospholipase D activ-

0

0

49

0

U.

50

-o 50 100 150 ZOO

PROTEIN (mg)

FiG. 1. Effect of protein concentration on phos-pholipase D activity. The incubation mixture contained20 nmoles of CL (20,000 counts/min of 2P) addedas an emulsion in 2% (v/v) Triton X-100 (8), 10 mMMgCl2, 0.07% (v/v) Triton X-100, and S0 mM potas-sium phosphate buffer (pH 7.5) in a total volume of50 ,uiters. Cell-free homogenate of H. parainfluenzaecontaining the phospholipase D was added io a finalvolume of 0.S ml. After incubation at 37 C for 30 min,the mixture was extracted, and the decrease in CL was

measured. Chromatography (8) indicated that the hy-drolysis products were PG and PA.

0

-i.J

0

0w

0

U)

w

0

HCUBATION TIME (MIN)

FIG. 2. Time course of CL hydrolysis by phos-pholipase D from H. parainfluenzae. The reaction mix-ture contained 100 jug ofprotein and was analyzed asin Fig. 1.

ity. The activity of phospholipase D in 1 mmMgC12 was activated by the divalent ions:Mg2 > Co2+ > Mn2+ > Ca2+ (Fig. 5). Maxi-mal activity occurred with 10 mM MgC12. TheKm for MgC12 was 1.33 mm, as measured in ex-

periments like those illustrated in Fig. 4. Heavymetals, EDTA, and the detergents SDS, Tween20, and hexadecylpyridinium chloride, werestrongly inhibitory (Table 1). Cyanide, fluoride,ferric ions, iodoacetate, and the detergents Triton,deoxycholate, and Sarcosyl, had little effect on theenzyme activity. Treatment of the incubation mix-ture with diethyl ether destroyed enzyme activity.

Separation of phosphodiesterase activity. Thegreat specificity for CL suggested the enzyme

VOL. 104, 1970 713

ONO AND WHITE

70[

60O

50

a

:049

w

0

40

30

20[

1o0

5 6 7 8 9

60114

12

5O

40

10 2

i

0

8 ft

w

6 cn-J

0

4c

a

a

t 300

a,20

10

2

(PH)

FIG. 3. Optimal pH level of phospholipase D fromH. parainfluenzae. CL hydrolysis was measured as inFig. I except that the pH was changed. The incubationmixture contained 100 jug ofprotein (cell-free homog-enate of H. parainfluenzae). Symbols: 0, 50 mmacetate buffer; *, indicates 50 mm phosphate buffer;A, 50 mM Tris-hydrochloride buffer.

(1/S)(a MOLET1)

FIG. 4. Effect of CL concentration on the rate ofhydrolysis by H. parainfluenzae phospholipase D.Assay conditions were as in Fig. 1 except that the CLconcentration was varied. The incubation mixturecontained 100 Mg of protein (cell-free preparation).

DEGRADATION OF CL

I0

4aJ

0

0.U,

4a

2

0. 100 2 4 6 8 10

mm

FIG. 5. Effect of metal ions on phospholipase Dfrom H. parainfluenzae. Assay conditions were as inFig. I with 100 ,sg of protein of the cell-free homog-enate. Symbols: 0, enzymatic activity in the presenceof I mM MgC12; *, activity in I mm MgCl2 plus higherconcentrations of MgCI2; V, activity in I mm MgCIlplus higher CoCl2 concentrations; *, activity in I mmMgCI2 plus higher MnCI2 concentrations; A, activityin I mM MgCl2 plus higher CaCl2 concentrations.

could be a relatively nonspecific phosphodiester-ase. However, gel filtration column chromatog-raphy clearly separated the phospholipase D ac-tivity from the phosphodiesterase activity (Fig. 6).The CL-specific phospholipase D activity elutedin the same volume as Blue Dextran of molecularweight about 2,000,000. B-PNPP did not inhibitthe CL-specific phospholipase D activity(Table 1).SubceDular localization of phospholipase D.

After sonic oscillation, the phospholipase D ac-tivity was much enriched in small membrane frag-ments which sedimented at 25,000 X g (Table 2).Some activity remained in the supernatant, possi-bly attached to smaller membrane fragments sincesome cytochrome b can be detected in this frac-tion after prolonged sonic oscillation.PS decarboxylase and phospholipases A and C

activities. PS decarboxylase was active in the cell-free homogenate (Table 3). Diglyceride and freefatty acids were formed when phospholipids wereincubated with a partially purified homogenate(Table 3). After the addition of 10 mm MgCl2, theCL-specific phospholipase D activity was readilydemonstrated in this preparation. The total activ-ities of the phospholipases A and C were not in-creased by adding detergents, calcium ions,diethyl ether, or changing the pH.

714

A TRIS H Cl* PHOSPHATE BUFFER0 ACETATE BUFFER

J. BACTERIOL.

i

CARDIOLIPIN-SPECIFIC PHOSPHOLIPASE D

TABLE 1. Inhibitors of H. parainfluenzaephospholipase D

Inhibitor Concn activityv

mm

MgC12 1 100BaC12 1 120ZnCl2 1 20HgCl2 1 16CuC12 1 13FeCl3b 1 117Ethylenediaminetetra- I 0

acetateKCN 10 108NaF 10 93Iodoacetate 10 86Bio-p-nitrophenylphos- 2 122phate

Sodium deoxycholate 10 121Sodium dodecyl sulfate 5 0Hexadecylpyridinium 0. d (w/v) 0

chlorideTween 20 0.ld (w/v) 15Sarcosyl NL-30 0.ld (w/v) 108Triton N-101 o.ld (w/v) 156Diethyl ether treatedc 30

a Incubation mixture contained 32P-cardiolipin(20 nmoles), 1 mM MgCl2, 0.07% (w/v) TritonX-100, 50 mm potassium phosphate buffer (pH7.5), and 100 jg of protein in the cell-free homog-enate to which the components listed here wereadded. Incubation was carried out at 37 C for30 min, and the amount of cardiolipin, phos-phatidic acid, and phosphatidylglycerol wasdetermined after chromatographic separation.bAssay as above except that 50 mm Tris-hydro-

chloride buffer was used.c To 0.5 ml of the reaction mixture, 0.5 ml of

diethyl ether was added, the supension was mixedvigorously, and the ether was removed in a streamof nitrogen before the incubation.

d Expressed as per cent.

Phospholipase D activity in vivo. It was possibleto inhibit phospholipase D activity in the cell-freehomogenate (Table 1). Radioactivity was incor-porated into the fatty acids, glycerols, and phos-phate of the phospholipids of H. parainfluenzaeduring 3.4 doublings, after which the cells werewashed and suspended into nonradioactive me-dium; thus, the replacement of the radioactivitywith unlabeled isotopes could be studied (Fig. 7).During growth in nonradioactive medium, theCL lost about 5% and the PG about 45% of theirradioactivities in 1 hr. The doubling time was 48min. In the presence of 1 mM CuCl2, which in-hibited phospholipase D activity in vitro by 87%the doubling time of the cells increased to 54 min(110% of normal), the CL accumulated and the

LI PDoi

N

40 S 0 0 8 9 0

64N

a,*0

wz

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40spat bu 0e 60H 705 60s 90ed 00iite w r

Dlte H.pranlunaeecolumnw2tSbye60s-ydcm)oid

Tris-hydrochiowride opH 0.S) Min and-m

fractions were collected. A 0.5-mi portion was removed

from each tube and assayed for phospholipase D activity

as in Fig. 1 (0), and phosphodiesterase was assayed

with 1.1 mMi B-PNPP in the assay mixture (a). The

elution volume of Blue Dextran (molecular weight,

2,000,000) was also determined by its absorption at

700nm (v).

TABLE 2. Localization ofphospholipase D

activity in H. parainfluenzae

SpecificFraction Total phospho-protein lipase D

activity'

mg

Total cell-free homogenateb..... 485 0.84Fraction Ic..................... 267 0.62Fraction Ild........... ........ 48 2.96Supernatante ................... 184 0.49

a Values expressed as nanomoles hydrolyzedper minute per milligram.bH. parainfluenzae suspended in 50 mm phos-

phate buffer (pH 7.5) with 10 mM MgCl2 weretreated with ultrasonic vibration at the maximallevel of the Branson LS-75 Sonifier at intervalsfor a total of 5 min (8). After the treatment, nointact cells were detected by phase-contrastmicroscopy.

c Fraction I indicated large membrane-wallfragments in the pellet after centrifugation at4,030 X g for 10 min.

d Fraction II indicated smaller membranefragments in the pellet after centrifugation at25,000 X g for 30 min.

e Supernatant of fraction II.

715VOL. 104, 1970

ONO AND WHITE

TABLE 3. Phospholipase and phosphatidylserinedecarboxylase activities of H. parainfluenzae

Substrate Cell-free Partially purifiedhomogenatea preparationb

PE............. ND 0.043PG............. ND 0.005CL............. 3.29 0.006 (3.40)PSc............. 1.88

a Values expressed as nanomoles degradedper minute per milligram of protein. Assay con-ditions: 50 juliters containing 100 nmoles of asubstrate either phosphatidylethanolamine (PE),phosphatidylglycerol (PG), or (cardiolipin CL;specific activity, about 1,000 counts/min of 3pper nmole) or PS (phosphatidylserine; specificactivity, 1,500 counts/min of 14C per nmole,serine-3-14C labeled) that had been suspended in2% (w/v) Triton X-100 was added to 10 mm potas-sium phosphate buffer (pH 7.5) containing 10 mMMgCl2 and 0.85 mg of protein of the cell-freehomogenate of H. parainfluenzae in 1 ml. Thereaction was assayed after 15 min at 37 C, and therate of nP loss was calculated. ND, not detected.

b Values expressed as nanomoles degraded perminute per milligram of protein. Assay conditions:50 jliters containing 50 nmoles of either PE, PG,or CL labeled in the fatty acids by growth withacetate-i 14C at specific activities of 1,250, 1,250,and 285 counts per min per ;umole, respectively,and suspended in 2% Triton X-100 was added to50 mM glycylglycine-NaOH buffer (pH 7.0) with2.25 mg of protein in a final volume of 1 ml. Theenzymes were prepared by sonic oscillation inthe presence of 0.5% (w/v) sodium deoxycholate,centrifugation at 30,500 X g for 30 min, and pre-cipitation of the supernatant with 22.8% am-monium sulfate. After a second centrifugation,the precipitate was utilized for enzyme assays.Samples were removed at intervals, trichloroaceticacid and deoxycholate were added, and the fattyacids and diglycerides were extracted with hexane.Thin-layer chromatography with Silica Gel Gwith a solvent of chloroform-methanol-water(65:35:4,v/v) was used to separate the fatty acids(RF 0.7) and the diglycerides (R, 1.0). CL-specificphospholipase D was assayed in the preparationafter adding 10 mM MgCI2 (number in paren-theses).

c PS decarboxylase was assayed as the rate ofPS disappearence and PE appearence after sepa-ration by chromatography on silicic acid-impreg-nated paper (8).

PG turnover remained about the same. In thepresence of 1 mm ZnCl2, which inhibited phos-pholipase D activity 80% in vitro, the doublingtime slowed to 150 min and CL accumulatedabout as rapidly as with CuCl2; the PG turnover,however, slowed to half its maximal rate. EDTA,which completely inhibited phospholipase D ac-tivity in vitro, stopped growth and caused an

increase in CL from 5 to 15% and a decrease inPG from 11 to 3.5% of the total lipids in 1 hr.

DISCUSSIONThe CL-specific phospholipase D activity of

H. parainfluenzae required divalent metal ions foractivity. The unusual substrate specificity (8) sug-gested the phospholipase D activity represented anonspecific phosphodiesterase. Since B-PNPP didnot inhibit phospholipaseD activity (Table 1) andthe phospholipase D and B-PNPP phosphodies-

CONTROL mM Cu-- ImM Zn** mnM EDTA

06 06o04 0

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I 40

IFT 20w

I09

4i

,-

0

a:CY

0

CARDIOLONN40

20

10: Is

6. 4

i 1 2

PHOSPHATIOYL GLYCEROL40 4

44

0 0 0 0

HOURS

FIG. 7. Activity of H. parainfluenzae phospholipaseD in vivo. Cells were grown with 200 pCi of H,3'PO4and 100 jCi of glycerol-1,3-14C (U) or 100 pCi ofH3uPO4 (a) and 100 ,uCi of acetate-1-14C (A) per

300 ml for 3.4 doublings. The acetate-1-14C labels thefatty acids ofthe phospholipids and the glycerol-i ,3-14Clabels both the fatty acids and the glycerols (14).After growth with 2P and 14C, the cells were washedand suspended in nonradioactive medium and allowedto grow for I hr. The nonradioactive medium was

supplemented with I mms CuCl2, I mm ZnCI2, or I mmEDTA as indicated at the top of the figure. Sampleswere withdrawn at the beginning and end of the incu-bation in nonradioactive medium; the lipids were ex-

tracted, separated by two-dimensional chromatographyon silica gel-loaded paper (8), and located by auto-radiography; and the proportions of 2P and 14C inthe CL (middle curves) and PG (lower curves) were

determined. The lipids contained between 2 X 106and 9 X 105 counts/min of 2P and 3 X 104 and 15 X104 counts/min of 14C. The upper curves represent thechanges in bacterial density measured as the changesin absorbance at 750 nm (13).

716 J. BACTERIOL.

CARDIOLIPIN-SPECIFIC PHOSPHOLIPASE D

terase activities were clearly separated by gelfiltration chromatography (Fig. 6), the phospho-lipase D activity was characteristic of a true phos-pholipase.The H. parainfluenzae phospholipase D had

additional properties that were not typical forother phospholipases. The specificity for CL wasunusual. PE, PG, PA, and PS from either E. colior H. parainfluenzae; phosphatidylcholine, phos-phomono- and dimethylethanolamine from yeast;and ceramide phosphoryl glycerol, ceramide phos-phoryl glycerol phosphate, and ceramide phos-phoryl ethanolamine from Bacteriodes mel-aninogenicus were not hydrolyzed. Although thephospholipase D activity was inhibited by someheavy metals, cyanide, fluoride, ferric ions oriodoacetate had no effect on its activity (Ta-ble 1). Some detergents, SDS, hexadecyl-pyr-idinium chloride, and polyoxyethylene sorbitanmonolaurate, were inhibitory although alkylphenoxypolyethoxyethanols or sodium laurylsarcosinate were stimulatory. Diethyl ether, whichis usually required or at least stimulatory to phos-pholipase activity, inhibited the H. parainfluenzaephospholipase D activity as utilized in this study(Table 1). A butanol treatment of this enzymepreparation also completely inactivated the en-zyme. Addition of either a total lipid preparationfrom H. parainfluenzae or PE or PG that weresuspended in Triton X-100 to the solvent inac-tivated enzyme did not restore activity. Calciumions which activate many phospholipase activitieswere not stimulatory in the presence of 1 mMMgCl2 (Fig. 5).The phospholipase D activity has a requirement

for a high Mg2+ ion concentration (Km of 1.3 mM).This high concentration is reasonable in vivo,since the calculated soluble Mg2+ concentrationin E. coli is 4 mM (4) and other enzymes like DNApolymerase (9) or the amino acid-activating en-zymes (1) require at least 1 mm Mg2+ for activity.

Phospholipase A has been shown to be mem-brane-bound in Mycobacterium phlei (6), suggest-ing its involvement in membrane function. TheH. parainfluenzae phospholipase D activity wasalso enriched in membrane fragments after cellrupture (Table 2), and a high Mg2+ ion concen-tration seems necessary for membrane stability(5). In H. parainfluenzae, the chelating agentEDTA inhibited CL-specific phospholipase Dactivity (Table 1) caused an accumulation of CLand a loss ofPG (Fig. 7), and, when added to cellsfrom the exponential growth phase suspended intris(hydroxymethyl)aminomethane (Tris) buffer,caused a release of membrane fragments fromviable cells (10). The membrane fragments re-leased from the cells in EDTA-Tris are particu-larly enriched in CL and to a lesser extent in PGwhen compared to the rest of the membrane (11).

In pulse-chase experiments with exponentiallygrowing cells, CL has a slow metabolism and is aminor lipid (3 to 5% of the total) in H. parain-fluenzae (12). The CL-specific phospholipase Dhas an unexpectedly high activity. This phospholi-pase D was more active than PS decarboxylase inhomogenates (Table 3), and PS decarboxylaseforms the PE which comprises 78 to 85% of thetotal phospholipid (12). At the Vmax, the CL-specific phospholipase D activity could degradeall of the CL in 1 or 2 min. Both phospholipase Aand C activities were predicted from the turnoverof PG and PE during exponential growth (14).Phospholipase C activity with PG was expected tobe four times more active than CL-hydrolyzingactivity (14). In the partially purified preparationsnecessary to demonstrate phospholipase A and Cactivities, the phospholipase D activity was ahundred times the maximal activities of phospho-lipase A and C thus far demonstrated (Table 3).The CL-specific phospholipase D was active in

vivo, as demonstrated by pulse-chase experimentwith exponentially growing cells performed in thepresence of enzyme inhibitors (Fig. 7). As inprevious experiments, 'IC and 32p in the fattyacids and glycerols were lost faster from PG thanfrom CL in the chase period. If 1 mM CuCl2 werepresent, the growth rate was decreased by 10%,rate of loss of glycerol from PG was decreased by10%, and CL was accumulated. In the presence of1 mm ZnC12, there was a doubling of the genera-tion time, a doubling of the turnover time for PG,and, again, an accumulation of CL. In the pres-ence of 1 mm EDTA, growth stopped, CL ac-cumulated (an increase of 10% in the 14C-glycerol)accompanied by an almost equal loss of PG (8%loss of "4C-glycerol) (Fig. 7). In vitro, 1 mM CuCl2,ZnCl2, and EDTA inhibited phospholipase D

P

-OH

P-OH

2 3

P CCH CH2 NH2

4

OH

OH

FIG. 8. Diagram ofthe metabolic interactions ofCL.

VOL. 104, 1970 717

ONO AND WHITE

activity by 87, 80, and 100%, respectively, andeach of these caused an accumulation of radio-activity in CL in exponentially growing H. para-influenzae. CuCl2 and ZnCl2, which slow thegrowth rate, also slow the turnover of PG. EDTA,which completely inhibited growth and phos-pholipase D activity, caused a rapid accumulationof CL, matched by a similar decrease in PG. Thisevidence strongly suggests that the CL-specificphospholipase D is functioning in vivo and thatthe rate of turnover of CL determined in a classicpulse-chase experiment is a gross underestimationof the true rate. The underestimation is because ofrecycling activity between PG and CL. CL musthave a high rate of metabolism and is both formedfrom and contributes to the PG pool. Since thePG pool is so large, the rapid metabolism of CLcannot be detected in classical pulse-chase experi-ments. Possible reactions are indicated in Fig. 8.The phospholipase D forms PA and PG from CL(reaction 1, Fig. 8). The PA can be transformedinto PE, PG, or CL, and the PG into CL viacytidine diphosphate diglyceride (reactions 2 to 4,Fig. 8).The membrane localization of the CL-specific

enzyme, the apparent high rate of CL metabolism,and the fact that the CL apparently is concen-trated in the 50S portion of the ribosome (K. F.Hedden and E. Cundliffe, Bacteriol. Proc., 1970,p. 66) makes further understanding of the func-tion of this enzyme imperative.

ACKNOWLEDGMENTS

This study was supported by Public Health Service grant GM10285 from the National Institutes of General Medical Sciences.

LITERATURE CITED1. Berg, P., F. H. Bergman, E. J. Offngand, and M. Dieckmann.

1963. The enzymatic synthesis of amino acyl derivatives ofribonucleic acid. I. The mechanism of leucyl-valyl-isoleucyl-and methionyl ribonucleic acid formation. J. Biol. Chem.236:1726-1734.

2. Futai, M., and Mizuno. 1967. A new phosphodiesteraseforming nucleoside 5'-monophosphate from rat liver. J.Biol. Chem. 242:5301-5307.

3. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Ran-dall. 1951. Protein measurement with the Folin phenolreagent. J. Biol. Chem. 193:265-275.

4. Lusk, J. E., R. J. P. Williams, and E. P. Kennedy. 1968.Magnesium and the growth of Escherichia coli. J. Biol.Chem. 243:2618-2624.

5. McQuillen, K. 1960. Bacterial protoplasts, p. 288-289. InI. C. Gunsalus and R. Y. Stainer (ed.), The bacteria, vol. 1.Academic Press Inc., New York.

6. Ono, Y., and S. Nojima. 1969. Phospholipase of the membranefraction of Mycobacterium phli. Biochim. Biophys. Acta176:111-119.

7. Ono, Y., and S. Nojima. 1969. Phospholipase A of Myco-bacterium phlei: a regulatory membrane enzyme withferric iron as effector. J. Biochem. (Tokyo) 65:979-981.

8. Ono, Y., and D. C. White. 1970. Cardiolipin-specific phos-pholipase D activity in Haemophilus parainfluenzae. J.Bacteriol. 103:111-115.

9. Richardson, C. C., C. L. Shildkraut, H. V. Aposhian, and A.Kornberg. 1964. Enzymatic synthesis of deoxyribonucleicacid. XIV. Further purification and properties of deoxyri-bonucleic acid polymerase of Escherichia coli. J. Biol.Chem. 239:222-232.

10. Tucker, A. N., and D. C. White. 1970. Release of membranecomponents from viable Haemophilus parainfluenzae. J.Bacteriol. 101:498-507.

11. Tucker, A. N., and D. C. White. 1970. Heterogeneity of thephospholipid composition in the bacterial membrane. J.Bacteriol. 101:508-513.

12. White, D. C. 1968. Lipid composition of the electron transportmembrane of Haemophilus parainfluenzae. J. Bacteriol.96:1159-1170.

13. White, D. C., and F. E. Frerman. 1967. Extraction, charac-terization and cellular localization of the lipids of Staphylo-coccus aureus. J. Bacteriol. 94:1854-1867.

14. White, D. C., and A. N. Tucker. 1969. Phospholipid me-tabolism during bacterial growth. J. Lipid Res. 10:220-233.

718 J. BACTrERIOL.