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 structure of the PH-domain truncated core of rat phosphoinositide-specific phospholipase C-delta 1 has been determined at 2.4 A resolution and compared to the structure previously determined in a different crystal form. The stereochemical relationship between the EF, catalytic, and C2 domains is essentially identical. The Ca2+ analogue Sm3+ binds at two sites between the jaws of the C2 domain. Sm3+ binding ejects three lysine residues which bridge the gap between the jaws and occupy the Ca2+ site in the apoenzyme, triggering a conformational change in the jaws. The distal sections of the C2 jaws move apart, opening the mouth by 9 A and creating a gap large enough to bind a phospholipid headgroup.
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PMID:C2 domain conformational changes in phospholipase C-delta 1. 878 53

Point mutagenesis, phosphatidylinositol (PI), and phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis assays and equilibrium centrifugation PIP2 assays were used to study the functional roles of four highly conserved arginine residues in the Y region of human phospholipase C delta1 (PLCdelta1) (Arg-527, -549, -556, -701). Most of the mutant enzymes were either partially defective or fully active in their abilities to catalyze the hydrolysis of PI or PIP2. However, upon substitution of Arg-549 by glycine or histidine, the mutant enzyme was defective in its ability to catalyze the hydrolysis of PIP2, but it is still able to hydrolyze PI. Replacing Arg-549 with lysine had little effect on the level of PI and PIP2 hydrolytic activities of the mutant enzyme. The residual PIP2 hydrolyzing activity of R549H is highly dependent on pH. R549H showed 5-10% of the PIP2-hydrolyzing activity of the native enzyme between pH 5 and 7 and nondetectable PIP2-hydrolyzing activity at pH 8. The PIP2-hydrolyzing activity of R549G was not detectable at all pH values. Kinetic analysis of PLCdelta1-catalyzed PIP2 hydrolysis revealed that the micellar dissociation constant Ks and interfacial Michaelis constant Km were similar in the native, R549K, and R549H enzymes; but the specific activity at the saturated substrate mole fraction and infinite level of substrate (Vmax) of the R549H mutant were reduced by a factor of 15. PIP2 competitively inhibits the native enzyme to hydrolyze PI at both pH 7 and 8. However, PIP2 inhibits R549H only at pH 7.0 and does not inhibit R549G at either pH. Taken together, these results suggest that positive charge at position 549 of PLCdelta1 protein is essential for the enzyme to recognize and catalyze the hydrolysis of PIP2 but not PI.
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PMID:Positive charge at position 549 is essential for phosphatidylinositol 4,5-bisphosphate-hydrolyzing but not phosphatidylinositol-hydrolyzing activities of human phospholipase C delta1. 879 11

A new method of affinity chromatography purification of the detergent-solubilized nicotinic acetylcholine receptor protein (nAChR) is presented, based on the reversible coupling of a chemically monomodified alpha-toxin from Naja nigricollis to a resin. The alpha-toxin was monothiolated on the epsilon-amino group of its lysine-15 by reaction with N-succinimidly-3-(2-pyridyldithio)propionate and was covalently linked in a reversible manner to a thiopropyl-activated agarose resin by thiol-disulfide exchange. We found that 50% of the immobilized toxin molecules were effective for purifying nAChR, indicating a high accessibility of resin-bound toxins to their binding sites on the receptor protein. Purified alpha-toxin/nAChR complexes were eluted with nearly 100% recovery by reduction of disulfide bridges with dithiothreitol. nAChR solutions of high purity were obtained, as shown by polyacrylamide gel electrophoresis. A comparison was made with two other procedures of affinity chromatography using: (1) alpha-bungarotoxin from Bungarus multicinctus polymodified on several amines and covalently linked to a resin in a reversible manner, and (2) a commercial agarose resin bearing irreversibly immobilized alpha-cobrotoxin from Naja naja kaouthia. We conclude that: (1) the use of a selected regioselective linking of a peptidic ligand to a chromatography resin results in an increased efficiency of protein binding, and (2) a high yield of protein recovery is obtained via reversible covalent linking.
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PMID:Purification of the nicotinic acetylcholine receptor protein by affinity chromatography using a regioselectively modified and reversibly immobilized alpha-toxin from Naja nigricollis. 905 96

To delineate ligand binding and functional characteristics of the human B1 kinin receptor, a stable clone of Chinese hamster ovary cells expressing a single class of binding sites for [3H]des-Arg10-lysylbradykinin with a Kd of 0.3 nM and a Bmax of 38 fmol/mg protein ( approximately 40,000 receptors/cell) was isolated. Studies with peptide analogs showed that a lysine residue at position 1 (based on the lysylbradykinin sequence) of ligands was essential for high affinity binding to the human B1 receptor. In marked contrast to cloned Chinese hamster ovary cells expressing the human kinin B2 receptor, which internalized approximately 80% of the ligand within 5 min upon exposure to 2 nM [3H]bradykinin, exposure of cells expressing the B1 receptor to 1 nM [3H]des-Arg10-lysylbradykinin resulted in minimal ligand internalization. Stimulation of the B1 receptor led to inositol phosphate generation and transient increases in intracellular calcium, confirming coupling to phospholipase C, while immunoprecipitation of photoaffinity-labeled G-proteins from membranes indicated specific coupling of the receptor to Galphaq/11 and Galphai1,2. The B1, unlike the B2, receptor does not desensitize (as demonstrated by continuous phosphoinositide hydrolysis), enhancing the potential role of this receptor during inflammatory events.
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PMID:Stable expression of the human kinin B1 receptor in Chinese hamster ovary cells. Characterization of ligand binding and effector pathways. 911 Oct 52

The pleckstrin homology (PH) domain has been postulated to serve as an anchor for enzymes that operate at a lipid/water interface. To understand further the relationship between the PH domain and enzyme activity, a phospholipase C (PLC) delta1/PH domain enhancement-of-activity mutant was generated. A lysine residue was substituted for glutamic acid in the PH domain of PLC delta1 at position 54 (E54K). Purified native and mutant enzymes were characterized using a phosphatidylinositol 4,5-bisphosphate (PI(4, 5)P2)/dodecyl maltoside mixed micelle assay and kinetics measured according to the dual phospholipid model of Dennis and co-workers (Hendrickson, H. S., and Dennis, E. A. (1984) J. Biol. Chem. 259, 5734-5739; Carmen, G. M., Deems, R. A., and Dennis, E. A. (1995) J. Biol. Chem. 270, 18711-18714). Our results show that both PLC delta1 and E54K bind phosphatidylinositol bisphosphate cooperatively (Hill coefficients, n = 2.2 +/- 0.2 and 2.0 +/- 0.1, respectively). However, E54K shows a dramatically increased rate of (PI(4, 5)P2)-stimulated PI(4,5)P2 hydrolysis (interfacial Vmax for PLC delta1 = 4.9 +/- 0.3 micromol/min/mg and for E54K = 31 +/- 3 micromol/min/mg) as well as PI hydrolysis (Vmax for PLC delta1 = 27 +/- 3.4 nmol/min/mg and for E54K = 95 +/- 12 nmol/min/mg). In the absence of PI(4,5)P2 both native and mutant enzyme hydrolyze PI at similar rates. E54K also has a higher affinity for micellar substrate (equilibrium dissociation constant, Ks = 85 +/- 36 microM for E54K and 210 +/- 48 microM for PLC delta1). Centrifugation binding assays using large unilamelar phospholipid vesicles confirm that E54K binds PI(4,5)P2 with higher affinity than native enzyme. E54K is more active even though the interfacial Michaelis constant (Km) for E54K (0.034 +/- 0.01 mol fraction PI(4,5)P2) is higher than the Km for native enzyme (0.012 +/- 0.002 mol fraction PI(4,5)P2). D-Inositol trisphosphate is less potent at inhibiting E54K PI(4,5)P2 hydrolysis compared with native enzyme. These results demonstrate that a single amino acid substitution in the PH domain of PLC delta1 can dramatically enhance enzyme activity. Additionally, the marked increase in Vmax for E54K argues for a direct role of PH domains in regulating catalysis by allosteric modulation of enzyme structure.
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PMID:A single amino acid substitution in the pleckstrin homology domain of phospholipase C delta1 enhances the rate of substrate hydrolysis. 919 25

The pathogenicity of lipoprotein(a) [Lp(a)] as a risk factor for cardiovascular disease may depend upon its lysine binding sites (LBS) which impart unique functions to Lp(a) not shared with low density lipoprotein. Biologically relevant modifications of Lp(a) were tested for alterations of LBS activity using two previously described functional assays, a LBS-Lp(a) immunoassay and a lysine-Sepharose bead assay. In the LBS-Lp(a) immunoassay, minimal changes in the LBS activity of Lp(a) were observed after modification with lipoprotein lipase, sphingomyelinase, or phospholipase C. In contrast, a significant (p<0.003) increase in the LBS activity of Lp(a) occurred after phospholipase A2 (PLA2) treatment, and this increase was confirmed using the lysine-Sepharose bead assay. The increase depended upon the release of fatty acids from Lp(a) by PLA2. A decrease in the LBS activity of Lp(a) occurred after oxidation of Lp(a) with 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) (44% decrease), but CuSO4 oxidation increased LBS activity (210%). N-acetylcysteine (NAC) treatment of Lp(a) decreased (48%) LBS activity while homocysteine treatment had no (89%) effect. Thus, modification of phospholipids and protein moieties can alter the LBS-activity of Lp(a). Such enzymatic and chemical modifications may contribute to the variability in LBS function of Lp(a) seen within the population.
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PMID:Enzymatic and chemical modifications of lipoprotein(a) selectively alter its lysine-binding functions. 959 30

A body of evidence has shown the existence of a nuclear phosphoinositide cycle in different cell types. The cycle is endowed with kinases as well as phosphatases and phospholipase C (PLC). Among the PLC isozymes, the beta family is characterized by a long COOH-terminal tail that contains a cluster of lysine residues responsible for nuclear localization. Indeed, PLC beta 1 is the major isoform that has been detected in the nucleus of several cells. This isoform is activated by insulin-like growth factor I, and when this isoform is lacking, as a result of gene ablation, the onset of DNA synthesis induced by this hormone is abolished. On the contrary, PLC beta 1 is down-regulated during the erythroid differentiation of Friend erythroleukemia cells. A key question is how PLC beta 1 signaling at the nucleus fits into the erythroid differentiation program of Friend erythroleukemia cells, and whether PLC beta 1 signaling activity is directly responsible for the maintenance of the undifferentiated state of erythroleukemia cells. Here we present evidence that nuclear PLC beta 1 but not the isoform located at the plasma membrane is directly involved in maintaining the undifferentiated state of Friend erythroleukemia cells. Indeed, when wild-type PLC beta 1 is overexpressed in these cells, differentiation in response to DMSO is inhibited in that the expression of beta-globin is almost completely abolished, whereas when a mutant lacking the ability to localize to the nucleus is expressed, the cells differentiate, and the expression of beta-globin is the same as in wild-type cells.
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PMID:Nuclear but not cytoplasmic phospholipase C beta 1 inhibits differentiation of erythroleukemia cells. 982 10

We previously showed that thrombospondin 1 (TSP-1) upregulates the plasminogen/plasmin system and promotes breast tumor cell invasion. Preliminary data from our laboratory using neutralizing antibodies suggested that the upregulation in breast tumor cell invasion seen in response to TSP-1 involved the urokinase plasminogen activator receptor (uPAR). To confirm these findings in MDA-MB-231 breast cancer cells, we developed three other strategies to study the role of uPAR in tumor cell adhesion and TSP-1-mediated tumor cell invasion: (a) enzymatic cleavage of uPAR with glycosylphosphatidylinositol-specific phospholipase C; (b) inhibition at the mRNA level with a uPAR antisense construct (cells named LKAS-MDA); (c) inhibition of plasminogen binding with the lysine analogue epsilon-aminocaproic acid. Adhesion to laminin and type I and type IV collagen with and without the addition of epsilon-aminocaproic acid was studied. Tumor cell invasion was studied in a modified Boyden chamber collagen invasion assay. Antisense uPAR inhibition decreased uPAR expression by 48-66% and cell-associated urokinase plasminogen activator (uPA) by 30-68%. Additionally, antisense uPAR inhibition induced a 68-70% reduction in uPA and plasmin activities. Antisense uPAR transfection increased tumor cell adhesion by 46-53%. A similar effect was observed in epsilon-aminocaproic acid-treated MDA-MB-231 cells. TSP-1-mediated tumor cell invasion was almost completely inhibited by either antisense uPAR inhibition or treatment with phospholipase C or epsilon-aminocaproic acid. We conclude that uPAR plays a crucial role in the regulation of tumor cell adhesion and TSP-1-mediated tumor cell invasion.
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PMID:Role of urokinase plasminogen activator receptor in thrombospondin 1-mediated tumor cell invasion. 1009 Aug 48

The P2Y1 receptor is a membrane-bound G protein-coupled receptor stimulated by adenine nucleotides. Using alanine scanning mutagenesis, the role in receptor activation of charged amino acids (Asp, Glu, Lys, and Arg) and cysteines in the extracellular loops (EL) of the human P2Y1 receptor has been investigated. The mutant receptors were expressed in COS-7 cells and measured for stimulation of phospholipase C induced by the potent agonist 2-methylthioadenosine-5'-diphosphate (2-MeSADP). In addition to single point mutations, all receptors carried the hemagglutinin epitope at the N- terminus for detection of cell-surface expression. The C124A and C202A mutations, located near the exofacial end of transmembrane helix 3 and in EL2, respectively, ablated phospholipase C stimulation by </=100 microM 2-MeSADP. Surface enzyme-linked immunosorbent assay detection of both mutant receptors showed <10% expression, suggesting that a critical disulfide bridge between EL2 and the upper part of transmembrane 3, as found in many other G protein-coupled receptors, is required for proper trafficking of the P2Y1 receptor to the cell surface. In contrast, the C42A and C296A mutant receptors (located in the N-terminal domain and EL3) were activated by 2-MeSADP, but the EC50 values were >1000-fold greater than for the wild-type receptor. The double mutant receptor C42A/C296A exhibited no additive shift in the concentration-response curve for 2-MeSADP. These data suggest that Cys42 and Cys296 form another disulfide bridge in the extracellular region, which is critical for activation. Replacement of charged amino acids produced only minor changes in receptor activation, with two remarkable exceptions. The E209A mutant receptor (EL2) exhibited a >1000-fold shift in EC50. However, if Glu209 were substituted with amino acids capable of hydrogen bonding (Asp, Gln, or Arg), the mutant receptors responded like the wild-type receptor. Arg287 in EL3 was impaired similarly to Glu209 when substituted by alanine. Substitution of Arg287 by lysine, another positively charged residue, failed to fully restore wild-type activity.
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PMID:The role of amino acids in extracellular loops of the human P2Y1 receptor in surface expression and activation processes. 1032 57

We recently cloned a novel signaling molecule, p122, that shows a GTPase-activating activity specific for Rho and the ability to enhance the phosphatidylinositol 4,5-bisphosphate-hydrolyzing activity of phospholipase C delta1 in vitro. Here we analyzed the in vivo function of p122. Microinjection of the GTPase-activating domain of p122 suppressed the formation of stress fibers and focal adhesions induced by lysophosphatidic acid, suggesting a GTPase-activating activity for Rho as in in vitro. Transfection of p122 also induced the disassembly of stress fibers and the morphological rounding of various adherent cells. Analyses using deletion and point mutants demonstrated that the GTPase-activating domain of p122 is responsible for the morphological changes and detachment and that arginine residues at positions 668 and 710 and a lysine residue at position 706 in the GTPase-activating domain are essential. Using Fluo-3-based Ca2+ microscopy, we found that p122 evoked a rapid elevation of intracellular Ca2+ levels, suggesting that p122 stimulates the phosphatidylinositol 4, 5-bisphosphate-hydrolyzing activity of phospholipase C delta1. These results demonstrate that p122 synergistically functions as a GTPase-activating protein specific for Rho and an activator of phospholipase C delta1 in vivo and induces morphological changes and detachment through cytoskeletal reorganization.
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PMID:Morphological changes and detachment of adherent cells induced by p122, a GTPase-activating protein for Rho. 1036 18


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