Acylation Of Glycerol 3-phosphate Research Articles

Phosphatidate metabolism in stimulated synaptosomes.

Acetylcholine increases the incorporation of 32P1 into phosphatidate of guinea-pig brain synaptosomes incubated in vitro (Durell & Sodd, 1966; Schacht & Agranoff, 1972; Yagihara & Hawthorne, 1972). Three pathways for the synthesis of phosphatidate operate in brain tissue: acylation of glycerol 3-phosphate, acylation of dihydroxyacetone phosphate (Pollock et al., 1975) and the phosphorylation of 1,2-diacylglycerol (Hokin & Hokin, 1959). It seems likely, though it has not yet been shown conclusively, that the increased synthesis caused by acetylcholine takes place by the thud of these pathways. The diacylglycerol kinase involved is more than ten times as active as the acylation enzymes (Lapetina & Hawthorne, 1971) and diacylglycerol is available from the hydrolysis of phosphatidylinositol. Further, Schacht & Agranoff (1974) have shown that acetylcholine does not increase the incorporation of 3H from [3H]glycerol or [3H]glucose into synaptosomal phosphatidate. This indicates that the synthesis of phosphatidate from glycerol 3-phosphate or dihydroxyacetone phosphate is unaffected by acetylcholine. Evidence from experiments with various tissues shows that the increased labelling of phosphatidate is associated with muscarinic rather than nicotinic cholinergic receptors (Michell, 1975) though electric organ of Torpedo marmorata is an exception (Bleasdale et al., 1976). If the major response of phosphatidate to acetylcholine is post-synaptic, it is difficult to understand why synaptosomal phosphatidate responds to this agonist. Synaptosomes are essentially pre-synaptic nerve terminals and though post-synaptic membranes are attached at the junctional complex they will be isolated from the metabolism of the synaptosome. There is also some confusion about the action of atropine, which blocks the action of acetylcholine on muscarinic receptors. Schacht & Agranoff (1 974) found that atropine blocked the increased labelling of phosphatidate due to acetylcholine when synaptosomes were incubated with 32PI. Yagihara et al. (1973) showed that the major increase in phosphatidate labelling was associated with the synaptic vesicle fraction, and more recent work (J. E. Bleasdale, unpublished observations) shows that atropine does not affect this increase. As pointed out previously (Hawthorne & Bleasdale, 1975) effects of acetylcholine in vitro on a mixed population of synaptosomes from brain cortex may not be

Positional and fatty acid specificity of monoacyl- and diacylglycerol 3-phosphate formation by rabbit heart microsomes

Fatty acid selectivity of acyl-CoA:glycerol-3-phosphate acyltransferase (EC2.3.1.15) and acyl-CoA:monoacylglycerol-3-phosphate acyltransferase (EC2.3.1.52) of the microsomal fraction prepared from rabbit heart was studied. 1. 1. The rate of acylation of glycerol 3-phosphate was increased proportionally with the concentration of acyl-CoA. The maximum rate was reached at 0.3 μmol acyl-CoA per ml. Palmitoyl-, oleoyl- and linoleoyl-CoA all served equally well as acyl donors, and produced approximately equal amounts of mono- and diacylglycerol 3-phosphate. The rate of reaction measured in the presence of two acyl-CoA esters was similar to that in the presence of a single acyl-CoA. 2. 2. Treatment of synthesized monoacylglycerol 3-phosphate with Crotalusadamanteus venom phospholipase A 2 (EC 3.1.1.4) and that with phosphatidate phosphatase (EC 3.1.3.4) indicated that the heart enzyme synthesized almost exclusively 1-acylglycerol 3-phosphate regardless of the kind of acyl donor. 3. 3. Hydrolysis of diacylglycerol 3-phosphate with phospholipase A 2 revealed that there was a slight preference for linoleoyl-CoA at position 2 and a slight discrimination against palmitoyl- and stearoyl-CoA at position 2, when diacylglycerol 3-phosphate was synthesized in the presence of a mixture of acyl-CoA esters. When diacylglycerol 3-phosphate was formed in the presence of a single acyl-CoA, the fatty acid distribution was random. 4. 4. The rate of acylation of 1-palmitoylglycerol 3-phosphate by rabbit heart microsomal fraction was increased proportionally to the increasing concentrations of 1-palmitoylglycerol 3-phosphate up to 50 nmol per ml; higher concentrations were inhibitory. Differences in the activities measured with palmitoyl-, oleoyl- and linoleoyl-CoA as acyl donors were negligible. When stearoyl-, arachidonoyl- and erucoyl-CoA acted as acyl donors, the rates of reaction were low. 5. 5. The acyl-CoA:1-palmitoylglycerol-3-phosphate acyltransferase activity increased proportionally to the increasing concentrations of acyl-CoA up to 10 nmol per ml; acyl donor specificity was similar to that found above [4]. The acyltransferase showed some but not marked fatty acid selectivity in the presence of a mixture of two acyl-CoA esters (palmitic, oleic or stearic acids) as substrates.

Positional Specificity of <italic>sn</italic>-Glycerol 3-Phosphate Acylation during Phosphatidate Formation by Rat Liver Microsomes

A microsomal preparation isolated from rat liver catalyzed the acylation of sn-glycerol 3-phosphate with both palmitoyl-CoA and oleoyl-CoA. The optimum pH was 7.5 for both acyl-CoA's, and the apparent Km values for glycerophosphate were 1.0 and 1.2 mM with palmitoyl-CoA and oleoyl-CoA as acyl donors, respectively. The major reaction product was diacyl-sn-glycerol 3-phosphate in the standard assay system with either acyl-CoA. Acyl-sn-glycerol 3-phosphate (monoacyl-GP) was added to the incubation system to trap newly synthesized radioactive intermediates. Radioactive monoacyl-GP was trapped effectively by the addition of nonlabeled 1-acyl-GP but ineffectively by adding nonlabeled 2-acyl-GP, regardless of whether palmitoyl-CoA or oleoyl-CoA was used as an acyl donor. The monoacyl-GP trapped in the presence of 1-acyl-GP was mostly the 1-acyl-GP isomer, regardless of which acyl-CoA was used. The system with added 2-acyl-GP produced various results as regards the isomeric monoacyl-GP accumulated. Although the pathway via the 2-acyl-GP could not be excluded completely in this experiment, diacyl-GP formation from glycerophosphate in rat liver microsomes appeared to occur primarily via the 1-acyl-GP as an intermediate, regardless of whether palmitoyl-CoA or oleoyl-CoA was used as an acyl donor.

The acylation of glycerol 3-phosphate in different rat organs and in the liver of different species (including man)

1. 1. The organ distribution of glycerol 3-phosphate acylating enzymes has been investigated in rat. The highest activities (per mg of protein) were found in adipose tissue, adrenal gland and liver. 2. 2. For mitochondria isolated from different rat organs and from the liver of different species, a preference for saturated fatty acids in the acylation of glycerol 3-phosphate was found. Monoacylglycerophosphate (lysophosphatidic acid) was a major mitochondrial reaction product. 3. 3. For microsomes, both saturated and unsaturated long-chain fatty acids were efficient substrates, and diacylglycerophosphate (phosphatidic acid) was the main reaction product.

Partial purification and properties of glycerophosphate acyltransferase from rat liver. Formation of 1-acylglycerol 3-phosphate from sn-glycerol 3-phosphate and palmityl coenzyme A.

1 Glycerophosphate acyltransferase was partially purified from rat-liver microsomes which were resolved with a nonionic detergent, Triton X-100, in glycine buffer pH 8.6. The purification procedure involves molecular-sieve chromatography and sucrose density gradient centrifugation. 2 The partially purified enzyme requires Ca2+ for its activity. Divalent cations, such as Mg2+, Mn2+ and Co2+, are able to substitute for Ca2+ with varying degrees of effectiveness. Ca2+ in higher concentrations is inhibitory, but this inhibition is abolished by the addition of small amounts of phospholipids. Moreover, phospholipids stimulate the enzyme activity at all concentrations of Ca2+ tested. The pH optimum of the enzyme is broad, extending over a pH range from 6.6 to 9.0. 3 The apparent Michaelis constant of the partially purified enzyme for sn-glycerol 3-phosphate is 0.2 mM, which approximates the value found with unresolved microsomes (0.5 mM). 4 Among the acyl donors examined, palmityl-CoA is utilized most efficiently by the partially purified enzyme. Unsaturated fatty acyl-CoA thioesters, such as oleyl-CoA, linoleyl-CoA and arachidonyl-CoA, are poor substrates. 5 The partially purified enzyme catalyzes the formation of monoacylglycerol 3-phosphate from sn-glycerol 3-phosphate and palmityl-CoA, esterifying preferentially position 1 of the glycerol moiety. Little phosphatidic acid is produced even in the presence of both palmityl-CoA and linoleyl-CoA. Thus the acyltransferase preparation is free of the enzyme responsible for acylation of 1-acylglycerol 3-phosphate. 6 The results presented indicate that phosphatidic acid synthesis in mammalian liver proceeds through a sequential acylation of sn-glycerol 3-phosphate mediated by two distinct acyltransferases. The data also show that acylation of sn-glycerol 3-phosphate to monoacylglycerol 3-phosphate occurs in a non-random manner, thus contributing to the asymmetric distribution of fatty acids in glycerolipids.

Resolution and reconstitution of the phosphatidate-synthesizing system of rat-liver microsomes.

The phosphatidate-synthesizing system of rat-liver microsomes was resolved into two component enzymes, glycerolphosphate acyltransferase and 1-acylglycerolphosphate acyltransferase. The resolution is effected by sucrose density gradient centrifugation in the presence of a nonionic detergent, Triton X-100. Combination of both enzymes results in reconstitution of the phosphatidate-synthesizing system. These results establish that two distinct enzymes, glycerolphosphate acyltransferase and 1-acylglycerolphosphate acyltransferase, are required for synthesis of phosphatidic acid from sn-glycerol 3-phosphate.Furthermore, the 1-acylglycerolphosphate acyltransferase preparation efficiently uses unsaturated (or saturated) fatty acyl-CoA as acyl donor. Our previous studies showed that the glycerolphosphate acyltransferase preparation catalyzes formation of 1-acylglycerol 3-phosphate, using preferentially saturated fatty acyl-CoA as acyl donor. These findings indicate that the reconstituted system is capable of yielding phosphatidic acid with an asymmetric fatty acid distribution.

Biosynthesis of Acyl Dihydroxyacetone Phosphate in Subcellular Fractions of Rat Liver

The activity of acyl-CoA:dihydroxyacetone phosphate acyltransferase in rat liver was studied by measuring the formation of labeled lipid from dihydroxyacetone [32P]phosphate and either palmitoyl-CoA or a mixture of palmitate, CoA, and ATP. Bovine serum albumin stimulated the activity of the enzyme several-fold. In the presence of albumin the acyltransferase activity in mitochondria was much higher than in microsomes. The labeled lipid was identified as acyl dihydroxyacetone phosphate by its chromatographic and chemical properties. The acyl dihydroxyacetone phosphate-synthesizing enzyme was also present in particulate fractions of rat kidney, heart, testis, spleen, brain, and adipose tissue. The formation of glycerolipid from dihydroxyacetone phosphate in rat liver mitochondria and microsomes was also studied by using [1-14C]palmitate, ATP, CoA, and other cofactors. [14C]Acyl dehydroxyacetone phosphate was the principal product in both mitochondria and microsomes. Phosphatidic acid and neutral glycerides were formed when either NADPH or NADH was also included in the reaction mixture. However, the major amount of glycerides formed in the presence of NADH was, possibly, due to the acylation of glycerol 3-phosphate. The glycerol 3-phosphate was formed in the reaction mixture by the reduction of the added dihydroxyacetone phosphate with NADH, catalyzed by the glycerophosphate dehydrogenase (EC 1.1.1.8) present in the mitochondrial or microsomal fractions. This activity of the glycerophosphate dehydrogenase could not be removed from mitochondrial or microsomal particles by repeated washings with 0.25 m sucrose.

Effect of Phospholipase C Hydrolysis of Membrane Phospholipids on Membranous Enzymes

The response of several Escherichia coli membranous enzymes to hydrolysis of up to 95% of membrane phospholipid has been investigated. Purified phospholipase C of Bacillus cereus was utilized in these studies. The rate and extent of digestion of E. coli phospholipids was independent of whether the lipid was associated with membrane protein or extracted from membranes and sonically dispersed. Phosphatidylethanolamine and phosphatidylglycerol were completely hydrolyzed, while cardiolipin was partially resistant to hydrolysis by phospholipase C. Acyl-CoA:glycerol 3-phosphate acyltransferase and NADH oxidase were inactivated at a rate very similar to the rate of hydrolysis of total lipids. Acyl-CoA:1-acylglycerol 3-phosphate acyltransferase was inactivated to an extent of 50% during hydrolysis of 50% of membrane phospholipid. The remaining activity was stable to continued hydrolysis of phospholipid. Glycerol 3-phosphate dehydrogenase and succinic dehydrogenase remained completely active after hydrolysis of 95% of membrane phospholipids. These results show the heterogeneity of membranous enzymes with respect to their dependence upon the presence of intact membrane phospholipids. The lack of effect of membranous lipid-protein interactions on the accessibility of phospholipids to phospholipase C hydrolysis was shown in general by the similarity in the rates of hydrolysis by phospholipase C of membranous and isolated phospholipids. More specifically, the similarity in the rate of hydrolysis of phospholipids and the rates of inactivation of certain membranous enzymes suggests that these enzymes are dependent on phospholipids whose susceptibility to phospholipase C hydrolysis is similar to the bulk of membrane phospholipids.

The Effect of Phospholipid Fatty Acid Composition on Membranous Enzymes in Escherichia coli

Abstract The shape of an Arrhenius plot of glycerol 3-phosphate acyltransferase activity of an unsaturated fatty acid auxotroph of Escherichia coli is identical in membranes containing cis-vaccenic, oleic, linoleic, or linolenic acid as sole unsaturated fatty acid. The curve is linear at low temperatures with a continuous decrease in slope above 15°. In membranes containing trans-unsaturated fatty acids as the sole unsaturated fatty acid, the decrease in slope occurs at 20°. Membranes from cells grown in the presence of oleic acid and then grown for one generation in the absence of unsaturated fatty acids contained 79% saturated fatty acids and 21% oleic acid. Temperature dependence of the enzyme activity in these membranes was intermediate between that observed in membranes containing trans-unsaturated fatty acids and those containing normal amounts of cis-unsaturated fatty acids. 1-Acylglycerol 3-phosphate acyltransferase activity exhibited a linear Arrhenius plot identical in slope in membranes containing cis-unsaturated fatty acids of varying degrees of unsaturation. Membranes from cells deprived of unsaturated fatty acids for one generation or those containing trans-unsaturated fatty acids exhibited a steeper slope. Membranous glycerol 3-phosphate dehydrogenase activity appeared to be independent of membrane fatty acid composition. Linear Arrhenius plots of identical slope were observed in all membrane preparations described above. The difference in the dependence of the temperature characteristics of various membranous enzymes on membrane fatty acid composition suggests a heterogeneity in the relationship between membranous enzymes and membrane phospholipids.

The effect of drugs on lipogenesis from glucose and palmitate in human adipose tissue.

Several drugs were tested for their ability to inhibit lipogenesis in human adipose tissue. Only fenfluramine was found to inhibit the incorporation of T-palmitate and 14C-glucose into neutral lipid in intact tissue. This effect was observed at drug concentrations above 1 mM. Fenfluramine inhibited lipogenesis in broken-cell preparations of human adipose tissue at concentrations of 1 mM and above. However, in this system the N-benzoyloxyethyl derivative of fenfluramine, S. 1513, was also found to inhibit lipogenesis. This drug was more potent than fenfluramine, a significant inhibition being observed at 0.4 mM During inhibition of lipogenesis in homogenates of adipose tissue by fenfluramine radioactitivity was found to accumulate in long-chain acyl-CoA which suggests that the drug may interfere with acylation of glycerol 3-phosphate. Evidence that fenfluramine may have a similar effect in vivo was considered, but the results were not statistically significant.

The Specific Acylation of Glycerol 3-Phosphate to Monoacylglycerol 3-Phosphate in Escherichia coli: EVIDENCE FOR A SINGLE ENZYME CONFERRING THIS SPECIFICITY

The Specific Acylation of Glycerol 3-Phosphate to Monoacylglycerol 3-Phosphate in Escherichia coli: EVIDENCE FOR A SINGLE ENZYME CONFERRING THIS SPECIFICITY

1-Acylglycerol 3-Phosphate Acyltransferase from Rat Liver

Abstract The substrate specificity of 1-acylglycerol 3-phosphate acyltransferase from rat liver microsomes for both acylcoenzyme A and 1-acylglycerol 3-phosphate has been determined at levels below the critical micelle concentrations of the substrates. The fatty acid of the acylglycerol 3-phosphate is not important, but for the acyl-CoAs the specificity is oleyl g palmitoleyl ≃ linoleyl ≃ palmityl g myristyl g stearyl g lauryl. 1-Acylglycerol 3-phosphate acyltransferase appears to work only with monomeric substrates. The Km for palmityl-CoA is less than 0.1 µm, while the Km values for the 1-acylglycerol 3-phosphates are in the low micromolar range (5 to 25 µm), but are difficult to measure accurately because micelles form at 4 to 8 µm 1-acylglycerol-3-P and the velocity ceases to increase. By contrast, acyl-CoA hydrolase appears to prefer micelles as substrate. This enzyme shows little activity at 3 µm acyl-CoA, and shows 8 times more activity with palmityl-CoA than with any other acyl-CoA tested. In order to plan the specificity studies, the critical micelle concentrations of the acyl-CoAs and 1-acylglycerol 3-phosphates used were determined by the dye adsorption technique with pinacyanol chloride. For acyl-CoAs in 6.7 mm phosphate, 10 mm K+, pH 6.9, the values were: lauryl, 9.5 µm; myristyl, 4 µm; palmityl, 3.6 (4 µm in 50 mm Tris-HCl, pH 7.4; 6 µm in deionized water); palmitoleyl, 2 µm; stearyl, 2 µm; oleyl, 4.5 µm; linoleyl, 5.5 µm. For 1-acylglycerol 3-phosphates in 50 mm Tris-HCl, pH 7.4, the values were: myristyl, 120 µm; palmityl, 35 µm; stearyl, 7 µm; oleyl, 23 µm; linoleyl, 34 µm; 1-acylglycerol 3-phosphate prepared from egg lecithin (80% palmitic), 37 µm.

Involvement of Acyl Carrier Protein in Acylation of Glycerol 3-Phosphate in Clostridium butyricum: II. EVIDENCE FOR THE PARTICIPATION OF ACYL THIOESTERS OF ACYL CARRIER PROTEIN

Abstract 1. In order to examine the involvement of acyl carrier protein in the acylation of glycerophosphate by bacterial extracts, a series of experiments with radioactive glycerol 3-phosphate, potential acyl donors, and enzyme preparations from Clostridium butyricum were carried out. 2. The conversion of 14C-glycerophosphate to lipid, mainly lysophosphatidic acid, in the presence of a particulate fraction from C. butyricum was found to be dependent on the addition of acyl carrier protein (ACP) isolated from either C. butyricum or Escherichia coli. The apparent Km was 1.2 x 10-6 m for C. butyricum ACP and 3.4 x 10-6 m for E. coli ACP. Since no source of acyl groups was added in this series of experiments, it is postulated that the acyl groups transferred to glycerophosphate were derived from the particles. 3. With this particulate fraction, chemically synthesized 3H-palmityl-ACP also markedly stimulated the acylation of glycerol 3-phosphate and was an efficient donor of palmitate in the synthesis of lysophosphatidic acid. Palmityl coenzyme A had little effect on glycerophosphate acylation with the particulate fraction as the sole enzyme source. 4. A two-fold stimulation of glycerophosphate acylation by palmityl-CoA was observed when a soluble protein fraction from C. butyricum was added to the particulate fraction. Under these conditions some transfer of labeled fatty acids from 3H-acyl-CoA to 14C-glycerophosphate was observed. The lysophosphatidic acid isolated had 0.3 3H-acyl group per 14C-glycerophosphate. Presumably, the remainder of the acyl groups was derived from the enzyme sources. When ACP was added, the labeled fatty acids derived from acyl-CoA were further diluted by endogenous fatty acids. 5. Sodium lauryl sulfate also stimulated glycerophosphate acylation in the presence of particles from C. butyricum plus the soluble protein fraction. Both palmityl-CoA and sodium lauryl sulfate stimulated glycerophosphate acylation more at 25° than at 30°. These results suggest that some of the effects of palmityl-CoA in this system are the result of its surfactant properties.

Biosynthesis of Phosphatidic Acid, Lysophosphatidic Acid, Diglyceride, and Triglyceride by Fatty Acyltransferase Pathways in Escherichia coli

Fatty acyl coenzyme A-glycerol 3-phosphate, fatty acyl coenzyme A-lysophosphatidic acid, fatty acyl coenzyme A-monoglyceride, and fatty acyl coenzyme A-diglyceride acyltransferase activities were found in a particulate fraction of broken cell preparations of Escherichia coli. The synthesis of phosphatidic acid and lysophosphatidic acid catalyzed by these enzymes was demonstrated to be dependent upon the presence of glycerol 3-phosphate, fatty acyl-CoA, and magnesium ion. Magnesium ion was partially replaceable by calcium ion. Isolated, radiochemically pure lysophosphatidic acid served as a substrate in the synthesis of phosphatidic acid in an enzymatic reaction with fatty acyl-CoA. This observation suggests that lysophosphatidic acid functions as an intermediary in the synthesis of phosphatidic acid from glycerol 3-phosphate and fatty acyl-CoA. Under similar conditions of incubation both monoglyceride and diglyceride functioned as acceptors of the fatty acyl moiety from fatty acyl-CoA. Glycerol, however, did not serve as a substrate in the acylation reaction. Glycerol was incorporated into phospholipid only when adenosine triphosphate was added to an incubation system capable of acylating glycerol 3-phosphate. Lauryl, myristyl, palmityl, stearyl, oleyl, linoleyl, linolenyl, cis-vaccenyl, and trans-vaccenyl coenzyme A derivatives were tested and found to be active substrates in vitro in the acylation of glycerol 3-phosphate. However, lauric, stearic, linoleic, linolenic, and trans-vaccenic acids have not been reported to exist as constituents of the phospholipids of E. coli. This finding suggests that the acyl transferase reaction does not possess the specificity required for excluding certain fatty acids which do not naturally occur in the phospholipids of E. coli.

Acylation of Glycerol 3-Phosphate in Bacterial Extracts: STIMULATION BY ACYL CARRIER PROTEIN

Abstract The conversion of 14C-glycerol 3-phosphate to lysophosphatidic acid in the presence of palmityl coenzyme A is catalyzed by particles plus a soluble protein fraction from sonic extracts of Clostridium butyricum. This conversion is markedly stimulated by the addition of 10-5 M acyl carrier protein isolated from Escherichia coli.

Palmityl-Acyl Carrier Protein as Acyl Donor for Complex Lipid Biosynthesis in Escherichia coli

Abstract An Escherichia coli membrane preparation catalyzes the formation of monopalmitin from glycerol 3-phosphate and palmityl acyl carrier protein. This preparation also catalyzes the formation of lysophosphatidic and phosphatidic acids from glycerol 3-phosphate and palmityl coenzyme A.