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 N and C terminals and tyrosine-phosphorylating site of the middle-sized tumor antigen of polyoma virus were chemically synthesized. The sequences of these peptides were Met-Asp-Arg-Val-Leu-Ser-Arg-Ala-Asp-Lys (N-MT), Met-Leu-Phe-Ile-Leu-Ile-Lys-Arg-Ser-Arg-His-Phe (C-MT), and Glu-Glu-Glu-Glu-Tyr-Met-Pro-Met-Glu (MT-Tyr), respectively. Among these peptides, the C-MT peptide inhibited phospholipase A2 (EC 3.1.1.4), phospholipase C (EC 3.1.4.3), and phospholipase D (EC 3.1.4.4). In addition, phosphatidylinositol-specific phospholipase C (EC 3.1.4.10) was also inhibited by this peptide. To study the mechanism of the inhibition, kinetic analysis was performed using phospholipase A2 from porcine pancreas. The degree of inhibition of phospholipase was dose dependent, and maximal inhibition was observed at pH 8.8. This peptide inhibited phospholipase A2 in a competitive manner for low-affinity sites of Ca2+, and in a noncompetitive manner for phospholipid substrates. When a fatty acid in the 2 position of the glycerol moiety of phosphatidylcholine was replaced by palmitic acid (C16:0), oleic acid (C18:1), linoleic acid (C18:2), eicosatrienoic acid (C20:3), or arachidonic acid (C20:4), the degree of inhibition of phosphatidylcholine hydrolysis by the C-MT peptide decreased. Inhibition of phospholipase A2 by the C-MT peptide was reversed by low concentrations of sodium deoxycholate but not by Triton X-100 or Nonidet P40, nonionic detergents. These detergents and the modification of acyl groups altered the micellar state of phospholipids. These results, taken together, suggest that the binding of the C-MT peptide near the low-affinity Ca2+ binding sites modifies the interaction of phospholipid substrates with the active center of phospholipase A2.
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PMID:Inhibition of phospholipases by Met-Leu-Phe-Ile-Leu-Ile-Lys-Arg-Ser-Arg-His-Phe, C terminus of middle-sized tumor antigen. 285 79

The attachment of glycolipid anchors to the Thy-1 glycoprotein during biosynthesis was followed by the change of detergent-binding properties of biosynthetically labelled Thy-1 precursors upon phospholipase C treatment in the murine thymoma lines BW5147 and S1A. In S1A, 80% of the Thy-1 molecules were phospholipase-C-sensitive after a 2 min pulse with [35S]methionine, indicating that these molecules were already anchored via a glycolipid tail. In BW5147, 47% of the Thy-1 molecules had phospholipase-C-sensitive anchors attached after a 1.5 min labelling and, with longer pulses, this percentage rose to 76%. Tunicamycin did not block the addition of glycolipid anchors, and glycolipid attachment also occurred at 21 degrees C. The findings suggest that the attachment of glycolipid anchors occurs in the rough endoplasmic reticulum.
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PMID:Glycolipid anchors are attached to Thy-1 glycoprotein rapidly after translation. 289 53

P30, the major surface antigen of the parasitic protozoan Toxoplasma gondii, can be specifically labeled with [3H]palmitic acid and with myo-[2-3H]inositol. The fatty acid label can be released by treatment of P30 with phosphatidylinositol-specific phospholipase C (PI-PLC). Such treatment exposes an immunological "cross-reacting determinant" first described on Trypanosoma brucei variant surface glycoprotein. PI-PLC cleavage of intact parasites metabolically labeled with [35S]methionine results in the release of intact P30 polypeptide in a form which migrates faster in polyacrylamide gel electrophoresis. These results argue that P30 is anchored by a glycolipid. Results from thin layer chromatography analysis of purified [3H] palmitate-labeled P30 treated with PI-PLC, together with susceptibility to mild alkali hydrolysis and to cleavage with phospholipase A2, suggest that the glycolipid anchor of T. gondii P30 includes a 1,2-diacylglycerol moiety.
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PMID:The major surface antigen, P30, of Toxoplasma gondii is anchored by a glycolipid. 292 21

Lipocortin I, a 35-kDa protein, has been detected in terminally differentiated monocytes and neutrophils. This calcium-phospholipid binding protein appears to be identical to a 35-kDa protein that can serve as a substrate for the EGF-receptor/tyrosine kinase. We have used the human myelocytic cell line HL-60 to explore whether differentiation of hematopoietic cells is associated with changes in the level of lipocortin I. We find that differentiation of HL-60 cells toward the macrophage lineage by the addition of phorbol esters or vitamin D3 or toward neutrophils with dibutyryl cyclic AMP or dimethyl sulfoxide is accompanied by an increase in the cellular content of lipocortin I. In comparison, treatment of HL-60 cells with bryostatin 1, a compound that activates protein kinase C but does not differentiate HL-60 cells, did not effect the level of 35 kDa protein. We have developed a radioimmunoassay to quantitate this protein by using a polyclonal antibody to a synthetic amino terminal peptide of the 35-kDa protein. This antibody recognizes purified pig lung 35-kDa protein as well as a single 35-kDa protein in HL-60 and A-431 cells as determined by Western blotting and immune precipitation. Differentiated HL-60 cells contain 2.6-fold the amount of 35-kDa protein found in undifferentiated HL-60 cells. Our findings that the addition of phorbol esters to HL-60 cells results in an increase in the mRNA for the 35-kDa protein and in an increase in the incorporation of 35S-methionine into the protein suggest that transcriptional activation or increased stability of the mRNA is responsible for the increased rate of synthesis and accumulation of lipocortin I during differentiation of these cells. In the absence of added divalent cations, we have determined that in differentiated HL-60 cells 79% of lipocortin I protein is located in the cytosol while 21% of the total cellular protein is bound to the particulate fraction. The 35-kDa protein can be removed from the particulate fraction by incubation with chelators or treatment with phospholipase A2 or phospholipase C. Addition of the calcium ionophore A23187 to intact differentiated HL-60 cells causes the 35-kDa protein to associate with the particulate fraction of the cell, suggesting that modulation of intracellular calcium levels may play a role in changing the intracellular location of this protein.
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PMID:Differentiation of HL-60 cells is associated with an increase in the 35-kDa protein lipocortin I. 297 67

Membranes prepared from DMSO-differentiated HL60 cells labeled with [3H]inositol hydrolyze polyphosphoinositides in a Ca2+-dependent manner, generating inositol 1,4-bisphosphate (IP2) and inositol 1,4,5-trisphosphate (IP3). Incubation of membranes with GTP or GTP gamma S reduces the concentration of Ca2+ required for activation. This nucleotide effect is potentiated by formyl-Met-Leu-Phe (FMLP). Pertussis toxin inhibits FMLP-induced augmentation, but not the induction of IP2/IP3 formation by GTP or GTP gamma S. These results suggest that differentiated HL60 cells contain a membrane-associated phospholipase C that degrades polyphosphoinositides and that activation of this enzyme is mediated by at least two guanine nucleotide binding proteins, one of which is linked to FMLP receptors and is pertussis toxin sensitive.
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PMID:Chemotactic peptide, calcium and guanine nucleotide regulation of phospholipase C activity in membranes from DMSO-differentiated HL60 cells. 303 41

The GTP-binding proteins involved in signal transduction now constitute a large family of so called 'G proteins'. Among them, Gs and Gi mediate the stimulation and inhibition of adenyl cyclase, respectively. Recently, another G protein (Go) abundant in brain was purified, but its function is still unknown. Like other G proteins, Go is a heterotrimer (alpha, beta, gamma) and the beta-gamma subunits seem to be identical to those of Gs and Gi. The alpha subunit of Go (Go-alpha) has a molecular weight of 39 kDa lower than those of Gi (41 kDa) or Gs (45-52 kDa). A positive immunoreativity with antibodies against Go-alpha was found in peripheral nervous tissues, adrenal medulla, heart, adenohypophysis and adipocytes. Go ressembles Gi in its ability to be ADP-ribosylated by pertussis toxin, and sequence analysis reveals a 68% homology between their alpha subunits. The GTPase activity of Go is several times higher than that of Gi. The affinity of the beta-gamma entity is about 3 times higher for Gi than for Go. In reconstitution studies, Go does not mimic the inhibitory effect of Gi on adenyl cyclase-stimulated by Gs. On the contrary, Go is as efficient as Gi in reconstituting the functional coupling with the muscarinic, alpha 2-adrenergic and chemotactic agent f-Met-Leu-Phe (fMLP), receptors. Recent studies seem to rule out Go as the coupling G protein of phospholipase C, the enzyme involved in phosphatidyl inositol trisphosphate hydrolysis. However, Go remains a putative candidate for transduction mechanisms coupled to a potassium channel or to a voltage-dependent calcium channel.
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PMID:Go, a major brain GTP binding protein in search of a function: purification, immunological and biochemical characteristics. 311 14

Amnion is believed to be a tissue of signal importance, anatomically and functionally, in the maintenance of pregnancy and during the initiation of parturition. Epidermal growth factor (EGF)-like agents cause a striking increase in the secretion of prostaglandin E2 (PGE2) in human amnion cells but only if arachidonic acid is present in the culture medium. To investigate the regulation of arachidonic acid metabolism by EGF-like agents in amnion, we used mEGF and human amnion cells in primary monolayer culture as a model system. The amount of PGE2 secreted into the culture medium was quantified by radioimmunoassay and the rate of conversion of [14C]arachidonic acid to [14C]PGE2 (PGH2 synthase activity) in cell sonicates was determined under optimal in vitro conditions. Treatment of amnion cells with mEGF led to a marked increase in the rate of production of PGE2. The specific activity of PGH2 synthase (viz. the combined activities of prostaglandin endoperoxide (PGH2) synthase and PGH2-PGE isomerase) was increased by 2-5-fold in cells treated with mEGF. Treatment of amnion cells with mEGF for 4 h did not affect the specific activities of phospholipase A2 or phosphatidylinositol-specific phospholipase C. By immunoisolation of newly synthesized, [35S]methionine-labeled PGH2 synthase, we found that mEGF stimulated de novo synthesis of the enzyme. Thus, mEGF acts in human amnion cells in primary monolayer culture to increase the rate of PGE2 biosynthesis by a mechanism that involves induction of PGH2 synthase; the manifestation of EGF action on PGE2 biosynthesis is dependent on the presence of nonesterified arachidonic acid.
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PMID:Epidermal growth factor stimulation of prostaglandin E2 biosynthesis in amnion cells. Induction of prostaglandin H2 synthase. 313 28

Using high-resolution 2-dimensional gel electrophoresis to separate proteins from cells labeled in vivo with either [32P]phosphate or [35S]methionine, the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) was shown to stimulate phosphorylation of at least 18 proteins in a subline of S49 mouse lymphoma cells deficient in cyclic AMP-dependent protein kinase. Phosphorylation of these proteins was not altered by phorbol acetate, a phorbol ester inactive in tumor promotion, and stimulation by TPA was half-maximal at less than 16 nM; therefore, these responses appeared to reflect specific interactions of TPA with high-affinity receptors. Treatment of cells with phospholipase C mimicked TPA in stimulating phosphorylation of some of these substrate proteins, thereby suggesting possible involvement of protein kinase C, the calcium-activated phospholipid-dependent protein kinase. Substrates differed in their relative responses to phospholipase C, the kinetics and concentration dependence of their phosphorylation in response to TPA, their extents of TPA-stimulated changes in phosphorylation, and their responses to tetracaine and retinal, two inhibitors of protein kinase C. Using these responses as criteria for classification, the TPA-mediated phosphorylations could be shown to fall into at least three distinct groups. The significance of these results to regulation of intracellular protein phosphorylation, to the relationship of protein kinase C and phorbol ester receptors, and to possible heterogeneity in kinases stimulable by phorbol ester tumor promoters is discussed.
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PMID:Phorbol ester-mediated protein phosphorylations in S49 mouse lymphoma cells. 315 48

There exists circumstantial evidence for activation of phospholipase D (PLD) in intact cells. However, because of the complexity of phospholipid remodeling processes, it is essential to distinguish PLD clearly from other phospholipases and phospholipid remodeling enzymes. Therefore, to establish unequivocally PLD activity in dimethyl sulfoxide-differentiated HL-60 granulocytes, to demonstrate the relative contribution of PLD to phospholipid turnover, and to validate the hypothesis that the formation of phosphatidylethanol is an expression of PLD-catalyzed transphosphatidylation, we have developed methodologies to label HL-60 granulocytes in 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkyl-PC) with 32P without labeling cellular ATP. These methodologies involve (a) synthesis of alkyl-lysoPC containing 32P by a combination of enzymatic and chemical procedures and (b) incubation of HL-60 granulocytes with this alkyl-[32P] lysoPC which enters the cell and becomes acylated into membrane-associated alkyl-[32P]PC. Upon stimulation of these 32P-labeled cells with the chemotactic peptide, N-formyl-Met-Leu-Phe (fMLP), alkyl-[32P]phosphatidic acid (alkyl-[32P]PA) is formed rapidly. Because, under these conditions, cellular ATP has not been labeled with 32P, alkyl-[32P]PA must be formed via PLD-catalyzed hydrolysis of alkyl-[32P]PC at the terminal phosphodiester bond. This result conclusively demonstrates fMLP-induced activation of PLD in HL-60 granulocytes. These 32P-labeled HL-60 granulocytes have also been stimulated in the presence of ethanol to produce alkyl-[32P]phosphatidylethanol (alkyl-[32P]PEt). Formation of alkyl-[32P]PEt parallels that of alkyl-[32P]PA with respect to time course, fMLP concentration, inhibition by a specific fMLP antagonist (t-butoxycarbonyl-Met-Leu-Phe), and Ca2+ concentration. These results strongly support the hypothesis that in HL-60 granulocytes, PEt is formed via PLD-catalyzed transphosphatidylation. Moreover, using HL-60 granulocytes double-labeled by incubation with [3H]alkyl-lysoPC and alkyl-[32P]lysoPC, it has been established that the early (30 s) appearance of alkyl-PA is due primarily to PLD, not phospholipase C as previously thought, and that alkyl-PEt is formed exclusively by PLD. These results constitute the first direct evidence for receptor-linked activation of PLD, leading to the generation of PA and PEt in an intact cell system.
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PMID:Phospholipase D catalyzes phospholipid metabolism in chemotactic peptide-stimulated HL-60 granulocytes. 316 77

The biosynthesis and post-translational modification of placental alkaline phosphatase were studied in human choriocarcinoma cells, JEG-3. Pulse-chase experiments with [35S]methionine demonstrated that placental alkaline phosphatase was synthesized as a major precursor form with Mr 63,000, which was then converted to a mature form with Mr 66,000, by processing of its N-linked oligosaccharides from the high-mannose type to the complex type. In addition, the two forms of the protein were found to be modified by a glycophospholipid, components of which were characterized by metabolic incorporation into placental alkaline phosphatase of 3H-labeled compounds such as myo-inositol, palmitic acid, stearic acid, mannose, glucosamine, and ethanolamine. When placental alkaline phosphatase labeled with these compounds was treated with phosphatidylinositol-specific phospholipase C or papain, the phospholipase C removed only the 3H-labeled fatty acids, whereas papain, that is known to cleave the C-terminal region, released all the radioactive glycolipid components including [3H]ethanolamine. More detailed analysis with shorter pulse-chase experiments demonstrated that placental alkaline phosphatase was primarily synthesized as a form with Mr 64,500 which was not yet labeled with [3H]palmitic acid. This form was converted by papain digestion to the above-mentioned major precursor with Mr 63,000. Taken together, these results suggest that placental alkaline phosphatase is initially synthesized as the precursor with Mr 64,500, which is immediately converted to the intermediate form with Mr 63,000 by simultaneously occurring proteolysis of the C terminus and replacement by the glycophospholipid, and finally to the mature form with Mr 66,000 by terminal glycosylation of its N-linked oligosaccharides. The glycophospholipid thus attached is considered to function as the membrane-anchoring domain of placental alkaline phosphatase.
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PMID:Biosynthesis of placental alkaline phosphatase and its post-translational modification by glycophospholipid for membrane-anchoring. 334 38


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