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.     

The effect of beta-chloro D-alanine and L-cycloserine on the serine, phosphorus and palmitic acid uptake and metabolism of Tetrahymena lipids

The serine palmitoyltransferase inhibitors beta-chloro-D-alanine and L-cycloserine resulted in the uptake and metabolism of 3H-serine, 3H-palmitic acid and 32P significant alterations in the unicellular Tetrahymena pyriformis GL as compared to the untreated cells. In contrast with the higher eukariotic cells, by these treatments - except 5 mM L-cycloserine - the ceramide formation were not inhibited in Tetrahymena. L-cycloserine inhibited the conversion of phosphatidylserine (PS) to phosphatidyl-ethanolamine (PE) by decarboxylation, and the conversion of PE to phosphatidylcoline (PC) by methylation. The shorter L-cycloserine treatments caused lower, and the longer treatments higher label in glycerophospholipids. beta-chloro-D-alanine resulted in the glycerophospholids higher lipid precursor incorporation both in the shorter and longer treatments. Presumably beta-chloro-D-alanine treatments inhibit the transaminase activity, and the higher concentration (5 and 10 mM) proved to be toxic for Tetrahymena. We found differences between the metabolism of serine and palmitic acid labeled lipids in the beta-chloro-D-alanine and L-cycloserine treated groups. This phenomenon is probably due to a difference in the uptake of phospholipid head group component serine and hydrophobic tail precursor palmitic acid: the incorporation of palmitic acid in Tetrahymena is extremely quick, on the other hand, the uptake of serine is slower, a clear time dependence was measured.     

Phospholipid substrate-specificity of the L-serine base-exchange enzyme in rat liver microsomal fraction

The specificity of the L-serine base-exchange enzyme towards the fatty acid composition of the phospholipid substrate was investigated with a rat liver microsomal fraction. The relative rates of L-serine incorporation into saturated-hexaenoic, saturated-pentaenoic, saturated-tetraenoic, saturated-trienoic, dienoic-dienoic, monoenoic-dienoic, saturated-dienoic and saturated-monoenoic + saturated-saturated phosphatidylserine molecular species were 42, 5, 23, 4, 5, 4, 5 and 11% respectively. This is similar to, but not identical with, the relative mass abundance of these molecular species in total liver cell phosphatidylserines. The results indicate that the substrate-specificity of the L-serine base-exchange enzyme can at least in part explain the observed fatty acid composition of rat liver phosphatidylserines.     

Composition and metabolism of phospholipids of human leukocytes

Human mononuclear (MN) and polymorphonuclear (PMN) leukocytes were analyzed for their phospholipid, triglyceride, cholesterol and fatty acid content. The phospholipid/cholesterol ratio was 1.24 for both cels. MN cells contain more phosphatidylcholine (PC), but less phosphatidylserine (PS), phosphatidylethanolamine (PE) and sphingomyelin (SPH) than PMN cells when expressed as percent of total phospholipid. When expressed on the basis of lipid content per cell, MN cells contain less PS, PE and SPH but more triglyceride than PMN cells. PMN cells incorporate palmitic, stearic, linoleic and linolenic acids into their phospholipids, triglycerides or cholesterol esters. The incorporation into triglycerides was highest for all fatty acids. Of the phospholipids, the incorporation was highest into PC. Labeled fatty acids also were found in proteins which had been delipidized by exhaustive extraction with organic solvents. These represent tightly or covalently bound fatty acids. The incorporation of [³H]palmitic acid into this protein fraction is stimulated by insulin.     

Dependence of the fatty acid composition of the lipids in fungi of the genus Penicillium on their natural habitat conditions

The fatty acid composition of lipids was studied in Penicillium fungi growing in different habitats. Saturated fatty acids were represented by lauric, margaric, stearic and palmitic acids (the latter prevailed-- 18%-26%). Unsaturated monoene fatty acids were represented by acids from C16:1 to C18:1, diene and triene fatty acids by linoleic and linolenic acids. The predominance of linoleic acid was not found in all cultures of the genus. Changes in the fatty acid composition of lipids may be attributed to different ecological habitats of the Penicillium genus species.     

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.     

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.     

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.     

Trans fatty acids in partially hydrogenated soybean oil inhibit prostacyclin release by endothelial cells in presence of high level of linoleic acid

Partially hydrogenated soybean oil (PHSBO) and natural soybean oil (SBO) were obtained from a commercial source and their fatty acids were fractionated into saturates, monoenes and diene fractions. The present study compared the effect of the total, monoene and diene fatty acid fractions of PHSBO with those of the SBO on the fatty acid composition of the cell phospholipids (PL) and the prostacyclin (PGI(2)) release by endothelial cells (EC) in culture. Results showed that arachidonic acid (AA) level decreased significantly and linoleic acid (LA) significantly increased in the cells incubated with the diene fraction or the monoene fraction of PHSBO plus 18:2 at 3:1 ratio compared to the cell incubated with those fractions of SBO. These changes were attributed to the inhibition of LA conversion to AA by trans 18:1 and 18:2 isomers present in the monoene or diene fractions of PHSBO leading to a significant decrease of PGI(2) released by the cells incubated with monoene or diene fractions of PHSBO. The cells incubated with the monoene of PHSBO or SBO plus 18:2 at a 1:1 ratio showed no inhibition of LA conversion to AA and the level of AA was almost equal in their PL, but the PGI(2) released by the cells incubated with the monoene of PHSBO was significantly less than the cells incubated with the monoene of SBO. This decrease was not related to the inhibition of PGI(2) synthesizing enzymes or phospholipase (PLA(2)) activities. Our data show that trans acids in PHSBO inhibited the PGI(2) release by the cells through controlling the level of AA as substrate, either by (a) inhibiting the conversion of LA to AA or (b) by shunting the free AA released by the PLA(2) action to metabolism by another pathway leaving less AA available for PGI(2) synthesis.     

The base-exchange enzyme activities of sarcolemma and sarcoplasmic reticulum from rat heart

The Ca2+ dependent incorporation of [14C]ethanolamine, L-[14C]serine and [14C]choline into phosphatidylethanolamine, phosphatidylserine and phosphatidylcholine, respectively, were investigated in membrane preparations from rat heart. The ethanolamine and serine base-exchange enzyme-catalyzed reactions were associated with the sarcolemma and sarcoplasmic reticulum. There was a 17.2-fold and 6.8-fold enrichment, respectively, of the serine and the ethanolamine base-exchange enzyme activities in the sarcolemma compared to the starting whole homogenate. The sarcoplasmic reticulum was enriched in the ethanolamine and serine base-exchange enzyme activities. The choline base-exchange enzyme activity of all membranes fractions was negligible compared to the ethanolamine or serine base-exchange enzyme activities. The apparent Km for the ethanolamine and serine base-exchange enzyme in sarcolemma was 14 microM and 25 microM, respectively. The pH optimum for these base-exchange activities was 7.5-8.0. There was a dependence upon Ca2+ for these reactions with a 1 or 4 mM concentration required for maximal activity. The properties of the sarcoplasmic reticulum base-exchange enzymes were similar to the sarcolemmal base-exchange enzymes.     

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.     

Ethanol specifically alters the synthesis, acylation and transbilayer movement of aminophospholipids in rat-liver microsomes

By experimenting with the aminoalcohols [3-3H]serine and [2-14C]ethanolamine we have been able to relate the effects of ethanol upon the biosynthesis of radioactive aminophospholipids (APL) in rat-liver microsomes and their distribution within the bilayer. The translocation of newly synthesized molecules of aminophospholipids labeled with different fatty acids was also investigated. The synthesis of phosphatidylserine (PS) and phosphatidylethanolamine (PE) by base-exchange reaction (BES) was inhibited in membranes exposed to ethanol in direct response to its concentration. In addition, 100 mM ethanol specifically inhibited the transport of newly synthesized PS to the inner leaflet, resulting in similar levels of PS in both leaflets of the bilayer. The inhibition of PE synthesis by ethanol caused a decrease in its distribution in both inner and outer leaflets. An in vitro study of the incorporation of radioactive palmitate and oleate into the PS and PE of microsomes incubated with ethanol showed a decrease in the radioactivity levels of PE, suggesting that ethanol was specifically inhibiting the corresponding acyltransferase. It specifically altered the transbilayer movement of newly acylated phospholipids, modifying the distribution of palmitoyl- and oleoyl-acylated PS and PE in both leaflets. These results demonstrate for the first time that ethanol interferes with both the synthesis and intramembrane transport of aminophospholipids in endoplasmic reticulum (ER) membranes. Bearing in mind that if a membrane is to function properly its structure must be in optimum condition; it is evident that the observed processes may be responsible to some degree for the pathophysiological effects of alcohol upon cells.     

Early alterations induced by carbon tetrachloride in the lipids of the membranes of the endoplasmic reticulum of the liver cell II. Distribution of the alterations in the various lipid fractions

The distribution of carbon tetrachloride-induced alterations of membrane lipids in various fractions of liver microsomal lipids was studied. The chromatographic spot (referred to as the “D” spot in the previous paper [1]) which has been shown to contain the compounds responsible for the diene conjugation absorption [1], was found in the fatty acid methyl esters prepared from the fraction containing phosphatidylethanolamine (PE) and also in those obtained from the fraction containing phosphatidylserine (PS) and phosphatidylinositol (PI). The absorption of conjugated dienes was very marked in PE and less intense in PS and PI. The fatty acid methyl esters prepared from the fraction containing phosphatidylcholine (PC) showed no presence of the “D” spot and minimal absorption of conjugated dienes.A decrease in arachidonic acid content was found in the fraction containing PE, while no change in content of this fatty acid was found in the fraction containing PC. Results similar to those observed for PC were also found for neutral lipids (NL).Analysis of the fatty acid methyl esters of the various lipid fractions by gas-liquid chromatography (GLC) with an electron capture detector (ECD) gave a qualitative index of the free radical attack by CCl₄ metabolites. Quantitative estimation was attained by study of the irreversible binding of ¹⁴C from ¹⁴CCl₄ to the various lipid fractions. It was found that the fraction containing PS had the highest specific activity, while the fraction containing PC had the lowest specific activity of all the phospholipids. Thin layer chromatography (TLC) of the fraction containing PS revealed that only 11% of the radioactivity was associated with the pure PS moiety, while the remainder was associated with uncharacterized lipids (probably oxidation products).The possible relevance of the alterations induced by carbon tetrachloride in the various phospholipid fractions of liver microsomes to functional changes is discussed.     

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.     

Electrical potential dissipation induced by free fatty acids in pea stem mitochondria.

Linolenic, linoleic, oleic, palmitic and stearic acids (FFA) collapse the electrical potential of pea stem mitochondria in the absence or in the presence of 0.5 mM Mg2+. Higher concentrations of this cation (5 mM) lower the rate of dissipation caused by linoleic, oleic and palmitic acids, while abolishing that induced by stearic acid. Carboxyatractyloside and ADP do not reverse the FFA-induced collapse both in the presence or absence of Mg2+. EDTA, EGTA or BHT do not influence the dissipation caused by FFA that, in addition, is not linked to lipid peroxidation evaluated as malondialdehyde or conjugated diene formation. Only linolenic acid sustains a peroxidation which, however, appears to be caused by its own oxidation catalysed by lipoxygenases rather than by membrane lipoperoxidation induced by this free fatty acid. These results suggest that neither the ATP/ADP exchanger nor lipid peroxidation appear to be involved in FFA-induced uncoupling in pea stem mitochondria.     

Changes in phospholipids of Microsporum species in the presence of ethanol

Ethanol supplementation to the growth medium of Microsporum gypseum and Microsporum cookie resulted in changes in phospholipid composition and degree of unsaturation of their fatty acids without affecting the growth rate of the organism. Phosphatidylethanolamine increased with a simultaneous decrease in phosphatidylserine. Unsaturated fatty acids of total phospholipids decreased significantly which was primarily due to the decreased levels of linoleic acid.Abbreviations CL Cardiolipin - LPC Lysophosphatidylcholine - PC Phosphatidylcholine - PE Phosphatidylethanolamine - PI Phosphatidylinositol - PS Phosphatidylserine - UK Unknown phospholipids     

Properties of particulate and solubilized phosphatidylserine synthase activity from Saccharomyces cerevisiae. Inhibitory effect of choline in the growth medium

When radiolabeled serine is incubated with a particulate fraction from Saccharomyces cerevisiae, radioactivity is incorporated initially into phosphatidylserine and gradually appears in phosphatidylethanolamine. Because decarboxylation of phosphatidylserine is blocked by hydroxylamine, phosphatidylserine synthase can be assayed separately. The yeast phosphatidylserine synthase activity 1) exhibits a divalent cation requirement; 2) is stimulated by exogenous CDP-diolein (apparent Km = 0.17 mM); 3) has an apparent Km = 4 mM for L-serine; 4) has a neutral pH optimum; 5) is inhibited by p-hydroxymercuribenzoate; and 6) is reversible in the presence of 5'-CMP, but not 2'-CMP, 3'-CMP, or 5'-AMP. The phospholipid-synthesizing activity is solubilized with Triton X-100 and the enzymatic parameters have been compared with the particulate form of the enzyme. Detergent extracts catalyze the conversion of exogenous purified [31P]CDP-diglyceride to [32P]phosphatidylserine in the presence of Mn2+ and L-serine. Enzyme preparations from cells grown in the presence of choline, that have reduced phospholipid methylation activity (Waechter, C. J., Steiner, M. R., and Lester, R. L. (1969) J. Biol. Chem. 244, 3419-3422), also have substantially less phosphatidylserine synthase activity compared to identical preparations grown in the absence of choline. When choline, phosphocholine, CDP-choline, and phosphatidylcholine are present in vitro, there is no direct inhibitory effect on phosphatidylserine synthase activity. While the inclusion of choline in the growth medium caused a significant reduction in phosphatidylserine synthase activity, it did not appreciably effect the apparent Km values for L-serine and CDP-diglyceride. These results are consistent with choline-grown cells containing less phosphatidylserine synthase activity because of lower amounts of enzyme present or perhaps less active enzyme due to covalent modification.     

Aminophospholipid molecular species asymmetry in the human erythrocyte plasma membrane

The transbilayer distribution of the molecular species of aminophospholipids in human red blood cell plasma membrane has been investigated using a covalent labelling technique. Separation and quantitative analysis of the molecular species of phosphatidylethanolamine (PE) and phosphatidylserine (PS) was performed using high-performance liquid chromatography with UV detection of the trinitrophenyl derivatives obtained after reaction with trinitrobenzenesulfonic acid (TNBS). When the molecular species distribution obtained with intact cells was compared to that of the whole membrane, a molecular species asymmetry was evident. This phenomenon was most clearly evident when the reaction was performed at low temperatures (0 degrees C) and was obscured by the excessive labelling or probe permeation associated with higher temperatures or longer incubation times. The monoene species were enriched in the outer leaflet, they comprised about 30% of the PE species in this leaflet. The polyunsaturates were preferentially localized in the inner leaflet and this was true of the arachidonyl species in particular as they represented up to 35% of this pool. The w-3 polyunsaturated fatty acids displayed a preferential localization in the plasmalogen subclass in comparison to the diacyl fraction, i.e., they comprised about 58 of the former and 42% of the latter subclass of cellular PE w-3 species. Data concerning the separation, identification and quantification of PS molecular species in human erythrocytes is also presented. The internal localization of the polyunsaturated species as well as the compartmentalization of the w-3 and w-6 pools will have metabolic, structural and physical implications for membrane function.     

Kinetics of long-chain (sphingoid) base biosynthesis in intact LM cells: effects of varying the extracellular concentrations of serine and fatty acid precursors of this pathway

Serine palmitoyltransferase (EC 2.3.1.50) catalyzes the condensation of L-serine and palmitoyl-CoA to yield 3-ketosphinganine in the first unique reaction of long-chain (sphingoid) base biosynthesis. The kinetic effects of changing the extracellular concentrations of the precursors for this pathway were studied with LM cells by following the incorporation of L-[3-14C]serine into the long-chain base (i.e., sphinganine and sphingenine) backbones of complex sphingolipids. [14C]Serine was taken up by the cells and rapidly reached steady-state concentrations similar to those of the medium. From the cellular [14C]serine concentrations and specific activities, the apparent Vmax [14 pmol min-1 (10(6) cells)-1] and Km (0.23 mM) values for long-chain base synthesis were determined and found to be essentially identical with those for serine palmitoyltransferase assayed in vitro [i.e., 13 pmol min-1 (10(6) cells)-1 and 0.27 mM, respectively]. The other precursor, palmitic acid, was also taken up rapidly and increased long-chain base biosynthesis in a concentration-dependent manner. This effect was limited to palmitic acid and matched the known specificity of serine palmitoyltransferase for saturated fatty acyl-CoA's of 16 +/- 1 carbon atoms. These studies delineate the influence of extracellular precursors on the formation of the sphingolipid backbone and suggest that the kinetic properties of serine palmitoyltransferase govern this behavior of long-chain base synthesis in intact cells.     

Regulation of liver base-exchange activity by acidic phospholipids

Liver microsomes were enriched in liposomal acidic lipids by Ca²⁺-dependent fusion of liposomes at pH 7.0. The extent of fusion was monitored by the transfer of radioactive cholesteryl oleate. The enrichment of membranes in phosphatidylserine inhibited ethanolamine base-exchange, whereas the fusion with phosphatidylinositol inhibited both ethanolamine and serine base-exchange reactions. In contrast, these two phospholipids had scarce effects on choline base-exchange. Phosphatidic acid did not suppress any of the three base-exchange activities. Possible functional implications are discussed.Abbreviations DTT dithiothreitol - HEPES 4-(2-hydroxyethyl)-1-piperazineethansulfonic acid - SHB suerose-HEPES buffer (0.25M sucrose, 3mM HEPES, pH 7.4)