The Role of Phosphatidylserine Decarboxylase in Brain Phospholipid Metabolism

In brain, phosphatidylethanolamine can be synthesized from free ethanolamine either by a pathway involving the formation of CDP-ethanolamine and its transfer to diglyceride, or by base-exchange of ethanolamine with existing phospholipids. Although de novo synthesis from serine has also been demonstrated, the metabolic pathway involved is not known. The enzyme phosphatidylserine decarboxylase appears to be involved in the synthesis of much of the phosphatidylethanolamine in liver, but the significance of this route in brain has been challenged. Our in vitro studies demonstrate the existence of phosphatidylserine decarboxylase activity in rat brain and characterize some of its properties. This enzyme is localized in the mitochondrial fraction, whereas the enzymes involved in base-exchange and the cytidine pathway are localized to microsomal membranes. Parallel in vivo studies showed that after the intracranial injection of L-[G-3H]serine, the specific activity of phosphatidylserine was greater in the microsomal fractions than in the mitochondrial fraction, whereas the opposite was true for phosphatidylethanolamine. When L-[U-14C]serine and [1-3H]ethanolamine were simultaneously injected, the 14C/3H ratio in mitochondrial phosphatidylethanolamine was 10 times that in microsomal phosphatidylethanolamine. The results demonstrate that serine is incorporated into the base moiety of phosphatidylethanolamine primarily through the decarboxylation of phosphatidylserine in brain mitochondria. A minimal value of 7% for the contribution of phosphatidylserine decarboxylase to whole-brain phosphatidylethanolamine synthesis can be estimated from the in vivo data.     

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.     

Biosynthesis of phosphatidylethanolamine via the CDP-ethanolamine route is an important pathway in isolated rat hepatocytes

In the present study pulse-label and pulse-chase experiments with isolated rat hepatocytes in suspension were designed to investigate the effects of the presence of either serine or ethanolamine in the medium on the rate of phosphatidylethanolamine synthesis via the CDPethanolamine pathway and by decarboxylation of phosphatidylserine. Addition of serine to the medium did not affect the incorporation of [1,2-14C]ethanolamine into phosphatidylethanolamine. Pulse-label experiments showed that the incorporation of [3H]serine into phosphatidylserine decreased in the presence of ethanolamine with a corresponding decrease of the incorporation of label into the ethanolamine base moiety of phosphatidylethanolamine. However, the radioactivity in the diacylglycerol part of phosphatidylethanolamine was considerably higher in the presence of ethanolamine than in its absence. Pulse-chase experiments with labelled serine demonstrated that the conversion of phosphatidylserine to phosphatidylethanolamine was not affected by varying concentrations of ethanolamine. Our observations indicate that in the presence of ethanolamine the biosynthesis of phosphatidylethanolamine via the CDPethanolamine pathway is enhanced relative to the synthesis by decarboxylation of phosphatidylserine.     

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.     

In vivo incorporation of l-[14C]serine into phospholipids and proteins of the subcellular fractions of developing rat brain

A study was conducted on the in vivo incorporation of l -[¹⁴C]-serine into the lipids and proteins of the various subcellular fractions of the developing rat brain before and during the stage of active myelination. The total radioactivity in the various fractions at 12 days of age was higher than that at 3 days, while the radioactive specific activity was reversed. The specific activities of the proteins and lipids were higher at 3 days of age with the exception of the subcellular fraction containing myelin. At both ages the lipids of the various cellular fractions had similar specific activities, a finding that suggests a common source for lipid biosynthesis. Incorporation of radioactivity into the various phospholipids was in the following order: phosphatidyl serine > phosphatidyl ethanolamine > phosphatidal serine > sphingomyelin and phosphatidyl choline. Of all the phospholipids, the plasmalogens increased most in total radioactivity during the period when meylination was most active. Serine-containing phospholipids appear to be most tightly bound to proteins. The brain mitochrondrial fraction contained most of the phosphatidyl serine decarboxylase activity with some activity in the nuclei. Biosynthesis of phosphatdyil ethanolamine through decarboxylation of phosphatidyl serine could take place in rat brain. Four unidentified radioactive metabolites were found in the acid-soluble fraction in addition to l -[¹⁴C]serine.     

Incorporation of [1-14C]Palmitic Acid into Neutral Lipids and Phospholipids of Rat Cerebral Cortex In Vitro

Abstract: Incorporation of [1-¹⁴C]palmitic acid into neutral lipids and phospholipids of rat cerebral cortex was examined in vitro in normal Krebs-Ringer bicarbonate buffer containing 3% (wthol) albumin and 0.75 mM palmitic acid. Under standard assay conditions, radioactivity in the triacylglycerol fraction increased rapidly during the first 30 min, and then decreased after 60 min, with corresponding increase in radioactivity in phosphatidyl choline, phosphatidyl ethanolamine, and a fraction of phosphatidyl inositol plus phosphatidyl serine. Diacylglycerol was shown to be an intermediate metabolite. Radioactivity increased in triacylglycerol, and decreased in phosphatidyl choline and phosphatidyl ethanolamine throughout incubation under NZ gas. In the fraction of phosphatidyl inositol plus phosphatidyl serine, radioactivity decreased after 30 min during incubation under N, gas. A possible acylation-deacylation cycle, in which triacylglycerol could be a source of free fatty acids for phospholipids, is discussed.     

Reconstitution of phosphatidylserine import into rat liver mitochondria

The synthesis translocation and decarboxylation of phosphatidylserine occurs in a cell-free system. The principal membrane components necessary are microsomes (source of phosphatidylserine synthase) and mitochondria (source of phosphatidylserine decarboxylase). The interorganelle translocation of phosphatidylserine can be measured by quantitating the decarboxylation of phosphatidyl[1'-14C]serine initially present in prelabeled microsomal membranes using a 14CO2 trapping assay. The decarboxylation of microsomal phosphatidylserine by intact mitochondria is 1) dependent upon substrate (microsomal membrane) concentration, 2) different from decarboxylation of liposomal phosphatidylserine, 3) resistant to proteases, 4) independent of soluble factors, and 5) unaffected by the addition of partially purified phospholipid exchange proteins but accelerated by purified nonspecific phospholipid exchange protein. The rate-limiting step in the reconstituted translocation-decarboxylation system is not the decarboxylation reaction but the initial translocation event between the microsomal membrane and the outer mitochondrial membrane. These data are interpreted to demonstrate that phosphatidylserine import into the mitochondria can occur via collision complexes formed between the endoplasmic reticulum or vesicles derived therefrom and the outer mitochondrial membrane.     

Serine regulates phosphatidylethanolamine biosynthesis in the hamster heart.

The role of serine as a precursor and metabolic regulator for phosphatidylethanolamine biosynthesis in the hamster heart was investigated. Hearts were perfused with 50 microM [1-3H]ethanolamine in the presence or absence of serine for up to 60 min. Ethanolamine uptake was attenuated by 0.05-10 mM serine in a noncompetitive manner, and the incorporation of labeled ethanolamine into phosphatidylethanolamine was also inhibited by serine. Analysis of the ethanolamine-containing metabolites in the CDP-ethanolamine pathway revealed that the conversion of ethanolamine to phosphoethanolamine was reduced. The reduction was a result of an inhibition of ethanolamine kinase activity by an elevated pool of intracellular serine. Perfusion of the heart with 1 mM serine caused a 5-fold increase in intracellular serine pool. In order to examine the action of serine on other phosphatidylethanolamine metabolic pathways, hearts were perfused with [1-3H]glycerol in the presence and absence of serine. Serine did not cause any enhancement of phosphatidylethanolamine hydrolysis. The base-exchange reaction for phosphatidylserine formation or the decarboxylation of phosphatidylserine was not affected by serine perfusion. We conclude that circulating serine plays an important role in the modulation of phosphatidylethanolamine biosynthesis via the CDP-ethanolamine pathway in the hamster heart but does not affect the contribution of the decarboxylase pathway for phosphatidylethanolamine formation.     

Cytolysis of nucleated cells by complement: inhibition of membrane-transmethylation enhances cell death by C5b-9

Inhibition of transmethylation, i.e., enzymatic transfer of methyl groups to phosphatidyl ethanolamine resulting in generation and translocation of phosphatidyl choline, enhances the killing of nucleated cells by complement. Furthermore, under complement attack, transmethylation measured as incorporation of [3H]methyl groups into phosphatidyl choline is enhanced, suggesting that transmethylation functions as a membrane defense mechanism either by increasing the phosphatidyl choline synthesis or by transducing a signal that might trigger another membrane repair process.     

Ethanolamine Glycerophospholipid Formation by Decarboxylation of Serine Glycerophospholipids in Myelinating Organ Cultures of Cerebellum

Abstract: Serine decarboxylation as a source of glycer-ophospholipid ethanolamine is known to occur in mammals. However, early investigators failed to demonstrate the pathway in brain. In the present study serine is shown to be decarboxylated to glycerophospholipid ethanolamine in myelinating organ cultures of rat cerebellum up to 32 days in vitro. The pattern of incorporation of l -[3-¹⁴C]serine into culture phospholipids strongly suggests a precursor-product relationship between serine glycero-phospholipids (SGP) and ethanolamine glycerophospho-lipids (EGP), with serine label appearing in the ethanolamine moiety of EGP. The time course of labelling was similar for both acid-stable and acid-labile EGP In contrast DL-[l-¹⁴C]serine failed to label EGP significantly due to the loss of serine carbon C₁ on decarboxylation. Through the systematic hydrolysis of phospholipids from cerebellar cultures incubated with l -[3-¹⁴C], it was clear that in SGP, acid-stable EGP, and acid-labile EGP >70% of radiolabel resides in the base moiety of each of these molecular species. It is proposed that serine decarboxylation as a source of EGP ethanolamine may be important in the early stages of brain development.     

Differential Utilization of the Ethanolamine Moiety of Phosphatidylethanolamine Derived from Serine and Ethanolamine during NGF-Induced Neuritogenesis of PC12 Cells

Neurite elongation involves the expansion of the plasma membrane and phospholipid synthesis. We investigated membrane phosphatidylethanolamine (PE) biosynthesis in PC12 cells during neurite outgrowth induced by nerve growth factor (NGF). When PE was prelabeled with [³H]ethanolamine and the radioactivity was chased by incubation with 1 mM unlabeled ethanolamine, the radioactivity of [³H]PE steadily declined and [³H]ethanolamine was released into the medium in NGF-treated cells during neurite outgrowth; in the absence of unlabeled ethanolamine, the radioactivity of [³H]PE remained relatively constant for at least 24 hr. In undifferentiated cells but not in NGF-treated cells, [³H]phosphoethanolamine accumulated in significant amounts during pulse labeling, and was converted partly to PE but largely released into the medium irrespective of incubation with unlabeled ethanolamine. The decline in the radioactivity of [³H]PE and release of [³H]ethanolamine following incubation with unlabeled ethanolamine were also observed in undifferentiated cells. Thus, the ethanolamine moiety of PE derived from ethanolamine is actively recycled in both differentiated and undifferentiated cells. When PE was derived from [³H]serine through phosphatidylserine (PS) decarboxylation, the decrease in radioactivity of [³H]PE and release of [³H]ethanolamine into the medium following incubation with unlabeled ethanolamine were observed only in NGF-treated cells, but not in undifferentiated cells, indicating that the ethanolamine moiety of PE derived from PS is actively recycled only in the cells undergoing NGF-induced neuritogenesis. Thus, in PC12 cells, the ethanolamine moiety of PE derived from PS is regulated differently from that of PE derived from ethanolamine.     

Adriamycin disrupts phosphatidylserine import into the mitochondria of permeabilized CHO-K1 cells

The action of adriamycin (an inhibitor of precursor protein import into mitochondria) upon phosphatidylserine (PtdSer) import into mitochondria was examined in permeabilized CHO-K1 cells. The decarboxylation of nascent PtdSer to phosphatidylethanolamine was used as an indicator reaction for the lipid translocation process. Adriamycin was without effect upon new PtdSer synthesis but blocked the time- and translocation-dependent decarboxylation of this lipid at the mitochondrial inner membrane of permeabilized cells. The effect of adriamycin was concentration-dependent with an IC50 of 150 microM and was not due to direct inhibition of PtdSer decarboxylase. To determine at which level of PtdSer transport adriamycin was working, the adriamycin-treated permeabilized cells were incubated with 1-acyl-2-[N-(6-[(7-nitrobenz-2-oxa-1,3-diazo-4-yl)] aminocaproyl)]phosphatidyl[1'-14C] serine (NBD-Ptd[1'-14C]Ser), and its decarboxylation was determined. Since the NBD-Ptd[1'-14C]Ser freely partitions into all cell membranes, it can partition into the outer mitochondrial membrane in an ATP-independent fashion. The NBD-Ptd[1'-14C]Ser was readily decarboxylated in an ATP-independent manner in permeabilized cells. Adriamycin inhibited the decarboxylation of NBD-Ptd[1'-14C]Ser, thereby indicating that it can act upon lipid transport processes between the outer and inner mitochondrial membrane.     

Effects of docosahexaenoic and arachidonic acids on the synthesis and distribution of aminophospholipids during neuronal differentiation of PC12 cells

We have shown previously that docosahexaenoic acid (DHA) promotes and arachidonic acid (AA) suppresses neurite outgrowth of PC12 cells induced by nerve growth factor (NGF) and that incorporation of [3H]ethanolamine into phosphatidylethanolamine (PE) is suppressed in PC12 cells by AA while DHA has no effect. In the present study, the effects of these fatty acids on PE synthesis via decarboxylation of phosphatidylserine (PS), another pathway of PE synthesis, and distribution of aminophospholipids were examined. Incorporation of [3H]serine into PS and PE was elevated in the course of NGF-induced differentiation and was further stimulated significantly by DHA, but not by AA. [3H]Ethanolamine uptake by PC12 cells was significantly suppressed by AA but not by DHA while these fatty acids did not affect [3H]serine uptake, indicating that the suppression by AA of [3H]ethanolamine incorporation into phosphatidylethanolamine is attributable, at least in part, to a reduction in [3H]ethanolamine uptake. The distribution of PE in the outer leaflet of plasma membrane decreased during differentiation, which is known to be accompanied by an increase in the surface area of plasma membrane. Supplementation of PC12 cells with DHA or AA did not affect the distribution of aminophospholipids. Thus, DHA and AA affected aminophospholipid synthesis and neurite outgrowth differently, but not the transport and distribution of aminophospholipids, while the PE concentration in the outer leaflet of the plasma membrane decreased in association with morphological changes in PC12 cells induced by NGF.     

Sequence of prolactin effects on phospholipid synthesis in mouse mammary gland explants

In cultured mouse mammary gland explants derived from 12-14 day pregnant mice, the effect of prolactin (PRL) on the rate of incorporation of several precursors into neutral lipids and phospholipids was determined. Employing [14C]-acetate as a substrate, PRL stimulates its incorporation into a) neutral lipids by 4-6 hours, b) phosphatidyl choline (PC) and phosphatidyl inositol-phosphatidyl serine (PI-PS) by 1-2 hours, and c) phosphatidyl ethanolamine (PE) by 2-4 hours. Using [3H]-glycerol as a substrate, the temporal response to PRL for its incorporation into the neutral lipids was the same as that for [14C]-acetate, however, PRL did not enhance the rate of [3H]-glycerol incorporation into the phospholipids at any time through 16 hours. PRL similarly had no effect on the rates of [3H]-choline, [3H]-serine, [3H]-ethanolamine, or [32P]O4 incorporation into the phospholipids at hormone exposure periods of 8 hours or more. And finally, PRL had no effect on the rates of [3H]-arachidonate or [14C]-linoleate incorporation into neutral lipids or phospholipids at culture periods up to 18 hours. These data suggest that the early effect of PRL on [14C]-acetate incorporation into the phospholipids is due to either the insertion of newly synthesized fatty acids and/or the extension of fatty acids contained in the phospholipids.     

Optically active aromatic amino acids. Part VI. Synthesis and properties of (Leu5)-enkephalin analogues containing O-methyl-L-tyrosine1 with ring substitution at position 3'.

Twelve new [Tyr(Me)1, Leu5]-enkephalin analogues with substituents at position 3' of the Tyr ring have been synthesized using traditional solution methods. The substituents were -CO2H, -CONH2, -CO2Me, -(E)-CH=NOH, -(E)-CH=NOMe and CH2OH. The analogues were C-terminated with methyl esters, amides or as free acids. In the in vitro biological assays a remarkable agonist activity to the opiate receptor mu in guinea pig ileum (GPI) relative to Leu-ENK was shown by the following: Leu-ENK, 100; [Tyr(Me)(3'-CO2Me)1, Leu-OMe5]-ENK (I), 8.1; [Tyr(Me)(3'-(E)-CH=NOH)1, Leu-OMe5]-ENK (VI), 26.2; [Tyr(Me)(3'-(E)-CH=NOH)1, Leu-OH5]-ENK (VII), 2.9; [Tyr(Me)(3'-(E)-CH=NOH)1, Leu-NH2(5)]-ENK (VIII), 4.7; and [Tyr(Me)(3'-CH2OH)1, Leu-OMe5]-ENK (X), 5.6. The agonist effect was naltrexone- or naloxone-reversible. The masking of the hydroxyl group in (E)-hydroxyiminomethyl group of analogue (VI) by O-methylation has totally abolished its GPI agonist activity. It seems that the (E)-CH=NOH group shows affinity and plays an analogous role to the phenol group Tyr1 in leucine-enkephalin and in the tyramine group of the opiate alkaloids. The analogues: [Tyr(Me)(3'-CO2Me)1, Leu-OMe5]-ENK (I), [Tyr(Me)(3'-CO2H)1, Leu-OMe5]-ENK (II), [Tyr(Me)(3'-CO2Me)1, Leu-NH2(5)]-ENK (III), [Tyr(Me)(3'-CO2H)1, Leu-NH2(5)]-ENK (IV), [Tyr(Me)(3'-CONH2)1, Leu-NH2(5)]-ENK (V), [Tyr(Me)(3'-(E)-CH=NOH)1, Leu-OMe5]-ENK (VI), [Tyr(Me)(3'-(E)-CH=NOH)1, Leu-OH5]-ENK (VII), [Tyr(Me)(3'-(E)-CH=NOH)1, Leu-NH2(5)]-ENK (VIII), [Tyr(Me)(3'-(E)-CH=NOMe)1, Leu-OMe5]-ENK (IX), [Tyr(Me)(3'-CH2OH)1, Leu-OMe5]-ENK (X), [Tyr(Me)(3'-CH2OH)1, Leu-OH5]-ENK (XI) and [Tyr(Me)(3'-CH2OH)1, Leu-NH2(5)]-ENK (XII) under testing had no significant agonist activity to the enkephalinergic receptor in mouse vas deferens (MVD). All methyl esters of synthesized analogues of [Leu5]-ENK showed higher activity to mu receptors than structurally identical C-terminal amides. It is a surprising result since usually C-terminate amides are stronger agonists than C-terminate esters.     

Contribution of different pathways to the supply of phosphatidylethanolamine and phosphatidylcholine to mitochondrial membranes of the yeast Saccharomyces cerevisiae

In the yeast, three biosynthetic pathways lead to the formation of phosphatidylethanolamine (PtdEtn): (i) decarboxylation of phosphatidylserine (PtdSer) by phosphatidylserine decarboxylase 1 (Psd1p) in mitochondria; (ii) decarboxylation of PtdSer by Psd2p in a Golgi/vacuolar compartment; and (iii) the CDP-ethanolamine (CDP-Etn) branch of the Kennedy pathway. The major phospholipid of the yeast, phosphatidylcholine (PtdCho), is formed either by methylation of PtdEtn or via the CDP-choline branch of the Kennedy pathway. To study the contribution of these pathways to the supply of PtdEtn and PtdCho to mitochondrial membranes, labeling experiments in vivo with [(3)H]serine and [(14)C]ethanolamine, or with [(3)H]serine and [(14)C]choline, respectively, and subsequent cell fractionation were performed with psd1Delta and psd2Delta mutants. As shown by comparison of the labeling patterns of the different strains, the major source of cellular and mitochondrial PtdEtn is Psd1p. PtdEtn formed by Psd2p or the CDP-Etn pathway, however, can be imported into mitochondria, although with moderate efficiency. In contrast to mitochondria, microsomal PtdEtn is mainly derived from the CDP-Etn pathway. PtdEtn formed by Psd2p is the preferred substrate for PtdCho synthesis. PtdCho derived from the different pathways appears to be supplied to subcellular membranes from a single PtdCho pool. Thus, the different pathways of PtdEtn biosynthesis play different roles in the assembly of PtdEtn into cellular membranes.     

Serine and ethanolamine incorporation into different plasmalogen pools: Subcellular analyses of phosphoglyceride synthesis in cultured glioma cells

In cultured glioma cells, plasma membrane (PM) is enriched in phosphatidylserine (PtdSer) and plasmalogens (1-O-alk-1-enyl-2-acyl-sn-glycero-3-phosphoethanolamine). Serine can be a precursor of headgroups of both ptdSer and ethanolamine phosphoglycerides (PE) including plasmalogens and non-plasmalogen PE (NP-PE). Synthesis of phospholipids was investigated at the subcellular level using established fractionation procedures and incorporation of [³H(G)]L-serine and [1,2-¹⁴C]ethanolamine. Specific radioactivity of PtdSer from [³H]serine was 2-fold greater in PM than in microsomes, reaching maximum by 2–4 h. Labeled plasmalogen from [³H]serine appeared in PM by 4 h and increased to 48 h, whereas almost no plasmalogen accumulated in microsomes within 12 h. In contrast, labeled plasmalogen from [1,2-¹⁴C]ethanolamine appeared in both PM and microsomes at early incubation times and became enriched in PM beyond 12 h. Thus, in glioma cells: (1) greater and faster accumulation of labeled PtdSer in PM may reflect direct synthesis from serine within PM; (2) PM is a major source of PtdSer for decarboxylation and PE synthesis; (3) NP-PE in both PM and microsome provides headgroup for synthesis of plasmalogen; and, (4) plasmalogen synthesis may involve different intracellular pools depending on headgroup origin.Abbreviations NP-PE nonplasmenylethanolamine phosphoglycerides including both diacyl and alkylacyl species - PE total ethanolamine phosphoglycerides: plasmalogen-plasmenylethanolamine or alkenylacyl ethanolamine phosphoglyceride (1-O-alk-1-enyl-2-acyl-sn-glycero-3-phosphoethanolamine) - PL phospholipid - PM plasma membrane - PtdCho phosphatidylcholine - PtdSer phosphatidylserine     

An anion-exchange radioassay for glucose 6-phosphate phosphatase: use in topological studies with endoplasmic reticulum vesicles.

A simple procedure is presented for the enzymatic preparation of [2-3H]mannose 6-phosphate (Man 6-P) with purified yeast hexokinase and unlabeled ATP. The enzymatically synthesized [2-3H]Man 6-P is utilized as the radiolabeled substrate in a new rapid assay for glucose 6-phosphate (Glc 6-P) phosphatase. The principle of the assay procedure is that the unreacted substrate, [2-3H]Man 6-P, is retained by the anion-exchange resin, AG 1-X8 (acetate), while the enzymatic product, [2-3H]-mannose, is eluted directly into a scintillation counting vial. When Glc 6-P phosphatase activity associated with mouse liver endoplasmic reticulum (ER) vesicles is assayed by the new chromatographic assay, the same characteristic latency and properties are observed, as determined by the commonly used colorimetric assay of inorganic phosphate produced. The anion-exchange radioassay described should be useful for a variety of topological studies on enzymes associated with membrane vesicles derived from liver and kidney ER.     

Total polar lipid biosynthesis during growth of Crotalaria juncea pollen tubes

[1-¹⁴C]-Acetate incorporation into total and polar lipids was studied in the growing pollen tubes of Crotalaria juncea. Ungerminated pollen had phosphatidyl inositol, phosphatidyl serine, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, monogalactosyl diglyceride, digalactosyl diglyceride, sulpholipid and steryl glycosides. In the growing pollen tubes considerable [1-¹⁴C]-acetate incorporation was observed into the individual polar lipids. The exogenous carbon source significantly influenced lipid biosynthesis. Boric acid (20mg/l.) promoted both pollen tube growth and acetate incorporation into phospholipids. In comparison to 5′-adenosine monophosphate, cyclic-3′,5′-adenosine monophosphate (cAMP) promoted tube growth and also enhanced phospho-and glycolipid biosynthesis. The regulation of membrane component biosynthesis by cAMP is suggested.     

Thin-layer chromatographic analysis of phospholipids in Echinostoma revolutum (Trematoda) adults.

Thin-layer chromatographic analyses were made of phospholipids extracted from adult Echinostoma revolutum (Trematoda) and from the nonnutrient medium in which worms were maintained. Identity of phospholipids was based on comparison with authentic lipid standards and on specific chemical detection tests. The most abundant phospholipids in the extract were phosphatidyl choline, phosphatidyl ethanolamine and phosphatidyl serine; lesser amounts of cerebrosides and lysophosphatidyl choline were detected also. Phospholipids detected in the incubation medium were essentially as described for extracts.