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)

We report that a 100 residue segment in the carboxy-terminus half of phosphatidylinositol-specific phospholipase C is similar to a segment in the amino-terminus half of protein kinase C. The computer-based comparison score is 9.5 standard deviations higher than that of 2500 comparisons of randomized sequences of these segments (P = 10(-21), suggesting that the two segments have similar biological properties. Phospholipase C has a segment that is homologous to part of the non-catalytic domain of src kinase and other tyrosine protein kinases. The similarity of phospholipase C with protein kinase C, a serine/threonine kinase suggests that novel exon shuffling occurred among serine/threonine and tyrosine kinases and phospholipase C.
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PMID:Similarity between phospholipase C and the regulatory domain of protein kinase C. 274 13

In lymphocytes, the Na+/H+ antiport can be stimulated by 12-O-tetradecanoylphorbol 13-acetate (TPA) and by osmotic shrinking. Since TPA acts by stimulating protein kinase C, we undertook experiments to determine if protein phosphorylation also underlies the osmotic stimulation of the antiport. We found that at least one of the membrane polypeptides labeled in cells treated with TPA is also phosphorylated by hypertonic shrinking. In both instances phosphorylation is alkali labile and associated with serine and threonine residues. We tested the possibility that shrinking activates phospholipase C, thereby stimulating protein kinase C through release of diacylglycerol. No decrease in phosphatidylinositol 4,5-bisphosphate levels was detected in hypertonically treated cells. Moreover, the concentrations of inositol phosphates, including inositol trisphosphate, were not altered in shrunken cells. Thus, shrinking does not appear to activate phospholipase C. Whereas TPA induced intracellular redistribution of soluble protein kinase C, no such effect was detected in osmotically activated cells. It was concluded that osmotic stimulation of the Na+/H+ antiport is associated with activation of protein phosphorylation by a kinase that is similar, but not identical to protein kinase C. Experiments in Na+-free or amiloride-containing media indicate that phosphorylation is not a consequence of activation of the antiport.
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PMID:Protein phosphorylation during activation of Na+/H+ exchange by phorbol esters and by osmotic shrinking. Possible relation to cell pH and volume regulation. 301 4

We have isolated a COOH-terminal tryptic peptide from the hydrophobic globular (5.6 S) form of Torpedo californica acetylcholinesterase that exhibits divergence in amino acid sequence from the catalytic subunit of the dimensionally asymmetric (17 S + 13 S) enzyme. The divergent peptide could be recovered from the glycophospholipid-modified 5.6 S enzyme only after treatment with phosphatidylinositol-specific phospholipase C. Upon reduction, carboxymethylation with [14C]iodoacetate, and trypsin digestion the resultant peptides were purified by gel filtration followed by high performance liquid chromatography. The high performance liquid chromatography profiles of 14C-labeled cysteine peptides from lipase-treated 5.6 S enzyme revealed unique radioactive peaks which had not been present in digests of the asymmetric form. These peaks all yielded identical amino acid sequences. The difference in chromatographic behavior of the individual peptides most likely reflects heterogeneity in post-translational processing. Gas-phase sequencing and composition analysis are consistent with the sequence: Leu-Leu-Asn-Ala-Thr-Ala-Cys. Composition includes 2-3 mol each of glucosamine and ethanolamine which is indicative of modification by glycophospholipid. Glucosamine is also present in an asparagine-linked oligosaccharide. The two forms of acetylcholinesterase diverge after the threonine residue within this peptide sequence; the hydrophobic form terminates with cysteine whereas the asymmetric form extends for 40 residues beyond the divergence. The locus of divergence and absence of any other amino acid sequence difference suggest that the molecular forms of acetylcholinesterase arise from a single gene by alternative mRNA processing.
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PMID:Divergence in primary structure between the molecular forms of acetylcholinesterase. 333 34

The COOH-terminal amino acid of carcinoembryonic antigen (CEA) is shown to covalently link with ethanolamine, evidence consistent with the anchorage of CEA to the plasma membrane through a phosphatidylinositol-glycan tail. Purified CEA was digested with trypsin, and the resulting peptides were isolated by reverse-phase HPLC. Tryptic hexapeptide T12, terminating atypically with alanine, corresponded in sequence (Ser-Ile-Thr-Val-Ser-Ala) with the last six residues (637-642) of the third repeating domain in the mature CEA protein. Mass determination of the hexapeptide by fast atom bombardment mass spectrometry suggested the presence of an additional ethanolamine moiety. This finding and the absence of the subsequent 26 hydrophobic residues predicted by cDNA sequence is evidence that hexapeptide T12 is the COOH-terminal peptide of mature CEA. A synthetic peptide identical to hexapeptide T12 was prepared, and ethanolamine was coupled to its COOH-terminal alanine; chromatographic properties of this synthetic ethanolamine-coupled peptide and peptide T12 were the same. B/E-linked-scan mass spectral analysis of the ethanolamine-coupled synthetic peptide and peptide T12 revealed a fragment ion series consistent with the presence of a COOH-terminal ethanolamine. Release of membrane-bound CEA from the CEA-expressing cell line LS 174T was shown by indirect immunofluorescence and flow cytometry after treatment with phosphatidylinositol-specific phospholipase C. We conclude that CEA is processed posttranslationally to remove the hydrophobic COOH-terminal residues (643-668) with subsequent addition of an ethanolamine-glycosylphosphatidylinositol moiety and that treatment of a colonic cell line with phosphatidylinositol-specific phospholipase C releases membrane-bound CEA.
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PMID:Carcinoembryonic antigen is anchored to membranes by covalent attachment to a glycosylphosphatidylinositol moiety: identification of the ethanolamine linkage site. 338 31

Previous attempts in several laboratories, including ours, to purify oligosaccharyl-transferase have met with limited success because of the lability of the membrane-associated enzyme after solubilization with detergents. In an effort to identify the enzyme in face of this lability, we recently developed a photoaffinity reagent to label the active site [J. K. Welply, P. Shenbagamurthi, F. Naider, H. R. Park, and W. J. Lennarz (1985) J. Biol. Chem. 260, 6459-6465]. In this report, the preparations of a more sensitive selective labeling probe, 125I-labeled N alpha-3-(4-hydroxyphenylpropionyl)-Asn-Lys-(N epsilon-p-azidobenzoyl)-Thr-NH2, is described. Using this new probe, we have confirmed, independently of catalytic activity, that hen oviduct oligosaccharyltransferase is tightly associated with the endoplasmic reticulum membrane. The 125I-labeled oligosaccharyltransferase was released from the membrane by detergent and strong alkali treatments but not by sonication, high salt, or hypotonic shock. However, all procedures that released the enzyme from the membrane resulted in a dramatic loss of enzyme activity. Treatment of sealed microsomal membrane vesicles with phospholipase A resulted in nearly complete enzyme inactivation; in contrast, phospholipase C or D had moderate or little effect, respectively. Taken together, these results suggest that the hydrophobic environment of the membrane is required for oligosaccharyltransferase activity. Trypsin treatment of intact vesicles diminished enzyme activity by nearly 70%, but it had no effect on the binding affinity of the enzyme for the 125I-labeled photoaffinity probe. This result suggests that the polypeptide acceptor portion of oligosaccharyltransferase is lumenally disposed, and that a trypsin-sensitive, cytoplasmically oriented domain or another subunit binds the carbohydrate donor, dolichol-PP-oligosaccharide.
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PMID:Studies on properties of membrane-associated oligosaccharyltransferase using an active site-directed photoaffinity probe. 370 33

The ability of dolichyl-P-P-oligosaccharide:peptide oligosaccharyltransferase to use exogenous substrates (a previously labeled oligosaccharide lipid and an Asn-X-Thr containing heptapeptide) is shown to require phospholipid. The enzyme was extracted from porcine thyroid rough microsomes using NaCl-Nonidet P-40. When measured at low concentration, in a neutral detergent-containing medium, it undergoes a rapid loss of activity, which renders impossible quantitative estimates in the range of 0-50 micrograms microsomal protein/50 microliters assay. We observed that inactivation could be prevented by supplementing the assay with a previously heat-treated suspension of microsomes in neutral detergent, or with the corresponding extract. Further investigation revealed that phospholipids are responsible for this enzyme stabilization, since phospholipase A2 and phospholipase C treatments were both able to abolish this effect. When individual phospholipids were compared for their protective efficiency, egg yolk phosphatidylcholine was found to be by far the most efficient. Phosphatidylglycerol, phosphatidylinositol and phosphatidylserine were only slightly effective, while phosphatidylethanolamine and lysophosphatidylcholine had no effect at all. Of those tested, partly unsaturated phosphatidylcholines with 16-18 carbon atom acyl chains were the most active, at an optimal concentration of 1-2 mM. Under these conditions a Km of 15 microM was measured for the acceptor, a synthetic ribonuclease heptapeptide, and a Km of 0.55 microM for the donor, dolichyl-P-P-GlcNAc2-Man9-Glc2-3. These findings were confirmed by subjecting a sodium deoxycholate extract to depletion of endogenous lipids by gel filtration. Enzyme activity was totally abolished and then restored (up to now only partially) by addition of phosphatidylcholine.
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PMID:Phosphatidylcholine requirement for the N-glycosylation of synthetic peptides by detergent-solubilized oligosaccharyltransferase. 654 Jan 20

Phospholipase C (heat-labile hemolysin) was purified from Pseudomonas aeruginosa culture supernatants to near homogeneity by ammonium sulfate precipitation followed by a novel application of DEAE-Sephacel chromatography. Enzymatic activity remained associated with DEAE-Sephacel even in the presence of 1 M NaCl, but was eluted with a linear gradient of 0 to 5% tetradecyltrimethylammonium bromide. Elution from DEAE-Sephacel was also obtained with 2% lysophosphatidylcholine, and to a lesser extent with 2% phosphorylcholine, but not at all with choline. The enzyme was highly active toward phospholipids possessing substituted ammonium groups (e.g., phosphatidycholine, lysophosphatidylcholine, and sphingomyelin); however, it had little if any activity toward phospholipids lacking substituted ammonium groups (e.g., phosphatidylethanolamine, phosphatidylserine, and phosphaditylglycerol). Collectively, these data suggest that phospholipase C from P. aeruginosa exhibits high affinity for substituted ammonium groups, but requires an additional hydrophobic moiety for optimum binding. The specific activity of the purified enzyme preparation increased 1,900-fold compared with that of culture supernatants. The molecular weight of the phospholipase C was estimated to be 78,000 by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Sephacryl S-200 column chromatography and was 76,000 by high-performance size exclusion chromatography. The isoelectric point was 5.5. Amino acid analysis showed that phospholipase C was rich in glycine, serine, threonine, aspartyl, glutamyl, and aromatic amino acids, but was cystine free.
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PMID:Phospholipase C (heat-labile hemolysin) of Pseudomonas aeruginosa: purification and preliminary characterization. 681 52

Src homology 2 (SH2) domains provide specificity to intracellular signaling by binding to specific phosphotyrosine (phospho-Tyr)-containing sequences. We recently developed a technique using a degenerate phosphopeptide library to predict the specificity of individual SH2 domains (src family members, Abl, Nck, Sem5, phospholipase C-gamma, p85 subunit of phosphatidylinositol-3-kinase, and SHPTP2 (Z. Songyang, S. E. Shoelson, M. Chaudhuri, G. Gish, T. Pawson, W. G. Haser, F. King, T. Roberts, S. Ratnofsky, R. J. Lechleider, B. G. Neel, R. B. Birge, J. E. Fajardo, M. M. Chou, H. Hanafusa, B. Schaffhausen, and L. C. Cantley, Cell 72:767-778, 1993). We report here the optimal recognition motifs for SH2 domains from GRB-2, Drk, Csk, Vav, fps/fes, SHC, Syk (carboxy-terminal SH2), 3BP2, and HCP (amino-terminal SH2 domain, also called PTP1C and SHPTP1). As predicted, SH2 domains from proteins that fall into group I on the basis of a Phe or Tyr at the beta D5 position (GRB-2, 3BP2, Csk, fps/fes, Syk C-terminal SH2) select phosphopeptides with the general motif phospho-Tyr-hydrophilic (residue)-hydrophilic (residue)-hydrophobic (residue). The SH2 domains of SHC and HCP (group III proteins with Ile, Leu, of Cys at the beta D5 position) selected the general motif phospho-Tyr-hydrophobic-Xxx-hydrophobic, also as predicted. Vav, which has a Thr at the beta D5 position, selected phospho-Tyr-Met-Glu-Pro as the optimal motif. Each SH2 domain selected a unique optimal motif distinct from motifs previously determined for other SH2 domains. These motifs are used to predict potential sites in signaling proteins for interaction with specific SH2 domain-containing proteins. The Syk SH2 domain is predicted to bind to Tyr-hydrophilic-hydrophilic-Leu/Ile motifs like those repeated at 10-residue intervals in T- and B-cell receptor-associated proteins. SHC is predicted to bind to a subgroup og these same motifs. A structural basis for the association of Csk with Src family members is also suggested from these studies.
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PMID:Specific motifs recognized by the SH2 domains of Csk, 3BP2, fps/fes, GRB-2, HCP, SHC, Syk, and Vav. 751 Dec 10

In the renal medulla during antidiuresis, the extracellular fluid becomes hyperosmotic. Madin-Darby canine kidney (MDCK) epithelial cells adapt in hyperosmotic conditions and serve as a useful tissue culture model for cellular responses to hyperosmolality. We demonstrate that hyperosmolality stimulates phospholipase C, Raf-1 kinase mitogen-activated protein (MAP) kinase kinase, MAP kinase, and S6 kinase activities and that it increases phosphorylation of Raf-1 kinase, and p42 MAP kinase in MDCK cells. Stimulation of these kinases is osmolality-dependent (from 300 to 600 mosm/kg H2O). The time course of activation is sequential; the peak stimulation for Raf-1 kinase is at 5 min, at 10 min for MAP kinase kinase and MAP kinase, and at 20 min for S6 kinase. The activation of Raf-1 kinase and MAP kinase is inhibited by phorbol 12-myristate 13-acetate pretreatment in the presence of calphostin C or H-7. Tyrosine kinase inhibitors (genistein, herbimycin) do not significantly suppress hyperosmolality-induced MAP kinase activity. The increase of Ins-1,4,5-P3 levels by hyperosmolality suggests that activation of these kinases is mediated at least partially via activation of phospholipase C. Thus, hyperosmolality stimulates the serine/threonine kinases, Raf-1 kinase, MAP kinase kinase, MAP kinase, and S6 kinase, via predominantly protein kinase C-dependent, tyrosine kinase-independent pathways in MDCK cells.
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PMID:Sequential activation of Raf-1 kinase, mitogen-activated protein (MAP) kinase kinase, MAP kinase, and S6 kinase by hyperosmolality in renal cells. 752 42

The substance P (neurokinin-1) receptor belongs to the family of seven putative transmembrane domain receptors that are coupled via G proteins to phospholipase C activation. Homologous desensitization of substance P-stimulated responses has been described in various systems. The rat neurokinin-1 receptor and a truncated mutant lacking the carboxyl-terminal region were expressed in Chinese hamster ovary cells to examine the mechanisms of substance P-induced desensitization. Wild-type and truncated receptor-bearing cells were indistinguishable in agonist binding affinity and EC50 of substance P-induced accumulation of 3H-inositol phosphates. Substance P-induced responses continued for 30-45 min in cells expressing wild-type and truncated receptors as well as in rat LRM-55 and human U373 cells, which express endogenous neurokinin-1 receptors. In transfected cells expressing the wild-type receptor, CP-96,345 added 15 min after substance P blocked further responses, demonstrating the continuing presence of responsive receptors. The rates of accumulation of 3H-inositol phosphates were four times greater in the initial 15 s of stimulation than for the next 20 min for both wild-type and truncated receptor types. This decrease in rate of substance P-stimulated phosphatidylinositol hydrolysis is therefore not dependent on the carboxyl-terminal region of the rat neurokinin-1 receptor, which contains 26 serine and threonine residues. These results are discussed in relation to current ideas regarding neurokinin-1 receptor desensitization.
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PMID:Similar rates of phosphatidylinositol hydrolysis following activation of wild-type and truncated rat neurokinin-1 receptors. 753 7


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