THE JOURNAL OF BIOLOGICAL CAEMISTRY Vol. 240, No. 4, April 1965

Printed in U.S.A.

Phospholipids of Clostridium butyricum

STUDIES ON PLASMALOGEN COMPOSITION AND BIOSYNTHESIS*

NICOLE A. BAUMANN,~ P-O. HAGEN, AND HOWARD GOLDFINE

From the Department of Bacteriology and Immunology, Harvard Medical School, Boston, Massachusetts 02115

(Received for publication, November 4, 1964)

Much has been learned about the chemistry and biosynthesis of bacterial fatty acids in the past decade (1). More recently attention has been focused on the complex lipids, which are im- portant constituents of the cell membranes of these organisms (2). This paper presents findings on the structure and metabo- lism of the phospholipids of Clostridium butyricum, a gram- variable, spore-forming anaerobe.

Two of the predominant phosphatides of this organism were found to exist in both diacylphosphatide and plasmalogen form. This finding stimulated an investigation of the biosynthetic relationships of these phospholipids. The origin of the vinyl ether-linked chain in plasmalogens and the mechanism of forma- tion of the vinyl ether bond are not fully understood. As a first approach to the study of this problem in bacteria, we have ex- amined the kinetics of incorporation of “Pi into the diacylphos- phatides and plasmalogens. In addition, we have measured the incorporation of a homologous series of carboxyl-labeled fatty acids into the aldehyde chains of plasmalogens and compared this with their incorporation into lipid-bound long chain fatty acids. The potential role of long chain fatty aldehydes in the biosynthesis of plasmalogens has also been tested.

EXPERIMENTAL PROCEDURE

Cells-C. butyricum ATCC 6015 was grown anaerobically at 37” in the Casamino acids (Difco) medium of Broquist and Snell (3). Methods for growing these cells have been described (4, 5) Occasionally, a lag phase of more than 12 hours was encountered if the media were prepared from concentrated stock salt solu- tions. This long lag period could be eliminated by using freshly dissolved salts in the medium.

Isolation of Lipids-Methods for harvesting and washing cells and for extracting and washing lipids are described in an earlier publication (4). Separation of the neutral lipid and phospholipid fractions was performed on columns of silicic acid (Unisil), which was obtained from the Clarkson Chemical Company, Inc. (6). Neutral lipids, including free fatty acids, were eluted with chloroform. Chloroform-methanol, 1: 1, was used to elute total phospholipids.

Thin Layer Chromatography-Both preparative and analytical

* This work was supported by Grant AI-05979 from the National Institutes of Health, United States Public Health Service. Parts of this work were presented before the American Society of Bio- logical Chemists, Atlantic City, New Jersey, April 1963, and in Chicago, Illinois, April 1964.

t Contrat de formation du Comite de Biologie Moleculaire, France.

separations were carried out on glass plates spread with a layer of silica gel G (C. A. Brinkmann and Company) approximately 0.4 mm thick. By applying the lipids dissolved in chloroform or chloroform-methanol (2 : 1) to the origin as a series of contigu- ous spots, up to 15 mg of lipids could be separated on a plate (200 x 200 mm). Syringes with a capacity of 50 ~1 (The Hamil- ton Laboratories, Inc.) were found to be useful for application of lipids to the thin layer plates.

Two solvent systems were used for the separation of phos- pholipids. Solvent A consisted of chloroform-methanol-7 N

NH40H (60 :35:5) (7). Solvent B consisted of benzene-pyri- dine-water (60:60: 10).

32P-Labeled lipids were located by radioautography. The plates were usually wrapped in Saran Wrap prior to exposure to x-ray film to protect the silica gel from abrasion by contact with the film. Silica gel containing the radioactive lipids was dis- lodged from the plate with a flat ended spatula and removed by the suction device illustrated in Fig. 1. The device, based on one described by Goldrick and Hirsch (S), is made from standard taper joints and a sintered glass funnel, which come apart for the elution step and for cleaning. Only one top is needed for a number of funnels. With the top (inner joint) removed, the lipids are eluted from the silica gel by approximately 125 ml of chloroform-methanol, 1: 2.

32Pi Incorporation Experiments-For these experiments cells were grown in approximately 18 liters of medium in 20.liter carboys to mid-log phase (Klett reading, approximately 80 (No. 66 filter)). 32Pi (5 mc) was then added and samples were re- moved at the times indicated. The medium (2 to 3 liters) was forced out through a siphon by introducing nitrogen gas into the carboy. The sample was cooled quickly by passage through a coil of metal tubing immersed in an ice bath. The cells were harvested by centrifugation at 2-4”, washed with ice-cold water, and finally suspended in approximately 150 ml of chloroform- methanol, 2: 1. The lipids were extracted as described previ- ously (4). They were then chromatographed on thin layer plates of silica gel G in Solvent A. The lipid in the lower band was rechromatographed in Solvent B. The procedure for ob- taining diacylphosphatide phosphorus and plasmalogen phos- phorus is described below. The phosphate esters were finally assayed for radioactivity and for phosphorus.

Deacylation of Phospholipids-The experiments with 32P- labeled lipids required a technique for the separation of the phosphorus-containing deacylation products of diacylphospha- tides from those arising from the deacylation of plasmalogens.

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1560 Biosynthesis of Clostridium butyricum Plasmalogens Vol. 240, No. 4

fritted disk

(medium)

FIG. 1. Suction device used for collection of fractions from thin layer plates.

The procedure described by Dawson (9), as modified by Daven- port and Dawson (lo), was used in pilot experiments. This method is based on the deacylation of diacylphosphatides by mild alkaline hydrolysis. The fatty acid ester bond in plasmalogens is also cleaved by base, but the remaining lysophosphatide re- mains lipid-soluble. The vinyl ether bond is then cleaved by mild acid. It soon became apparent that the yield of phos- phorus in the aqueous phase after mild acid hydrolysis was usually much lower than predicted on the basis of an independent assay for plasmalogens (11). Similar inconsistencies have been noted by Rapport and Norton (12). Thin layer chromatography of the lipid-soluble material aft,er mild alkaline hydrolysis showed that less than half of the plasmalogen had been converted to the expected monoalkenyl lysophosphatide. The fatty acid ester bond in the remainder appeared to be resistant to base under the conditions used. Upon mild acid hydrolysis, these unchanged plasmalogens were cleaved at the vinyl ether bond, but remained lipid-soluble owing to the remaining fatty acid chain. This stability of the fatty acid ester bond in plasmalogens to mild alkaline hydrolysis was observed in two other labora- tories while our work was in progress (13, 14). This difficulty was overcome by increasing the volume of 0.4 N sodium hydrox- ide from 0.25 to 1.0 ml/2 to 5 mg of phospholipid. The volumes of carbon tetrachloride (0.8 ml) and ethanol (7.5 ml) were not changed. We did not add 0.65 ml of water since the additional sodium hydroxide solution supplied an almost identical volume of water. The incubation time was estended to 23 hours to ensure complete hydrolysis of the fatty acid ester bonds in the plasmalogens. Neutralization with 0.4 ml of ethyl formate and the remainder of the procedure described by Davenport and Dawson (10) were retained.

Paper Chromatography-Chromatography of the water-soluble products of strong acid hydrolysis of the phospholipids has been described (15). The products of mild alkaline hydrolysis were passed through columns of IRC-50 (H+) ion exchange resin prior to paper chromatography (9). Omission of this step resulted in interference by the excess salts remaining in the hydrolysate.

Gas-Liquid Chromatography of Fatty Acid Methyl Esters and Aldehyde Dimethylacetals-These derivatives were obtained by methanolysis of total lipids or phospholipids in anhydrous methanolic hydrochloric acid. The mixed fatty acid methyl esters and aldehyde dimethylacetals were separated following saponification of the esters by extraction of the dimethylacetals into petroleum ether (5). The fatty acids were re-esterified in ether by reaction with freshly prepared diazomethane in the presence of approximately 5% methanol (v/v).

A Jarrell-Ash, Inc. (Newtonville, Massachusetts), model 700 chromatograph, equipped with an argon diode ionization de- tector, was used in these studies. Most of the analyses were

performed on a column of Apiezon M, lo’%, on alkaline Gas- Chrom P at 198”. This column tended to cause breakdown of dimethylacetals. Injections of ethanolamine reversed this tendency.1 Methods for collection of fractions and radioassay have been described (4). Relative specific activities are derived from the counts per minute of 14C in a fraction divided by the area under the chromatographic curve during the collection. Identifications of the fatty acids (4) and aldehydes (5) have been published.

Analytical Procedures-The phosphorus assay used has been described in a previous publication (16). Ester bonds in phos- pholipids were determined by the hydroxamate procedure of Rapport and Alonzo (17). Plasmalogens were determined both by the modification of Dawson’s procedure described above and by a calorimetric procedure (11). Glycerol was determined by periodate oxidation and formaldehyde assay (18). Mannitol was used as a standard in the periodate oxidation.

Materials-l-l%‘-Palmitaldehyde was synthesized from l-i4C- palmitic acid (19), which was obtained, as were the other car- boxyl-labeled fatty acids, from New England Nuclear Corpora- tion. 1-*4C-Palmitic acid (10 mg) was refluxed with an excess of freshly distilled thionyl chloride for 30 minutes. The thionyl chloride remaining was removed by distillation under reduced pressure, and dry air was permitted to re-enter the reaction vessel. To the acid chloride cooled in an ice-salt freezing mixture was added a solution of ethyleneimine (0.05 ml) and triethylamine (0.15 ml) in 1.0 ml of cold dry ethyl ether. The addition was carried out with continuous shaking and cooling for a period of 15 minutes. After an additional 30 minutes the precipitate was filtered off and washed thoroughly with dry ether. The com- bined ether solutions were reduced in volume under reduced pressure and cooled in an ice-salt freezing mixture. A suspen- sion of 5 mg of LiA1H4 in 2.0 ml of cold dry ether was added over a period of 10 minutes with continuous cooling and shaking. After an additional 30 minutes, cold 5 N sulfuric acid was added and the ether layer was separated. The aqueous layer was re- extracted with ether. The combined ether layers were washed twice with water, twice with a saturated sodium bicarbonate solution, and twice with water, and were dried over sodium sul- fate. The 1-I%-aldehyde was purified by gas-liquid chromatog- raphy on a column of ethylene glycol adipate polymer, 15ci,, on Chromosorb W, 100 to 200 mesh, at 184”. The collected ma- terial was dissolved in ether and washed with water-ethanol-3 N NaOH (40 : 10: 1) in order to remove any free fatty acid eluted from the gas-liquid column along with the aldehyde.

Phosphatidylglycerol for use as a reference standard was iso- lated from Escherichia coli (20). The cells were extracted with chloroform-methanol, 2: 1, and the lipid extract was washed with 0.05 N sodium chloride (21). The washed lipids were sepa- rated into neutral lipid and phospholipid fractions by column chromatography on silicic acid (see above). The phospholipids in a chloroform-methanol (2: 1) solution were put on a column of diethylaminoethyl cellulose-acetate. The cephalin fraction was eluted with the same solvent mixture. After elution with methanol, the acidic lipids, mainly phosphatidylglycerol, were eluted with a chloroform-methanol-ammonia-ammonium acetate solution (22). The phosphatidylglycerol thus obtained had 1.7 pmoles of glycerol per pmole of phosphorus. It was ninhydrin- negative on thin layer plates. On deacylation it, yielded a water-

r G. M. Gray, personal communication.

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April 1965 N. A. Baumann, P-O. Hagen, and H. Goldjine 1561

\ \

-PG

-PE, PME

5 minutes 13 minutes 44 minutes 90 minutes FIG. 2. Radioautographs of thin layer chromatograms of 3zP-labeled lipids isolated from log phase cells of C. butyricum. Separation

was achieved with a solvent containing chloroform-methanol-7 N NHdOH (60:35:5). PG, phosphatidylglycerol; PE, ethanolamine phosphatides; PME, N-methylethanolamine phosphatides.

,PE

\PME

5 minutes 13 minutes 22 minutes 33 minutes 45 minutes 90 minutes

FIG. 3. Radioautographs of thin layer chromatograms of 32P-labeled lipids recovered from the lower major band in the first chroma- togram (Fig. 1). Separation was achieved with a solvent containing benzene-pyridine-water (60:60: 10). The aliquot chromatographed from the &-minute sample had much less total 32P than that of the 3% and go-minute samples. For abbreviations, see Fig. 2.

soluble compound which behaved as glycerylphosphorylglycerol Solvent B, and radioautographs of these are shown in Fig. 3. in several solvent systems on paper chromatography. Dr. E. P. Only one fraction was labeled in the first two radioautographs, Kennedy kindly provided glycerylphosphorylglycerol for com- but another major fraction appeared at 22 minutes. Minor parison. fractions appeared in later samples.

Cardiolipin was purchased from Arnel Products Company (New York). Synthetic phosphatidylethanolamine was origi- nally obtained from Dr. Erich Baer. Later samples were pur- chased from commercial sources. Dr. Baer also kindly provided a sample of synthetic phosphatidyl-N-methylethanolamine.

RESULTS

Separation and I&nti$cation of C. butyrkum Phospholipio%-- About 4.5% of the dry weight of log phase cells was found to be lipid, and of thii 70% was phospholipid. Although the greater part of our exploratory work on the chemistry of C. butytim phospholipids was performed on lipids separated by column chromatography on silicic acid (15, 23), thin layer chromatogra- phy has now enabled us to separate fractions which had not separated well on columns. To illustrate the separations achieved by thin layer chromatography, radioautographs of lipids isolated from log phase cells exposed to 32Pi for varying periods are shown in Figs. 2 and 3. Two rapidly labeled frac- tions were resolved by Solvent A (Fig. 2). Some fractions, which migrate more slowly, begin to appear in radioautographs of lipids from cells exposed to 32Pi for 44 minutes. The lipids from the lower major band in Solvent A were rechromatographed in

Phosphatidylglycerol-The lipid in the upper band in Solvent A (Fig. 2) has been identified as phosphatidylglycerol on the basis of a number of criteria. It migrated on thin layer plates with phosphatidylglycerol isolated from E. COG, gave a positive test for phosphate ester, and was ninhydrin-negative. Deacylation with mild base yielded a phosphate ester which was indistinguish- able from the deacylation product of E. coZi phosphatidylglycerol in three solvent systems (Table I). It was also compared chromatographically with the deacylation product of cardiolipin (25) because cardiolipin migrates close to phosphatidylglycerol on thin layer chromatography in Solvent A and has the same staining properties. The phospholipid isolated from C. butyricum in the exponential phase of growth had a molar ratio of glycerol to phosphate of 2.0, which is the theoretical value for phos- phatidylglycerol.2 On hydrolysis in 90% acetic acid (27), the

2 In stationary phase, however, it appears that C. butyricum accumulates cardiolipin. The glycerol to phosphate mole ratio of the same thin layer chromatography fraction was 1.5. The ester to glycerol mole ratio was 1.2 (expected for phosphatidyl- glycerol, 1.0; expected for cardiolipin, 1.33). Acetic acid hy- drolysis of this fraction from stationary phase cells yielded glyc- erol diphosphate. Dr. R. L. Lester has analyzed the deacylation products of stationary phase C. butyricum lipids. Determinat.ion

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1562 Biosynthesis of Clostridium butyricum Plasmalogens Vol. 240, No. 4

TABLE I Paper chromatography of deacy2ation product

of phosphatidylglycerol

The solvents used were: Solvent 1, 1-butanol-acetic acid-water (5:4: 1) (24) ; Solvent 2, phenol-water-acetic acid-ethanol (50:22: 3:3) (24); and Solvent 3, methanol-formic acid (88’%)-water (80: 15: 15) (9). All ratios by volume, except phenol,which is by weight.

Solvent system 1

RQP”

Glycerylphosphorylglycerol. 0.90 Glycerylphosphorylglycerylphos-

phorylglycerol. . . . . 0.35 0.26 0.79 Glycerylphosphorylethanolamine 0.57 0.63 0.66 Deacylation product. 0.90 0.45 0.88

- 2

RF

0.44

3

RBP”

0.91

(1 Relative to L-a-glycerophosphate.

TABLE II Proportions of C. butyricuma phosphatides in plasmalogen form

Compound Plasmalogen form

% Et s.d.

N-Methylethanolamine phosphatides 78 f 5.6 Ethanolamine phosphatides 55 f 7.4 Phosphatidylglycerol 9f2

a Phosphatides isolated from log phase cells.

unknown phospholipid yielded glycerophosphate as shown by paper chromatography in ethanol-ammonium acetate, pH 7.5 (65:35).

Ethanolamine Phosphatides-The lipid from the upper band after chromatography in Solvent B (Fig. 3) proved to be a mix- ture of almost equal amounts of diacylphosphatide and plasmalo- gen (Table II). On hydrolysis in 1 N HCl it yielded ethanol- amine (15). Deacylation in mild base yielded a phosphate ester which migrated with glycerylphosphorylethanolamine in three solvent systems (Table I). The intact lipids were ninhydrin- positive and had the same RF as synthetic phosphatidylethanol- amine or phosphatidylethanolamine isolated from E. coli, on thin layer chromatography in Solvents A and B and in chloroform- methanol-water (65 : 25 : 4).

N-Methylethanolamine Phosphatides-This major phosphatide of C. butyricum was found to be present almost, entirely in the plasmalogen form (Table II). The details of the identification of its base in phospholipid hydrolysates have been published (15). Briefly, the base was obtained on hydrolysis of the phospholipid in 1 N HCl. It was chromatographed on paper in several solvent systems along with synthetic N-methylethanolamine. It was also isolated by column chromatography on ion exchange resin and converted chemically to choline which was crystallized as the reineckate.

The deacylation product obtained by successive mild alkaline and acid hydrolysis has been compared by paper chromatography with the deacylation product of synthetic phosphatidyl-N- methylethanolamine. In Solvent 2 (Table I) the deacylation

of phosphate after separation of the deacylation products by ion exchange chromatography (26) indicated a ratio of glycerylphos- phorylglycerylphosphorylglycerol to glycerylphosphorylglycerol of 2.5. His identification of the deacylation products was based only on the time of emergence from the ion exchange system.

product of the synthetic lipid had an RF of 0.85, while that of C. butyricum N-methylethanolamine phosphatide was 0.90. In Solvent 3 the respective Rp values were 0.55 and 0.61.

Minor Fractions-At least two slowly migrating lipids were seen on thin layer chromatograms in Solvent A. Two peaks, corresponding to these two bands, were partially resolved by column chromatography on silicic acid with a gradient of metha- nol in chloroform (28). The first gave 1.3 moles of ester per mole of phosphorus, and the second, 1.0 mole of ester per mole of phosphorus. The first had 0.17 mole of vinyl ether per mole of phosphorus; vinyl ether in the second was practically undetect- able. Together they had a molar ratio of nitrogen to phosphorus of 0.91 and contained 4.6y0 phosphorus by weight.

After mild alkaline hydrolysis 81% of the phosphorus of the second fraction remained lipid-soluble. This lipid-soluble ma- terial gave four polar lipid spots on thin layer chromatography in chloroform-methanol-water (65 : 25 :4). These results suggested that saturated glycerol ethers were present in the second fraction. The lipid was therefore subjected to acetolysis followed by sa- ponification. Only 21 y. of the lipid was found in the nonsaponi- fiable fraction. On periodate oxidation and formaldehyde determination by the method of Karnovsky and Brumm (29), the yield of formaldehyde was only 10% of that expected for an a-glycerol ether. We conclude that there is little, if any, satu- rated ether in this fraction. The phosphorus analysis of this fraction argued against a lysophosphatide structure, as did the lipid solubility after mild alkaline hydrolysis. Assay on rabbit red blood cells for hemolytic activity showed that the lipid had only about one-tenth of the hemolytic activity of lysolecithin. The structure of these lipids remains unclear.

Lipid hydrolysates revealed small amounts of serine. When C. butyricum was grown in the presence of uniformly labeled 14C-L-serine, less than 4% of the radioactivity in the phospholipid bases was associated with serine. The remainder was found in ethanolamine and N-methylethanolamine. We have not suc- ceeded in isolating the parent lipid, phosphatidylserine, by either column or thin layer chromatography of the phosphatJides.3

Lipid hydrolysates have also been examined for sugars. A small amount of sugar which has the same RF as glucose on paper chromatography in l-butanol-pyridine-water (20 : 20 : 10) has been found. This sugar appeared to be lipid-bound since much less sugar was found in chromatograms of unhydrolyzed lipid. Based on the intensity of staining with silver nitrate, we estimate that there is less than 2y, glycolipid in the extract- able lipids of C. butyricum.

Phosphatide Composition of Log Phase Cells-Table III gives the phosphatide composition of log phase cells based on the recoveries of lipid phosphorus from thin layer plates. If the

3 A fraction containing 2% of the total lipid phosphorus was isolated by thin layer chromatography on silica gel G in Solvent A, RF approximately 0.2. Mild alkaline hydrolysis (9) and hy- drolvsis in 1 N HCl of this fraction vielded alanine, which was ide&ified by paper chromatography in Solvents 1 to’3 (Table I) and by thin layer chromatography in ethanol-water (70:3, v/v). Another compound was observed, which stained yellow with ninhydrin and blue with isatin. These color reactions are also given by proline. The unknown compound had the same RF as proline on paper in Solvent 2 (Table I) and on thin layers of silica gel in ethanol-water (70:30, v/v). Macfarlane has isolated from Clostridium welchii O-amino acid esters of phosphatidylglycerol which yield alanine, lysine, and other amino acids on hydrolysis (30).

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April 1965 N. A. Baumann, P-O. Hagen, and H. Goldfine 1563

TABLE III

Composition of phosphatides of C. butyricum”

Compound Content

% f s.d.

N-Methylethanolamine phosphatides Phosphatidylglycerol. Ethanolamine phosphatides. Other components

Phosphatidylserine. Phosphatidic acid. Lysophosphatides Remainder unidentified

38 f 1.6

26 f 1.8 14 f 1.6

22

<4

Trace <3

a Lipids isolated from log phase cells.

cells are grown from a small inoculum in the presence of 32Pi and harvested in the log phase, the distribution of radioactivity agrees well with the figures given in Table III. Table II gives the proportion of each phosphatide in the plasmalogen form. These have, been determined by our modification of Dawson’s procedure (see “Experimental Procedure”). ilbout 10% of the phosphorus of each phosphatide was recovered in the organic solvent phase after mild acid hydrolysis. This fraction has been disregarded in these calculations.

Some samples were assayed for vinyl ether bonds by a color- imetric iodine uptake procedure (11). This assay gave about 10 to 15 ye higher ratios of vinyl ether to phosphorus than estima- tions based on selective hydrolysis.

Phospholipid Biosynthesis

Time Course of Incorporation of 32P Inorganic Phosphate into Phospholipids-The incorporation of 32Pi into phosphatides by growing cells provides some clues concerning their biosynthetic relationships. The results of one such experiment are shown in Figs. 2 to 5. A similar experiment in which one less sample was taken yielded essentially identical results. Fig. 4 shows the incorporation of 3’Pi into the total extractable phosphatidylglyc- erol, ethanolamine phosphatides, and N-methylethanolamine phosphatides. These values were obtained prior to hydrolysis with mild base and acid. Incorporation of radioactive phos- phorus into the ethanolamine and glycerol phosphatides is de- tectable in the first. sample, taken at 5 minutes after addition of isotope to the medium. It proceeds linearly for about 30 min- utes. Incorporation into N-methylethanolamine phosphatides is barely detectable in the first sample and does not approach linearity for 20 to 30 minutes.

To compare the rate of 32P incorporation into the diacylphos- phatides and plasmalogens, the data shown in Fig. 5 were ob- tained after selective hydrolysis of the ethanolamine and N- methylethanolamine phosphatides. It can be seen that incorporation of 3’Pi into the ethanolamine diacylphosphatide occurred with no detectable lag. On the other hand, incorpora- tion into ethanolamine plasmalogen is barely detectable in the first sample and does not become linear for at least 10 minutes. Incorporation into phosphatidyl-N-methylethanolamine also displays a distinct lag, but incorporation into the corresponding plasmalogen is delayed even longer. The results with phos- phatidylglycerol and the corresponding plasmalogen also indi- cated rapid incorporation into the diacylphosphatide and a delayed incorporation into the corresponding plasmalogen.

u) 16,000 ZJ t

Ethonolamine Phosphatides 1

"0 5 13 22 33 45 90

TIME (Minutes)

FIG. 4. Time course of incorporation of 32P-labeled inorganic phosphate into the major phospholipid groups of log phase cells of C. butyricum.

I8,OOO

2 4,000 z

2 " 2,000

/ Ethanolamine Plasmalogens

Monomethyl Ethanolamine -

Diacyl Phosphaiides -

Monomethyl Ethanolamine Monomethyl Ethanolamine

Plasmalagens -

Oil 0 5 13 22 33 45 90

TIME ( Minutes I

FIG. 5. Time course of incorporation of 32P-labeled inorganic phosphate into phosphatidylethanolamine, phosphatidyl-l\:- methylethanolamine, and the corresponding plasmalogens. Since these measurements were made 7 days after those used in Fig. 4, the specific activities must be multiplied by 1.4 in order to correct for the decay of 32P.

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Biosynthesis of Clostdium butyricum Plasmalogens Vol. 240, No. 4

These data are not shown because the small amount of plasmalo- gen (Table II) made accurate measurement of specific activities difficult.

Incorporation of Carboxyl-labeled Fatty Acids into Lipids of C. b&y&urn-Previous st.udies on fatty acid synthesis in this organism revealed that exogenous long chain, saturated fatty acids are incorporated into cellular lipids either unchanged or after elongation. Saturated fatty acids CIQ or longer were not converted to unsaturated fatty acids. On the other hand, both octanoic and decanoic acids were converted intact into long chain saturated, unsaturated, and cyclopropane acids (4, 31). These studies indicated that long chain monounsaturated fatty acids were formed by retention of a double bond in either As-decenoic acid or A3-dodecenoic acid, leading, by chain elongation, to the isomeric Al- and AQ-hexadecenoic acids and AQ- and Ail-octa-

decenoic acids, found in C. butyricum. As Zalkin, Law, and Goldfine (32) and Chung and Law (33) have shown, the cyclo- propane acids are formed by addition of a methyl group from

TABLE IV

Incorporation of l*C’ from carboxyl-labeled saturated fatty acids into C. butyricum lipids

The cells were grown in l- or a-liter volumetric flasks in the Casamino acid medium of Broquist and Snell (3) at 37“. Labeled

compounds were added at the start of the culture. The cells were harvested near the end of the log phase.

Carboxyl-labeled fatty acid added t

growth medium

8:O

lo:o 12:o 16:O

-7

0 “C added

i

PC

40 (3.3 mg) 40 (2.0 mg) 20 (0.4 mg) 10 (0.26 mg)

is

-

Lipid olatel

w w %

65 0.72 81 30 14.4 61 56 4.6 59 23 5.6 64

-

14C incor- porated

WI! in atty aci6

XT in fatty

kldehydes

% 19 39 41 36

TABLE 1’

Incorporation of carboxyl-labeled fatty acids into C. butyricum

Aldehyde isolated

16:0 0.62 70 (91)b 16:l 0.82 126 (92) 17: cyc 0.68 43 (28) 18:O 0.77 120 (128) 18:l 0.66 72 (90) 19:cyc 0.64 50 (45)

long chain aldehydes

Relative specific activities5 with various precursor acids

8:O lo:o 12:o

20 (20) 1.2 (0.8) 0.9 (1.6)

16 (9.1) 1.9 (2.5) 1.1 (1.4)

-

-

16:O

69” (73) 7.7 (4.4)d 4.7

23 (15) 4.7 (2.2) 0.54 (0.59)

0 Relative specific activities are comparable in all experiments They are based on counts in each fraction divided by the area under the chromatographic peak.

b Figures in parentheses are relative specific activities of cor- responding fatty acids.

c The compounds isolated in this experiment were from the phospholipid fraction. In all other experiments the aldehydes and acids were isolated from the total lipids.

d Resolution of 16:0 and 16:l fatty aldehydes was usually not complete on gas-liquid chromatography. When saturated and unsaturated fatty acids were separated prior to gas-liquid chroma- tography, much less 14C was found in 16:l fatty acid when cells were grown on carboxyl-labeled 16: 0 (4).

S-adenosylmethionine to an unsaturated fatty acid, when the acceptor acid is in a phospholipid such as phosphatidylethanol- amine. At the time of the aforementioned studies, the presence of plasmalogens in C. butyricum lipids was not known. The present study afforded an opportunity to obtain similar informa- tion on the origin of the vinyl ether-linked chains in plasmalogens. On acid hydrolysis these chains become aldehydes, and we shall refer to them as such for the sake of convenience.

Incorporation of several carboxyl-labeled fatty acids into the fatty acid and aldehyde chains of C. butyricum lipids is shown in Table IV. The relative specific activities of the long chain alde- hydes isolated as the dimethylacetals by gas-liquid chromatog- raphy are given in Table V. In the experiments with carboxyl- labeled decanoic, lauric, and palmitic acids, the relative specific activities of the cellular fatty acids were also determined and are shown in parentheses. Exogenous fatty acids were readily in- corporated into cellular long chain aldehydes. Earlier work had shown that there is little or no breakdown of these precursors prior to utilization by C. butyricum (4, 31). As in the case of incorporation into long chain fatty acids, octanoic and decanoic acids were incorporated into saturated, unsaturated, and cyclo- propane aldehydes, but the precursors of longer chain length were incorporated primarily into saturated fatty aldehydes (Table V). With few exceptions, the relative specific activities of the fatty aldehydes and the corresponding acids were equal within the experimental error encountered in these measure- ments ( 120 %) .

Incorporation of Pdmitaldehyde into Phospholipids-The in- corporation of fatty acids and their precursors into both acid and aldehyde chains of the lipids of growing cells led us to ask whether exogenous long chain aldehydes could be utilized by growing cells, and, if they were, whether they would be preferen- tially incorporated into plasmalogen aldehyde chains. Table VI shows that I-14C-palmitaldehyde was incorporated into both fatty acids and fatty aldehydes of phospholipids. There seemed to be little preference. The specific activity of the isolated palmitaldehyde was slightly higher than that of the isolated palmitic acid, whereas the specific activity of stearaldehyde was slightly lower than that of stearic acid. A parallel culture was grown in the presence of labeled palmitaldehyde plus unlabeled palmitic acid. The relative specific activities of the saturated fatty acids and aldehydes isolated from the phospholipids are given in Table VI, Line 2. There was a 50% dilution of 1% in palmitic acid and a 37% dilution of i4C! in palmitaldehyde. There was no significant dilution of either the 18 :0 acid or alde- hyde chains.4

The converse experiment is shown in Table VI, Lines 3 and 4. The cells were grown in the presence of trace amounts of l-14C- palmitic acid in the absence and the presence of unlabeled palmit- aldehyde. As shown in Table V, palmitic acid was incorporated into both aldehyde and acid chains of the phospholipids. The addition of unlabeled palmitaldehyde diluted the labeled pre- cursor markedly. The specific activities of both 16:0 aldehyde and acid were diluted 75y0. The specific activity of stearic acid was diluted 53 y0 and that of stearaldehyde was diluted 70 %.

Interconversion of Free Fatty Acid and Aldehyde-The ability of cells to interconvert these compounds was tested. In one

4 In the abbreviations of fatty acids and aldehydes, the number preceding the colon indicates the number of carbon atoms in the chain; the number following the colon represents the number of double bonds; “cyc” denotes cyclopropane aldehydes.

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April 1965 1565 N. A. Baumann, P-O. Hagen, and H. Goldjine

TABLE VI Incorporation of l-‘4C-palmitaldehyde into C. butyricum phospholipids

The cells were grown in Z-liter flasks in the Casamino acid medium (3) at 37”. Add’t’ I ions were made at the start of the culture. The cells were harvested near the end of the log phase.

Addition to medium

1. l-IGPalmitaldehyde, 16.5 X lo6 c.p.m. (58 X lo6 c.p.m. per mg) _. _.

2. I-I%-Palmitaldehyde, 16.5 X lo6 c.p.m. (58 X lo6 c.p.m. per mg), f palmitic acid.

Dilution. 3. 1-14C-Palmitic acid, 14.5 X lo6 c.p.m. (58 X

lo6 c.p.m. per mg). _. 4. l-14C-Palmitic acid, 14.5 X lo6 c.p.m. (58 X

IO6 c.p.m. per mg), + palmitaldehyde.. Dilution.

Incogkd

phospholipids

c.p.m.

0.74 x 106

0.62 X lo6

4.23 X lo6

1.51 x 100

Relative specific activity”

Fatty acid isolated

16:O

3.1 f 0.4 1.4 f 0.1

1.6 f 0.2 1.8 f 0.6 52% 0%

73 f 6.1

18 f 0.3 75%

15 f 2.5

6.9 f 0.8

54%

l&O

Fatty aldehyde isolated

-

16:0

4.1 f 0.7

2.6 f 0.1 37%

69 f 1.1

17 f 1.1 75%

18:0

1.4 f 0.6

1.1 f 0.2 21%

23 f: 7.4

6.9 f 1.8

70%

0 Values are in counts per minute per unit area under chromatographic curve with standard deviations.

experiment in which cells were grown in the presence of l-l%- palmitic acid (14.5 x lo6 c.p.m.; 58 x lo6 c.p.m. per mg) plus approximately 10 mg of unlabeled palmitaldehyde, the neutral lipids were isolated by silicic acid chromatography. To these were added an additional 10 mg of palmitaldehyde and the mix- ture allowed to reacted with excess acidic 2,4-dinitrophenyl- hydrazine. The resulting precipitate was recrystallized three times from hot ethanol, m.p. 101-103” (uncorrected); palmital- dehyde 2,4-dinitrophenylhydrazone melts at 105-107”. The recrystallized material had a constant specific activity of 19,100 c.p.m. per mg.5

In another experiment, cells were grown in the presence of 23 X lo6 c.p.m. (58 X lo6 c.p.m. per mg) of l-14C-palmitaldehyde. The neutral lipids isolated from the cells contained approxi- mately 7.7 X lo6 c.p.m. Of this, 1.47, was in the free fatty acid fraction, 79% in the nonsaponifiable fraction, and 19% in esterified fatty acids. Presumably the nonsaponifiable fraction is largely free palmit,aldehyde.

DISCUSSION

The phospholipids of C. butyricum are unusual for bacteria in two respects. The first is the presence of N-methylethanolamine phosphatides. In most bacteria, phosphatidylglycerol, O- amino acid esters of phosphatidylglycerol, and phosphatidyl- ethanolamine constitute the major phosphatides (2). Phos- phatidylcholine (lecithin) is rarely a major component of bacterial lipids, with the exception of the genus dgrobacterium (16). The partial methylation products of phosphatidylethanolamine, N- methylethanolamine and N, N’-dimethylethanolamine phos- phatides, are also rare in bacteria (16).

The other unusual aspect of the chemistry of C. butyricum phosphatides is the presence of large proportions of plasmalo- gens, especially in the N-methylethanolamine and the ethanol- amine phosphatides (Table II). Plasmalogens have been found only recently in bacteria, and thus far only in anaerobes. Apart

6 Palmitaldehyde 2,4-dinitrophenylhydrazone, recrystallized from the commercial lJ*C-palmitic acid to which unlabeled palmitaldehyde was added in a control experiment, had a specific activity of 1400.

from C. butyricum, the remaining known plasmalogen-containing species are rumen bacteria (34, 35). We are unaware of any previous report on the presence of substantial amounts of N- methylethanolamine plasmalogen in either animal or bacterial cells.

In experiments with growing cells we found that ethanolamine and N-methylethanolamine were derived from exogenous serine but not from exogenous ethanolamine (15). These results sug- gested that ethanolamine and N-methylethanolamine phospha- tides were derived from phosphatidylserine by a pathway similar to one for phosphatidylethanolamine biosynthesis found in E. coli by Kanfer and Kennedy (20).

The methyl group of N-methylethanolamine can be formed from exogenous methionine in growing cells of C. butyricum (15). Kaneshiro and Law (36) have purified a soluble enzyme from extracts of Agrobacterium tumefaciens which catalyzes the con- version of phosphatidylethanolamine to phosphatidyl-N-methyl- ethanolamine with S-adenosylmethionine as the methyl donor. The delay seen in the incorporation of 32Pi into N-methylethanol- amine phosphatides, compared to the rapid labeling of the ethanolamine phosphatides (Fig. 4), is consistent with a product- precursor relationship for the two compounds.

Kanfer and Kennedy have studied the biosynthesis of phos- phatidylglycerol in extracts of E. coli, and have shown that this lipid, like phosphatidylethanolamine, arises from CDP-diglyc- eride (20). The two diverge at the next step, where reaction of CDP-diglyceride with either L-serine or L-a-glycerophosphate determines the fate of the diglyceride and the attached phosphate residue. The simultaneous rapid labeling of phosphatidyletha- nolamine and phosphatidylglycerol in growing C. butyricum

(Figs. 4 and 5) is in agreement with either completely inde- pendent or branched pathways to these compounds.

The 3’Pi incorporation into C. butyricum plasmalogens, by the same token, suggests a precursor-product relationship between the diacylphosphatides and the corresponding plasmalogens. In each case there is a distinct lag between the incorporation of phosphate into a diacylphosphatide and incorporation into the corresponding plasmalogen (Fig. 5).

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1566 Biosynthesis of Clostridium butyricum Plasmalogens Vol. 240, No. 4

Our experiments with carboxyl-labeled fatty acid precursors indicate that the aldehyde chains of plasmalogens can arise from fatty acid precursors. Not only are the specific activities of the long chain fatty acids and aldehydes similar in each experiment, but the relative incorporation into the saturated, unsaturated, and cyclopropane aldehyde chains closely mirrors that seen in the corresponding fatty acids. The mechanism of long chain fatty acid synthesis has not been studied in detail at the enzy- matic level in C. butyricum. The incorporation data of Goldfine and Bloch (4) and Scheuerbrandt et al. (31) led to the postulate that exogenous octanoic and decanoic acids serve as precursors of long chain monounsaturated fatty acids by retention of the double bonds in A3-decenoic acid or As-dodecenoic acid, followed by chain elongation. The saturated fatty acids are thought to arise from octanoic and decanoic acids by reduction of the cu,/3- unsaturated intermediates at all stages of chain elongation. The data presented here show that the monounsaturated and cyclo- propane aldehydes in plasmalogens can also be synthesized from exogenous octanoic and decanoic acids, but not at all well from the longer chain saturated acids. The saturated fatty alde- hydes, on the other hand, can be synthesized from all of the precursors tested from C’s to Ci,. The detailed mechanism of unsaturated aldehyde synthesis cannot be inferred at present because we lack information on the positions of the double bonds in these compounds. The derivation of the aldehyde chains of animal plasmalogens from long chain fatty acids has been re- ported (37, 38).

Since the vinyl ether-linked (aldehyde) chains in plasmalogens are chemically equivalent to the dehydration product of a hemiacetal, the derivation of these chains from long chain alde- hydes was considered. We tested for this specific derivation by growing cells in the presence of a long chain aldehyde labeled in the carbonyl carbon. The results given in Table VI show that long chain aldehyde was incorporated into the lipids of C. butyri- cum. In several experiments, at least 20% of the radioactive material found in the washed cells was associated with com- pounds other than free fatty aldehydes. These included phos- pholipids, free fatty acids, and esterified fatty acids. The data indicate little, if any, preference for incorporation into the alde- hyde chains of plasmalogens as opposed to the fatty acids of phospholipids in general. When unlabeled palmitaldehyde was added to a culture growing in the presence of trace amounts of l-I%-palmitate, there was a marked dilution of radioactivity in both acid and aldehyde chains of phospholipids (Table VI, Lines 3 and 4). Apparently, relatively large amounts of palmitalde- hyde in the medium are utilized efficiently in competition with palmitate. When unlabeled fatt.y acid was added to the me- dium of cells growing in the presence of I-14C-palmitaldehyde, the dilution of radioactivity in the aldehyde and acid chains of phospholipids was not as marked. The acid chains were diluted somewhat more (52”) than the aldehyde chains (37%) (Table VI, Lines 1 and 2).

These results argue against a special role for palmitaldehyde as a precursor of the vinyl ether-linked chains in plasmalogens. It should be noted that palmitaldehyde carbonyl carbon is also found in the Cl8 acid and aldehyde in these experiments. Break- down of the precursor and reutilization of the breakdown prod- ucts is unlikely since no radioactivity was found in the Cl4 acid. A chain elongation, implying prior oxidation, seems to have occurred. On the other hand, these findings do not completely

rule out free aldehydes as direct precursors of the alde’:yde chains in plasmalogens. A rapid equilibration of free fatty acids and aldehydes followed by incorporation of the aide’- yde chains into plasmalogens could also account for these observations.

Equilibration of exogenous fatty acid and alde’ yde is also indicated by experiments in which labeled free fatty acids and labeled non-phosphatide-bound aldehydes were c’etected in cells grown in the presence of labeled palmitaldehyde and palmitate, respectively.

Two biosynthetic routes to animal plasmalcgens have re- ceived consideration. On the basis of the diacylphosphatide and plasmalogen content of various tissues, Thiele 1 as proposed the derivation of the latter from the former and 1 as postulated reduction of the ester bond to a hemiacetal, folk wed by a de- hydration to the vinyl ether bond (39, 40). Kiyasu and Ken- nedy have demonstrated the conversion of a “plasmalogenic diglyceride,” i.e. an ol-alkenyl-&acyldiglyceride, to choline and ethanolamine plasmalogens by reaction of the “plasmalogenic diglyceride” with CDP-choline and CDP-ethanclamine in the presence of a particulate fraction of rat liver (41). b-0 pathway for phospholipid biosynthesis involving cytidine nucleotide derivatives of ethanolamine or choline has been found in bacteria. Further, even in animal tissues, the reactions obcerved with the “plasmalogenic diglyceride” may only be a reflection of lack of specificity of the enzymes for the structure of the diglyceride acceptor, as Kiyasu and Kennedy have pointed out. No path- way to the ol-alkenyl+acyldiglycerides has been found in animal tissues. Of course these diglycerides could be formed by hydrol- ysis of the glycerol-phosphate bond in preformed plasmalogens. They may also arise from dietary sources. In thece cases, reac- tion with activated choline or ethanolamine would serve to regenerate the plasmalogen structure.

Our experiments with growing cells of C. b&y&urn lead us to conclude that long chain fatty acids, presumably formed by the normal pathway for fatty acid synthesis, are precursors of both the aldehyde and acid chains of the phospholipids. On teleo- nomic grounds it is unlikely that a separate biosynthetic pathway to long chain aldehydes would coexist with that for fatty acids. If the diacylphosphatides are indeed precursors of the correspond- ing plasmalogens in C. b&y&urn, as the “Pi incorporation data suggest, two classes of pathways must be considered. In one, the acyl group attached to the CY’ position of glycerol would be retained and the ester bond would be converted in some way to a vinyl ether bond.6 In the second possible pathway, this acyl group would be removed and would be replaced by an aldehyde group. This could occur by preliminary hydrolysis to form a lysophosphatide, followed by condensation with another chain to form the vinyl ether bond, or by a mechanism in which no free lysophosphatide is formed and an interchange between the acyl group and a group destined to form the vinyl ether bond takes place. No labeled lysophosphatides were detected in our ‘2Pi incorporation experiments, but a small rapidly metabolized pool of these compounds cannot be ruled out. We are unable to choose among these alternative mechanisms at present.

6 In the plasmalogens of animal tissues, the aldehydogenic chain is found in the 01’ position of glycerol (12). Unpublished work in this laboratory indicates a similar position for at least a ma- jority, if not all, of the aldehydogenic chains in C. buly&um plasmalogens.

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April 1965 N. A. Baumann, P-O. Hagen, and H. Gold&e

SUMMARY

Phospholipids isolated from Clostridium butyricum in the exponential phase of growth have been separated by thin layer chromatography. The major phosphatides have been identified as N-methylethanolamine phosphatides, which are predomi- nantly in the plasmalogen form; phosphatidylglycerol, which is predominantly in the diacylphosphatide form; and ethanolamine phosphatides, which are approximately equally divided between the diacylphosphatide and plasmalogen forms. Several minor phosphatides have also been partially purified but not identified.

The biosynthesis of the major phosphatides has been studied in growing cultures. The time course of incorporation of 32P- labeled inorganic phosphate into phosphatidylglycerol, ethanol- amine, and N-methylethanolamine phosphatides is consistent with precursor-product relationships between the ethanolamine and N-methylethanolamine phosphatides, and also between each diacylphosphatide and the corresponding plasmalogen.

The incorporation of a series of carboxyl-labeled fatty acids into the lipid-bound fatty acids and aldehydes of growing cells of C. butyricum was measured, and in each case the cellular fatty acids and aldehydes with the same hydrocarbon chain length and structure had equal or nearly equal specific activities.

The potential role of free long chain aldehyde as a precursor of the vinyl ether-linked (aldehyde) chains of plasmalogens was test,ed. Growing cells incorporated I-W-palmitaldehyde into both the fatty acid and aldehyde chains of phospholipids. When unlabeled palmitaldehyde was added to cultures growing in the presence of I-X-palmitate, a marked dilution of radioactivity in both the acid and aldehyde chains of the phospholipids was observed. These results do not support a special role for free long chain aldehydes as precursors of plasmalogen aldehyde chains, nor do the data completely rule out this possibility.

Acknowledgments-We wish to express our gratitude to Drs.

Edward A. Kravitz, John H. Law, and N. C. Johnston for help- ful discussions, and to acknowledge the able technical assistance of Miss Martha E. Ellis.

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Nicole A. Baumann, P-O. Hagen and Howard GoldfineCOMPOSITION AND BIOSYNTHESIS

: STUDIES ON PLASMALOGENClostridium butyricumPhospholipids of

1965, 240:1559-1567.J. Biol. Chem. 

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