Biosynthesis of cardiolipin in the membranes of Micrococcus lysodeikticus

280 BBA

BIOCHIMICA

ET BIOPHYSICA

ACTA

55889

BIOSYNTHESIS MICROCOCCUS

AUGUST

J. DE

OF CARDIOLIPIN

SIERVO*

AND

Department of Microbiology, (Received

January

IN THE

MEMBRANES

OF

LYSODEIKTICUS

rgth,

M. R. J. SALTON

New York University. School of Medicine,

New, York, N.Y.

(U.S.A.)

1971)

SUMMARY

An enzyme which cardiolipin (diphosphatidyl

catalyzes the conversion of phosphatidyl glycerol to glycerol) has been found to reside solely in the membrane

of Micvococcus lysodeikticus. This enzyme has been tentatively designated as “cardiolipin synthetase”, since its reaction mechanism has not been defined. It appears to synthesize one molecule of cardiolipin from two molecules of phosphatidyl glycerol and the conversion has been followed by using an assay employing 3aP-labelled phosphatidyl glycerol as the only added substrate. Higher than go% conversion of phosphatidyl glycerol to cardiolipin has been achieved with isolated membranes as the enzyme source and by stimulation with Triton X-100. A low-density, small particle fraction

bearing

“cardiolipin

synthetase”

activity

has been released from whole mem-

branes. Such preparations exhibit activity in the absence of detergent and are devoid of phosphatidic acid cytidyltransferase activity found in whole membranes. In contrast to several other phospholipid synthesizing enzymes reported1-4, the synthesis of cardiolipin by this enzyme(s) is not dependent upon added Mg2+ or K+. Cardiolipin synthesis proceeds in the absence of detectable CDP-diglyceride and without significant degradation of phosphatidyl glycerol to phosphatidic acid.

INTRODUCTION

Bacterial

phospholipids

are almost

exclusively

localized

in the membrane

sys-

tems of the celW, where they undoubtedly perform the general barrier functions found for this class of lipid in all biomembranes ‘. In addition, the phospholipids probably enter into specific associations with certain membrane enzymes where they are required for optimum enzymatic activities as in mammalian cell membraness~e. Investigations in this laboratory have been directed towards the elucidation of the organization of the multi-functional membrane of Micrococc~s lysodeikticus. In view of the earlier reports of the presence in particulate fractions of enzymes responsible t This paper was presented in part at the 70th Annual Meeting of the American Society Microbiology, Boston, Mass., 26 April-r May, 1970. * Present address: Department of Microbiology, University of Maine, Orono, Me., U.S.A. Biochim. Biophys. Acta, 239 (1971) 28o--292

for

281

MEMBRANE SYNTHESIS OF CARDIOLIPIN

for the biosynthesis of bacterial phospholipids1-4120,it appeared likely that such enzymes would be of membrane origin. Membranes of M. lysodeikticus have accordingly been examined for the presence of enzymes involved in phospholipid biosynthesis and the results on the synthesis of cardiolipin are reported in this paper. MATERIALS AND METHODS Growth conditions and membrane isolation M. lysodeikticus (NCTC 2665) was grown in a peptone-yeast extract medium as

previously described10 and aerated by shaking at 30’ in a New Brunswick incubator shaker. Cells were harvested in the stationary phase (approx. 24 h) by centrifugation and were washed twice with cold 0.05 M Tris-HCl buffer, pH 7.5, and incubated at room temperature (about 24”) for 30 min to I h in the Tris buffer containing 200 pg/ml lysozyme and 0.6% NaCl. Upon complete lysis of the cells, ~oopg/ml deoxyribonuclease was added and incubated for an additional 30 min. Membranes were deposited by centrifugation at 30000 x g for 30 min and the membrane pellets usually washed 3 times with cold 0.05 M Tris-HCl buffer (pH 7.5) to remove cytoplasmic components. Final preparations were stored at -70’ in the Revco apparatus. Extraction,

identijication, and isolation of membrane Phospholipids Lipid was extracted from whole cells, membranes, or assay fractions with chloroform-methanol (I : 2, v/v) by the method of BLIGHANDDYER’~. The chloroform phase was separated and dried under a stream of N, gas. The lipid was redissolved in chloroform and stored at -70~. Phospholipids were separated and identified on silica gel loaded paper (Whatman SG-81) using the methods described by KATES~ and solvent systems of WUTHIER’~.Both one and two dimensional ascending chromatographs were made. Rhodamine 6G at a concentration of 0.0012% (w/ v ) in distilled water was used in staining for lipids. Tests were also performed to detect amino and vicinal hydroxyl groups12. Identification of the phospholipids was further verified by the mild alkaline methanolysis procedure described by WHITE ANDFRERMAN~~. Phospholipid standards used were cardiolipin, phosphatidic acid and phosphatidy1 inositol purchased from General Biochemicals, Chagrin Falls, Ohio, and phosphatidyl glycerol obtained from Supelco Inc., Bellafonte, Pa. 32P-labelled M. lysodeikticus phospholipids were prepared by growing cells in the presence of [3aP]orthophosphate. Labelled-lipid was extracted, streaked out on silica gel paper and separated with the chloroform-methanoldiisobutyl ketoneacetic acid-water (45 : 15 : 20: 30 : 4, by vol.) solvent system13. The phospholipid bands were cut out, eluted with chloroforn-methanol (I : 2, v/v) followed by 95% methanol. The purified phospholipids were weighed and stored in chloroform at -70”. Preparation

of membrane fractions for enzymatic analysis

In several experiments, membranes subjected to three washes in 0.05 M Tris buffer were used as the enzyme source without any further treatment. Membrane fractions with specific activities higher than those of untreated membranes were obtained with the relatively mild procedures of adding EDTA to a final concentration of 0.005 M in the 0.05 M Tris buffer (referred to as the EDTA wash) or by the “shock wash” procedure’s, with and without 0.005 M EDTA in 0.005 M Tris-HCl buffer. This was Biochinz.Biophys.

x‘fcta. 239

(1971)

280-292

282

A. J. DE SIERVO, M. R. J. SALTON

accomplished by diluting a concentrated preparation of membranes (10-12 mg/ml protein) I : IO with distilled water (“shock”) to yield a Tris-HCl concentration of 0.005 M or by diluting I : IO with 0.005 M EDTA in distilled water (subsequently referred to as EDTA-shock). Membranes thus treated were left in an ice bath for 18 h and subsequently centrifuged at IZZOOOx g for z h. Under these conditions of centrifugation, three fractions were obtained and separated: a large reddish-brown, high-density pellet; a small low-density yellow pellet, which was deposited above the dense pellet and could be easily removed with a Pasteur pipette; and a clear soluble supernatant. At a lower centrifugal force of IOOOOOx g for 2 h, the low-density yellow material did not separate from the soluble fraction. A control sample consisting of membranes diluted I : IO in the same buffer was usually included with the other preparations for comparison, since even mild treatment cf the membrane can sometimes have a profound effect on the release of protein and/or enzymatic activity.

Assay for cardioli~in

sy~~thes~s 32P-labelled pllosphatidyl glycerol, obtained from M. L~s~d~~k~~~~sgrown in the presence of 32P, was used as the substrate for the synthesis of cardiolipin. Because the enzyme fraction obtained from membranes contained fairly high endogenous levels of the same phospholipids, the assay method was based on measuring the transfer of label(s) from phosphatidyl glycerol to cardiolipin using relatively small amounts of enzyme material to minimize large changes in substrate concentration. To determine the degree of conversion of phosphatidyl glycerol to cardiolipin during the incubation of the enzyme-substrate mixture, samples were extracted for total lipid and the lipids separated on silica gel loaded paper with the chlorofo~-methanol-diisobutyl ketoneacetic acid-water solvent system. The phosphatidyl glycerol and cardiolipin spotswere located by Rhodamine 6G staining, cut out, counted in a gas-flow counter, and the amounts of cardiolipin synthesized were calculated. Phosphatidyl glycerol was prepared for use in the assay by sonicating a weighed amount of dried, purified phospholipid in distilled water. To obtain a uniform opalescent dispersion of the lipid, sonication for 2-3 min was necessary. The most active enzyme-substrate mixture so far developed consisted of 0.2 mM ~32Plphosphatidyl glycerol, 0.2 M Tris-HCl buffer (pH 7.01, 0.250/bTriton X-100 (where used, a nonionic detergent generously supplied by Rohm and Haas, Philadelphia, Pa.), and 5-15 pg enzyme protein, in a final vclume of 0.1 ml. Incubation was carried out in a water bath at 35”. Proteins were determined by the method of LOWRY et aLlA using bovine serum albumin, dissolved in the appropriate concentration of Tris-HCl buffer, as a standard. Total phosphorus was determined by the method described by AMES AND DUBIN’? and glycerol by the method of ~~NKoN~N~~,using glycerol phosphate as standards for both determinations.

of~hos~hat~d~c acid Cyt~dyLtra~~~erase Phosphatidic acid cytidyltransferase which synthesized CDP-diglyceride from phosphatidic acid and CTP, was assyed by a method modified from those of MCCAMAN AND PINNERTY~and CARTERS.The assay contained I mM phosphatidic acid, 2 mM [8-l*C]CTP (Schwarz Bioresearch Inc.), 44 mM KCl, IO mM MgCl,, 0.5% Triton XIOO, 0.1 M Tris-HCl buffer (pH 7.5), 5 mM mercaptoethanol, and whole washed memAssay

Biocki~~.Biop&s. Acta, 239 (1971) 28o--292

MEMBRANE SYNTHESIS

283

OF CARDIOLIPIN

branes at a level of 10-15 pug protein, in a final volume of 0.1 ml. After incubation at 35”, the assay mixture was extracted and the lipid fraction isolated. Activity is based on the incorporation of labelled cytidine into the lipid fraction. Controls of boiled membranes (100’ for IO min) were always included in the assay. RESULTS

The phospholipids of M. ~yso~~~kt~c~~ constituted about 82% of the total extractable lipid in stationary phase cells. Of this, approx. 36% was accounted for by cardiolipin, 34% by phosphatidyl glycerol, and 12% by a compound tentatively identi~ed as phosphatidyl inositol. based on its co-chromatography with authentic phosphatidyl inositol and identity of their deacylation products (see ref. xg). Glycolipid (60/,) and neutral lipids (12%) accounted for the remaining lipid content of the cells. The identification of these phospholipids is supported by chemical and chromatographic data obtained on the intact phospholipids and their deacylated derivatives, and is in agreement with the results reported by ~ACFARLANE~S. Fig. x is an autoradiogram of M. Zysadeiktims lipids labelled with sodium [ldC]acetate (Amershamj Searle Corp.) and 32P. The glycolipid, phospholipids and neutral lipids can all be demonstrated in the 14C-labelled sample as shown in Fig. I. Fig. z is an autoradiogram of a twodimensional chromatogram prepared to detect any phospholipids which may be co-cllromatographing with the major 32P-labelled compounds shown in Fig. I.

Fig. I. Autoradiogram of the total lipids of M. Zysodcikticus labelled by growing the cells in the presence of [s*P]orthophosphate or [%]sodium acetate in the growth medium. Cultures were grown to the stationary phase and total lipid extracted and separated on silica gel loaded paper as described in MATERIALS AND METHODS. Lipids in this and subsequent figures designated as follows: NL, neutral Iipid; CL, cardiolipin: PG, phosphatidyl glycerol; GL, glycolipid; PI, phosphatidyl inositol. Two unknown compounds were also present as shown in Fig. 2. Biockim. Biophys.

A&z,

239 (1971) z8o--292

284

A. J. DE SIERVQ, M. R. J. SALTON

Only two minor unidentified phospholipids X, and X,, (less than 1% of the total counts) were detected, Phosphatidic acid and CDP-diglyceride were not detected in the total lipid under the conditions used in our experiments, although they are known to be precursors of phosphatidyl glycerol and cardiolipinzO. [aaP]Phosphatidyl glycerol was isolated from A!,!.~~~~~~~~~~&~s total lipid (see MATERIALS AND METHODS) for use as a substrate in cardiolipin synthesis. Fig. 3 is an

Fig. 2. Autoradiogram of a two-dimensional chromatogram of the total [Y?]lipids of M. lysodeiRiicus. First dimension solvent was: chloroform-methanol-diisobutyl ketone-acetic acid-water (45 : 15: 30 : 20: 4, by vol.) ; second dimension solvent: chloroform-rnethan~~-diisobutyl ketonepyridine-o.5 M ammoninm chloride buffer, pH lo.4 130 : I 7-5 : 25 : 35 : 6, by vol.). Unlabdled glycolipid (GL) was detected with Rhodamine 6G staining. Two minor, unidentified phospholipids (X, and X,) were also detected in addition to the major phospholipids. CL, cardiolipin; PG, phosphatidyl glycerol. Fig. 3. Autoradiogram of total and isolated phosphol~pids of M. lysodeikticus. Phospholipids were isolated by eluting respective bands from silica gel loaded paper and the purity of the fractions checked by chromatography with the chloroform-methanol-diisobutyl ketone-acetic-water solvent as described in MATERIALS AND METHODS (see aiso Fig. 2). The [SaP]phosphatidyl glycerol so prepared was used as the substrate in assaying for cardiolipin synthesis. CL, cardiohpin; PG, phosphatidyl glycerol; PI, phosphatidyl inositol.

autoradiogram of the total and purified phospholipids. The phosphatidyl glycerol isolated corresponded to an authentic preparation and agreed with published RF data12s13AIt gave a positive vicinal hydroxyl group reaction and on analysis had a glycerol to phosphate ratio of zoo. Synthesis of cardioli$in by membrane bound, EDTA-shock released proteilz and Triton X-xao stimulated fractions When [3zP~ph~sphatidyl glycerol was incubated with whole washed membrane in 0.1 ‘Iris-HCl buffer (pII 7.5), a small amount of [3*P]cardiolipin was synthesized. The addition of Triton X-rou, a nonionic detergent, at a final concentration of 0.5% increased the synthesis of cardiolipin more than 4o-fold as indicated in Table I. The presence of Mgei or K+ ions had little or no effect on this activity. It was subsequently found that a “soluble” supernatant fraction containing the low-density, yellow partic-

MEMBRANE TABLE

SYNTHESIS

2&J

OF CARDIOLIPIN

I

SYNTHESIS OF CARDIOLIPIN BY MEMBRANE-BOUND ENZYME FROM M. lysodeikticus Assay conditions: o.I M Tris-HCI, pH 7.5; 0.2 miM ~3~P]phosphatidyl total vol. 0.1 ml; incubated I h at 35”. A say

(I) (2) (3) (4) (5) (6)

system

Complete system (0.57; Triton X-100) KC1 (44 mM) added M&l, (4 mM) added KC1 + MgCI, added Triton X-100 omitted Control (boiled membrane)

glycerol,

14 flcg protein in

nmoles pho$hatidyL glycerol converted to cardiolipin 18.6 18.8 17.8 18.6 0.5 0.1

ulate material on centrifugation at IOOOOOx g for 2 h after the EDTA-shock treatment (see MATERIALS AND METHODS), exhibited considerable activity in the absence of detergent. In fact, as shown in Table II, the activity was inhibited by the higher level (0.5%) of detergent. In addition, Mg2f but not K+ was inhibitory in the absence of Triton X-100. The difference in the effects of Triton X-100 on the membrane-bound and the low-density particulate (EDTA-shock fraction) form of “cardiolipin synthetase” was TABLE

II

SYNTHESIS OF CARDIOLIPIN BY ENZYME FRACTION RELEASED BY EDTA-SHOCK 34. Zysodeikticus MEMBRANES

TREATMENT*

Assay conditions: 0.1 M Tris-HCI, pH 7.5, 0.2 mM [3ZP]phosphatidyl total vol. 0.1 ml; incubated I h at 35’.

13 pg protein in

Assay system

glycerol,

OF

nmoles phosphatidyl glycerol. co~v~yted to cardiolippin

(I) (2) (3) (4)

Complete system (0.50,6 Triton X-100) I.0 Triton X-100 omitted 6.6 Triton X-100 omitted, Mgs+ (4 mM) added I.9 Triton X-100 omitted, Kf (44 mM) added 7.0 __.* Supernatant fraction from Iooooo x g, 2 h centrifugation, material.

containing the low-density

particulate

investigated by varying the final concentration of detergent in the assay and the results are shown in Fig. 4. At 0.5% Triton X-100, the membrane-bound form is at its peak of activity whereas the EDTA-shock released form is inhibited. In the absence of Triton X-100, the membrane-bound enzyme is only slightly active but the released form exhibits considerable activity. Both enzyme fractions showed high levels of activity at 0.25% Triton X-100 and consequently this concentration of detergent was used in later experiments. The mode of stimulation of “cardiolipin synthetase” by Triton X-100 is unknown. It may be due to the dispersion of the substrate or a more favorable conformation of active site of the enzyme protein or a combination of both. ~~StY~~~t~o~of l~C~Y~~O1~~~~ synt~et~se’~ in M. lyso~~~~c~s The cytoplasm obtained after cell lysis and the three membrane washes were assayed for cardiolipin synthesis activity. These fractions were first centrifuged at 122000 x g for 2 h to remove any particulate contamination (e.g. the active EDTAshock yellow pellet fraction). Table III gives the specific activities obtained in the Biochim. Biophys.

Acta, 239 (1971) 280-292

286

A. J. DE SIERVO, &I. R. J. SALTON

0

0.1

0.2

0.3

Percent

0.4

0.5

Triton

0.6 X-100

0.7

0.8

0.9

1.0

(w/v)

Fig. 4. Stimulation of cardiolipin (CL) synthesis from phosphatidyl glycerol (PG) by the non-ionic detergent, Triton x-100. Membrane-bound activity (a---# and EDTA-shock released activity in the IOOOOOxg, 2 h, supernatant (e--o) were assayed in 0.1 M Tris-HCI buffer, pH 7.5, for 30 min at 35”. Reaction mixtures contained 14 ,ug protein/o.1 ml.

TABLE

III

DISTRIBUTIOK

OF“CARDIOLIPIN

SYNTHETASE"

IN

Specijic activity (pnoles

Cytoplasm Wash I * Wash 2 Wash 3 Membrane

n/r.

lysodeikticus

cardiolipinlg protein per h)

Without T&on X-100

With Triton X-100

5 I7 27 93 244

‘4 19 19 48 3730

* Successive membrane washes with 0.05 M Tris-HCl buffer (pH 7.5). Similar amounts of protein were used in assaying the fractions and where necessary the fractions were diluted with the TrisHCl buffer to the desired level of protein.

presence and absence of Triton X-100. In both cases the cytoplasm had the lowest activity and the membrane the highest. There is, however, a small release of enzyme from the membrane with successive washing of the membrane, but this could be attributed to progressive fragmentation of the structures. Again, this activity appeared to be sensitive to detergent as the third wash indicated. The membranes appear to become less stable, at least with respect to this enzyme, during the washing procedure. However, this experiment does indicate that “cardiolipin clusively localized in the membranes. Release

synthetase”

is almost ex-

of “cardiolipin synthetase” from the membrane Various mild wash procedures were tested in order to release cardiolipin

synthe-

tase from the membrane in a form which is active in the absence of detergent. It was found that the low-density, yellow pellet (i.e. the EDTA-shock yellow pellet) released by the procedure described in MATERIALS AND METHODS, had the highest specific activity when compared with similar pellets obtained from a control, shock-washed or EDTA washed membranes (Table IV). EDTA-shock washing was similar to the control Biochim. Biophys. Acta,

239

(1971) 280-292

MEMBRANE TABLE

SYNTHESIS

287

OF CARDIOLIPIN

IV

CARDIOLIPIN

SYNTHESIS

BY

MEMBRANE

FRACTIONS

ASSAYED

IN THE

ABSENCE

OF DETERGENT

Assay conditions: [3ZP]phosphatidyl glycerol, 0.2 mM; Tris-HCl, 0.2 M, pH 7.0; final vol. 0.1 ml. Incubation for 30 min at 35”; assay tubes contained 2-7 pg protein. Fractions derived from washing whole membranes are indicated. Protein (I&

Fraction from membrane

Control yellow low-density pellet EDTA-wash yellow low-density pellet Shock yellow low-density pellet EDTA-shock yellow low-density pellet Control sol. protein* EDTA-wash sol. protein Shock sol. protein EDTA-shock sol. protein * Soluble protein =

122000

x 9.2

260 420 690 235 850 I020

980 400

Specijk activity (pmoles cardiolipin /g protein per h)

Total activity (mmoleslh)

346 I480 2780 44 400

90 620 I 920 10450


286 800 I 360


292 784 545

h supernatant.

washing in that the lowest amount of protein was released by this procedure. The EDTA-shock yellow pellet fraction was also very active and similar to whole membrane in showing maximal activity in the presence of 0.25% Triton X-100. It also had the highest phospholipid/protein ratio (w/w) of the fractions (2.6 compared to about 0.3 for whole membrane9). Electron microscopy of negatively-stained preparations (Fig. 5)

Fig. 5. Electron micrograph of the enzymatically active EDTA-shock yellow pellet fraction. Negative staining was performed with 296 magnesium many1 acetate. The average diameter of the vesicles is approximately 300 nm. x 50 700. Biochim. Biophys. Acta, 239 (1971) z8o-zgz

288

A. J. DE SIERVO, &I. R. J. SALTON

indicated that the EDTA-shock yellow pellet fraction consisted of small vesicles possessing an average diameter of approximately 300 nm. The small particulate or vesicular nature of this fraction is not surprising in view of its high lipid content and its loose packing as a pellet at IZZOOOx g. That the EDTA-shock yellow pehet fraction is not simply composed of small fragments representative of the whole membrane was also indicated by the observation that there was no detectable phosphatidic acid ~ytidy~transferas~ activity when assayed as described in ZATERIALS AND METHODS. Attempts to release additional “cardiolipin synthetase” in the EDTA-shock yellow pellet fraction by sonication were unsuccessful. Although the total activity released was increased, the specific activity was considerably reduced because of concommitant release of other proteins from the membrane.

An autoradiogram showing the production of cardiolipin from [32P]phosphatidyl glycerol with the EDTA-shock yellow pellet fraction as the enzyme source and in the absence of Triton X-100 is presented in Fig. 6. An increase in an unidentified

Fig. 6. Au~radiogram showing the course of synthesis of [s~P]~rdiolip~ (CL) from [3aPJphosphatidy1 glycerol (PG) from o to 135 min of incubation at 35’. An unidentified phospholipid (X) was also formed in the reaction. The assay contained [azP]phosphatidyl glycerol (PG), 0.2 mM; 0.1 M Tris-HCI buffer (pH 7.5), and ~~~gprotein (EDTA-shock yellow pellet) in a total of o. I ml. Samples of the assay-mixture were removed at intervals, extracted and chromatographed with chloroformmethanol-diisobutyl ketone-acetic acid-water solvent as described in MATERIALS AND METHODS.

phospholipid (X), which is presumably derived from phosphatidyl glycerol, chromatographs just above phosphatidyl glycerol. In 135 min of incubation at 35”, 38% of phosphatidyl glycerol was converted into cardiolipin under these conditions, and 8% into the unidenti~ed phospholipid. Two-dimensional chromatography (Figs. 7 and 8) of these reaction products in the presence and absence of Triton X-100, indicated that the unknown phospholipid is not phosphatidic acid, a possible breakdown product of BiocBim. Biophys.

Acta,

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MEMBRANE SYNTHESIS OF CARDIOLIPIN

either phosphatidyl glycerol or cardiolipin. However, a trace amount of a phospholipid which chromatographed similarly to authentic phosphatidic acid was formed in the reaction and was detected in the autoradio~ams (see Figs. 7 and 8).

Fig. 7. Autoradiogram of a two-dimensional chromatogram of the products of conversion of [a2P]phosp~atidyl glycerol (PC) to [SzP]cardiolipin (CL) by the enzyme system incubated in the presence of Triton x-100 for I h at 35”. The assay system consisted of [3*P]phosphatidyl glycerol (PG), 0.2 mM; 0.2 MTris-HCl buffer (pH 7.0); 0.25% Triton X-100; enzyme (EDTA-shock yellow pellet), 24 pg protein/ml. 81% of the [azP] phosphatidyl glycerol (PG) was converted to [aaP]cardiolipin. Minor amounts of labelled unidentified phospholipids (X, and X,) and labelled phosphatidic acid (PA) were detected on the autoradiogram. A significant amount of phospholipid X (see Fig. 0) was formed. Unlabelled glycolipid (GL) and phosphatidyl inositol (PI) were detected by staining with Rhodamine 6G and were derived from the lipid-rich enzyme fraction. T = Triton X-100. Chromatography conditions as for Fig. 2. Very small amounts of labelled Xi, X, and phosphatidic acid were just detectable on the autoradiogram and their location is indicated by broken lines. Fig. 8. Autoradiogram of a two-dimensional chromatogram of the products of conversion [a*P]phosphatidyl glycerol (PG) to [a*P]cardiolipin (CL) by the synthesizing system incubated in the absence of Triton x-100. See Fig. 7 for conditions of assay. 67% of the [3eP]phosphatidyl glycerol was converted to [38P]cardiolipin. Only a minor amount of phospholipid X was synthesized under these conditions. Trace amounts of phospholipid X, and phosphatidic acid (PA) were also detected on the autoradio~am but they were too faint for reproduction and their location is indicated by broken lines as in Fig. 7. Unlabelled glycolipid (GL) and phosphatidyl inositol (PI) detected on the stained chromatogram were derived from EDTA-shock yellow pellet enzyme fraction.

“Cardiolipin synthetase” in the form released from the membranes (EDTAshock yellow pellet) exhibited a broad pH optimum at about pH 7 (Fig. 9). The enzyme reaction was more tolerant of acidic than alkaline conditions but was, however, quite active throughout the range of pH studied. The molarity of the Tris-HCI buffer (Fig. IO) had a more pronounced effect on activity than did pH and the optimal activity under the conditions of the assay was observed in 0.2 M Tris-HCl. DISCUSSION

As anticipated from earlier reports of the presence of phospholipid synthesizing systems in particulate fractions of bacteria, we have demonstrated the enzymatic synthesis of cardiolipin in isolated, washed membranes of M. lysodeikticus. Moreover, Biochina.Biophys.

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290

SIERVO,M.R.J.SALTON

3Or

6.5

7.0

7.5

6.0

6.5

9.0

”

0.1 Molar

PH

0.2

0.3

concentration

0.0 of

0.5

Tris

Fig. g. Effect of pH of 0.1 M Tris-HCl buffer on the synthesis of cardiolipin (CL) from phosphatidy1 glycerol (PC). Assay conditions were similar to those described in Fig. 7, except that Triton X-100 was omitted. Incubation performed for 15 min at 35”. Fig. 10. Effect of molar concentration of Tris-WC1 buffer (pH 7.0) on the synthesis of cardiolipin (CL) from phosphatidyl glycerol (PG). Assays were performed in the absence of Triton X-100. otherwise conditions as for Fig. 7; incubation for r5 min at 35”.

the enzyme

system

(“cardiolipin

synthetase”)

is almost

exclusively

localized

in the

membrane of this organism, with less than 3% of the activity being found in the cytoplasmic fraction (Table III). A relatively simple assay for the enzymatic synthesis of cardiolipin was developed employing as a substrate the [32P]phosphatidyl glycerol isolated from M. Zysodeikticus cells grown in the presence of [32P]orthophosphate. The use of a radioactive precursor was an essential feature of the assay as it enables us to distinguish between the cardiolipin synthesized and the endogenous phospholipids of the membranes and fractions derived from these structures (e.g. EDTA-shock yellow pellet). Isolated, washed membranes suspended in 0.2 8l TrissHCl buffer (pH 7.0) and Triton X-100 catalyzed a rapid conversion of as much as 96% of the phosphatidy1 glycerol to cardiolipin. High conversions were dependent upon the addition of Triton X-100 (see Fig. 4). Thus the “cardiolipin synthetase” behaves similarly to other membrane enzymes in exhibiting stimulation by detergents. By lowering the molarity of the Tris-HCI buffer in the presence of EDTA and subsequent high-speed 0.25%

centrifugation (IOOOOO x g, 2 h) of the membrane suspensions so treated, a “soluble” supernatant fraction was obtained. As material in this supernatant fraction formed a loosely packed yellow pellet on centrifugation at IZZOOO x g for z h, it must still be in the form of a low-density, lipid-protein particle and is not strictly speaking a truely soluble fraction. Indeed, the small particulate nature of this fraction was confirmed by electron microscopy which revealed particles of about 300 nm in diameter (see Fig. 5). The EDTA-shock yellow pellet preparation, in contrast to whole membranes, was quite active in the absence of Triton X-100 and exhibited a high phospholipid to protein ratio which was approx. 8 times that of the membrane. This fraction also responded to the addition of Triton X-100 and the activity was approximately doubled at the optimal Triton X-100 concentration of 0.25% (Fig. 4). B&him.

Biophys. Ada, 239 (1971) z8o--292

MEMBRANE

291

SYNTHESIS OF CARDIOLIPIN

The properties of membrane-bound and released forms of “cardiolipin synthetase” appear to be very similar except for the requirement of detergent with whole membranes. Both preparations, at the concentrations of enzyme protein employed, exhibited maximal stimulation with 0.25% Triton X-100. Whether the released enzyme activity is identical to that observed with the membrane enzyme is unknown at present and we have no estimate of recoveries of total membrane and released activities. The fact that enzyme can be released from the membrane by procedures rather similar to those which release ATP.ase15 suggests that at least some “cardiolipin synthetase” may be loosely bound to the membrane. However, the major difference from the ATPasels is the high ratio of lipid to protein in the fractions containing the “cardiolipin synthetase” activity. The identity of cardiolipin as the major enzymatic product when phosphatidyl glycerol was used as substrate was verified by both one and two-dimensional paper chromatographic systems run simultaneously with known standards. Mild alkaline methanolysis also yielded identical compounds from beef heart cardiolipin, cardiolipin isolated from whole cells of M. lysodeikticus and the enzymatically formed cardiolipin. The possibility that phosphatidic acid, a potential derivative of phosphatidyl glycerol, was the major product formed has also been ruled out. The formation of cardiolipin from phosphatidyl glycerol can proceed without the addition of other substrates and there was no loss of 32P-label during its formation. Evidence that the enzymatic formation of cardiolipin from only phosphatidyl glycerol is possible has recently been published by STANACEV AND STUHNE-SEKALE@~ in experiments using phospholipase D from cabbage. However, in contrast to their results with phospholipase D in which over 95% of the phosphatidyl glycerol was converted to phosphatidic acid and apparently less than 2% converted to cardiolipin, the enzyme(s) obtained from M. lysodeiktiws membranes can convert greater than 90% of the phosphatidyl glycerol to cardiolipin without the formation of significant amounts of phosphatidic acid. In the light of these results and considering the known structure of cardiolipin, it appears that cardiolipin can be formed from the condensation of two molecules of phosphatidyl glycerol with the loss of a three carbon segment. A possible role for CDP-diglyceride in the synthesis of cardiolipin has been ruled out in the enzyme(s) studied in this investigation. CDP-diglyceride cannot be detected in any of the enzyme preparations nor is it formed in the reaction, although it is synthesized by M. lysodeikticus membranes if CTP, phosphatidic acid, and Mgz-+ are present. However, these results in no way eliminate the possibility that an alternate pathway for the synthesis of cardiolipin, as described by STANACEV et al.20 in Escherichtia coli employing CDP-diglyceride and phosphatidyl glycerol as substrates, also operates in M. lysodeikticus. This possibility is presently being investigated. The relationships between the enzyme(s) which synthesize(s) cardiolipin and the series of enzymes which result in the accumulation of phosphatidyl glycerol in the membrane of M. lysodeikticus may reveal further details of membrane biosynthesis and organization. An understanding of the biosynthesis of the phospholipid synthesizing enzymes is also extremely relevant to membrane synthesis since their major products, cardiolipin and phosphatidyl glycerol, account for the bulk of the membrane lipid and as such provide essential elements of the membrane structure.

Biochim. Biophys. Acta, 239

(1971)

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A. J. DE SIERVO, M. R. J. SALTON

ACKNOWLEDGEMENTS

These investigations were supported by a grant (GB 17107) from the National Science Foundation and by the General Research Support Grant (FR 05399). The work was carried out during the tenure of a National Institutes of Health postdoctoral fellowship (I-Fa-AI-q, 416-01) to A. J. De Siervo. We are indebted to Dr. Kwang S. Kim for the electron micrograph and Mr. Charles Harman for the photography. REFERENCES Y. Y. R. J. M. M. D.

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Biochim. Biophys. Acta, 239 (1971) 280-292