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 possibility that thrombin-induced platelet reactivity could occur via both a receptor-related and a proteolytic process was examined. Thrombin elicited the formation of considerably more [32P]phosphatidic acid (an index of phospholipase C catalysed phosphoinositide metabolism) than did platelet activating factor, 5-hydroxytryptamine, ADP, and the thromboxane A2 analogue EP171, when these agents were added either alone or in combination. Co-addition of thrombin and EP171 did not evoke significantly more [32P]phosphatide acid than did thrombin alone. The protease inhibitor leupeptin, decreased but did not abolish [32P]phosphatidic acid formation elicited by either thrombin alone or thrombin in combination with EP171. The serine protease, trypsin, stimulated an increase in [32P]phosphatidic acid and this effect was additive with that of EP171. This augmentation by trypsin of EP171-induced [32P]phosphatidic acid formation was inhibited by leupeptin. These results are consistent with the concept that thrombin-induced activation of phospholipase C occurs by two distinct mechanisms: one via proteolysis, which is sensitive to leupeptin, and the other via receptor activation, a process shared by EP171. The individual components of this dual mechanism can be mimicked by the co-addition of a receptor-directed agonist (EP171) and a proteolytic agent (trypsin).
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PMID:Evidence for two mechanisms of thrombin-induced platelet activation: one proteolytic, one receptor mediated. 255 47

Investigations on the turnover of the membrane-form variant surface glycoprotein (mfVSG) of Trypanosoma brucei during cultivation in vitro of the monomorphic variant clones MIT at 1.2 and MIT at 1.4 showed that bloodstream forms slowly released the surface coat into the medium (time required to decline to half the initial amount, t50% = 32 +/- 3 h). VSG appeared in the medium in its soluble form (sVSG) which lacked the dimyristoylglycerol membrane anchor as judged by electrophoretic mobility and exposure of the cross-reacting determinant. The total VSG in the culture was very stable with a t50% = 189 +/- 24 h, compared to the other cellular proteins with a t50% approximately 28 h. Coat release during differentiation of bloodstream forms to procyclic cells could be distinguished from this turnover both by its more rapid kinetics (t50% = 13 +/- 1 h) and by the appearance in the medium of a predominant proteolytic fragment in addition to sVSG. Coat release during the transition to procyclic forms was not inhibited by the lysosomotropic agents ammonium chloride or chloroquine, by the proton ionophore monensin, or by the protease inhibitor tosyl-L-lysine chloromethyl ketone. The experiments demonstrate that coat release during differentiation is a specific cellular event distinct from simple turnover. The possibility is discussed that VSG release under both conditions occurs by endocytosis of mfVSG, degradation by a phospholipase C or a protease or both in a non-acidic intracellular compartment and recycling to the surface by exocytosis.
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PMID:Release of the variant surface glycoprotein during differentiation of bloodstream to procyclic forms of Trypanosoma brucei. 291 Dec 81

The serine protease inhibitors diethyl p-nitrophenyl phosphate and phenylmethylsulfonyl fluoride (chemical modifiers of serine residue) and N-acetyl-l-tryptophan ethyl ester (competitive inhibitor of chymotryptic protease) inhibited 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC; platelet-activating factor)-induced platelet aggregation and secretion. The inhibition was dependent on the preincubation time with the serine protease inhibitor and on the concentration of AGEPC and inhibitor. The IC50 value of diethyl-p-nitrophenyl phosphate, phenylmethylsulfonyl fluoride, and N-acetyl-l-tryptophan ethyl ester towards 5 X 10(-10) M AGEPC in serotonin release was 2.2 X 10(-4), 8.0 X 10(-4), and 5.0 X 10(-4) M, respectively. In experiments where platelets were incubated with these inhibitors and then washed with buffer, the inhibition of AGEPC stimulation was not observed. Prostaglandin H2 analog U46619 (10(-6) to 10(-5) M)- and thrombin (0.1 unit/ml)-induced platelet activation were also blocked by 1 mM diethyl p-nitrophenyl phosphate and 1 mM N-acetyl-l-tryptophan ethyl ester. The binding of AGEPC (1.5 X 10(-11) to 9.4 X 10(-10) M) to platelets and the platelet cyclic AMP level were not affected by diethyl p-nitrophenyl phosphate, phenylmethylsulfonyl fluoride, and N-acetyl-l-tryptophan ethyl ester. However, 1 mM diethyl p-nitrophenyl phosphate and 1 mM N-acetyl-l-tryptophan ethyl ester suppressed 10(-9) M AGEPC-induced breakdown of phosphatidylinositol 4,5-bisphosphate and formation of phosphatidic acid to 10-12 and 39-42%, 40-kDa protein phosphorylation to 4 and 30%, and arachidonic acid release to 17 and 28% of controls, respectively. On the other hand, 5 mM diethyl p-nitrophenyl phosphate did not inhibit diacylglycerol production and arachidonic acid release initiated by 2.5 mM deoxycholate treatment, suggesting that receptor-mediated phospholipase C and phospholipase A2 activation were inhibited by the serine protease inhibitor, but the deoxycholate (physicochemical stimulant)-initiated activation was not. AGEPC-induced 20-kDa protein phosphorylation and the inhibitory action of AGEPC on cyclic AMP accumulation were abolished in the presence of diethyl p-nitrophenyl phosphate and phenylmethylsulfonyl fluoride. However, a tryptic protease inhibitor, 1 mM p-aminobenzamidine and 1 mM benzoyl-l-arginine methyl ester, did not prevent the AGEPC-induced platelet secretion.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Platelet-activating factor stimulation of rabbit platelets is blocked by serine protease inhibitor (chymotryptic protease inhibitor). 310 43

A maximally effective dose of indomethacin does not prevent serotonin release and aggregation in human platelets stimulated with thrombin. Thrombin induces rapid activation of inositol phospholipids-specific phospholipase C, which is reflected by the degradation of inositides and the phosphorylation of the resultant 1,2-diacylglycerol to phosphatidic acid. Thrombin also activates protein kinase C and myosin light chain kinase as indicated by phosphorylation of the 40,000 and 20,000 dalton proteins, respectively. Leupeptin, a protease inhibitor that does not inhibit thrombin's proteolytic activity or its binding to platelet surface, is able to reverse platelet activation by thrombin when it is administered after the addition of the agonist and indomethacin. The results suggest a proteolytic-mediated pathway in transmembrane signalling involved in platelet activation by thrombin.
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PMID:Sustained proteolysis is required for human platelet activation by thrombin. 371 2

a cDNA library transfer system based on retroviral vectors has been developed for expression cloning in mammalian cells. The use of retroviral vectors results in stable cDNA transfer efficiencies which are at least 100-fold higher than those achieved by transfection and therefore enables the transfer and functional screening of very large libraries. In our initial application of retroviral transfer of cDNA libraries, we have selected for cDNAs which induce oncogenic transformation of NIH 3T3 fibroblasts, as measured by loss of contact inhibition of proliferation. Nineteen different transforming cDNAs were isolated from a total of 300,000 transferred cDNA clones. Three of these cDNAs were derived from known oncogenes (raf-1, lck, and ect2), while nine others were derived from genes which had been cloned previously but were not known to have the ability to transform fibroblasts (beta-catenin, thrombin receptor, phospholipase C-gamma 2 and Spi-2 protease inhibitor genes). The Spi-2 cDNA was expressed in antisense orientation and therefore is likely to act as an inhibitor of an endogenous transformation suppressor. Seven novel cDNAs with transforming activities, including those for three new members of the CDC24 family of guanine nucleotide exchange factors, were also cloned from the retroviral cDNA libraries. Retroviral transfer of libraries should be generally useful for cloning cDNAs which confer selectable phenotypes on many different types of mammalian cells.
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PMID:Expression cloning of oncogenes by retroviral transfer of cDNA libraries. 782 39

Tumor necrosis factor (TNF) is one of the most potent physiological inducers of the nuclear transcription factor kappa B (NF-kappa B). A key event in the activation of NF-kappa B is the rapid release of the inhibitory subunit I kappa B-alpha. Various inhibitors of serine-like proteases are shown to block TNF-mediated NF-kappa B activation as well as the disappearance of I kappa B-alpha immunoreactivity in primary murine T lymphocytes and in various human leukemic cell lines. The protease inhibitors did not block TNF-induced activation of either phosphatidylcholine-specific phospholipase C or acidic sphingomyelinase (SMase), indicating that the putative protease operates rather downstream of TNF signal transduction processes. I kappa B-alpha degradation could be directly induced by addition of sphingomyelinase or synthetic ceramide to a cell-free system, indicating a stringent coupling of SMase to the NF-kappa B activation pathway. SMase-induced I kappa B-alpha degradation was suppressed by the protease inhibitor dichloroisocoumarin. Together, the data suggest that a TNF-responsive sphingomyelinase triggers the rapid degradation of I kappa B-alpha through a serine-like protease, which appears to be crucial to the control of NF-kappa B activation.
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PMID:Sphingomyelinase activates proteolytic I kappa B-alpha degradation in a cell-free system. 818 52

We have recently reported that the activated serine protease and blood coagulation Factor VII (FVIIa) can induce Ca2+ oscillations in Madin-Darby canine kidney cells. We now demonstrate a similar response by Madin-Darby canine kidney cells to the active coagulation Factor X (FXa), which is also a serine protease and a substrate of the tissue factor (TF).FVIIa complex in the initiation of the coagulation cascade. The phosphatidyl inositol-specific phospholipase C inhibitor U73122 inhibited the signals elicited by both FVIIa and FXa. Lack of sensibility to the tyrosine kinase inhibitors herbimycin A, genistein, and the tyrphostin AG18 and discordance between TF expression and FVIIa responsiveness argued against TF acting as a cytokine-like receptor, with tyrosine kinase-mediated activation by FVIIa. As demonstrated using the protease inhibitor benzamidine and by specific active site inhibition with 1,5-dansyl-Glu-Gly-Arg chloromethyl ketone, both FVIIa and FXa lost their ability to elicit a calcium response when devoid of their proteolytic activity. Consistent with this, the native (zymogen) form of Factor X did not induce Ca2+ transients. Homologous but not heterologous inhibition of FVIIa- and FXa-evoked Ca2+ signals by 1,5-dansyl-Glu-Gly-Arg chloromethyl ketone-inactivated FVIIa and FXa suggested that each factor had its own specific cell surface anchoring receptor. The two coagulation factors did not show homologous desensitization as seen for thrombin stimulation. Studies with hirudin excluded involvement of the established activation pathway through thrombin itself. Lack of desensitization of the response to FVIIa or FXa by thrombin ruled out any involvement of proteinase activated receptor-1 (PAR-1), the thrombin receptor. We speculate that FXa and FVIIa may work via a receptor (possibly common) analogous to PAR-1 or its functional homologue PAR-2. Although TF is essential for the FVIIa-induced signaling event, its role in the phosphatidyl inositol-specific phospholipase C-mediated Ca2+ signal may be in anchoring FVIIa to the cell surface rather than in transmembrane signal mediation.
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PMID:Coagulation factors VII and X induce Ca2+ oscillations in Madin-Darby canine kidney cells only when proteolytically active. 891 May 56

We recently demonstrated that the engagement of HLA class I alpha1 domain induced Fas-independent apoptosis in human T and B lymphocytes. We analyzed the signaling pathway involved in HLA class I-mediated apoptosis in comparison with Fas (APO-1, CD95)-dependent apoptosis. The mouse mAb90 or the rat YTH862 monoclonal antibodies which bind the human HLA class I alpha1 domain induced the production of ceramide which was blocked by addition of the phosphatidylcholine-dependent phospholipase C inhibitor, D609. Furthermore, HLA class I-mediated apoptosis involved at least two different caspases, an interleukin-1 converting enzyme-like protease and another protease inhibited by the CPP32-like protease inhibitor Ac-DEVD-CHO. Despite similarity between Fas and HLA class I signaling pathways, we failed to demonstrate any physical association between these two molecules. We also report that the pan-caspase inhibitory peptide zVAD-fmk, but not Ac-DEVD-CHO and Ac-YVAD-CHO, inhibited decrease of mitochondrial transmembrane potential and generation of ceramide induced by anti-HLA class I and anti-Fas monoclonal antibodies, whereas all three peptides efficiently inhibited apoptosis. Altogether these results suggest that signaling through Fas and HLA class I involve caspase(s), targeted by zVAD-fmk, which act upstream of ceramide generation and mitochondrial events, whereas interleukin-1 converting enzyme-like and CPP32-like proteases act downstream of the mitochondria.
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PMID:Caspase-dependent ceramide production in Fas- and HLA class I-mediated peripheral T cell apoptosis. 947 56

The contributions of several Ca(2+)-dependent processes to neurotoxicity were examined in primary cultures of rat cortical neurons. The Ca2+ ionophore ionomycin induced a rapid loss of axonal morphology and concomitant release of inositol phosphates that preceded morphological alterations of neuronal cell bodies, choline and arachidonate release, and protein degradation. These events were followed by a degree of neuronal lysis proportional to the external Ca2+ concentration and exposure time. The phospholipase inhibitor neomycin decreased both arachidonate release and the phospholipid hydrolysis catalysed by phospholipases C and D. Proteolysis was abated by the protease inhibitor leupeptin, but not by lysosomal proteolysis inhibitors. Neuronal lysis was inhibited partially by either leupeptin or neomycin and almost completely by both in combination. However, neither agent, alone or in combination, affected the morphological derangements. The diacylglycerol lipase inhibitor RHC-80267 reduced arachidonate release, but not neuronal lysis. Phospholipase A2 inhibitors had no effect on either arachidonate release or lysis. Treatment of mixed cultures of neurons and glia with a Ca(2+)-dependent glutamate challenge caused similar morphological changes and a delayed neuronal lysis that was also diminished by leupeptin and neomycin, but not by inhibitors of lysosomal proteolysis. These data describe several distinct stages of Ca(2+)-dependent injury to cortical neurons, a key feature of which is the stimulation of protease, and phospholipase C and D activities. The initial stage is characterized by a rapid loss of axonal morphology and increased phosphatidylinositol hydrolysis. An intermediate stage involves changes in cell body morphology plus the degradation of neuronal protein and phosphatidylcholine. In a later stage, the loss of plasma membrane integrity denotes neuronal death.
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PMID:Ca(2+)-dependent mechanisms of cell injury in cultured cortical neurons. 969 20

To identify a sperm-surface component that is highly antigenic, we immunized female cynomolgus macaques with glycosylphosphatidylinositol (GPI)-anchored sperm surface proteins that were released following treatment with phosphatidylinositol-specific phospholipase C (PI-PLC). Five different adjuvants were used in combination with the PI-PLC-released proteins, and three of these proteins (24, 48, and 53 kDa) were shown to be potent antigens for immunization of female monkeys. The 53 kDa protein was found to be a surface coating protein and not a GPI-anchored protein. Polyclonal antibodies to the 24 kDa protein and the 48 kDa protein were produced in rabbits. The two antibodies recognized both proteins on Western blots. The same rabbit antibodies recognized 28, 18, and 10 kDa bands on a Western blot of chemically reduced PI-PLC-released proteins, suggesting that the 48 kDa protein is a dimer of the 24 kDa protein, which we refer to as MAK248. Rabbit polyclonal antibodies developed to reduced fragments of the 24 kDa protein showed that the 18 and 10 kDa bands are proteolytic peptide fragments of the 24 kDa protein. Screening of tissues from male macaques showed that MAK248 is expressed only in the epididymis. Microsequencing of two proteolytic fragments of the 18 kDa component showed 100% amino acid homology to a 233 deduced amino acid sequence previously identified in human testes genome. Antibodies to MAK248 recognized a 24 kDa protein released from human sperm exposed to PI-PLC. Antibodies to MAK248 recognized the equatorial segment and posterior head regions of capacitated cynomolgus macaque sperm. Structural analysis suggests that MAK248 is a novel CRISP protein and a member of the CAP (CRISP, Ag 5, PR-1) family of proteins. Based on amino acid sequence homology, it is possible that MAK248 functions as a protease inhibitor.
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PMID:Identification of a novel GPI-anchored CRISP glycoprotein, MAK248, located on the posterior head and equatorial segment of cynomolgus macaque sperm. 1241 52


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