Fatty acid metabolism in sn-glycerol-3-phosphate acyltransferase (plsB) mutants.

Fatty acid metabolism was examined in Escherichia coli plsB mutants that were conditionally defective in sn-glycerol-3-phosphate acyltransferase activity. The fatty acids synthesized when acyl transfer to glycerol-3-phosphate was inhibited were preferentially transferred to phosphatidylglycerol. A comparison of the ratio of phospholipid species labeled with 32Pi and [3H]acetate in the presence and absence of glycerol-3-phosphate indicated that [3H]acetate incorporation into phosphatidylglycerol was due to fatty acid turnover. A significant contraction of the acetyl coenzyme A pool after glycerol-3-phosphate starvation of the plsB mutant precluded the quantitative assessment of the rate of phosphatidylglycerol fatty acid labeling. Fatty acid chain length in membrane phospholipids increased as the concentration of the glycerol-3-phosphate growth supplement decreased, and after the abrupt cessation of phospholipid biosynthesis abnormally long chain fatty acids were excreted into the growth medium. These data suggest that the acyl moieties of phosphatidylglycerol are metabolically active, and that competition between fatty acid elongation and acyl transfer is an important determinant of the acyl chain length in membrane phospholipids.     

Fatty acid synthesis in Escherichia coli is indirectly inhibited by phenethyl alcohol.

Experiments were performed to determine how phenethyl alcohol inhibits phospholipid synthesis in E. coli. At a nonbacteriostatic concentration, the drug reduces the rate of de novo fatty acid and phospholipid synthesis by 60 to 70%. The inhibition of fatty acid synthesis was found to be a secondary consequence of the inhibition of phospholipid synthesis. Phenethyl alcohol reduces the rate of incorporation of exogenous fatty acids into the phospholipids of a fatty acid auxotroph by 60%. These results indicate that this drug controls phospholipid synthesis beyond the level of fatty acid synthesis. Phenethyl alcohol inhibits the synthesis of phospholipids containing saturated fatty acids to a greater extent than it does the synthesis of phospholipids containing unsaturated fatty acids. It controls the synthesis of phospholipids containing saturated fatty acids at both the level of fatty acid synthesis and the level of incorporation of the saturated fatty acids into phospholipids. The synthesis of phospholipids containing unsaturated fatty acids is inhibited at the level of incorporation of the fatty acids into phospholipids.     

Glycerol 3-phosphate analogues as metabolic inhibitors in Escherichia coli, 3-hydroxy-4-oxobutyl-1-phosphonate, a drug that interferes with normal phosphoglyceride metabolism.

3-Hydroxy-4-oxobutyl-1-phosphonate, the phoshonic acid analogue of glyceraldehyde 3-phosphate, enters Escherichia coli via the glycerol 3-phosphate transport system. There is no differential effect upon the accumulation of deoxyribonucleic acid, ribonucleic acid, or phosphoglycerides, although the accumulation of proteins was less effected. Examination of the phospholipids revealed that phosphatidylglycerol accumulation was most severely inhibited and cardiolipin accumulation was least affected. Concentrations of glyceraldehyde 3-phosphate and its phosphonic acid analogue that markedly inhibit macromolecular and phosphoglyceride biosynthesis have no effect upon the intracellular nucleoside triphosphate pool size. The phosphonate is a competitive inhibitor of sn-glycerol 3-phosphate in reactions catalyzed by acyl coenzyme A:sn-glycerol-3-phosphate acyltransferase and CDP-diacylglycerol:sn-glycerol-3-phosphate phosphatidyltransferase. A Km mutant for the former enzyme was susceptible to the phosphansferase activity. Studies with mutant strains ruled out the aerobic glycerol-3-phosphate dehydrogenase, glycerol-3-phosphate synthase, and fructose-1,6-biphosphate aldolase as the primary sites of action.     

Partial purification of glycerophosphate acyltransferase from Escherichia coli.

Glycerophosphate acyltransferase, a membrane-bound enzyme catalyzing the initial step of phospholipid biosynthesis in Escherichia coli, has been extracted with Triton X-100, a nonionic detergent, and purified 20- to 40-fold. This preparation is free from lysophosphatidate acyltransferase. Glycerophosphate acyltransferase is inactive in detergent extracts, but can be reconstituted by the addition of phospholipid. Under such conditions, the enzyme is associated with phospholipid. The sole product of the reaction with acyl coenzyme A as substrate is 1-acyl-sn-glycero-3-phosphate. Furthermore, the enzyme shows a marked preference for saturated fatty acyl conenzyme A, implying that this enzyme is responsible for the predominance of saturated moieties in position 1 of E. coli phospholipids. Acyltransferase from two mutants, plsA and plsB, was partially purified and characterized. Results support the view that plsB is a structural gene for the acyltransferase, but suggest that the plsA gene product is not directly involved in phospholipid biosynthesis.     

Regulation of phospholipid synthesis in Escherichia coli by guanosine tetraphosphate

Phospholipid synthesis has been reported to be subject to stringent control in Escherichia coli. We present evidence that demonstrates a strict correlation between guanosine tetraphosphate accumulation and inhibition of phospholipid synthesis. In vivo experiments designed to examine the pattern of phospholipid labeling with (32)P-inorganic phosphate and (32)P-sn-glycerol-3-phosphate suggest that regulation must occur at the glycerol-3-phosphate acyltransferase step. Assay of phospholipid synthesis by cell-free extracts and semipurified preparations revealed that guanosine tetraphosphate inhibits at least two enzymes specific for the biosynthetic pathway, sn-glycerol-3-phosphate acyltransferase as well as sn-glycerol-3-phosphate phosphatidyl transferase. These findings provide a biochemical basis for the stringent control of lipid synthesis as well as regulation of steady-state levels of phospholipid in growing cells.     

Pathways for the incorporation of exogenous fatty acids into phosphatidylethanolamine in Escherichia coli

Two distinct pathways for the incorporation of exogenous fatty acids into phospholipids were identified in Escherichia coli. The predominant route originates with the activation of fatty acids by acyl-CoA synthetase followed by the distribution of the acyl moieties into all phospholipid classes via the sn-glycerol-3-phosphate acyltransferase reaction. This pathway was blocked in mutants (fadD) lacking acyl-CoA synthetase activity. In fadD strains, exogenous fatty acids were introduced exclusively into the 1-position of phosphatidylethanolamine. This secondary route is related to 1-position fatty acid turnover in phosphatidylethanolamine and proceeds via the acyl-acyl carrier protein synthetase/2-acylglycerophosphoethanolamine acyltransferase system. The turnover pathway exhibited a preference for saturated fatty acids, whereas the acyl-CoA synthetase-dependent pathway was less discriminating. Both pathways were inhibited in mutants (fadL) lacking the fatty acid permease, demonstrating that the fadL gene product translocates exogenous fatty acids to an intracellular pool accessible to both synthetases. These data demonstrate that acyl-CoA synthetase is not required for fatty acid transport in E. coli and that the metabolism of exogenous fatty acids is segregated from the metabolism of acyl-acyl carrier proteins derived from fatty acid biosynthesis.     

Altered acyltransferase activity in Escherichia coli associated with mutations in acyl coenzyme A synthetase

Growth of a temperature-sensitive general fatty acid synthesis mutant of Escherichia coli K12 at its restrictive temperature in the presence of exogenous palmitate results in lysis of the bacterium. Under these conditions, palmitate is incorporated into membrane phospholipid to a high level. Mutants of bacteria restricting this incorporation (having a palmitate-resistant phenotype) have been isolated and one such mutant, strain L8-2/3, has been further characterized. This mutant has lowered acyl-CoA synthetase (fadD) activity (25-33% of normal) and consequently is defective in fatty acid uptake. This lowered uptake could explain the palmitate-resistant phenotype of strain L8-2/3. However, both in vivo (fatty acid composition and positional distribution data) and in vitro (acyltransferase activity measurements) experiments suggest that this mutant is also altered in its acyltransferase activities. The mutation(s) of strain L8-2/3 appears to allow increased (approximately 2-fold) incorporation of myristate (and possible unsaturated fatty acids) into position 2 of 1-acyl-sn-glycerol 3-phosphate but normal palmitate incorporation into the same position. The incorporation of palmitate, myristate, and oleate into position 1 of sn-glycerol 3-phosphate by strain L8-2/3 is also higher than that observed with the parent, strain L8-2. Replacing the partially defective fadD gene of strain L8-2/3 with a wild type allele conferred on this strain the palmitate sensitivity and the acyltransferase activity of the parent strain L8-2. This finding, taken together with other data, suggests that acyl-CoA synthetase interacts with the acyltransferase(s) in some manner to influence the fatty acid specificity of the acyltransferase.     

Inhibition of T lymphocyte activation by cyclosporin A: interference with the early activation of plasma membrane phospholipid metabolism

Rabbit lymph node and thymus lymphocytes were stimulated with concanavalin A (Con A). Cyclosporin A (CSA) inhibited in a dose-dependent way the induction of RNA and DNA synthesis; nearly complete inhibition was observed at a concentration of 200 ng/ml. Results of kinetic studies suggested that the immunosuppressive drug interfered with an early event occurring in activated lymphocytes. Among the earliest changes detectable in activated lymphocytes, the turnover of plasma membrane phospholipids is increased, predominantly of their fatty acid moieties, catalyzed by the membrane-bound lysophosphatide acyltransferase. CSA, at concentrations identical with those inhibiting macromolecular synthesis, also inhibited the Con A-stimulated specific increase in the incorporation of labeled fatty acids into plasma membrane phospholipids. When lymphocytes were stimulated with Con A for 1 hr, incorporation of labeled oleic acid and arachidonic acid approximately doubled in plasma membrane phospholipids. CSA at a concentration of 200 ng/ml prevented the elevated incorporation of labeled fatty acids into plasma membrane phospholipids of Con A-stimulated thymocytes. Concomitantly, the activation of lysolecithin acyltransferase, the key enzyme for the incorporation of long-chain fatty acids into phospholipids, was strongly inhibited. Up to high concentrations, CSA had no effect on the phospholipid metabolism of unstimulated lymphocytes. The results suggest that CSA inhibits the activation of T lymphocytes by interfering with the early activation of plasma membrane phospholipid metabolism.     

Lysophosphatidylcholine concentrations and metabolism in aortic intima plus inner media: effect of nutritionally induced atherosclerosis

The concentration of lysophosphatidylcholine (monoacyl sn-glycerol 3-phosphorylcholine) in intima plus inner media of atherosclerotic aorta from squirrel monkeys was nearly eight times that in comparable control tissue. Plasma levels of the same compound were somewhat elevated in the atherosclerotic group. The metabolism of fatty acyl CoA's and lysophosphatides was studied in cell-free preparations of intima plus inner media from squirrel monkey aorta. Linoleic acid was incorporated predominantly into phosphatidylcholine (as opposed to other phospholipids) when linoleoyl-1-(14)C CoA was the substrate. The extent of this reaction was dependent on the concentration of lysophosphatidylcholine. Lysophosphatidylethanolamine (monoacyl sn-glycerol 3-phosphorylethanolamine) stimulated the incorporation of linoleate into phosphatidylethanolamine. 1-Palmitoyl-1'-(14)C sn-glycerol 3-phosphorylcholine ((14)C-lysophosphatidylcholine) was incorporated into phosphatidylcholine only in the presence of acyl CoA's or ATP plus CoA. Incorporation of (14)C with (14)C-lysophosphatidylcholine plus linoleoyl CoA equaled that with linoleoyl-1-(14)C CoA and lysophosphatidylcholine. Various other lines of evidence are presented to support the importance of the fatty acyl CoA:lysophosphatide fatty acyl transferase mechanism in aortic phospholipid metabolism. Cell-free preparations of aortic intima plus inner media from squirrel monkeys with early, nutritionally-induced atherosclerosis utilized linoleoyl-1-(14)C CoA more than preparations from control monkeys when incubations were carried out without added lysophosphatidylcholine and for long periods (30 min). With optimum levels of labeled linoleoyl CoA and unlabeled lysophosphatidylcholine, or unlabeled linoleoyl CoA and labeled lysophosphatidylcholine, there were no differences in substrate utilization between control and atherosclerotic tissues. We conclude that the concentrations of lysophosphatidylcholine, which are higher in atherosclerotic than in control aortic tissues, could be a factor controlling rates of fatty acid incorporation into phosphatidylcholine.     

Comparison of the acylation of sn-glycerol 3-phosphate and membrane-bound lipid in the microsomal fraction from rabbit brain throughout maturation.

1. Age-related changes in the specific activity of palmitoyl-CoA synthetase, sn-glycerol 3-phosphate acyltransferase (EC 2.3.1.15) and the esterification of [3H]palmitate into endogenous lipid in the microsomal fraction from rabbit brain have been determined throughout development. 2. The increased specific activity of sn-glycerol 3-phosphate acyltransferase at the onset of myelination (rising in parallel with other lipogenic enzymes) is consistent with a direct role of the acyltransferase in promoting the accumulation of cerebral lipid. In adult brain microsomes, although the specific activity was low, the total activity was only 20% lower than during active myelination. 3. Palmitoyl-CoA, synthesized by the palmitoyl-CoA synthetase in the microsomal membrane, was the preferred substrate for the esterification of sn-glycerol 3-phosphate. There was no evidence for a pool of palmitoyl-CoA formed from palmitate. 4. The esterification of [3H]palmitate into membrane-bound lipid remained high throughout development and may be part of an acyl-exchange cycle via lysophospholipids. [3H]palmitate was incorporated into both neutral lipids and phospholipids, while phosphatidic acid was the major product of sn-[1(3)-3H]-glycerol-3-phosphate esterification. 5. The microsomal fraction contained a pool of unesterified fatty acid, which was activated and esterified into sn-glycerol 3-phosphate.     

Interaction of sn-glycerol 3-phosphorothioate with Escherichia coli. In vitro and in vivo incorporation into phospholipids

sn-Glycerol 3-phosphorothioate, a bacteriocidal analog of sn-glycerol 3-phosphate in strains of Escherichia coli with a functioning glycerol phosphate transport system, was investigated for its ability to be incorporated into phospholipid under in vitro and in vivo conditions. A cell-free particulate fraction from E. coli strain 8 catalyzes the transfer of sn-[3H]glycerol 3-phosphoro[35S]thioate to chloroform-soluble material in the presence of either CDP-diglyceride or palmitoyl coenzyme A. With CDP-diglyceride as the co-substrate, the product of the reaction was tentatively identified as phosphatidylglycerol phosphorothioate. No formation of phosphatidylglycerol was observed, suggesting that the specific phosphatase required for the synthesis of phosphatidylglycerol does not catalyze, or else at a greatly reduced rate, the hydrolysis of the phosphorothioate monoester linkage. The kinetics of incorporation of sn-[3H]glycerol 3-phosphate and phosphorothioate into chloroform-soluble material in the presence of CDP-diglyceride are almost identical. In the presence of palmitoyl coenzyme A, sn-[3H]glycerol 3-phosphoro[35S]thioate was converted to the phosphorothioate analog of phosphatidic acid. Kinetic analysis showed that the apparent Km values for the incorporation of the phosphate and the phosphorothioate derivatives into phospholipid were 0.4 and 0.8 mM, respectively. The Vmax for the phosphorothioate analog was approximately half that for the phosphate derivative. Chemically synthesized thiophosphatidic acid was not a substrate for CTP:phosphatidic acid cytidylyltransferase. sn-[3H]Glycerol 3-phosphoro[35S]thioate was incorporated into phospholipid by cultures of E. coli strain 8. The major phosphorothioate-containing phospholipid synthesized in vivo was identified as 1,2-diacyl-sn-[3H]glycerol 3-phosphoro[35S]thioate. The phosphorothioate analog of phosphatidylglycerol phosphate was not observed despite our observations that this analog can be synthesized in vitro. Our results indicate that the phosphorothioate analog is an effective sn-glycerol 3-phosphate surrogate and suggest that a major reason for its toxicity toward E. coli strain 8 may be due to a total blockade of endogenous phospholipid biosynthesis.     

Rapid cessation of phospholipid synthesis in fructose-1,6-diphosphate aldolase mutants of Escherichia coli.

Escherichia coli GH352, which was originally described as a temperature-sensitive strain containing a thermolabile acyl coenzyme A:monoacylglycerol 3-phosphate acyltransferase, does not now contain a thermolabile form of this enzyme. It has a defect in fructose-1,6-diphosphate aldolase and at least one additional temperature-sensitive lesion. Both strains GH352 and NP315, a temperature-sensitive aldolase mutant, show rapid cessation of 32-P1 incorporation into nucleic acids and phospholipids at 42 C. These characteristics of strain GH352 are therefore no longer attributed to thermolabile phospholipid synthesis, but can be attributed to the fructose-1,6-diphophate aldolase lesion.     

Comparison of glycerolipid biosynthesis in non-green plastids from sycamore (Acer pseudoplatanus) cells and cauliflower (Brassica oleracea) buds.

The availability of methods to fractionate non-green plastids and to prepare their limiting envelope membranes [Alban, Joyard & Douce (1988) Plant Physiol. 88, 709-717] allowed a detailed analysis of the biosynthesis of lysophosphatidic acid, phosphatidic acid, diacylglycerol and monogalactosyl-diacylglycerol (MGDG) in two different types of non-green starch-containing plastids: plastids isolated from cauliflower buds and amyloplasts isolated from sycamore cells. An enzyme [acyl-ACP (acyl carrier protein):sn-glycerol 3-phosphate acyltransferase) recovered in the soluble fraction of non-green plastids transfers oleic acid from oleoyl-ACP to the sn-1 position of sn-glycerol 3-phosphate to form lysophosphatidic acid. Then a membrane-bound enzyme (acyl-ACP:monoacyl-sn-glycerol 3-phosphate acyltransferase), localized in the envelope membrane, catalyses the acylation of the available sn-2 position of 1-oleoyl-sn-glycerol 3-phosphate by palmitic acid from palmitoyl-ACP. Therefore both the soluble phase and the envelope membranes are necessary for acylation of sn-glycerol 3-phosphate. The major difference between cauliflower (Brassica oleracea) and sycamore (Acer pseudoplatanus) membranes is the very low level of phosphatidate phosphatase activity in sycamore envelope membrane. Therefore, very little diacylglycerol is available for MGDG synthesis in sycamore, compared with cauliflower. These findings are consistent with the similarities and differences described in lipid metabolism of mature chloroplasts from 'C18:3' and 'C16:3' plants (those with MGDG containing C18:3 and C16:3 fatty acids). Sycamore contains only C18 fatty acids in MGDG, and the envelope membranes from sycamore amyloplasts have a low phosphatidate phosphatase activity and therefore the enzymes of the Kornberg-Pricer pathway have a low efficiency of incorporation of sn-glycerol 3-phosphate into MGDG. By contrast, cauliflower contains MGDG with C16:3 fatty acid, and the incorporation of sn-glycerol 3-phosphate into MGDG by the enzymes associated with envelope membranes is not limited by the phosphatidate phosphatase. These results demonstrate that: (1) non-green plastids employ the same biosynthetic pathway as that previously established for chloroplasts (the formation of glycerolipids is a general property of all plastids, chloroplasts as well as non-green plastids), (2) the envelope membranes are the major structure responsible for the biosynthesis of phosphatidic acid, diacylglycerol and MGDG, and (3) the enzymes of the envelope Kornberg-Pricer pathway have the same properties in non-green starch-containing plastids as in mature chloroplasts from C16:3 and C18:3 plants.     

Glycerolipid labelling kinetics in isolated intact chloroplasts.

Glycerolipid synthesis was studied in intact chloroplasts isolated from three different plant species. The sequential acylation of sn-glycerol 3-phosphate and lysophosphatidate (1-acyl-sn-glycerol 3-phosphate) was confirmed by monitoring the incorporation of oleate synthesized in situ into lysophosphatidate, phosphatidate and diacylglycerol. Lysophosphatidate was not only readily detected in these experiments, but was also present in the chloroplasts at the beginning of the time courses. The rate of glycerolipid synthesis depended primarily on sn-glycerol 3-phosphate supply, and given adequate sn-glycerol 3-phosphate, the proportion of newly synthesized fatty acids diverted into glycerolipids appeared to be determined by differing acyltransferase activities in the chloroplasts isolated from different plant species.     

Incorporation of extracellular fatty acids by a fatty acid kinase‐dependent pathway in Staphylococcus aureus

Acyl‐CoA and acyl‐acyl carrier protein (ACP) synthetases activate exogenous fatty acids for incorporation into phospholipids in Gram‐negative bacteria. However, Gram‐positive bacteria utilize an acyltransferase pathway for the biogenesis of phosphatidic acid that begins with the acylation of sn‐glycerol‐3‐phosphate by PlsY using an acyl‐phosphate (acyl‐PO₄) intermediate. PlsX generates acyl‐PO₄ from the acyl‐ACP end‐products of fatty acid synthesis. The plsX gene of Staphylococcus aureus was inactivated and the resulting strain was both a fatty acid auxotroph and required de novo fatty acid synthesis for growth. Exogenous fatty acids were only incorporated into the 1‐position and endogenous acyl groups were channeled into the 2‐position of the phospholipids in strain PDJ39 (ΔplsX). Extracellular fatty acids were not elongated. Removal of the exogenous fatty acid supplement led to the rapid accumulation of intracellular acyl‐ACP and the abrupt cessation of fatty acid synthesis. Extracts from the ΔplsX strain exhibited an ATP‐dependent fatty acid kinase activity, and the acyl‐PO₄ was converted to acyl‐ACP when purified PlsX is added. These data reveal the existence of a novel fatty acid kinase pathway for the incorporation of exogenous fatty acids into S. aureus phospholipids.     

Acyl coenzyme A:cholesterol acyltransferase in neonatal chick brain

An acyl coenzyme A:cholesterol acyltransferase activity which directly incorporates palmitoyl coenzyme A into cholesterol esters using endogenous cholesterol as substrate was demonstrated in microsomal preparations from neonatal chick brain. The enzyme showed, at pH 7.4, about 2-fold greater activity than that observed at pH 5.6. Nearly 10-times higher esterifying activity was found in brain microsomes using palmitoyl coenzyme A than that with palmitic acid. The acyltransferase activity was clearly different from the other cholesterol-esterifying enzymes previously found in brain, which incorporated free fatty acids into cholesterol esters and did not require ATP or coenzyme A as cofactors. Chick brain microsomes also incorporated palmitoyl coenzyme A into phospholipids and triacylglycerols. However, most of the radioactivity from this substrate was found in the fatty acid fraction, due to the presence of an acyl coenzyme A hydrolase activity in the enzyme preparations. Therefore, the formation of palmitate was tested during all the experiments. The brain acyltransferase assay conditions were optimized with respect to protein concentration, incubation time and palmitoyl coenzyme A concentration. Microsomal activity was independent of the presence of dithiothreitol in the incubation medium and microsomes can be stored at -40 degrees C for several weeks without losing activity. Addition of fatty acid-free bovine serum albumin to brain microsomal preparations produced a considerable increase in the acyltransferase activity, while acyl coenzyme A hydrolase was clearly inhibited. Results obtained show the existence in neonatal chick brain of an acyl coenzyme A:cholesterol acyltransferase activity similar to that found in a variety of tissues from different species but not previously reported in brain.     

Acyl coenzyme A: cholesterol acyltransferase in neonatal chick brain

An acyl coenzyme A:cholesterol acyltransferase activity which directly incorporates palmitoyl coenzyme A into cholesterol esters using endogenous cholesterol as substrate was demonstrated in microsomal preparations from neonatal chick brain. The enzyme showed, at pH 7.4, about 2-fold greater activity than that observed at pH 5.6. Nearly 10-times higher esterifying activity was found in brain microsomes using palmitoyl coenzyme A than that with palmitic acid. The acyltransferase activity was clearly different from the other cholesterol-esterifying enzymes previously found in brain, which incorporated free fatty acids into cholesterol esters and did not require ATP or coenzyme A as cofactors. Chick brain microsomes also incorporated palmitoyl coenzyme A into phospholipids and triacylglycerols. However, most of the radioactivity from this substrate was found in the fatty acid fraction, due to the presence of an acyl coenzyme A hydrolase activity in the enzyme preparations. Therefore, the formation of palmitate was tested during all the experiments. The brain acyltransferase assay conditions were optimized with respect to protein concentration, incubation time and palmitoyl coenzyme A concentration. Microsomal activity was independent of the presence of dithiothreitol in the incubation medium and microsomes can be stored at −40°C for several weeks without losing activity. Addition of fatty acid-free bovine serum albumin to brain microsomal preparations produced a considerable increase in the acyltransferase activity, while acyl coenzyme A hydrolase was clearly inhibited. Results obtained show the existence in neonatal chick brain of an acyl coenzyme A:cholesterol acyltransferase activity similar to that found in a variety of tissues from different species but not previously reported in brain.     

Control of fatty acid distribution in phosphatidylcholine of spinach leaves

The acylation of lysophosphatidylcholine by enzyme preparations from spinach leaves was studied. The acylation reaction was followed by the incorporation of (14)C-labeled fatty acids from the respective coenzyme A derivatives into phosphatidylcholine. The subcellular fraction with the highest specific activity was the microsomal fraction. Contaminating thioesterase activity which was encountered was inhibited by treatment with sodium dodecyl sulfate. The acyltransferase activity was only mildly inhibited by sulfhydryl reagents. Labeled fatty acid was primarily incorporated into phosphatidylcholine. When saturated and unsaturated fatty acyl CoA derivatives were used, the saturated derivatives were incorporated primarily into the 1-position of the glycerol moiety, and the unsaturated fatty acids went primarily to the 2-position. This pattern of incorporation agrees with the fatty acid distribution in vivo.     

Lipid metabolism in Chinese hamster V79-R membranes composed of unusual phospholipid molecular species

The relationship between the inhibition of cell growth and the changes in phospholipid metabolism in the presence of erucic acid was studied in Chinese hamster V79-R cells. 1. The addition of erucic acid to the medium inhibited cell growth. The degree of inhibition by erucic acid at a given concentration was dependent on cell density. 2. Exogenous erucic acid was incorporated into cellular phospholipids to form new phospholipid molecular species, which were identified to be the erucoyl/oleoyl, erucoyl/gondoyl and erucoyl/erucoyl species. 3. Synthesis of phosphatidylcholine and phosphatidylethanolamine in endoplasmic reticulum was reduced by erucic acid. Erucic acid had no effect on membrane flow of phospholipids from endoplasmic reticulum to plasma membrane. 4. The specific activity of sn-glycerol-3-phosphate acyltransferase in the membrane fraction from the cells supplemented with erucic acid was lower than that from the control cells. The reduction of phospholipid synthesis was attributed to the decrease in sn-glycerol-3-phosphate acyltransferase activity.     

Biosynthesis in Escherichia coli of sn-glycerol 3-phosphate, a precursor of phospholipid. Palmitoyl-CoA inhibition of the biosynthetic sn-glycerol-3-phosphate dehydrogenase.

Homogeneous biosynthetic sn-glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) of Escherichia coli was potently inhibited by palmitoyl-CoA and other long chain acyl-CoA thioesters. The concentration dependence of this inhibition was not cooperative. Enzyme activity was inhibited 50% at 1 microM palmitoyl-CoA; thus, this inhibition occurred at concentrations below the critical micellar concentration of palmitoyl-CoA. Palmitoyl-CoA was a reversible, noncompetitive inhibitor with respect to both NADPH and dihydroxyacetone phosphate. Palmitoyl-CoA did not affect the quaternary structure of the enzyme. This inhibition could be prevented or reversed by the addition of phospholipid vesicles prepared from E. coli phospholipids. Palmitoyl-CoA did not alter the kinetics of inhibition by sn-glycerol 3-phosphate, which is a proven physiological regulator of this enzyme. Decanoyl-CoA, dodecanoyl-CoA, myristoyl-CoA, palmitoyl-(1,N6-etheno)CoA, stearoyl-CoA, and oleoyl-CoA inhibited sn-glycerol-3-phosphate dehydrogenase at concentrations below their critical micellar concentrations. Palmitate inhibited sn-glycerol-3-phosphate dehydrogenase activity 50% at 200 microM. Palmitoyl-carnitine, deoxycholate, taurocholate, and dodecyl sulfate were more potent inhibitors than Triton X-100, Tween-20, or Tween-80. Palmitoyl-acyl carrier protein at concentrations up to 50 microM had no effect on sn-glycerol-3-phosphate dehydrogenase activity. The possible physiological role of long chain fatty acyl-CoA thioesters in the regulation of sn-glycerol 3-phosphate and phospholipid biosynthesis in E. coli is discussed.