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)

Thiol:protein-disulfide oxidoreductase catalyzes the GSH reduction of protein disulfides to sulfhydryls. Chromatography of solubilized hepatic microsomes on Mono Q yielded two peaks, Q-2 and Q-5, which contained all the thiol:protein-disulfide oxidoreductase activity. These were further purified by chromatofocusing giving specific activities of 14.4 and 45.9 nmol/mg of protein/min, respectively with purifications of 45.0- and 143.6-fold. Amino acids 1-18 of Q-5 were the same as previously reported for Thiol:protein-disulfide oxidoreductase (Edman, J. C., Ellis, L., Blacher, R. W., Roth, R. A., and Rutter, W. J. (1985) Nature 317, 267-270), except amino acid 1 was leucine instead of aspartate and amino acid 6 was asparagine instead of glutamate. The N-terminal amino acid sequence of Q-2 differed markedly from Q-5 but Q-2 showed 100% identity at amino acids 25-54, 258-269, 285-310, 347-350, 412-419, and 434-463 for the reported sequence of rat, hepatic, cytosolic phosphatidylinositol-specific phospholipase C form 1a (PLC) (Bennett, C. F., Balcarek, J. M., Varrichio, A., and Crooke, S. T. (1988) Nature 334, 268-270). PLC activity was found in the elution from the Mono Q column, but none was found in purified Q-2 or Q-5. Antibodies to Q-5 reacted with Q-2, but anti-Q-2 did not react with Q-5. Anti-Q-2 antibody showed immunoreactivity with 55- and 60-kDa microsomal proteins, whereas Q-5 antibody reacted with a number of microsomal proteins. Although Q-2 was immunoreactive with a polyclonal antibody to guinea pig, uterine cytosolic PLC, partially purified PLCs from rat liver cytosol did not react to this antibody. Our data would suggest that the published sequence for PLC form 1a may actually be the sequence for Q-2.
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PMID:Purification and characterization of a new isozyme of thiol:protein-disulfide oxidoreductase from rat hepatic microsomes. Relationship of this isozyme to cytosolic phosphatidylinositol-specific phospholipase C form 1A. 165 21

Protection against the pore-forming activity of the human C5b-9 proteins was conferred on a nonprimate cell by transfection with cDNA encoding the human complement regulatory protein CD59. CD59 was stably expressed in Chinese hamster ovary cells using the pFRSV mammalian expression vector. After cloning and selection, the transfected cells were maintained in media containing various concentrations of methotrexate, which induced surface expression of up to 4.2 x 10(6) molecules of CD59/cell. Phosphatidylinositol-specific phospholipase C removed greater than 95% of surface-expressed CD59 antigen, confirming that recombinant CD59 was tethered to the Chinese hamster ovary plasma membrane by a lipid anchor. The recombinant protein exhibited an apparent molecular mass of 21-24 kDa (versus 18-21 kDa for human erythrocyte CD59). After N-glycanase digestion, recombinant and erythrocyte CD59 comigrated with apparent molecular masses of 12-14 kDa, suggesting altered structure of asparagine-linked carbohydrate in recombinant versus erythrocyte CD59. The function of the recombinant protein was evaluated by changes in the sensitivity of the CD59 transfectants to the pore-forming activity of human C5b-9. Induction of cell-surface expression of CD59 antigen inhibited C5b-9 pore formation in a dose-dependent fashion. CD59 transfectants expressing greater than or equal to 1.2 x 10(6) molecules of CD59/cell were completely resistant to human serum complement. By contrast, CD59 transfectants remained sensitive to the pore-forming activity of guinea pig C8 and C9 (bound to human C5b67). Functionally blocking antibody against erythrocyte CD59 abolished the human complement resistance observed for the CD59-transfected Chinese hamster ovary cells. These results confirm that the C5b-9 inhibitory function of the human erythrocyte membrane is provided by CD59 and suggest that the gene for this protein can be expressed in xenotypic cells to confer protection against human serum complement.
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PMID:Amplified gene expression in CD59-transfected Chinese hamster ovary cells confers protection against the membrane attack complex of human complement. 171 84

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

Cd2+ provokes an immediate production of inositol trisphosphate and the release of Ca2+ from internal stores in human fibroblasts and some other mammalian cells. Ni2+, Co2+, Fe2+, and Mn2+ evoke the release of stored Ca2+, but are less potent than Cd2+ (apparent K0.5 = 40 nM). Zn2+ and Cu2+ competitively inhibit Ca2+ release evoked by Cd2+ without affecting Ca2+ release by hormones such as bradykinin. Zn2+ has the same apparent Ki value (80-90 nM) towards the five agonist metals, which suggests that the metals interact with the same site. Many other divalent cations neither released stored Ca2+ nor affected Cd(2+)-evoked Ca2+ release. The agonist metals appear to activate phospholipase C via a G protein rather than a tyrosine kinase. The production of reactive oxygen species is probably not involved in Ca2+ release by the metals. Cd2+ and other stimuli that raise cytosolic-free Ca2+ induce cyclic (AMP) production, apparently by activating a calmodulin-dependent adenylyl cyclase. We suggest that an orphan receptor mediates the hormonelike responses to Cd2+ and the other agonist metals. The receptor is referred to as an orphan because its physiological stimulus is unknown. Growth of the fibroblasts in high Zn2+ desensitizes them to the five agonist metals without affecting Ca2+ release by bradykinin or histamine. A several hour incubation in culture medium with normal Zn2+ fully restores responsiveness to the five active metals. Growth in high Zn2+ appears to repress the synthesis of the putative orphan receptor because inhibitors of RNA or protein synthesis, or asparagine-linked glycosylation, prevented the restoration of metal responsiveness.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Transmembrane signals and protooncogene induction evoked by carcinogenic metals and prevented by zinc. 784 95

Insulin-like growth factor II (IGF II)/mannose 6-phosphate (Man-6-P) receptor, which has two binding sites, was efficiently purified using a phospho-pentamannan affinity column. The molecular mass of the receptor purified from rat fibrosarcoma cells (KMT-17) was 240,000 (240K), whereas that of the receptor from normal rat liver and fibroblasts (NRK-49F) was 250K. However, when the receptor was subjected to deglycosylation by treatment with tunicamycin, the receptors of the KMT-17 and MRK-49F cells gave the same molecular mass of 225K, indicating that the difference in molecular mass between two cell lines resulted from the different oligosaccharide sizes. Analysis of the number of receptor site and binding affinity with IGF II on cell surface showed that KMT-17 cells expressed about 10 times more sites than in NRK-49F cells, whereas the binding affinity of KMT cells was lower than that of NRK-49F cells. Since it is known that asparagine-linked oligosaccharides in the IGF II/Man-6-P receptor are essential for IGF II binding, the types of oligosaccharides in the receptor were investigated by concanavalin A affinity chromatography. It was revealed that a ratio (38%) of tetra- and triantennary (multiantennary) complex-type oligosaccharides in the receptor of KMT-17 cells was lower than that (61%) in NRK-49F cells. These results indicate that the receptor of KMT-17 cells possesses predominantly less branched complex-type oligosaccharides and that multiantennary complex-type oligosaccharides of the receptor are most likely to contribute to higher affinity binding of IGF II. To determine whether IGF II activates phosphatidyl inositol-specific phospholipase C (PLC) in KMT-17 cells, the production of inositol trisphosphate (IP3) was measured. Treatment with IGF II increased the level of IP3 in a concentration-dependent manner. The level of IP3 reached the maximum after 15 seconds of incubation. When the cells were pretreated with anti-receptor antibody, no activation of the PLC was observed. These findings suggest that IGF II stimulates PLC through binding to the IGF II/Man-6-P receptor.
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PMID:[Insulin-like growth factor II/mannose-6 phosphate receptors in a rat fibrosarcoma cell line]. 831 38

The NH2-terminal domain of the alpha-toxin of Clostridium perfringens is highly homologous to the complete phospholipase C from Bacillus cereus (PC-PLC), for which a high-resolution crystal structure is available. This structural information was used as the basis of a site-directed mutagenesis strategy in which critical amino acid residues of alpha-toxin involved in zinc binding, interaction with substrate, or catalysis were replaced. Biochemical studies with the corresponding toxin variants indicate that there is probably a single active site endowed with lecithinase, sphingomyelinase, and hemolytic activities. By using a highly purified variant in which the catalytic aspartate residue at position 56 was replaced by asparagine, it was shown that phospholipase activity was essential for lethality in vivo and for mediating platelet aggregation in vitro.
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PMID:Use of site-directed mutagenesis to probe structure-function relationships of alpha-toxin from Clostridium perfringens. 869 64

The nucleotide sequences of the hly and plcA genes (encoding listeriolysin, a thiol-activated cytolysin and a non-specific phospholipase C, respectively) were determined for two strains of Listeria monocytogenes serotype 1/2b. The deduced amino acid sequences for listeriolysin were identical for the two strains and identical to that of listeriolysin from serotype 4b. The two serotype 1/2b strains differed in one amino acid (histidine vs asparagine) in the deduced amino acid sequence for phosopholipase C.
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PMID:Nucleotide sequence analysis of two virulence-associated genes in Listeria monocytogenes serotype 1/2b and comparison with the same genes in other serotypes important in human disease. 908 Jun 92

A protective surface antigen (200 kDa) on C. salmositica was detected using a monoclonal antibody (mAb-001). Enzymatic studies on the epitope indicated that it was sensitive to nonspecific protease K and to site-specific trypsin and protease V8 but not to alpha-chymotrypsin. The reactivity of the epitope with mAb-001 was not affected when the antigen was denatured with 8 M urea; however, reduction of the antigen with dithiothreitol destroyed the epitope. The epitope was susceptible to sodium m-periodate oxidation and N-glycosidase F, but not to O-glycosidase or neuraminidase. It was also sensitive to mild potassium hydrochloride hydrolysis and to phospholipase C, which is specific for phosphatidylinositol. These results suggest that the epitope consists of a polypeptide, a carbohydrate, and probably a phospholipid. The asparagine-bound N-glycosidically linked hybrid-type carbohydrate chain has the minimum length of a chitobiose core unit. There is probably a phosphatidylinositol residue which anchors the polypeptide to the surface membrane. The antigen is extensively posttranslationally modified.
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PMID:Biochemical characterisation of an epitope on the surface membrane antigen (Cs-gp200) of the pathogenic piscine haemoflagellate Cryptobia salmositica Katz 1951. 950 43

Double mutant cycles provide a method for analyzing the effects of a mutation at a defined position in the protein structure on the properties of an amino acid at a second site. This approach was used to map potential interactions between aspartates 69, 97, and 103 in the m2 muscarinic acetylcholine receptor transmembrane helices 2 and 3. Receptors containing single and double aspartate to asparagine mutants were expressed in Chinese hamster ovary cells and their effects on ligand binding, signal transduction, and thermal stability determined. Analysis of the double mutant cycles showed that the mutations had approximately additive effects on ligand binding, signal transduction, and thermal stability. Ligand binding and thermal inactivation results support the conclusion that aspartate-103 is the ligand amine counterion. Effector coupling properties of the mutant receptors showed that aspartate-103 was also required for signal transduction activity. The mutation of aspartate-69 to asparagine completely eliminated signal transduction by the agonists acetylcholine, carbachol, and pilocarpine but not oxotremorine M, which caused reduced but significant inhibition of adenylyl cyclase and stimulation of phospholipase C. In contrast, adenylyl cyclase stimulation by the asparagine-69 mutant was elicited only by acetylcholine and carbachol but not by oxotremorine M. The variation in agonist-dependent effector coupling properties provides evidence that the asparagine-69 mutant can exist in activated receptor states that are different from the wild-type m2 muscarinic receptor.
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PMID:Double mutant cycle analysis of aspartate 69, 97, and 103 to asparagine mutants in the m2 muscarinic acetylcholine receptor. 988 58

The solution structure of growth factor receptor-bound protein 2 (Grb2) SH2 complexed with a Shc-derived phosphotyrosine (pTyr)-containing peptide was determined by nuclear magnetic resonance (NMR) spectroscopy. The pTyr binding site of Grb2 SH2 was similar to those of other SH2 domains. In contrast, the amino acid residues C-terminal to pTyr did not form an extended structure because of steric hindrance caused by a bulky side-chain of Trp121 (EF1). As a result, the peptide formed a turn-structure on the surface of Grb2 SH2. The asparagine residue at the pTyr+2 position of the Shc-peptide interacted with the main-chain carbonyl groups of Lys109 and Leu120. The present solution structure was similar to the crystal structure reported for Grb2 SH2 complexed with a BCR-Abl-derived phosphotyrosine-containing peptide. Finally, the structure of Grb2 SH2 domain was compared with those of the complexes of Src and phospholipase C-gamma1 with their cognate peptides, showing that the specific conformation of the peptide was required for binding to the SH2 domains.
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PMID:Solution structure of the SH2 domain of Grb2 complexed with the Shc-derived phosphotyrosine-containing peptide. 1035 20


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