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)

ADP-stimulation of washed human platelets suspended in Tyrode/albumin solution containing Ca2+ (2 mM) and fibrinogen (0.4 mg/ml) causes extensive, reversible aggregation without appreciable secretion of granule contents. Under these conditions ADP (10 microM) stimulation decreased the amounts of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) and phosphatidylinositol 4-phosphate (PtdInsP) at 10 s. Omitting fibrinogen from the suspending medium or blocking fibrinogen binding to the platelets using Arg-Gly-Asp-Ser (RGDS, 0.23 mM) inhibited these decreases in PtdInsP2 and PtdInsP. In contrast, ADP-induced decreases in PtdInsP2 and increases in PtdInsP at 60 s compared to 10 s were not affected by RGDS or the absence of fibrinogen. In platelets prelabelled with [3H]glycerol and [32P]phosphate, changes in labelling of the inositol phospholipids paralleled the changes in amount. The ADP-induced changes in phosphatidic acid (PtdOH) at 10 s were unaffected by RGDS; this finding supported previous reports that phospholipase C was not the cause of the early decreases in PtdInsP2 and PtdInsP. These results indicate that the early decreases in PtdInsP2 and PtdInsP at 10 s are dependent on fibrinogen binding to the platelets and occur after fibrinogen binding which is activated by ADP stimulation. It is proposed that the fibrinogen-dependent changes in PtdInsP2 and PtdInsP may have a feedback role augmenting platelet aggregation or other responses of platelets that might occur after fibrinogen binding, possibly due to effects on actin polymerisation.
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PMID:ADP-stimulated fibrinogen binding is necessary for some of the inositol phospholipid changes found in ADP-stimulated platelets. 839 29

A series of site-specific mutants of the phosphatidylcholine-preferring phospholipase C from Bacillus cereus (PLCBc) was prepared in which the glutamic acid residue at position 146 was replaced with glutamine, aspartic acid, histidine, and leucine to elucidate what role Glu146 might play in catalysis. An expression system for the native enzyme in Escherichia coli was first developed to provide PLCBc that was fused via an intervening factor Xa protease recognition sequence at its N-terminus to maltose binding protein (MBP). This MBP-PLCBc fusion protein was isolated at levels of 50-70 mg/L of culture; selective trypsin digestion of the MBP-PLCBc fusion protein followed by chromatographic purification yielded recombinant PLCBc at levels of ca. 10 mg/L. Polymerase chain reaction (PCR) mutagenesis on the PLCBc gene (plc) was then used to replace the Glu146 codon with those for glutamine (E146Q), aspartic acid (E146D), histidine (E146H), and leucine (E146L). The catalytic efficiency of the E146Q mutant was 1.6% that of native PLCBc, while the other mutants each possessed activities of 0.2-0.3% of the wild type. The kcat/Km vs pH profiles for both E146Q and native PLCBc have ascending acidic limbs, suggesting that Glu146 does not serve as the general base in the hydrolysis reaction. As measured by circular dichroism, all of the mutant proteins contained less helical structure and underwent denaturation at lower temperatures than the wild type in the order: wild type > E146Q > E146D approximately E146H approximately E146L. Atomic absorption analyses indicated that the mutant proteins also exhibited lower Zn2+ content than the wild type. Thus, the Glu146 residue in PLCBc stabilizes the secondary and tertiary structure of the enzyme and serves as a critical ligand for Zn2, but it does not appear to have any specific catalytic role.
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PMID:Expression and site-directed mutagenesis of the phosphatidylcholine-preferring phospholipase C of Bacillus cereus: probing the role of the active site Glu146. 884 Nov 44

We have previously demonstrated that thrombin possesses an active yet cryptic Arg-Gly-Asp (RGD) site which upon exposure induces endothelial cell (EC) adhesion via alpha nu beta 3 integrin [Bar-Shavit et al. (1991): J Cell Biol 112:335]. This was achieved in the presence of cell surface-associated heparan sulfate proteoglycans (HSPG) and exceedingly low concentrations of plasmin [Bar-Shavit et al. (1993): J Cell Biol 123:1279]. A portion of the cell surface-associated HSPG (glypican) is anchored via a covalently linked glycosyl-phosphatidylinositol (PI) residue, which can be released by treatment with glycosyl-PI-specific phospholipase C (PI-PLC). We report here that exposure of either bovine aortic EC, smooth muscle cells (SMC), or wild-type CHO cells to PI-PLC released HSPG involved in the conversion of thrombin to an adhesive molecule. The adhesion-promoting activity of the released HSPG was abolished following treatment with heparinase but not chondroitinase ABC. Incubation of thrombin with heparan sulfate-deficient CHO cells or cells that were pretreated with PI-PLC failed to induce its conversion to an adhesive molecule, indicating that glypican was playing a major role in this conversion. Moreover, affinity-purified glypican, but not syndecan or fibroglycan, elicited efficient conversion of plasmin-treated thrombin into an adhesive molecule. Antibodies raised against the RGD site in thrombin failed to interact with native thrombin, prothrombin, or the RGD site in other adhesive proteins such as vitronectin, fibrinogen, or fibronectin. Anti-thrombin-RGD antibodies which blocked the adhesion-promoting activity of thrombin were also capable of recognizing thrombin that was first incubated with a suboptimal concentration of plasm in in the presence of PI-PLC-released HSPG. Heparin, heparan sulfate, and PI-PLC-released HSPG had no effect on other cellular properties of thrombin such as receptor binding and growth-promoting activity. Altogether we have demonstrated that the heparin binding domain in thrombin plays a specific role in promoting thrombin adhesive properties and that membrane-associated glypican is likely to be the major physiological inducer of this property.
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PMID:Specific involvement of glypican in thrombin adhesive properties. 917 91

A new substrate analogue, (2R)-1,2-dipalmitoyloxypropanethiophospho-1-D-myo-inositol (DPsPI), has been used in a new, continuous assay for phosphatidylinositol-specific phospholipase C (PI-PLC). DPsPI is superior to other substrate analogs that have been used for assaying PI-PLC since it is synthesized as a pure diastereomer and maintains both acyl chains of the natural substrate, dipalmitoylphosphatidylinositol (DPPI). The assay that has been developed using this new analogue has allowed us to elucidate detailed kinetic data so far lacking in the field. In addition, several mutants of PI-PLC were constructed and assayed. The results show that Arg-69 is essential for catalysis, since mutations at this position led to a 10(3)- 10(4)-fold decrease in activity with respect that of to the wild-type (WT) enzyme. An alanine mutant of Asp-67, a residue also found at the active site, displays activity similar to that of WT. We have also used nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy to analyze the structural integrity and conformational stability of the mutants. The results show that the overall global conformation of the enzyme is not perturbed by the mutants. The 15N-1H HSQC NMR spectrum of WT PI-PLC is also reported at 600 MHz. The stereoselectivity of the reaction toward the stereoisomers of another analogue, 1,2-dipalmitoyl-sn-glycero-3-thiophospho-1-myo-inositol (DPPsI), was used to probe whether Arg-69 interacts with the phosphate moiety of the substrate. We have calculated that the WT enzyme shows a stereoselectivity ratio of 160000:1 in favor of the Rp isomer versus the Sp isomer. The R69K mutant displayed a significant 10(4)-fold relaxation of stereoselectivity. Our data support the role of Arg-69 in stabilizing the negative charge on the pentacoordinate phosphate in the transition state during catalysis.
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PMID:Phosphatidylinositol-specific phospholipase C: kinetic and stereochemical evidence for an interaction between arginine-69 and the phosphate group of phosphatidylinositol. 918 43

The current study has investigated the role of D-56, D-130, and E-152 in zinc ion binding properties, as well as the hemolytic, phospholipase C (PLC), and sphingomyelinase (SMase) activities of Clostridium perfringens alpha-toxin, based upon crystallography studies of the Bacillus cereus PLC, which had suggested these residues might be important for these functional activities. The replacement of D-56 in alpha-toxin resulted in complete loss of hemolytic, PLC, and SMase activities. The variant toxins at D-130 showed an approximately 100-fold reduction of biological activities compared to that of the wild-type toxin. The substitution of glutamine or glycine for E-152 caused complete loss of these activities, but substitution of aspartic acid for E-152 reduced but did not completely inhibit these activities. The variant toxins at D-56 and D-130, as well as the wild-type toxin, possessed approximately 2 mol of zinc atoms per mol of the protein, but E152G and E152Q contained approximately 1 mol of zinc metal per mol of the protein. On the other hand, the zinc content in E152D was calculated as about 1.4 mol in the toxin molecule. The replacement of D-56, D-130, or E-152 had no effect on binding to sheep erythrocytes and uptake of free zinc ion from the solution. The variant toxins at D-130 showed partial antigenic identity with the wild-type toxin on a double gel diffusion test. These observations suggest that D-56 in alpha-toxin is required for catalytic activity of alpha-toxin, D-130 is essential for maintenance of structure, and the carboxyl group of E-152 tightly ligands one zinc ion, which is essential for catalytic activity of the toxin.
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PMID:Site-specific mutagenesis of Clostridium perfringens alpha-toxin: replacement of Asp-56, Asp-130, or Glu-152 causes loss of enzymatic and hemolytic activities. 923 19

We previously reported that endothelin-1 (ET-1) stimulates phosphatidylcholine-hydrolyzing phospholipase D independently of phosphoinositide hydrolysis in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the characteristics of the receptors mediating ET-1-induced intracellular signaling pathway in MC3T3-E1 cells. Cyclo-D-Trp-D-Asp-Pro-D-Val-Leu (BQ123), a selective ETA receptor antagonist, significantly inhibited the ET-1-induced formation of inositol phosphates in a dose-dependent manner in the range between 22 nmol/L (IC50) and 2.2 mumol/L (IC50 x 100). On the contrary, N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma MeLeu-D-Trp(COOMe)-D-Nle-ONa (BQ788), a selective ETB receptor antagonist, had no effect on the ET-1-induced formation of inositol phosphates in the range between 1.2 nmol/L (IC50) and 120 nmol/L (IC50 x 100). BQ123 significantly suppressed the ET-1-induced formation of choline dose-dependently, however, BQ788 did not affect the choline formation. BQ123 also inhibited the ET-1-induced release of arachidonic acid, but BQ788 had little effect. The results strongly suggest that ETA receptor mediates the three intracellular signaling pathways of ET-1: (1) phosphoinositide hydrolysis by phospholipase C; (2) phosphatidylcholine hydrolysis by phospholipase D; (3) arachidonic acid release in osteoblast-like cells.
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PMID:ETA receptor mediates the signaling of endothelin-1 in osteoblast-like cells. 926 89

The mechanism of phosphatidylinositol-specific phospholipase C (PI-PLC) has been suggested to resemble that of ribonuclease A. The goal of this work is to rigorously evaluate the mechanism of PI-PLC from Bacillus thuringiensis by examining the functional and structural roles of His-32 and His-82, along with the two nearby residues Asp-274 and Asp-33 (which form a hydrogen bond with His-32 and His-82, respectively), using site-directed mutagenesis. In all, twelve mutants were constructed, which, except D274E, showed little structural perturbation on the basis of 1D NMR and 2D NOESY analyses. The H32A, H32N, H32Q, H82A, H82N, H82Q, H82D, and D274A mutants showed a 10(4)-10(5)-fold decrease in specific activity toward phosphatidylinositol; the D274N, D33A, and D33N mutants retained 0. 1-1% activity, whereas the D274E mutant retained 13% activity. Steady-state kinetic analysis of mutants using (2R)-1, 2-dipalmitoyloxypropane-3-(thiophospho-1d-myo-inositol) (DPsPI) as a substrate generally agreed well with the specific activity toward phosphatidylinositol. The results suggest a mechanism in which His-32 functions as a general base to abstract the proton from 2-OH and facilitates the attack of the deprotonated 2-oxygen on the phosphorus atom. This general base function is augmented by the carboxylate group of Asp-274 which forms a diad with His-32. The H82A and D33A mutants showed an unusually high activity with substrates featuring low pKa leaving groups, such as DPsPI and p-nitrophenyl inositol phosphate (NPIPs). These results suggest that His-82 functions as the general acid with assistance from Asp-33, facilitating the departure of the leaving group by protonation of the glycerol O3 oxygen. The Bronsted coefficients obtained for the WT and the D33N mutant indicate a high degree of proton transfer to the leaving group and further underscore the "helper" function of Asp-33. The complete mechanism also includes activation of the phosphate group toward nucleophilic attack by a hydrogen bond between Arg-69 and a nonbridging oxygen atom. The overall mechanism can be described as "complex" general acid-general base since three elements are required for efficient catalysis.
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PMID:Mechanism of phosphatidylinositol-specific phospholipase C: a unified view of the mechanism of catalysis. 952 77

The highly conserved aspartate residue in the second transmembrane domain of G protein-coupled receptors is present in position 113 in the type 1 neurotensin receptor (NTR1) but is replaced by an Ala residue in position 79 in the type 2 neurotensin receptor (NTR2). NTR1 couples to Galphaq to stimulate phospholipase C and its binding affinity for neurotensin is decreased by sodium ions and GTP analogs. By contrast, NTR2 does not seem to couple to any G protein in eukaryotic cells, and its binding of neurotensin is insensitive to sodium and GTP analogs. By using site-directed mutagenesis, we substituted Asp113 of the NTR1 by alanine and the homologous residue Ala79 of NTR2 by aspartate. Both mutant receptors display similar affinity for neurotensin as compared with their respective wild type. We demonstrate that the presence of the Asp residue determines by itself the occurrence of the sodium effect on neurotensin affinity for both wild-type and mutated NTR1 and -2. The introduction of an Asp in the second transmembrane domain of NTR2 is not enough to restore a functional coupling to G proteins. In contrast, replacement of Asp113 by Ala residue in NTR1 strongly decreases its ability to activate inositol turnover, indicating that the functionally active conformation of NTR1 is maintained by interaction of sodium ions with aspartate 113.
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PMID:Pivotal role of an aspartate residue in sodium sensitivity and coupling to G proteins of neurotensin receptors. 992 10

Fertilization includes sperm-oocyte recognition, adhesion, binding, fusion and egg activation. Integrin receptors, which are adhesion molecules, are expressed on sea urchin, mouse, hamster and human unfertilized oocytes. Potential sperm ligands have been identified. A role for integrins during fertilization is supported by inhibition of sperm-egg adhesion and/or fusion by means of anti-integrin monoclonal antibodies, Arg-Gly-Asp (RGD) or disintegrin-like peptides. In several cell-cell interactions, such as lymphocyte activation, viral fusion, bacterial infection or macrophage phagocytosis, integrins act as co-receptors after activation and, by clustering in a multimolecular complex, are able to transduce signals through cytoskeletal proteins and adaptor kinases. Experimental data suggest that they may act in a similar way during fertilization and may participate to initiation and/or propagation of the calcium signal via stimulation of phospholipase C gamma and inositol trisphosphate production.
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PMID:Role of integrins during fertilization in mammals. 1009 Oct 56

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


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