Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

The effects of low density lipoprotein (LDL) and high density lipoprotein (HDL3) on second messenger systems were investigated in cultured human vascular smooth muscle cells (VSMC) and compared with those of angiotensin II (Ang II) and platelet-derived growth factor (PDGF-BB). Phosphoinositide metabolism was studied in myo-[2-3H]-inositol prelabelled VSMC using high performance liquid anion-exchange chromatography. The spectra of inositol phosphate isomers increased after stimulation with either Ang II, LDL, HDL3 or PDGF-BB were qualitatively identical. Major increases occurred in 4-IP1, 1,4-IP2, 1,3,4-IP3 and 1,3,4,5-IP4. These are metabolic conversion products of 1,4,5-IP3 for which only a minor increase was found. Thus lipoproteins, like Ang II and PDGF-BB, activate polyphosphatidylinositol-specific phospholipase C. Intracellular Ca2+ concentrations ([Ca2+]i) were studied in fura-2 loaded VSMC. In monolayer cultures LDL and HDL3 increased [Ca2+]i with kinetics comparable to those for Ang II. Relative to the effects of these agonists, the PDGF-BB-induced increase in [Ca2+]i was slower in onset and the decay from peak [Ca2+]i levels more gradual. Fluorescence recordings from single cells exposed to LDL and HDL3 revealed a prolonged series of transient oscillations of [Ca2+]i, a phenomenon typical for calcium-mobilizing hormones. Additionally, as found for Ang II, preincubation of VSMC with either phorbol 12-myristate, 13-acetate, forskolin or 8-bromo-cyclic GMP inhibited LDL- and HDL-induced accumulation of [3H]-inositol monophosphate. We propose that LDL and HDL3 stimulate signal transduction in VSMC via mechanisms analogous to those of Ca(2+)-mobilizing hormones.
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PMID:Phosphoinositide and calcium signalling responses in smooth muscle cells: comparison between lipoproteins, Ang II, and PDGF. 133 16

An economical enzymatic assay for HDL phosphatidyl choline is described, as adapted for the Cobas-Bio analyser (Hoffmann LaRoche). This method entails the enzymatic cleavage of phosphatidyl choline by phospholipase C from B. cereus, hydrolysis of phosphoryl choline and enzymatic determination of choline with choline oxidase by an enzymatic colour test. This method provides consistent values and is, by comparison to the enzymatic UV method (assaying choline with choline kinase in an optical test procedure), simpler to perform, more precise, and less expensive.
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PMID:An economical assay for HDL phosphatidyl choline. 635 3

A routine method is described for the enzymatic determination of phosphatidyl choline in the apolipoprotein B-free supernatant after precipitation of blood sera with phosphotungstic acid/MgCl2. The principle of this method is based on the specific cleavage of phosphatidyl choline by purified phospholipase C from B. cereus, and the enzymatic determination of choline by choline kinase after hydrolysis of phosphoryl-choline. The enzymatic method provides HDL phosphatidyl choline values which coincide with those of the conventional chemical method. Furthermore, the values obtained with the enzymatic method for the HDL fraction isolated by ultracentrifugation (1.063--1.21 kg/l) also closely coincide with those of the apolipoprotein B-free supernatant fraction. The precision, linearity and sample stability were also checked. The findings obtained show that the enzymatic assay introduced here is suitable for the routine determination of phosphatidyl choline in the apolipoprotein B-free supernatant.
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PMID:Determination of HDL phosphatidyl choline by an enzymatic method. 685 25

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

Combined treatment with trypsin, cholesterol esterase, and neuraminidase transforms LDL, but not HDL or VLDL, to particles with properties akin to those of lipid extracted from atherosclerotic lesions. Single or double enzyme modifications, or treatment with phospholipase C, or simple vortexing are ineffective. Triple enzyme treatment disrupts the ordered and uniform structure of LDL particles, and gives rise to the formation of inhomogeneous lipid droplets 10-200 nm in diameter with a pronounced net negative charge, but lacking significant amounts of oxidized lipid. Enzymatically modified LDL (E-LDL), but not oxidatively modified LDL (ox-LDL), is endowed with potent complement-activating capacity. As previously found for lipid isolated from atherosclerotic lesions, complement activation occurs to completion via the alternative pathway and is independent of antibody. E-LDL is rapidly taken up by human macrophages to an extent exceeding the uptake of acetylated LDL (ac-LDL) or oxidatively modified LDL. After 16 h, cholesteryl oleate ester formation induced by E-LDL (50 micrograms/ml cholesterol) was in the range of 6-10 nmol/mg protein compared with 3-6 nmol/mg induced by an equivalent amount of acetylated LDL. At this concentration, E-LDL was essentially devoid of direct cytotoxic effects. Competition experiments indicated that uptake of E-LDL was mediated in part by ox-LDL receptor(s). Thus, approximately 90% of 125I-ox-LDL degradation was inhibited by a 2-fold excess of unlabeled E-LDL. Uptake of 125I-LDL was not inhibited by E-LDL. We hypothesize that extracellular enzymatic modification may represent an important step linking subendothelial deposition of LDL to the initiation of atherosclerosis.
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PMID:On the pathogenesis of atherosclerosis: enzymatic transformation of human low density lipoprotein to an atherogenic moiety. 750 42

We compared HDL3- and LDL-induced signal transduction in normal and Tangier fibroblasts to elucidate whether impaired signal transduction responses to lipoproteins might contribute to disturbed cellular lipid and lipoprotein metabolism in Tangier disease, a rare autosomal disorder of cellular lipid and lipoprotein metabolism. In several cell types HDL and LDL activate a currently unknown isoform of phosphatidylinositol-specific phospholipase C (PI-PLC) that results in the generation of 1,2-diacylglycerol and inositol 1,4,5-trisphosphate. Compared with normal fibroblasts, Tangier fibroblasts stimulated with HDL3 or LDL resulted in a significantly reduced accumulation of inositol phosphates and 1,2-diacylglycerol formation. Furthermore, in Tangier fibroblasts both lipoproteins failed to mobilize calcium from internal pools, and the cytosol-to-membrane redistribution of protein kinase C (in both the alpha and epsilon isoforms) was markedly reduced. Thus, the data indicate an impaired PI-PLC activation in response to lipoproteins in Tangier fibroblasts.
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PMID:Activation of phosphatidylinositol-specific phospholipase C in response to HDL3 and LDL is markedly reduced in cultured fibroblasts from Tangier patients. 767 Sep 51

We have isolated the core aldehydes (aldehydes still bound to parent molecules) of phosphatidylcholine (PC) and cholesteryl esters (CE) from copper-catalyzed peroxidation of human plasma low (LDL) and high (HDL) density lipoproteins. The aldehydes were isolated by extraction with acidified chloroform-methanol containing 2,4-dinitrophenylhydrazine. The 2,4-dinitrophenylhydrazone (DNPH) derivatives formed were resolved by reversed phase high performance liquid chromatography (HPLC) and identified by on-line quadrupole mass spectrometry (LC/MS). The major PC core aldehydes from oxidized LDL and HDL were identified as 1-palmitoyl-(1-stearoyl) 2-(9-oxononanoyl)-, 1-palmitoyl-(1-stearoyl) 2-(8-oxooctanoyl)-, and 1-palmitoyl-(1-stearoyl) 2-(5-oxovaleroyl)-sn-glycerols after phospholipase C digestion of the DNPH derivatives of the phospholipids. The major aldehydes from peroxidation of cholesteryl esters were the 9-oxononanoyl, 8-oxooctanoyl, and 5-oxovaleroyl esters of cholesterol and 7-ketocholesterol. The core aldehydes were estimated to account for a minimum of 1-2% of the consumed linoleate and arachidonate esters. A relatively smaller yield of the PC core aldehydes from LDL compared to HDL was attributed to the presence of greater amounts of phospholipases in LDL than in HDL. More comparable yields of PC core aldehydes were obtained in the presence of phenylmethylsulfonylfluoride, which inhibits phospholipases. We conclude that peroxidation of LDL and HDL results in formation of detectable amounts of cholesteryl and glycerophospholipid esters containing aldehyde functions. The yield of PC aldehydes varies with the activity of the platelet activating factor (PAF) acetyl hydrolase.
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PMID:Lipid ester-bound aldehydes among copper-catalyzed peroxidation products of human plasma lipoproteins. 855 76

The enzyme hepatic lipase may play several roles in lipoprotein metabolism. Recent investigation has suggested a role for the enzyme in lipoprotein and/or lipoprotein lipid uptake. To study this, a simple isolated system that mimics the in vivo system would be desirable. The enzyme is secreted by the hepatic parenchymal cell but exists, and presumably exerts its effects, while bound to capillary endothelial cells in the liver, adrenal gland, and the ovary. We constructed a cDNA that encodes the expression of a chimeric protein composed of rat hepatic lipase and the signal sequence for the addition of the glycophosphatidylinositol (GPI) anchor from human decay-accelerating factor. When transfected into Chinese hamster ovary (CHO) cells this gave rise to a cell population that had immunoreactive hepatic lipase on the cell surface. Cloning of the transfected cells produced several cell lines that expressed the chimeric protein bound to the cell surface by a GPI anchor. This was documented by demonstrating incorporation of [3H]ethanolamine into anti-hepatic lipase immunoprecipitable material; in addition, hepatic lipase was released from the cells by phosphatidylinositol-specific phospholipase C but not by heparin. Phosphatidylinositol-phospholipase C treatment of cells expressing the anchored lipase released material that comigrated with hepatic lipase on SDS-polyacrylamide gel electrophoresis and was immunoreactive with antibody to the cross-reacting determinant of GPI anchors. Cell lysates containing the anchored protein contained salt-resistant lipase activity, a known feature of the secreted hepatic lipase; thus it appears that these cells have a surface-anchored hepatic lipase molecule. Although it was not possible to demonstrate lipolysis by the enzyme while it was on the cell surface for technical reasons, the protein produced by these cells was active when studied in cell membranes. The ability of the cells to take up lipoproteins was studied. The cells demonstrated an increased affinity for low density lipoprotein (LDL) receptor mediated uptake of LDL. They did not, however, demonstrate any enhanced binding or removal of chylomicron remnants. With respect to LDL and remnants, the cells expressing anchored lipase behaved similarly to CHO cell that expressed secreted hepatic lipase. The cells expressing anchored hepatic lipase had a marked increase in the uptake of high density lipoprotein and high density lipoprotein cholesteryl ester when compared to that seen with CHO cells secreting hepatic lipase. This increase occurred primarily via the selective pathway, and was not reduced by addition of anti-LDL receptor or anti-hepatic lipase antibodies or the receptor-associated protein. Together the results suggest that hepatic lipase, when bound to the cell surface by a GPI anchor, plays a role in enhancing lipoprotein uptake. For LDL this may involve the provision of a second foot for particle binding, thus enhancing affinity for the LDL receptor. For chylomicron remnants an additional molecule or molecules are necessary to mediate this effect. For HDL, the enzyme facilitates uptake of cholesteryl ester primarily by the selective pathway.
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PMID:Chinese hamster ovary cells expressing a cell surface-anchored form of hepatic lipase. Characterization of low density lipoprotein and chylomicron remnant uptake and selective uptake of high density lipoprotein-cholesteryl ester. 866 89

The negative correlation between coronary heart disease and plasma levels of HDL has been attributed to the ability of HDL to take up cellular cholesterol. The HDL3-induced removal of cellular cholesterol was reported to be impaired in fibroblasts from patients with familial HDL deficiency (Tangier disease, TD). In addition, we have recently shown that HDL3 stimulates the hydrolysis of phosphatidylcholine (PC) in cholesterol-loaded fibroblasts. To investigate whether this cell signaling pathway is involved in cholesterol efflux mechanisms, we compared the HDL3-induced PC hydrolysis in normal fibroblasts and in fibroblasts from a TD kindred, in whom the HDL3- and apolipoprotein A-I (apo A-I)-induced mobilization of cellular cholesterol was found to be reduced by 50%. The HDL3-induced formation of phosphatidic acid (PA) via PC-specific phospholipase D (PC-PLD) was markedly reduced by 60-80% in these cells, whereas the formation of diacylglycerol (DG) via PC-specific phospholipase C (PC-PLC) was two- to threefold enhanced. Defective regulation of PC-PLC and PC-PLD was similarly observed in response to apo A-I and endothelin, but not in response to the receptor-independent stimulation of PC hydrolysis by PMA. A Tangier-like PA and DG formation pattern could be induced in normal cells after preincubation with pertussis toxin, suggesting the involvement of a G-protein. The impaired mobilization of radiolabeled cellular cholesterol in TD cells could completely be overcome by increasing the PA levels in the presence of the PA phosphohydrolase inhibitor propranolol. Conversely, the inhibition of PA formation in the presence of 0.3% butanol as well as the inhibition of DG formation in the presence of the PC-PLC inhibitor D 609 reduced the mobilization of cellular cholesterol both in normal and in TD cells. Our data indicate that the coordinate formation of PA and DG via PC-PLD and PC-PLC is essential for efficient cholesterol efflux. The molecular defect in this TD kindred appears to affect an upstream effector of protein kinase C responsible for the G-protein-dependent regulation of PC-specific phospholipases.
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PMID:Defective regulation of phosphatidylcholine-specific phospholipases C and D in a kindred with Tangier disease. Evidence for the involvement of phosphatidylcholine breakdown in HDL-mediated cholesterol efflux mechanisms. 894 49

To identify the HDL3-binding proteins on human macrophages, we examined the involvement of GPI-anchored protein in the binding of HDL3, and tried to purify HDL3-binding protein. From membrane fractions of macrophages, we obtained 80- and 130-kDa HDL3-binding proteins by ligand blotting. Treatment of macrophages with phosphatidylinositol-specific phospholipase C (PI-PLC) significantly decreased the specific HDL3-binding in a dose-dependent manner. Furthermore, treatment with mannosamine, which blocks GPI-anchor formation, decreased specific HDL3-binding in a dose-dependent manner. PI-PLC treatment released from the cells the proteins with an M(r) of 80 kDa, which could also bind HDL3. PI-PLC as well as mannosamine treatment markedly reduced cholesterol efflux from macrophages in association with the decreased HDL-binding. Using HDL3-affinity chromatography, we purified 80-kDa GPI-anchored type HDL3-binding protein. In summary, we demonstrate the implication of 80-kDa GPI-anchored protein in the binding of HDL3 to human macrophages, which might have some role in reverse cholesterol transport.
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PMID:Identification of a GPI-anchored type HDL-binding protein on human macrophages. 1086 Aug 43


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