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Query: EC:3.1.4.3 (phospholipase C)
 18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Investigations on the cholic acid CoA ligase activity of rat liver microsomes were made possible by the development of a rapid, sensitive radiochemical assay based on the conversion of [3H]choloyl-CoA. More than 70% of the rat liver cholic acid CoA ligase activity was associated with the microsomal subcellular fraction. The dependencies of cholic acid CoA ligase activity on pH, ATP, CoA, Triton WR-1339, acetone, ethanol, magnesium, and salts were investigated. The hypothesis that the long chain fatty acid CoA ligase activity and the cholic acid CoA ligase activity are catalyzed by a single microsomal enzyme was investigated. The ATP, CoA, and cholic (palmitic) acid kinetics neither supported nor negated the hypothesis. Cholic acid was not an inhibitor of the fatty acid CoA ligase and palmitic acid was not a competitive inhibitor of the cholic acid CoA ligase. The cholic acid CoA ligase activity utilized dATP as a substrate more effectively than did the fatty acid CoA ligase activity. The cholic acid and fatty acid CoA ligase activities appeared to have different pH dependencies, differed in thermolability at 41 degrees, and were differentially inactivated by phospholipase C. Moreover, fatty acid CoA ligase activity was present in microsomal fractions from all rat organs tested while cholic acid CoA ligase activity was detected only in liver microsomes. The data suggest that separate microsomal enzymes are responsible for the cholic acid and the fatty acid CoA ligase activities in liver.
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PMID:Characterization of liver cholic acid coenzyme A ligase activity. Evidence that separate microsomal enzymes are responsible for cholic acid and fatty acid activation. 1 45

The phospholipase C of clostridium welchii (alpha toxin) has an absolute requirement for trace quantities of Ca2+. It attacks pure phosphatidylcholine particles (smectic mesophases) having a close-packed bilayer structure only when their surface zeta potential is made positive by the addition of certain divalent ions (e.g., Ca2+) to the aqueous phase or by the presence of low concentrations of long chain cations to the lipid. Alternatively, if the rotational freedom of individual phospholipid molecules is increased by the insertion of short n-alkanols (e.g., hexanol) into the bilayer or when a monolayer of the substrate at an air/water interface is expended, enzymic hydrolysis can occur without any requirement for a net postive charge on the surface.
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PMID:On the question of an electrokinetic requirement for phospholipase C action. 97 17

Human plasma platelet-activating factor (PAF) acetylhydrolase hydrolyzes the sn-2 acetyl residue of PAF, but not phospholipids with long chain sn-2 residues. It is associated with low density lipoprotein (LDL) particles, and is the LDL-associated phospholipase A2 activity that specifically degrades oxidatively damaged phospholipids (Stremler, K. E., Stafforini, D. M., Prescott, S. M., Zimmerman, G. A., and McIntyre, T. M. (1989) J. Biol. Chem. 264, 5331-5334). To identify potential substrates, we synthesized phosphatidylcholines with sn-2 residues from two to nine carbon atoms long, and found the V/k ratio decreased as the sn-2 residue was lengthened: the C5 homolog was 50%, the C6 20%, while the C9 homolog was only 2% as efficient as PAF. However, the presence of an omega-oxo function radically affected hydrolysis: the half-life of the sn-2 9-aldehydic homolog was identical to that of PAF. We oxidized [2-arachidonoyl]phosphatidylcholine and isolated a number of more polar phosphatidylcholines. We treated these with phospholipase C, derivatized the resulting diglycerides for gas chromatographic/mass spectroscopic analysis, and found a number of diglycerides where the m/z ratio was consistent with a series of short to medium length sn-2 residues. We treated the polar phosphatidylcholines with acetylhydrolase and derivatized the products for analysis by gas chromatography/mass spectroscopy. The liberated residues were more polar than straight chain standards and had m/z ratios from 129 to 296, consistent with short to medium chain residues. Therefore, oxidation fragments the sn-2 residue of phospholipids, and the acetylhydrolase specifically degrades such oxidatively fragmented phospholipids.
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PMID:Human plasma platelet-activating factor acetylhydrolase. Oxidatively fragmented phospholipids as substrates. 204 Jun 20

Cyclic nucleotide phosphodiesterase activity in rat heart microsomes is attributable to several isoenzymatic forms: a cyclic AMP-specific, a cyclic GMP-specific, and a cyclic GMP-stimulated enzyme. Incubation of microsomes with an exogenous phospholipase C (C. welchii) induced a marked stimulation (+126%) of cyclic AMP phosphodiesterase and a moderate stimulation (+49%) of cyclic GMP-phosphodiesterase in the membrane-bound fraction. Besides, a notable fraction of activity was solubilized by the treatment. A parallel decrease in the activating effect of cyclic GMP on the hydrolysis of cyclic AMP was observed in the membranes (down to 18% of the control effect). It resulted from a marked stimulation of the basal activity, while the activated level was unaffected. The treatment by an exogenous phospholipase D induced more moderate modifications. The addition to microsomes of oleyl,acetyl-glycerol, but not of long chain-diacylglycerols, partly reproduced the phospholipase C effect. Phosphatidate also induced variations in phosphodiesterase activity, and could thus participate in the phospholipase effects. These results suggest that endogenous phospholipases, the activity of which is modulated by hormonal stimuli, might influence phosphodiesterase activity in cardiac membranes by producing phospholipid metabolites, with potential consequences on heart contractility.
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PMID:Phospholipid metabolism modulates cyclic nucleotide phosphodiesterase activity in rat heart microsomes. 216 7

This study reports the application of modern methods of molecular species analysis in determination of the structure of both major and minor glycerophospholipids and sphingomyelins of human erythrocytes. Individual phospholipid classes were resolved from total lipid extracts by thin-layer chromatography. Diradylglycerols were released by phospholipase C and converted into trimethylsilyl ethers, which were resolved into the alkenylacyl, alkylacyl and diacylglycerol subclasses by normal phase high performance liquid chromatography. Molecular species of diradylglycerols and ceramides were quantitated according to carbon and double bond number by gas liquid chromatography using a fused silica capillary column wall-coated with bonded RTx-2330. The molecular species of ceramides were determined by GC/MS. The diradyl glycerophosphocholines contained 93.0% diacyl, 4.6% alkylacyl and 2.5% alkenylacyl, while the diradyl glycerophosphoethanolamines were made up of 48.8% diacyl, 47.8% alkenylacyl and 3.4% alkylacyl subclasses. Analysis of the molecular species showed that the long chain polyunsaturated acids were mainly combined with C16 in all diradyl GPC subclasses and in diacyl GPE, while in the alkylacyl and alkenylacyl GPE and in diacyl glycerophosphoinositol and diacyl glycerophosphoserine they were combined mainly with C18 saturated fatty chains. In addition to the C16 and C18 alkyl and alkenyl, the ether fractions also contained significant proportions of C20, C22 and C24 chains. The molecular species of the ceramide moieties of the SPH were made up largely of mono- and diunsaturated species. Over 200 molecular species were identified and quantitated in a representative sample of human red blood cells.
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PMID:Molecular species of glycerophospholipids and sphingomyelins of human erythrocytes: improved method of analysis. 275 17

Transmembrane signaling in CTL is found to be extremely sensitive to short term exposure to long chain free fatty acids (FFA). Both alloantigen specific target cells and the lectin Con A were used to stimulate cloned murine CTL. This stimulation was monitored by changes in intracellular calcium concentrations ([Ca2+]i) using the fluorescence indicator fura-2. Treatment of the CTL cells with oleic acid (18:1) at concentrations corresponding to less than 10% (mol/mol) bound to the cell, completely inhibits target cell or Con A-mediated rise in [Ca2+]i. The inhibitory effect of oleic acid is observed within seconds of addition and the inhibition is completely reversed by treating cells with fatty acid free BSA. In addition, using the fluorescence indicator 2',7'-bis(carboxyethyl)carboxyfluorescein to monitor intracellular pH, it was found that oleic acid itself acidifies the cytosol by about 0.3 to 0.4 pH units. Acidification is probably necessary, but is not sufficient to inhibit the calcium rise. Stearic acid (18:0), even at concentrations that correspond to a factor of two to three more bound to the cell than for oleic acid, had no effect on either the [Ca2+]i or intracellular pH responses. Oleic acid was found to bind to cells with single site kinetics and with a number of sites and affinity corresponding to membrane lipid binding sites. Esterification of added oleic acid was negligible in the time (seconds to minutes) required to induce inhibition of the [Ca2+]i response. Inasmuch as added FFA primarily binds to membrane lipid, is not appreciably esterified, and the inhibition is reversed by treatment with fatty acid free BSA, it is likely that the oleic acid effects are due to a physical perturbation of membrane lipid. Furthermore, oleic acid does not affect Con A binding or the production of inositol phosphate metabolites, suggesting that the inhibition of the response is distal to surface recognition events or receptor-phospholipase C coupling. Given the relatively low levels of FFA at which these effects occur it is possible, under conditions in which FFA levels are elevated, that FFA perturbation may modulate CTL activity.
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PMID:Free fatty acid perturbation of transmembrane signaling in cytotoxic T lymphocytes. 278 60

The activity of rat brain protein kinase C, measured in the presence of diacylglycerol, phosphatidylserine and Ca+2, was found to be greatly increased by micromolar amounts of long chain acyl-CoAs, using two different assay systems (lipids added as sonicated dispersion or as mixed micelles with Triton X-100). The potentiation phenomenon required the presence of both diacylglycerol and phosphatidylserine; it was observed at low and saturating concentrations of these effectors, and it was inhibited at high, non physiological Ca+2 concentrations. Under similar conditions, fatty acids alone or coenzyme A were ineffective. The data strongly suggest that acyl-CoAs at the intracellular concentration levels, are important in the modulation of protein kinase C, after activation of the enzyme by the phospholipase C/phosphatidylinositol pathway.
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PMID:Diacylglycerol activation of protein kinase C is modulated by long-chain acyl-CoA. 337 82

Chicken egg yolk phospholipids were subjected to mild alkaline hydrolysis. The resulting alkali-stable phospholipids were characterized by chemical chromatographic, and enzymatic methods. Two major phospholipids, 1 - O - alkyl - sn - glycero - 3 - pohosphoethanolamine and sphingomyelin; and two minor phospholipids, 1-O-alkenyl-sn-glycero-3-phosphoethanolamine and 1-O-alkyl-sn-glycero-3-phosphocholine; were identified. The sphingomyelins were converted into ceramides by enzymatic hydrolysis with phospholipase C. Ceramides derived from sphingomyelins with non-hydroxy fatty acids (99% of total ceramides) consisted predominantly of the N-palmitoyl, N-stearoyl, and N-nervonyl species. Ceramides derived from sphingomyelins with hydroxy fatty acids (1% of total ceramides) consisted almost exclusively of the N-alpha-hydroxyeicosanoyl species. The long chain bases of ceramides derived from both species of sphingomyelins (hydroxy and non-hydroxy fatty acids) consisted of 97% D-erythro-sphing-4-enine and 3% D-erythro-sphinganine, 1-O-alkyl-sn-glycero-3-phosphoethanolamine and 1-O-alkyl-sn-glycero-3-phosphocholine were converted to corresponding 1-O-alkyldiacetylglycerols. The 1-O-alkyldiacetylglycerols derived from both ether phospholipids consisted largely of the hexadecyl and octadecyl species. Smaller quantities of the heptadecyl, cis-9-octadecenyl and eicosanyl derivatives were also present.
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PMID:Mild alkali-stable phospholipids in chicken egg yolks: characterization of 1-alkenyl and 1-alkyl-sn-glycero-3-phosphoethanolamine, sphingomyelin, and 1-alkyl-sn-glycero-3-phosphocholine. 719 4

Individual and pooled samples of plasma from normolipemic and hyperlipemic subjects were separated into very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein fractions (HDL2 and HDL3) by conventional ultracentrifugation and total lipid extracts prepared by standard methods. The composition of the molecular species of the sphingomyelins in each lipoprotein class was determined by packed column and capillary gas-liquid chromatography of the trimethylsilyl (TMS) and t-butyldimethylsilyl (t-BDMS) ethers and by gas chromatography - mass spectrometry of t-BDMS ethers of ceramides derived by phospholipase C hydrolysis of the corresponding parent compounds. It was demonstrated that the molecular weight of the species of the sphingomyelins increases with the density of the lipoprotein fraction in normolipemic subjects, and that this increase is due to an increase in the chain length of the fatty acids in the ceramide molecules. In contrast, patients with type IV hyperlipoproteinemia possessed similar species in the LDL and HDL fractions, while maintaining normal differences between HDL and VLDL. Type III patients possessed normal HDL and VLDL differences, but had variable LDL. Type II patients had ceramide profiles for VLDL, LDL, and HDL fractions that were very similar to those of normals. The differential distribution of the molecular species of the sphingomyelins is rationalized on the basis of a lateral phase separation of the short and long chain sphingomyelins during the shedding of the excess VLDL or chylomicron surface material and a subsequent preferential transformation of the long chain species into HDL. The LDL sphingomyelins in type III hyperlipemia are variable and approximate either the VLDL or HDL composition.
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PMID:Differential distribution of sphingomyelins among plasma lipoprotein classes. 729 45

The dependence of acyl-CoA synthetase on the lipid composition of rat liver plasma membranes has been investigated. For this purpose the composition of the membranes was modified by incorporation of different phospholipids in the presence of partially purified lipid transfer proteins. Another approach to the modification of the membrane phospholipid composition was treatment with exogenous phospholipase C and subsequent enrichment with different phospholipids. The experiments performed in vitro indicated that the presence of certain phospholipids such as phosphatidylnositol, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylserine was essential for the activation of long chain fatty acids by acyl-CoA synthetase. However, some differences were observed when oleate and palmitate were used as substrates. Sphingomyelin was found to inhibit this activity especially when oleic acid served as substrate. In addition, we tried to modify in vivo the membrane lipid composition by treatment with D-galactosamine, which is known to induce acute hepatitis and cause biochemical and biophysical alterations in liver membranes. The results thus obtained confirmed the idea that the augmentation of the membrane lipids and especially of PI, PE and PG was accompanied by acyl-CoA synthetase activation. The presence of two different enzymes, activating the saturated and unsaturated fatty acids is discussed.
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PMID:Acyl-CoA synthetase activity depends on the phospholipid composition of rat liver plasma membranes. 772 15


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