Biosynthesis Of Phosphatidylserine Research Articles

Agonist-induced inhibition of phosphatidylserine synthesis is secondary to the emptying of intracellular Ca2+ stores in Jurkat T-cells.

The biosynthesis of phosphatidylserine (PtdSer) by the serine base-exchange enzyme system, in Jurkat T-lymphocytes, was inhibited in intact cells maintained in low-Ca(2+)-containing buffer (< 10 microM-Ca2+) by using Ca2+ ionophores (A23187 or ionomycin). The rise in cytosolic Ca2+ concentration under these experimental conditions was only due to the release of Ca2+ from intracellular compartments, suggesting that the inhibition of PtdSer synthesis was correlated with the emptying of intracellular Ca2+ pools. This was further studied in saponin-permeabilized cells, in which PtdSer synthesis was found to be inhibited by EGTA, Ca2+ ionophores (A23187 or ionomycin) and Ca(2+)-ATPase inhibitors [thapsigargin or 2,5-di-(t-butyl)-benzohydroquinone]. Since Ca(2+)-ATPase inhibitors impaired refilling of the Ca2+ stores with Ca2+, and since in CD3-activated Jurkat T-cells the Ca2+ stores remained empty after 1 h of treatment with anti-CD3 monoclonal antibodies, we suggest that PtdSer synthesis is mainly regulated by the level of Ca2+ in the intracellular compartments and that the Ca(2+)-dependent serine base-exchange system responsible for PtdSer synthesis is probably located within or close to a Ca(2+)-storage organelle.

In utero ethanol exposure decreases the biosynthesis of phosphatidylserine in rat pup cerebrum.

Phosphatidylserine is enriched in the brain and has been implicated to play a role in regulating neuronal membrane functions. In this study, three experimental protocols were used to examine the effects of in utero ethanol exposure on phosphatidylserine biosynthesis in rat pup brain, namely, (1) assay of the serine base-exchange enzyme activity in brain microsomes, (2) incubation of brain slices with [3H] serine, and (3) incorporation in vivo of [3H]serine into phosphatidylserine as well as serine-related phospholipids in brain. Results from all three protocols point to a decrease in phosphatidylserine biosynthesis in newborn rat pup cerebrum on exposure to ethanol in utero compared with the pair-fed controls. When in utero ethanol-exposed pups were nursed by mothers given a chow diet, the differences gradually returned to control levels by 17 days of age. The decrease in phosphatidylserine biosynthesis may be important in explaining some of the neuronal deficits associated with in utero ethanol exposure.

Secretion of VLDL, but not HDL, by rat hepatocytes is inhibited by the ethanolamine analogue N-monomethylethanolamine.

The role of phospholipids in the assembly and secretion of very low density lipoproteins (VLDL) has been investigated by incubation of monolayer cultures of rat hepatocytes with monomethylethanolamine, an analogue of ethanolamine and choline. The cellular concentration of phosphatidylmonomethylethanolamine was increased 17-fold in response to treatment of hepatocytes with monomethylethanolamine. The secretion of phosphatidylcholine, triacylglycerol, and the apolipoproteins BH, BL, and E into VLDL was inhibited by approximately 50% in hepatocytes incubated with monomethylethanolamine, compared to untreated cells. Cell viability was unaffected by treatment with the ethanolamine analogue, as was cellular protein synthesis. The mechanism by which monomethylethanolamine reduced VLDL secretion was examined. Since monomethylethanolamine is a structural analogue of ethanolamine and choline, an obvious hypothesis for explanation of the effect on VLDL secretion was that phosphatidylcholine biosynthesis, which is required for VLDL secretion (Z. Yao and D. E. Vance. 1988. J. Biol. Chem. 263: 2998-3004) was inhibited. However, the biosynthesis of phosphatidylcholine from [3H]choline or from [3H]glycerol was not significantly reduced in the analogue-treated, compared with the untreated, hepatocytes. Nor was the incorporation of [3H]glycerol into cellular triacylglycerol altered in the monomethylethanolamine-treated cells. Furthermore, addition of monomethylethanolamine to hepatocytes did not reduce the rate of biosynthesis of phosphatidylethanolamine either from CDP-ethanolamine or from phosphatidylserine, nor was phosphatidylserine biosynthesis from [3-3H]serine affected. The 50% inhibition of VLDL secretion elicited by monomethylethanolamine was apparently specific for VLDL because there was no difference in secretion of HDL (lipid or apoprotein moieties) or albumin by cells incubated with or without the ethanolamine analogue. The experiments showed that inhibition of VLDL secretion by monomethylethanolamine was not the result of decreased biosynthesis of phospholipids, triacylglycerols, or cholesteryl esters. More subtle effects of the ethanolamine/choline analogue, for example interference by the increased amount of phosphatidylmonomethylethanolamine, in the process of assembly of lipids with apoB remain a possibility.

Regulation of phosphatidylserine decarboxylase in Saccharomyces cerevisiae by inositol and choline: Kinetics of repression and derepression

The biosynthesis of phosphatidylserine (PS) and its conversion to phosphatidylcholine (PC) are regulated coordinately by inositol and choline in Saccharomyces cerevisiae ( G. M. Carman and S. A. Henry, 1989, Annu. Rev. Biochem. 58, 635–669). In this study, PS decarboxylase activity is shown to be partially repressed when inositol is added to the medium of cells in the log phase of growth, and the extent of repression is augmented by the inclusion of choline, but not ethanolamine. The kinetics of repression and derepression of PS decarboxylase, PS synthase, and phospholipid N-methyltransferase (PNMT) activities, as regulatory responses to the availability of exogenous inositol and choline, have been characterized. When inositol was added to the medium of cell cultures growing exponentially, the three biosynthetic enzyme activities reached an intermediate level of repression (50–85% of control) within 60 min. After the addition of the combination of inositol and choline, PS decarboxylase, PS synthase, and PNMT activities decreased to the intermediate levels of repression in 60 min and were subsequently reduced to 15–40% of control values during a later stage of regulation (2–3 h). In a derepression study, the three enzyme activities remained relatively stable for approximately 60 min following the removal of choline and/or inositol from the growth medium, but the specific activities of PS decarboxylase, PS synthase, and PNMT increased to maximally derepressed levels within 2–3 h. The induction of the three biosynthetic activities was blocked by cycloheximide, but not by chloramphenicol. In summary, the level of PS decarboxylase activity in S. cerevisiae is partially and reversibly suppressed by inositol and further diminished by the combination of inositol and choline. The biphasic kinetics of repression by inositol and choline suggest that the effect of choline is dependent on earlier events mediated by inositol and possibly involves a separate regulatory factor(s).

A cloned gene encoding phosphatidylserine decarboxylase complements the phosphatidylserine biosynthetic defect of a Chinese hamster ovary cell mutant.

A phosphatidylserine-auxotrophic mutant of cultured Chinese hamster ovary cells, PSA-3, manifests a defect in phosphatidylserine synthase I activity (Kuge, O., Nishijima, M., and Akamatsu, Y. (1986) J. Biol. Chem. 261, 5790-5794). We cloned a Chinese hamster gene, designated pssC, which was able to transform the PSA-3 cell line to a phosphatidylserine prototroph. The resultant transformant contained phosphatidylserine in normal amounts but remained defective in phosphatidylserine synthase I activity, indicating that pssC is a suppressor gene. Using the genomic fragment of pssC as a probe, a cDNA clone of pssC was isolated, and its nucleotide sequence was determined. A computer search through a protein data bank revealed that pssC had homology with the Escherichia coli psd gene encoding the proenzyme of phosphatidylserine decarboxylase at the amino acid level. Introduction of the cloned pssC gene into PSA-3 resulted in a 2-fold increase in phosphatidylserine decarboxylase activity. When the pssC cDNA was placed downstream of the yeast GAL1 promoter and introduced into yeast Saccharomyces cerevisiae cells, the phosphatidylserine decarboxylase activity increased in a galactose-dependent manner. These results indicate that pssC encodes phosphatidylserine decarboxylase. The mechanism by which pssC complements the defect of PSA-3 in phosphatidylserine biosynthesis is discussed.

Serine utilization as a precursor of phosphatidylserine and alkenyl-(plasmenyl)-, alkyl-, and acylethanolamine phosphoglycerides in cultured glioma cells

In several tissues and cell lines, serine utilized for phosphatidylserine (PS) synthesis is an eventual precursor of the base moiety of ethanolamine phosphoglycerides (PE). We investigated the biosynthesis and decarboxylation of PS in cultured C6 glioma cells, with particular attention to 1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine (plasmenylethanolamine) biosynthesis. Incorporation of [3H]serine into PS reached a maximum within 4-8 h, and label in nonplasmenylethanolamine phosphoglyceride (NP-PE) and plasmenylethanolamine was maximal by 12-24 h and 48 h, respectively. After 8 h, label in PS decreased even though 40-60% of initial label remained in the culture medium. Serial additions of fresh [3H]serine restored PS synthesis to higher levels of labeled PS accumulation followed by a subsequent decrease in 4-8 h. High performance liquid chromatographic analyses confirmed that medium serine was depleted by 8 h, and thereafter metabolites, including acetate and formate, accounted for radioactivity in the medium. The rapid but transient appearance of labeled glycine and ATP inside the cells indicated conversion of serine by hydroxymethyltransferase. 78-85% of label from serine was in headgroup of PS or of PE formed by decarboxylation. A precursor-product relationship was suggested for label from [3H]serine appearing in the headgroup of diacyl, alkylacyl, and alkenylacyl subclasses of PE. By 48 h, a constant specific activity, ratio of approximately 1:1 was reached between plasmenylethanolamine and NP-PE, similar to the molar distribution of these lipids. In contrast, equilibrium was not achieved in cells incubated with [1,2-14C]ethanolamine; plasmenylethanolamine had 2-fold greater specific activity than labeled NP-PE by 72-96 h. These observations indicate that in cultured glioma cells 1) serine serves as a precursor of the head group of PS and of both plasmenyl and non-plasmenyl species of PE; 2) exchange of headgroup between NP-PE and plasmenylethanolamine may involve different donor pools of PE depending on whether the headgroup originates with exogenous serine or ethanolamine; 3) serine is rapidly converted to other metabolites, which limits exogenous serine as a direct phospholipid precursor.

Chinese hamster ovary cells deficient in peroxisomes lack the nonspecific lipid transfer protein (sterol carrier protein 2).

Chinese hamster ovary cells deficient in intact peroxisomes were compared with wild type cells for the presence of the nonspecific lipid transfer protein (nsL-TP; sterol carrier protein 2). With the immunoblotting technique using the affinity purified antibody against rat liver nsL-TP, this protein was shown to be present in the homogenates from wild type cells, but could not be detected in mutant cells. In agreement with a previous study using livers from Zellweger patients it appears that there is a positive, as yet unknown, correlation between peroxisomes and the occurrence of nsL-TP in the cell. As a control using the affinity-purified antibody against the phosphatidylinositol transfer protein from bovine brain, levels of this protein were found to be normal in mutant cells. By use of metrizamide density gradients, nsL-TP was shown to cosediment with a membrane fraction different from peroxisomes. A protein of 58,000 daltons cross-reactive with the antibody against nsL-TP did cosediment with the peroxisomes in wild type cells and possibly with a "peroxisomal remnant" in the mutant cells. Incubation of wild type and mutant cells with L-[3-14C]serine showed that the biosynthesis of phosphatidylserine and the subsequent conversion into phosphatidylethanolamine was comparable in both cell types. This indicates that nsL-TP is not involved in the translocation of phosphatidylserine from the endoplasmic reticulum to the mitochondria as the site of decarboxylation.

Isolation and characterization of a Chinese hamster ovary cell mutant with altered regulation of phosphatidylserine biosynthesis

We have screened approximately 10,000 colonies of Chinese hamster ovary (CHO) cells immobilized on polyester cloth for mutants defective in [14C]ethanolamine incorporation into trichloroacetic acid-precipitable phospholipids. In mutant 29, discovered in this way, the activities of enzymes involved in the CDP-ethanolamine pathway were normal; however, the intracellular pool of phosphorylethanolamine was elevated, being more than 10-fold that in the parental CHO-K1 cells. These results suggested that the reduced incorporation of [14C]ethanolamine into phosphatidylethanolamine in mutant 29 was due to dilution of phosphoryl-[14C]ethanolamine with the increased amount of cellular phosphorylethanolamine. Interestingly, the rate of incorporation of serine into phosphatidylserine and the content of phosphatidylserine in mutant 29 cells were increased 3-fold and 1.5-fold, respectively, compared with the parent cells. The overproduction of phosphorylethanolamine in mutant 29 cells was ascribed to the elevated level of phosphatidylserine biosynthesis, because ethanolamine is produced as a reaction product on the conversion of phosphatidylethanolamine to phosphatidylserine, which is catalyzed by phospholipid-serine base-exchange enzymes. Using both intact cells and the particulate fraction of a cell extract, phosphatidylserine biosynthesis in CHO-K1 cells was shown to be inhibited by phosphatidylserine itself, whereas that in mutant 29 cells was greatly resistant to the inhibition, compared with the parental cells. As a conclusion, it may be assumed that mutant 29 cells have a lesion in the regulation of phosphatidylserine biosynthesis by serine-exchange enzyme activity, which results in the overproduction of phosphatidylserine and phosphorylethanolamine as well.

Cluster Headache: Incorporation of (1-14C)oleic Acid into Phosphatidylserine in Polymorphonuclear Cells

As recently demonstrated by our group, polymorphonuclear cells (PMNs) from cluster headache patients have an increased ability to incorporate arachidonic acid (AA) and L-serine into phosphatidylserine (PS). To evaluate whether there is an increased incorporation into PS also from fatty acids not involved in eicosanoid metabolism, PMNs from controls (n = 14) and cluster headache patients (n = 12) were incubated with (1-14C)oleic acid. After 1 h 2.7% +/- 1.1 (mean value +/- SD) of the glycerophospholipid radioactivity was found in PS in controls, whereas 4.2% +/- 1.2 was found in cluster headache patients (p less than 0.005). For phosphatidylcholine (PC) the corresponding figures were 74.2 +/- 5.4 in controls and 66.7 +/- 7.6 in cluster headache patients (p less than 0.01). The results suggest that the de novo biosynthesis of PS is increased and the biosynthesis of PC is decreased in cluster headache. The results may have an effect on the role of PS as an obligate protein kinase C activator.

白血球細胞膜のｔｒａｎｓｍｅｔｈｙｌａｔｉｏｎ，ｐｈｏｓｐｈｏｌｉｐａｓｅ Ａ２活性以外のｍａｊｏｒおよびｔｈｉｒｄ ｐａｔｈｗａｙの燐脂質酵素活性 炎症反応との関与についての検討

The biosynthesis of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS) by base-exchange reactions, and of PC and PE by the major pathway (ethanolaminephosphotransferase (EPT) and cholinephosphotransferase (CPT) ), was assessed in the membranes of human leukocyte subsets. Among the three base-exchange activities, ethanolamine-exchange was the highest, and choline-exchange the lowest. In major pathway, EPT and CPT had comparable activities. Among leukocyte subpopulations, T lymphocytes showed the highest levels of each enzyme activity, and neutrophils showed the least. Base-exchange activities were increased by addition of Ca++ but inhibited by Mg++ while EPT and CPT were inhibited by Ca++ but enhanced by Mg++. However, each base-exchange was also enhanced by calmodulin inhibitors and inhibited by calmodulin, suggesting that base-exchange reaction is dependent on Ca++ but inhibited by Ca-calmodulin complex. Among the enzyme activities tested, PE formation alone was increased by the presence of low concentrations of bioactive stimulants. Our recent reports showed increased ethanolamine-exchange activity in the patients with various inflammatory disorders. These postulate that although its biological significance is unknown, base-exchange activities may be related to cell activation and that PE formation in base-exchange is a precursor of N-transmethylation of PE, leading to the production of arachidonic acid cascade through PC by phospholipase A2 activity.

Genetic and biochemical studies on biosynthesis and physiological roles of phosphatidylserine in mammalian cells

The base-exchange enzymes which catalyze exchange of free serine, choline, and ethanolamine with polar head groups of phospholipids are known to occur in eukaryotic cells. To understand the physiological roles of the base-exchange reactions, a rapid autoradiographic in situ enzyme assay has been developed for detecting the base-exchange activities in Chinese hamster ovary (CHO) cell colonies immobilized on filter paper or polyester cloth and obtained several mutants strikingly defective in the choline-exchange activity. CHO cell mutants that required exogenously added phosphatidylserine for cell growth were also isolated by using the replica technique with polyester cloth. Biochemical characterization of these mutants has revealed the following results. 1) There are at least two kinds of serine-exhange enzyme in CHO-K1 cells ; one of the enzymes (serine-exchange enzyme I) can catalyze the base-exchange reaction of phospholipids with serine, choline and ethanolamine, and the other enzyme (serine-exchange enzyme II) does not use choline as a substrate. 2) Phosphatidylserine in CHO cells is biosynthesized through the following sequential reactions : phosphatidylcholine→phosphatidylserine→phosphatidylethanolamine→phsophatidylserine. The three reactions are catalyzed by serine-exchange I, phosphatidylserine decarboxylase, and serine-exchange enzyme II, respectively. 3) Serine-exchange enzyme I is essential for the growth of CHO cells. In addition, we have found that exogenous phosphatidylserine can be efficiently incorporated into CHO cells and utilized for membrane biogenesis, endogenous phosphatidylserine biosynthesis thereby being suppressed.

Phospholipid base exchange in human leukocyte membranes: Quantitation and correlation with other phospholipid biosynthetic pathways

The biosynthesis of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS) by base-exchange reactions, and of PC and PE by the CDP pathways, was assessed in the membrane phospholipids of human leukocytes (neutrophils, lymphocytes, T lymphocytes, non-T lymphocytes, and monocytes). Of the three base-exchange activities, ethanolamine exchange was the highest and choline exchange the lowest in each leukocyte membrane. In the CDP pathways, ethanolaminephosphotransferase (EPT) and cholinephosphotransferase (CPT) had comparable activities. Among subpopulations of leukocytes, T lymphocytes showed the highest levels of each enzyme activity, and neutrophils showed the least. In contrast to the enzymes of the CDP pathways, each base-exchange activity was directly proportional to the Ca 2+ concentration, but markedly inhibited by Mg 2+. Despite this Ca 2+ dependence, the base-exchange activities were increased in a dose-dependent manner by calmodulin antagonists and, except for ethanolamine exchange, inhibited by the addition of calmodulin; EPT and CPT activities were only slightly inhibited by calmodulin antagonists and were unaffected by calmodulin. PE formation in both neutrophil and lymphocyte base-exchange reactions was enhanced in a dose-dependent manner by the presence of low concentrations of bioactive stimulants (zymosan, 0.05–0.2 mg/ml; Con A, 0.5–2μg/ml), while EPT and CPT activities were not increased by these cell stimulants. Taken together, our data suggest that base-exchange activity, the biological significance of which has been hitherto unclear, may be related to cell activation; in contrast, the CDP pathways appear primarily to involve the constitutive biosynthesis of phospholipids. Our data further suggest that ethanolamine required for base-exchange reactions is a precursor of PE, N-transmethylation of which can serve as a source of cell activation, leading to production of arachidonic through PC by mediation of phospholipase A 2 activity.

Metabolism of the phospholipid precursor inositol and its relationship to growth and viability in the natural auxotroph Schizosaccharomyces pombe.

Phospholipid metabolism in the fission yeast Schizosaccharomyces pombe was examined. Three enzymes of phospholipid biosynthesis, cytidine diphosphate diacylglycerol synthase (CDP-DG), phosphatidylinositol (PI) synthase, and phosphatidylserine (PS) synthase, were characterized in extracts of S. pombe cells. Contrary to an earlier report, we were able to demonstrate that CDP-DG served as a precursor for PI and PS biosynthesis in S. pombe. S. pombe is naturally auxotrophic for the phospholipid precursor inositol. We found that S. pombe was much more resistant to loss of viability during inositol starvation than artificially generated inositol auxotrophs of Saccharomyces cerevisiae. The phospholipid composition of S. pombe cells grown in inositol-rich medium (50 microM) was similar to that of S. cerevisiae cells grown under similar conditions. However, growth of S. pombe at low inositol concentrations (below 30 microM) affected the ratio of the anionic phospholipids PI and PS, while the relative proportions of other glycerophospholipids remained unchanged. During inositol starvation, the rate of PI synthesis decreased rapidly, and there was a concomitant increase in the rate of PS synthesis. Phosphatidic acid and CDP-DG, which are precursors to these phospholipids, also increased when PI synthesis was blocked by lack of exogenous inositol. The major product of turnover of inositol-containing phospholipids in S. pombe was found to be free inositol, which accumulated in the medium and could be reused by the cell.

Phosphatidylserine biosynthesis in cultured Chinese hamster ovary cells. II. Isolation and characterization of phosphatidylserine auxotrophs.

Chinese hamster ovary (CHO) cell mutants that required exogenously added phosphatidylserine for cell growth were isolated by using the replica technique with polyester cloth, and three such mutants were characterized. Labeling experiments on intact cells with 32Pi and L-[U-14C]serine revealed that a phosphatidylserine auxotroph, designated as PSA-3, was strikingly defective in phosphatidylserine biosynthesis. When cells were grown for 2 days without phosphatidylserine, the phosphatidylserine content of PSA-3 was about one-third of that of the parent. In extracts of the mutant, the enzymatic activity of the base-exchange reaction of phospholipids with serine producing phosphatidylserine was reduced to 33% of that in the parent; in addition, the activities of base-exchange reactions of phospholipids with choline and ethanolamine in the mutant were also reduced to 1 and 45% of those in the parent, respectively. Furthermore, it was demonstrated that the serine-exchange activity in the parent was inhibited approximately 60% when choline was added to the reaction mixture whereas that in the mutant was not significantly affected. From the results presented here, we conclude the following. There are at least two kinds of serine-exchange enzymes in CHO cells; one (serine-exchange enzyme I) can catalyze the base-exchange reactions of phospholipids with serine, choline, and ethanolamine while the other (serine-exchange enzyme II) does not use the choline as a substrate. Serine-exchange enzyme I, in which mutant PSA-3 is defective, plays a major role in phosphatidylserine biosynthesis in CHO cells. Serine-exchange enzyme I is essential for the growth of CHO cells.

Phosphatidylserine biosynthesis in cultured Chinese hamster ovary cells. III. Genetic evidence for utilization of phosphatidylcholine and phosphatidylethanolamine as precursors.

In the preceding paper, we reported that Chinese hamster ovary (CHO) cells contain two different serine-exchange enzymes (I and II) which catalyze the base-exchange reaction of phospholipid(s) with serine and that a phosphatidylserine-requiring mutant (strain PSA-3) of CHO cells is defective in serine-exchange enzyme I and lacks the ability to synthesize phosphatidylserine (Kuge, O., Nishijima, M., and Akamatsu, Y. (1986) J. Biol. Chem. 261, 5790-5794). In this study, we examined precursor phospholipids for phosphatidylserine biosynthesis in CHO cells. When mutant PSA-3 and parent (CHO-K1) cells were cultured with [32P]phosphatidylcholine, phosphatidylserine in the parent accumulated radioactivity while that in the mutant was not labeled significantly. On the contrary, when cultured with [32P]phosphatidylethanolamine, the mutant incorporated the label into phosphatidylserine more efficiently than the parent. Furthermore, we found that mutant PSA-3 grew normally in growth medium supplemented with 30 microM phosphatidylethanolamine as well as phosphatidylserine and that the biosynthesis of phosphatidylserine in the mutant was biosynthesis of phosphatidylserine in the mutant was normal when cells were cultured in the presence of exogenous phosphatidylethanolamine. The simplest interpretation of these findings is that phosphatidylserine in CHO cells is biosynthesized through the following sequential reactions: phosphatidylcholine----phosphatidylserine----phosphatidylethanolamine--- - phosphatidylserine. The three reactions are catalyzed by serine-exchange enzyme I, phosphatidylserine decarboxylase, and serine-exchange enzyme II, respectively.

Phosphatidylserine biosynthesis in cultured Chinese hamster ovary cells. I. Inhibition of de novo phosphatidylserine biosynthesis by exogenous phosphatidylserine and its efficient incorporation.

The effect of phosphatidylserine exogenously added to the medium on de novo biosynthesis of phosphatidylserine was investigated in cultured Chinese hamster ovary cells. When cells were cultured for several generations in medium supplemented with phosphatidylserine and 32Pi, the incorporation of 32Pi into cellular phosphatidylserine was remarkably inhibited, the degree of inhibition being dependent upon the concentration of added phosphatidylserine. 32Pi uptake into cellular phosphatidylethanolamine was also partly reduced by the addition of exogenous phosphatidylserine, consistent with the idea that phosphatidylethanolamine is biosynthesized via decarboxylation of phosphatidylserine. However, incorporation of 32Pi into phosphatidylcholine, sphingomyelin, and phosphatidylinositol was not significantly affected. In contrast, the addition of either phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, or phosphatidylinositol to the medium did not inhibit endogenous biosynthesis of the corresponding phospholipid. Radiochemical and chemical analyses of the cellular phospholipid composition revealed that phosphatidylserine in cells grown with 80 microM phosphatidylserine was almost entirely derived from the added phospholipid. Phosphatidylserine uptake was also directly determined by using [3H]serine-labeled phospholipid. Pulse and pulse-chase experiments with L-[U-14C] serine showed that when cells were cultured with 80 microM phosphatidylserine, the rate of synthesis of phosphatidylserine was reduced 3-5-fold whereas the turnover of newly synthesized phosphatidylserine was normal. Enzyme assaying of extracts prepared from cells grown with and without phosphatidylserine indicated that the inhibition of de novo phosphatidylserine biosynthesis by the added phosphatidylserine appeared not to be caused by a reduction in the level of the enzyme involved in the base-exchange reaction between phospholipids and serine. These results demonstrate that exogenous phosphatidylserine can be efficiently incorporated into Chinese hamster ovary cells and utilized for membrane biogenesis, endogenous phosphatidylserine biosynthesis thereby being suppressed.

Isolation of a somatic-cell mutant defective in phosphatidylserine biosynthesis.

Mutant clones of Chinese hamster ovary (CHO) cells defective in the base-exchange reaction of phospholipids with choline were isolated by using an in situ enzymatic assay for the reaction in cell colonies immobilized on polyester cloth. The specific activities of the choline-exchange reaction in extracts of one of the mutants (designated 64) grown at 33 degrees C and 40 degrees C were 13% and 6% of those in parental (CHO-K1) cells, respectively. The choline-exchange activity in the mutant was more thermolabile in cell extracts than that in the parent, suggesting that a mutation in the structural gene for the choline-exchange enzyme might have been induced in this mutant. In culture medium supplemented with lipoprotein-deficient serum, mutant 64 grew almost normally at 33 degrees C but divided only twice at 40 degrees C and then stopped growing. Labeling of intact cells with [32P]Pi showed that mutant 64 was also strikingly defective in the biosynthesis of phosphatidylserine at 40 degrees C but was normal at 33 degrees C. Most temperature-resistant revertants of mutant 64 exhibited nearly normal ability to synthesize phosphatidylserine at 40 degrees C and also showed choline-exchange activity similar to that in parental cells. The addition of phosphatidylserine to medium supplemented with newborn calf serum, in which mutant 64 grew more slowly than parental cells at 40 degrees C, restored the growth rate of the mutant to the parental level. Our findings suggest that the choline-exchange enzyme functions as the major route for the formation of phosphatidylserine and that the temperature-sensitive growth of mutant 64 is due to a defect in phosphatidylserine biosynthesis at 40 degrees C.

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.

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.

Effect of undecanoic acid on phospholipid metabolisminTrichophyton rubrum

At its minimum inhibitory concentration, undecanoic acid (UDA) inhibits biosynthesis of phosphatidyl serine, phosphatidyl ethanolamine and polyphosphoinositol but does not inhibit the synthesis of phosphatidyl glycerol, phosphatidyl choline, phosphatidyl inositol and phosphatidic acid in Trichophyton rubrum. At higher concentration, however UDA inhibits biosynthesis of all phosphatides present in this dermatophyte. UDA also affects catabolism of these phosphatides. This inhibitory effect of UDA may be partially responsible for its toxic action on T. rubrum.