Gene/Protein Disease Symptom Drug Enzyme Compound
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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)

Gas-liquid chromatography-mass spectrometry (GLC-MS), employing an open-tubular Silanox-type glass column, has been applied to the products of phospholipase C hydrolysis of natural and synthetic phospholipid mixtures. The materials studied were egg lysolecithin, synthetic L-alpha-l-stearoyl-2-oleoyl lecithin, bovine brain sphingomyelin, and phospholipids derived from human arterial tissue. l-Monoglycerides and ceramides were analysed as methaneboronates, and 1,2-diglycerides as trimethylsilyl ethers. The results indicate the potential value of open-tubular GLC-MS in a rapid procedure for the concurrent analysis of the major classes or polar lipids after enzymic dephosphorylation.
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PMID:Gas-liquid chromatography-mass spectrometry of phospholipid mixtures after enzymic hydrolysis. 91 30

Plasma or serum [ 0.1-1.0 ml] was digested with phospholipase C and total lipid extracts were prepared and silylated in the presence of tridecanoylglycerol as internal standard. The neutral lipid and free fatty acid profiles were determined by means of an automated GLC system equipped with an unheated on-column inlet, time actuated liquid injector, programmed heating, cooling and equilibration cycles, and an electronic peak area integrator. The separations were accomplished on a 50 cm x 2 mm i.d. steel column packed with 3% OV-1 on100-120 mesh Gas Chrom Q using nitrogen as a carrier gas in the temperature range 175-350 degrees C. The tube number, peak retention time and peak area were recorded on a punched paper tape, which was subsequently read into a computer via a time-share terminal. The composition of the sample was calculated in relation to the internal standard using a modification of a commercially available computer program and the results were expressed as mg or mole % and characteristic molar ratios of lipid classes. In addition to estimates for total cholesterol and triglyceride, the method provides a detailed account of individual or small groups of molecular species of various lipid classes, which is a major advantage over other automated methods of plasma lipid analyses.
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PMID:Determination of plasma lipid profiles by automated gas chromatography and computerized data analysis. 115 32

The relative proportions and compositions of the diacyl molecular species of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (PA), and diacylglycerol (DG) from rat retinal membranes were determined. Two membrane fractions were derived by discontinuous sucrose gradient floatation: rod outer segment (ROS) and 'rest of the retina' (ROR). ROR is defined as those membranes pelleted as 100,000 g following removal of the ROS. Diacylglycerols were prepared from PC, PE and PS by phospholipase C treatment and were converted into the corresponding 1,2-diacylglycerobenzoates (DGBZ). PI, PA, and DG were converted into 1,2-diacylglyceroacetates (DGAC) by acetolysis. The molecular species of the DGBZ and DGAC were resolved by reverse-phase HPLC and detected by UV absorption at 230 and 210 nm, respectively. Fatty acid methyl esters of PC, PE, PS, PI and DG from ROS and ROR were prepared and analysed by GLC. The fatty acid and molecular species patterns of PC, PE and PS were similar in both membrane fractions, although the levels of docosahexaenoic acid (22:6 omega 3) and 22:6-containing molecular species were lower in ROR than in ROS, PE and PS were enriched in 22:6 omega 3 and 18:0, an evidenced by the high levels of 18:0-22:6 and 22:6-22:6 molecular species. PC contained relatively more saturated and monoene species, such as 16:0-16:0, 16:0-18:0, 16:0-18:0, 16:0-18:1 and 18:0-18:1. The fatty acids and molecular species patterns of DG, PI and PA in ROS and ROR differed from those of PC, PE and PS.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Fatty acid and molecular species compositions of phospholipids and diacylglycerols from rat retinal membranes. 201 3

We have investigated the effects of the specific platelet-activating factor (PAF; 1-alkyl-2-acetyl-glycerophosphocholine) antagonist BN52021 on free fatty acid (FFA) and diacylglycerol (DG) accumulation and on the loss of fatty acids from phosphatidylinositol-4,5-bisphosphate (PIP2) in mouse brain. Mice were pretreated with BN52021 (10 mg/kg, i.p.) 30 min before electroconvulsive shock (ECS) or postdecapitation ischemia. These procedures cause rapid breakdown of PIP2 and accumulation of FFA and DG. Lipid extracts were prepared from microwave-fixed cerebrum and fractionated by TLC, and the fatty acid methyl esters were prepared by methanolysis and quantified by capillary GLC. In saline or vehicle (dimethyl sulfoxide)-treated mice, ECS caused marked accumulation of FFA and DG and loss of mainly stearic (18:0) and arachidonic (20:4) acids from PIP2. BN52021 pretreatment of ECS-treated mice decreased the accumulation of free palmitic (16:0), 18:0, 20:4, and docosahexaenoic (22:6) acids with no effect on the fatty acids in DG or the loss of PIP2. BN52021 had no effect on basal levels of FFA, DG, or PIP2. One minute of postdecapitation ischemia induced PIP2 loss and accumulation of FFA and DG. BN52021 attenuated the accumulation of free 20:4 and 22:6 acids, decreased the content of oleic (18:1), 20:4, and 22:6 acids in DG, but had no effect on PIP2 loss. These data indicate that BN52021 reduces the injury-induced activation of phospholipase A2 and lysophospholipase, which mediate the accumulation of FFA in brain, while having a negligible effect on phospholipase C-mediated degradation of PIP2.
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PMID:Platelet-activating factor antagonist BN52021 decreases accumulation of free polyunsaturated fatty acid in mouse brain during ischemia and electroconvulsive shock. 284 88

Brain cell membranes are known to abound in polyphosphoinositides (PPI) which contain large amounts of arachidonic acid and stearic acid. When a state of cerebral ischemia comes about, there occurs severe energy depletion and decomposition of PPI into diglyceride (DG) and inositol triphosphate (IP3) through activation of phospholipase C. Previous studies clarified rapid postischemic degradation of PPI, a time during which the metabolically active fraction of PPI is lost, but there have been no reports on PPI metabolism after the establishment of recirculation following ischemia. The authors examined relationship between the duration of the ischemia and the reversibility of PPI metabolism in rats with cerebral ischemia lasting 5 or 30 min that was followed by recirculation, and, further studied acyl group composition of PPI and DG in rats with 30 min of ischemia. Global cerebral ischemia was produced in male Wistar rats (220-250 g) by occlusion of basilar and bilateral common carotid arteries. The brains were frozen in situ at 1, 5, or 30 min of ischemia, or at 30 or 60 min of recirculation following either 5 or 30 min of ischemia. Phosphatidylinositol (PI), phosphatidylinositol, 4-phosphate (PIP), phosphatidylinositol, 4, 5-bisphosphate (PIP2), and DG were measured by TLC, and GLC. And also their acyl group compositions were determined. PI showed no significant changes. In contrast, both PIP and PIP2 sharply decreased immediately after onset of cerebral ischemia. then continued to fall gradually from 5 min onwards. And PIP and PIP 2 increased after onset of recirculation in both 5 and 30 min ischemia groups.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:[Polyphosphoinositide metabolism in temporary cerebral ischemia--the reversibility after recirculation]. 285 44

Two physicochemical methods have been developed for the quantitative analysis of lyso-platelet activating factor (lyso-PAF) based on gas-liquid chromatography-mass spectrometry (GLC/MS) and fast atom bombardment-mass spectrometry (FAB/MS) using stable isotope dilution. After addition of deuterated internal standards, lyso-PAF produced from neutrophils was purified by silicic acid chromatography and thin-layer chromatography (TLC). The GLC/MS assay employed phospholipase C or hydrofluoric acid for hydrolysis of the phosphocholine moiety to yield ether monoglycerides. Condensation of monoglycerides with acetone yielded the 1-O-alkyl-2,3-isopropylidene glycerol which could be analyzed by GLC/MS. The ions corresponding to M-15 fragments for both the labeled and unlabeled derivatives were monitored in a selected ion recording mode. Standard curves were found to be linear over the range tested (10-2000 ng) with a limit of detection found to be below 200 pg injected on column. For the FAB/MS assay, the unmodified lyso-PAF was well suited for direct analysis; however, the limit of detection (S/N greater than 3) using a glycerol matrix was found to be 5 ng placed on the probe tip. It was found that human neutrophils contain approximately 300 pg/10(6) cells which increased 2-3-fold during the 5-min period following challenge with 1.9 microM calcium ionophore, A23187. Two molecular species of lyso-PAF were identified as hexadecyl and octadecyl ethers at sn-1 with the octadecyl molecular species of lyso-PAF predominating in abundance after stimulation.
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PMID:Quantitation of lyso-platelet activating factor molecular species from human neutrophils by mass spectrometry. 310 21

Ischemia gives rise to severe energy depletion and influx of Ca from the extracellular space, and it is suggested that increased intracellular Ca leads to the activation of phospholipase C and A, and to liberation of free fatty acids (FFA) in particular arachidonic acid. Phenytoin has been reported not only to maintain the intra- and extracellular cation balance but blockade the Ca channel. The purpose of the present study is to investigate the effect of phenytoin on the liberation of FFA, energy metabolism and mononucleotide metabolism in ischemic brain. Male Wistar rats were subjected to global cerebral ischemia induced by the occlusion of basilar and bilateral common carotid arteries. The brains were frozen in situ by the funnel technique after 5 or 30 min of ischemia or after 10, 30, or 60 min of recirculation following 30 min of ischemia. Purine and pyrimidine nucleotides, FFA, and glycolytic intermediates were measured by HPLC, GLC, and fluoro-enzymatic method. In non-treated rats, ATP reached a nadir after 5 and 30 min of ischemia. Phenytoin significantly attenuated ATP depletion after 5 and 30 min of ischemia. And also E.C. is higher in phenytoin treated rats than in non-treated rats in ischemia. After 60 min of recirculation, ATP recovered to 1.93 +/- 0.02 mumol (72.3% of pre-ischemia) in treated rats but 1.60 +/- 0.07 mumol/g (60% of pre-ischemia) in non treated rats. In E.C., there are significant differences between non-treated and treated rats after 10 and 30 min of recirculation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:[The effect of phenytoin on free fatty acid liberation and mononucleotide metabolism in transient ischemia]. 321 41

Inositol phospholipids (IPL) from epimastigote forms of Trypanosoma cruzi have been investigated by metabolic labelling with [3H]palmitic acid and by GLC-MS analysis of the lipids obtained from non-labelled parasites. The IPL fraction was separated into phosphatidylinositol (PI) and inositol-phosphoceramide subfractions, the latter accounting for 80-85% of the total IPL. The neutral lipids released from the IPLs by PI-specific phospholipase C (PI-PLC) from Bacillus thuringiensis were analysed by silica-gel and reverse-phase TLC for the radioactive lipids and by GLC-MS for the non-radioactive samples. Ceramides containing dihydrosphingosine and sphingosine with C16:0 and C18:0 fatty acids were identified. The main component in the [3H]palmitic acid-labelled ceramides was palmitoyldihydrospingosine, while in the non-labelled sample the ceramides contained mainly sphingosine. This could reflect partial uptake of phospholipid from the medium. The PI contain both alkylacyl- and diacyl-glycerol lipids, with the ether lipid being more abundant. The latter was identified as 1-O-hexadecylglycerol esterified by C18:2 and C18:1 fatty acids. Interestingly, the same lipid had been identified in the anchor of the 1G7 glycoprotein of T. cruzi metacyclic forms.
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PMID:Structural analysis of inositol phospholipids from Trypanosoma cruzi epimastigote forms. 764 54

Several studies have shown the potential role of phosphatidic acid (PA) as a second messenger in different cell types. Thus, PA has been shown to mimic physiological agonists leading to various cellular responses, such as neurotransmitter and hormone release, cell proliferation by modulating DNA or RNA synthesis, the expression of several proto-oncogenes and growth factors, and the stimulation of enzyme activities such as phospholipase C (PLC), protein kinases and cyclic AMP (cAMP) phosphodiesterase. Stimulation of [3H]arachidonate-labelled rat thymocytes with the mitogen lectin concanavalin A (con A) resulted in enhanced production of radiolabelled PA after only 5 min of activation. The radiolabelled PA increase corresponded to a real increase in PA mass as determined by GLC quantification of its fatty acid content. In the presence of ethanol (0.5%), formation of phosphatidylethanol was not observed after 5 min of con A activation. Pretreatment of cells with R 59022 (10 microM), a diacylglycerol (DAG) kinase inhibitor, showed an inhibition in the formation of radiolabelled PA and in PA mass. These results suggest that the PLC-DAG kinase may be the pathway for PA synthesis in the first minutes of mitogenic thymocyte activation. A detailed analysis of the fatty acid composition showed that the relative amount of unsaturated fatty acids was increased in PA from stimulated cells concomitantly with a decrease in saturated ones; in particular, arachidonic acid was increased approximately 2-fold only 2 min after con A addition whereas palmitic acid was decreased for the whole period investigated (20 min). These changes favour the hydolysis of phosphoinositides rather than phosphatidylcholines by PLC. As PA remains a minor phospholipid, these changes are unlikely to affect cell membrane fluidity; but PA being now well recognized as a potential second messenger, its increased content as well as its increased unsaturation in the fatty acyl moiety might modulate several signalling pathways or the activity of enzymes such as cyclic nucleotide phosphodiesterase, controlling in this way the cellular level of cAMP, a negative regulator of blastic transformation.
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PMID:Time-course changes in content and fatty acid composition of phosphatidic acid from rat thymocytes during concanavalin A stimulation. 775 52

We performed detailed chromatographic analyses on the molecular species of the major glycerophospholipids (GPLs) and free sn-1,2-diacylglycerols (DAGs) from SH-SY5Y human neuroblastoma cells following incubation with or without LiCl. For this comparison the inositol, choline, ethanolamine and serine GPLs were dephosphorylated with phospholipase C and the released sn-1,2-diacylglycerols along with the DAGs were subjected to high-temperature GLC on polar and non-polar capillary columns as their trimethylsilyl and tert.-butyl-dimethylsilyl ethers. A 30-min incubation with 10 mM LiCl increased the total amount of human neuroblastoma DAGs by 32-58% (P < 0.05) to 2.6 pmol/micrograms cell protein. This was accompanied by a limited qualitative shift in the molecular species pattern, the most obvious of which was the increase (13%) in the major saturated-polyunsaturated molecular species and the ca. 46% increase in the minor 18:1-18:1 species over control levels. The DAGs originated mainly from the inositol GPLs (IGPLs), as indicated by the high levels of the characteristic 18:0-20:4n6 (18:0-20:3n9) species in both IGPLs and DAGs, and to a lesser extent from the choline GPLs (CGPLs), as indicated by the high proportion in CGPLs of the oligoenoic species, which were largely absent from IGPLs. Alkenylacylglycerols were not detected in DAGs, although they made up some 60% of the total ethanolamine GPLs (EGPLs). No significant changes in the molecular species composition of the cellular GPLs, including IGPLs, were detected after exposure to LiCl.
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PMID:Complementary chromatographic analysis of free diacylglycerols and potential glycerophospholipid precursors in human SH-SY5Y neuroblastoma cells following incubation with lithium chloride. 782 Feb 50


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