Phosphatidylserine Synthesis Research Articles

Effects of calmodulin antagonists on serine phospholipid base-exchange reaction in rabbit platelets.

Effects of the calmodulin antagonists chlorpromazine, trifluoperazine, and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide on phospholipid metabolism were examined in rabbit platelets using [3H]serine, [3H]ethanolamine, [3H]choline, and [3H]glycerol. All these drugs markedly stimulated the incorporation of [3H]serine into phosphatidylserine. On the other hand, these drugs had only a slight effect on the rate of incorporation of [3H]ethanolamine and [3H]choline into the corresponding phospholipid. When [3H]glycerol was used as a precursor of the phospholipids, 3H-labeled phospholipids were mainly composed of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol. Although the phosphorus content of phosphatidylserine was about 40% of that of phosphatidylcholine in rabbit platelets, the amount of phosphatidylserine labeled with [3H]glycerol was less than 2% of that of the labeled phosphatidylcholine, and calmodulin antagonists slightly stimulated the incorporation of [3H]glycerol into phosphatidylserine. Treatment with calmodulin antagonists caused a marked decrease in the content of endogenous free serine with concomitant increase in the contents of endogenous free ethanolamine and choline. On the other hand, the contents of other free amino acids, including essential and non-essential amino acids, were unchanged. These results suggest that the calmodulin antagonists we used did not affect de novo synthesis of phosphatidylserine, but did stimulate the serine phospholipid base-exchange reaction in rabbit platelets.

Isolation and characterization of a Chinese hamster ovary cell line requiring ethanolamine or phosphatidylserine for growth and exhibiting defective phosphatidylserine synthase activity.

A mutant cell line (designated M.9.1.1) requiring ethanolamine for growth was derived from Chinese hamster ovary (CHO-K1) cells using 5-bromodeoxyuridine enrichment. The ethanolamine requirement was readily replaced by 20 microM phosphatidylserine and 10 microM lysophosphatidylethanolamine. When M.9.1.1 cells were supplemented with phosphatidyl[3H]serine it was rapidly taken up, and subsequently decarboxylated to form phosphatidyl[3H]ethanolamine. The incorporation of [3H]serine into phosphatidylserine in the mutant cells was 57% of that in the parental cells. Phosphatidylethanolamine synthesis from [3H]serine in the mutant cells was 35% of that in parental cells. When M.9.1.1 cells were deprived of ethanolamine for 48 h the level of phosphatidylserine decreased 34% and the level of phosphatidylethanolamine decreased 26% compared to parental cells. At the same time the rate of turnover of phosphatidylserine was reduced to half that found in parental cells. Examination of the enzymes of phosphatidylserine metabolism indicated defective phosphatidylserine synthase activity in the mutant. When exogenous phosphatidylcholine was used as the phospholipid substrate for the reaction the apparent kinetic constants were Vmax (mutant) = 5.7 pmol/min/mg protein and Vmax (parental) = 17.5 pmol/min/mg protein. Measurement of the back reaction (ATP-independent incorporation of choline into phospholipid) gave no detectable activity in the mutant cells. The data indicate that the phosphatidylcholine-dependent synthesis of phosphatidylserine is the primary lesion in M.9.1.1.

Effect of 1,25-dihydroxyvitamin D3 on phospholipid metabolism in a clonal osteoblast-like rat osteogenic sarcoma cell line.

The effect of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on phospholipid metabolism was examined in clonal rat osteogenic sarcoma cells, UMR 106, of osteoblastic phenotype. Treatment of UMR 106 cells with 10(-8)M 1,25-(OH)2D3 for 48 h caused an increase in [14C]serine incorporation into phosphatidylserine (PS) and a decrease in [3H]ethanolamine, [3H]linositol, and [14C]choline incorporation into phosphatidylethanolamine (PE), phosphatidylinositol, and phosphatidylcholine, respectively; the decrease in [3H]ethanolamine incorporation into PE was the largest. The total contents of phospholipids were similarly affected by 10(-8)M 1,25-(OH)2D3 treatment, suggesting that the effects of 1,25-(OH)2D3 are due largely to alterations in the synthesis of these phospholipids. The effects of 1,25-(OH)2D3 were evident at 10(-10) M 1,25-(OH)2D3, and 10(-8)M 1,25-(OH)2D3 caused a maximal stimulation of [14C]PS synthesis (167% of control) and a maximal reduction in the [3H]PE synthesis (41% of control). The [14C]PS/[3H]PE ratio increased gradually and reached a maximum after 70 h of treatment with 10(-8)M 1,25-(OH)2D3. When the cells were cultured in calcium-free medium containing 0.5 mM EGTA or when 5 microM cycloheximide was added to the medium, the effect of 1,25-(OH)2D3 on phospholipid metabolism was almost completely inhibited. Neither 25-hydroxyvitamin D3 nor 24,25-dihydroxyvitamin D3 caused significant changes in phospholipid metabolism. These results suggest that 1,25-(OH)2D3 alters phospholipid metabolism by enhancing PS synthesis through a calcium-dependent stimulation of the base exchange reaction of serine with other phospholipids and that the effect of 1,25-(OH)2D3 requires the synthesis of new proteins. Because PS is thought to be important for apatite formation and bone mineralization by binding calcium and phosphate to form calcium-PS-phosphate complexes, the present data suggest that 1,25-(OH)2D3 may stimulate bone mineralization by a direct effect on osteoblasts, stimulating PS synthesis.

Two recessive suppressors of Saccharomyces cerevisiae cho1 that are unlinked but fall in the same complementation group.

Phenotypic reversion of ethanolamine-requiring Saccharomyces cerevisiae cho1 mutants is predominantly due to recessive mutations at genes unlinked to the chromosome V cho1 locus. The recessive suppressors do not correct the primary cho1 defect in phosphatidylserine synthesis but circumvent it with a novel endogenous supply of ethanolamine. One suppressor (eam1) was previously mapped to chromosome X, and 135 suppressor isolates were identified as eam1 alleles by complementation analysis. Additional meiotic recombination studies have identified a second genetic locus, eam2, that falls in the eam1 complementation group but maps close to the centromere of chromosome IV. Although the normal EAM1 and EAM2 alleles are fully dominant over recessive mutant alleles, their dominance fails in diploids heterozygous for defects in both genes simultaneously. The unusual complementation pattern could be explained by interaction of the gene products in formation of the same enzyme.

Biochemical and Morphological Investigation on the Ataxic Mutant Shambling Mouse

ABSTRACT Ataxic Shambling mouse was investigated biochemically as well as morphologically to find any specific changes which would be compatible with their ataxic behaviors. Biochemical studies revealed that calcium uptake by the brain synaptosomes of ataxic mouse was decreased to about 30% at 30 mM potassium medium of the control and that the neuron specific enolase activity was lowered a half the value of control synaptosomes, while morphological investigations by Baker's staining for phospholipids disclosed little evidences of phospholipidosis in the nervous tissues and in other organs of the ataxic Shambling mouse. NAD dehydrogenase as well as myosin ATPase staining on the hindlimb muscles of the ataxic Shambling mouse showed a local atrophy and a certain derangement of muscle fibers.These results were discussed in relation to our previous finding of lowered phosphatidylserine synthesis by the brain microsomes which participated at the neuronal transport and transmission in the synaptosomes. Consequently, the ataxic behaviors of Shambling mouse would be ascribed to defective metabolism of phspholipids and decreased brain specific enolase in the synaptosomal fractions which may possibly result in reduced calcium uptake by the synaptosomes regulating neural transmission processes.

The biosynthesis of phosphatidylserine and phosphatidylethanolamine from l-[3- 14C]serine in isolated rat hepatocytes

Incorporation of l-[3- 14C]serine into phosphatidylserine (PS) and phosphatidylethanolamine (PE) has been studied in isolated rat hepatocytes. Ethanolamine inhibited the incorporation, indicating competition with serine in the base-exchange reaction. Choline, monomethylethanolamine, dimethylethanolamine and dimethyl-3-aminopropan-1-ol had no such effect. The observed rate of PS biosynthesis corresponded to 7–17 nmol/min per liver at 0.55 mM l-serine. The results indicate that only a small fraction ( 1 25 to 1 70 ) of the PS pool equilibrates with the base-exchange enzyme, and that decarboxylation to PE occurs preferentially from this pool. The rate of PS synthesis and decarboxylation can therefore not be calculated by methods which assume random, homogeneous labelling of the total PS pool. The apparent rate of PS decarboxylation increased approx. 4-fold when l-serine increased from 0.5 to 2.25 mM, suggesting that decarboxylation of PS to PE might be regulated by the concentration of l-serine or by the amount of PS present in the hepatocyte cell membranes. Lauric, palmitic, stearic, oleic and linoleic acid decreased the rate of PS synthesis. At 0.5 mM, lauric and palmitic acid were most inhibitory. At 1.0 mM, linoleic acid was the least inhibitory fatty acid. The saturated hexaenoic and saturated tetraenoic species of PS contained 51 and 29%, respectively, of the incorporated l-[3- 14C]serine. The combined monoene dienoic/diene dienoic fraction had the highest rate of synthesis judged by its relative specific activity. At 0.9 mM concentration, linoleic acid doubled the relative specific activity of the combined monoene dienoic/diene dienoic fraction of PS. Incorporation of l-[3- 14C]serine into molecular species of PE resembled that into PS, both in the absence and presence of linoleic acid, suggesting that the phosphatidylserine decarboxylase (EC 4.1.1.65) has a low specificity towards the fatty acid composition of PS. The results indicate that biosynthesis of PS from l-serine occurs mainly by the base-exchange with only negligible contribution from direct incorporation of phosphatidic acid or diacylglycerol. Furthermore, the deacylation-reacylation pathway seem to contribute only little to the determination of the fatty acid composition of hepatocyte PS. Active PS turnover seems to be confined to a small fraction of the PS pool.

Lysophosphatidylserine dependent incorporation of acyl CoA into phospholipids in rat brain microsomes

[ 14C]OleoylCoA was incorporated into phosphatidylinositol 4 1 2 times more efficiently than into phosphatidylserine in rat brain and liver microsomes when incubated with various levels of 1-acyl-sn-glycero-3-phosphoserine. In contrast, 1-acyl-sn-glycero-3-phosphocholine dependent incorporation of oleoylCoA was only into phosphatidylcholine. When [ l- 3H]serine labeled 1-acyl-sn-glycero-3-phosphoserine was used as the labeled substrate, no phosphatidylserine synthesis could be detected in rat brain microsomes. OleoylCoA incorporation in phospholipids in the presence of lysophosphatidylserine was primarily at the 2-position while stearoylCoA was incorporated at the 1-position. These results are interpreted to suggest that there is no acylCoA:1-acyl-sn-glycero-3-phosphoserine acyltransferase in rat brain microsomes and the lysophosphatidylserine dependent position-specific incorporation of acylCoA into various phospholipids may be due to an exchange reaction. A simple highly reproducible one dimensional thin-layer chromatographic system is described for the separation of all the major phospholipids of brain and liver.

SACCHAROMYCES CEREVISIAE RECESSIVE SUPPRESSOR THAT CIRCUMVENTS PHOSPHATIDYLSERINE DEFICIENCY

Phenotypic reversion of six independent Saccharomyces cerevisiae cho1 mutants was shown to be due predominantly to mutation of an unlinked gene, eam1. The eam1 gene was located very close to ino1 on chromosome X by meiotic tetrad analysis. Recessive eam1 mutations did not correct the primary cho1 defect in phosphatidylserine synthesis but made endogenous ethanolamine available for sustained nitrogenous phospholipid synthesis. A novel biochemical contribution to nitrogenous lipid synthesis is indicated by the eam1 mutants.

Phosphatidylserine synthesis in Saccharomyces cerevisiae. Purification and characterization of membrane-associated phosphatidylserine synthase.

Membrane-associated phosphatidylserine synthase (CDP-diacylglycerol:L-serine O-phosphatidyltransferase, EC 2.7.8.8) was purified from the microsomal fraction of Saccharomyces cerevisiae strains S288C and VAL2C(YEpCHO1). VAL2C(YEpCHO1) contains a hybrid plasmid bearing the structural gene for phosphatidylserine synthase and overproduces the enzyme 6-7 fold (Letts, V. A., Klig, L. S., Bae-Lee, M., Carman, G. M., and Henry, S. A. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 7279-7283) compared to wild-type S288C. The purification procedure included Triton X-100 extraction of the microsomal membranes, CDP-diacylglycerol-Sepharose affinity chromatography, and DE-53 chromatography. The procedure yielded a preparation from each strain containing a major peptide band (Mr = 23,000) upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phosphatidylserine synthase was dependent on manganese and Triton X-100 for maximum activity at pH 8.0. The apparent Km values for serine and CDP-diacylglycerol were 0.58 mM and 60 microM, respectively. Thioreactive agents inhibited enzyme activity. The enzyme was thermally labile above 40 degrees C. Results of isotopic exchange reactions between substrates and products suggest that the enzyme catalyzes a sequential Bi Bi reaction.

Isolation of the yeast structural gene for the membrane-associated enzyme phosphatidylserine synthase.

The structural gene (CHO1) for phosphatidylserine synthase (CDPdiacylglycerol:L-serine O-phosphatidyltransferase, EC 2.7.8.8) was isolated by genetic complementation in Saccharomyces cerevisiae from a bank of yeast genomic DNA on a chimeric plasmid. The cloned DNA (4.0 kilobases long) was shown to represent a unique sequence in the yeast genome. The DNA sequence on an integrative plasmid was shown to recombine into the CHO1 locus, confirming its genetic identity. The cho1 yeast strain transformed with this gene on an autonomously replicating plasmid had significantly increased activity of the regulated membrane-associated enzyme phosphatidylserine synthase. Partial purification of phosphatidylserine synthase from microsomes of this transformed strain confirmed that the membrane-bound enzyme was overproduced 6- to 7-fold as compared with the wild-type strain. The strain also synthesized the product phospholipid, phosphatidylserine, at an increased rate. The transformed strain had altered proportions of a variety of other phospholipids, suggesting that their synthesis is affected by the rate of synthesis of phosphatidylserine in yeast.

In vivo metabolic intermediates of phospholipid biosynthesis in Rhodopseudomonas sphaeroides.

The in vivo metabolic pathways of phospholipid biosynthesis in Rhodopseudomonas sphaeroides have been investigated. Rapid pulse-chase-labeling studies indicated that phosphatidylethanolamine and phosphatidylglycerol were synthesized as in other eubacteria. The labeling pattern observed for N-acylphosphatidylserine (NAPS) was inconsistent with the synthesis of this phospholipid occurring by direct acylation of phosphatidylserine (PS). Rather, NAPS appeared to be kinetically derived from an earlier intermediate such as phosphatidic acid or more likely CDP-diglyceride. Tris-induced NAPS accumulation specifically reduced the synthesis of PS. Treatment of cells with a bacteriostatic concentration of hydroxylamine (10 mM) greatly reduced total cellular phospholipid synthesis, resulted in accumulation of PS, and stimulated the phosphatidylglycerol branch of phospholipid metabolism relative to the PS branch of the pathway. When the cells were treated with a lower hydroxylamine dosage (50 microM), total phospholipid synthesis lagged as PS accumulated, however, phospholipid synthesis resumed coincident with a reversal of PS accumulation. Hydroxylamine alone was not sufficient to promote NAPS accumulation but this compound allowed continued NAPS accumulation when cells were grown in medium containing Tris. The significance of these observations is discussed in terms of NAPS biosynthesis being representative of a previously undescribed branch of the phospholipid biosynthetic sequence.

Reversibility of propranolol-induced changes in the biosynthesis of monoacylglycerol, diacylglycerol, triacylglycerol, and phospholipids in the retina.

The biosynthesis and metabolism of phospholipids and neutral glycerides were studied in the bovine retina. Radioactive glycerol was used as a precursor. Phentolamine and d- and dl-propranolol were found to produce similar effects on lipid metabolism in the retina. Marked stimulation of phosphatidylinositol (PhI) synthesis and maximal inhibition of phosphatidylcholine (PhC), diacylglycerol (DG), and triacylglycerol (TG) formation were observed within 5 min after exposure to 0.5 mM dl-propranolol. Pulse-chase experiments showed a high turnover rate in DG and a reversibility of the propranolol-induced changes produced during the synthesis of PhC, TG, DG, monoacylglycerol (MG), and phosphatidylserine. All reversals of the drug-induced biosynthetic profiles approached control values 60 min after incubation in drug-free medium. However, complete reversal was not achieved in any of the cases under these conditions. Propranolol appeared to inhibit both the formation of DG from phosphatidic acid and the further metabolism of DG, probably to MG. Phosphatidylethanolamine biosynthesis showed some recovery from this inhibition. Synthesis of PhI was greatly stimulated by preincubation with propranolol and was further enhanced by reincubation in the presence of propranolol. However, this effect was not reversed by reincubation without the drug. The active de novo biosynthesis of retinal phospholipids and glycerides is a very dynamic pathway that may be redirected by amphiphilic drugs. In addition, the partial reversal of modifications induced in the flux of [2-3H]glycerol through the lipids can occur during short-term reincubations of retinas in drug-free medium.

Biosynthesis and transport of phosphatidylserine in the cell.

This chapter presents the biosynthesis and transport of phosphatidylserine in the cell. Numerous biochemical differences between prokaryotic and eukaryotic organisms are well established. The two main pathways for phosphatidylserine synthesis are often described as characteristic for the two classes of living organisms. The Base Exchange reaction involving L-serine and phosphatidylethanolamine seems to be typical in eukaryotes. In bacteria, considerable progress has been made toward understanding the pathway and mechanism of phosphatidylserine biosynthesis. In animal cells, it is still not known whether phosphatidylserine in mitochondria is exclusively of microsomal origin, being transported into mitochondria by cytoplasmic transfer proteins, or whether this phospholipid is formed inside mitochondria by an ATP-dependent process. The mechanism of this process is still uncertain and its physiological significance is not clear. The occurrence of phosphatidylserine transfer proteins in the intermembranous space of mitochondria is also unknown.

Topography of glycerolepid synthetic enzymes Synthesis of phosphatidylserine, phosphatidylinositol and glycerolipid intermediates occurs on the cytoplasmic surface of rat liver microsomal vesicles

The topography of glycerolipid biosynthetic enzymes within the transverse plane of rat liver microsomal vesicles was investigated: (1) by use of the impermeant inhibitor, mercury-dextran; (2) by use of proteases; and (3) by determining whether the enzyme activities are latent. The seven enzyme activities investigated (dihydroxyacetone-phosphate acyltransferase, acyldihydroxyacetone-phosphate oxidoreductase, phosphatidic acid : CTPcytidyl-transferase, CDPdiacylglycerol : inositol phosphatidyltransferase, 2-monoacylglycerol acyltransferase, diacylglycerol kinase, and the serine base exchange enzyme) function in phosphatidylinositol and phosphatidylserine synthesis and at intermediate levels in glycerolipid synthesis including steps of ether lipid synthesis. Mercurydextran inhibited four of these enzymes greater than 60% in intact microsomal vesicles. One or more of the proteases employed (chymotrypsin, trypsin and pronase) inactivated each of the seven enzyme activities in intact microsomal vesicles. These two approaches indicate that each of these enzymes has important domains located on the cytoplasmic surface of microsomal vesicles. These enzyme activities could be assayed in intact microsomal vesicles. None appeared to be highly latent, indicating that substrates have free access to active sites. One substrate for each of these enzymes had been shown previously to be unable to cross the microsomal membrane. These data indicate that the active sites of these enzymes are located on the cytoplasmic surface of microsomal vesicles. It is concluded that the synthesis of phosphatidylserine and phosphatidylinositol, intermediates of ether lipid formation and other intermediates of glycerolipid synthesis occurs asymmetrically on the cytoplasmic surface of the endoplasmic reticulum. These findings and our previous investigations on the topography of seven enzymes of triacylglycerol, phosphatidylcholine and phosphatidylethanolamine biosynthesis (Ballas L.M. and Bell, RM., Biochim. Biophys. Acta 602, (1980) 578–590) indicate that the synthesis of the major cellular glycerolipids occurs asymmetrically on the cytoplasmic surface of the endoplasmic reticulum.

Characterization of a membrane-associated cytidine diphosphate-diacylglycerol-dependent phosphatidylserine synthase in bacilli.

The synthesis of phosphatidylserine in two gram-positive aerobic bacteria has been partially characterized. We have located a cytidine 5'-diphospho-diacylglycerol:L-serine O-phosphatidyltransferase (phosphatidylserine synthase) activity in the membrane fraction of Bacillus licheniformis and Bacillus subtilis. The activity was demonstrated to be membrane associated by differential centrifugation, sucrose gradient centrifugation, and detergent solubilization. The direct involvement of cytidine 5'-diphospho-diacylglycerol in the reaction was demonstrated by the conversion of the liponucleotide phosphatidyl moiety to phosphatidylserine. This activity is dependent on divalent metal ion (manganese being optimal) and is stimulated by nonionic detergent and its product phosphatidylserine. Based on studies with various combinations of products and substrates, the reaction appears to follow a sequential BiBi kinetic mechanism.

Biosynthesis of phosphatidylserine in rat brain microsomes

1. 1. Rat brain microsomes incorporated L-serine into phosphatidylserine in the presence of 2 mM ATP. This reaction was stimulated 2-fold by the addition of phosphatidic acid (0.2 mM) and 5-fold by the addition of nickel (0.5 mM). 2. 2. This phosphatidylserine synthesis was inhibited completely by p-hydroxy-mercuribenzoate (0.1 mM) and N-ethylmaleimide (1 mM), whereas the Ca 2+-dependent phosphatidylserine synthesis was unaffected by these sulfhydryl reagents. 3. 3. The specific activity of the ATP-Ni 2+-dependent phosphatidylserine was increased more than 2-fold during active myelination, whereas the Ca 2+-dependent system remained unchanged. 4. 4. Preliminary data indicate that pyrophosphatidic acid ( p, P'-bis'(1,2-diacyl- sn-glycero-3-) pyrophosphate) is the immediate precursor of phosphatidylserine synthesis.

An improved procedure for the synthesis of 14C-labeled phosphatidylserine from cerebral phosphatidic acid

A complete procedure to prepare a highly labeled phosphatidyl-L-[U-14C]serine possessing the same fatty acid composition of brain phospholipids is reported. CDP-diglyceride was synthesized by reaction between phosphatidic acid and CMP-morpholidate as the dicyclohexylcarboxamidium salt. The reaction between CDP-diglyceride and L-[U-14C]serine to produce the labeled phosphatidylserine was catalyzed by the CDP-diglyceride: L-serine phosphatidyl transferase (EC 2.7.8.8) from E. coli. A selective inhibition of phosphatidylserine decarboxylase activity, present as contaminant in the enzyme extract, was introduced in order to avoid a low yield of product. Traces of phosphatidylethanolamine (about 1%) were easily removed by preparative thin-layer chromatography. The yield of the labeled product was as high as 87% and it specific radioactivity was 170 mCi/mmol.

Membrane mutants: a yeast mutant with a lesion in phosphatidylserine biosynthesis.

A single-gene nuclear choline-requiring mutant of Saccharomyces cerevisiae was studied. Choline as a growth supplement to synthetic media could be substituted by low concentrations of dimethylethanolamine, monomethylethanolamine or ethanolamine. DL-Serine also supported growth, but only at high concentrations: on a molar basis it was approximately one hundred times less effective than choline. When cultured in unsupplemented medium the mutant cells soon ceased to grow. The growth-arrested cells contained less than one fifth of the phosphatidylethanolamine present in wild-type cells and only traces of phosphatidylserine. The relative content of the two phospholipid species was raised by growing the mutant cells in the presence of choline of the other supplements but still remained lower than in wild-type cells. The mutant cells depleted of phosphatidylethanolamine and phosphatidylserine had greatly diminished ability to fuse with other cells in mating and their protoplasts showed increased resistance to hypotonic lysis. Respiration was not substantially affected by the deficit of the two phospholipid species in the mutant. In cell-free preparations, the affinity of the phosphatidylserine synthesizing system for serine was found to be almost two orders of magnitude lower in the mutant than in the wild-type. The impairment of phosphatidylserine synthesis accounts for growth requirement and the abnormal phospholipid composition of the mutant cells.

Yeast mutant defective in phosphatidylserine synthesis.

Phospholipid biosynthesis in a mutant of Saccharomyces cerevisiae (cho1) which lacks phosphatidylserine (Atkinson, K. D., Jensen, B., Storm, E., Kolat, A. I., Henry, S. A. & Fogel, S. (1980) J. Bacteriol. 141, 558-564) has been examined. The ability of cells of this strain to synthesize phosphatidylserine in vitro in a cell-free system is reduced at least 10-fold, whereas other phospholipid-synthesizing activities are present at normal or slightly elevated levels. While all phospholipid biosynthetic activities, except phosphatidylserine synthesis, can be demonstrated in vitro in the cho1 mutant, the entire pattern of phospholipid synthesis, accumulation, and turnover in vivo is distorted. Phosphatidylinositol synthesis is elevated, as is phosphatidylcholine synthesis. In addition, the turnover of phosphatidylcholine is more rapid in the cho1 mutant. The cho1 mutant appears to use almost exclusively the alternative pathway described by Kennedy and Weiss (1956) J. Biol. Chem. 222, 193-214) for the production of phosphatidylethanolamine and phosphatidylcholine, bypassing phosphatidylserine as an intermediate.

A rapidly metabolizing pool of phosphatidylglycerol as a precursor of phosphatidylethanolamine and diglyceride in Bacillus megaterium.

Pulse-chase experiments in Bacillus megaterium ATCC 14581 with [U-14C]palmitate, L-[U-14C]serine, and [U-14C]glycerol showed that a large pool of phosphatidylglycerol (PG) which exhibited rapid turnover in the phosphate moiety (PGt) underwent very rapid interconversion with the large diglyceride (DG) pool. Kinetics of DG labeling indicated that the fatty acyl and diacylated glycerol moieties of PGt were also utilized as precursors for net DG formation. The [U-14C]glycerol pulse-chase results also confirmed the presence of a second, metabolically stable pool of PG (PGs), which was deduced from [32P]phosphate studies. The other major phospholipid, phosphatidylethanolamine (PE), exhibited pronounced lags relative to PG and DG in 14C-fatty acid, [14C]glycerol, and [32P]phosphate incorporation, but not for incorporation of L-[U-14C]serine into the ethanolamine group of PE or into the serine moiety of the small phosphatidylserine (PS) pool. Furthermore, initial rates of L-[U-14C]serine incorporation into the serine and ethanolamine moieties of PS and PE were unaffected by cerulenin. The results provided compelling in vivo evidence that de novo PGt, PS, and PE synthesis in this organism proceed for the most part sequentially in the order PGt yields PS yields PE rather than via branching pathways from a common intermediate and that the phosphatidyl moiety in PS and PE is derived largely from the corresponding moiety in PGt, whereas the DG pool indirectly provides an additional source for this conversion by way of the facile PGt in equilibrium or formed from DG interconversion.