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)

In the present study, the roles of Ca2+ and fibrinogen receptor occupancy in the regulation of phospholipase C by G protein-coupled and tyrosine kinase-linked receptor pathways in human platelets have been investigated. Agonist stimulation of phospholipase C was not altered significantly in the absence of stirring or in the presence of the fibrinogen receptor antagonist arginine-glycine-aspartate-serine, conditions that prevent platelet aggregation. Similarly, elevation of intracellular Ca2+ levels by the ionophores A23187 or ionomycin did not induce formation of inositol phosphates. In contrast, chelation of extracellular Ca2+ by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) reduced formation of inositol phosphates by G protein receptor (thrombin)- and tyrosine kinase (Fc receptor and peroxovanadate)-regulated pathways. Similarly, short term exposure to Ni2+ ions, which also prevent Ca2+ entry, inhibited thrombin-stimulated formation of inositol phosphates. Loading of platelets with the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) markedly suppressed elevation of intracellular Ca2+ and formation of inositol phosphates in platelets stimulated by G protein receptor- and tyrosine kinase-regulated pathways. The greater inhibition of phospholipase C by BAPTA, relative to that induced by EGTA, is consistent with the more pronounced inhibition of intracellular Ca2+ elevation. The tyrphostin tyrosine kinase inhibitor ST271 also reduced intracellular Ca2+ levels and inhibited activation of phospholipase C. The degree of inhibition of phospholipase C by ST271 was slightly greater than that induced by EGTA but was not additive with the effect of EGTA, suggesting a common mode of action. It is concluded that elevation of intracellular Ca2+ regulates agonist-induced activation of phospholipase C and that this contributes to the inhibition of thrombin-induced formation of inositol phosphates by the tyrphostin ST271.
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PMID:Ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and the tyrphostin ST271 inhibit phospholipase C in human platelets by preventing Ca2+ entry. 772 44

Substitution of His-35 with arginine in staphylococcal alpha-toxin leads to loss of its membrane perturbating properties in vitro. In this study, we report that the inactive mutant toxin is also devoid of toxic properties when injected intravenously into rabbits. Whereas a single application of native toxin at a dose of 20 micrograms/kg proved uniformly lethal within 3 hours (n = 3), all animals receiving 20, 100 or 200 micrograms/kg mutant toxin (n = 3 for each group) remained in unaltered, healthy states over a 15 day period of observation. Furthermore, all animals receiving inactive toxin developed antibody titers on day 10-15. No dose dependency was noted in the tested range. Thus, the site-directed alpha-toxin mutant (His-35:Arg) is an excellent vaccine candidate.
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PMID:Functionally inactive S. aureus alpha-toxin containing a single amino acid substitution: potential usefulness as a vaccine. 775 12

Receptor-mediated activation of heterotrimeric guanine nucleotide-binding proteins (G proteins) results in the dissociation of alpha from beta gamma subunits, thereby allowing both to regulate effectors. Little is known about the regions of effectors required for recognition of G beta gamma. A peptide encoding residues 956 to 982 of adenylyl cyclase 2 specifically blocked G beta gamma stimulation of adenylyl cyclase 2, phospholipase C-beta 3, potassium channels, and beta-adrenergic receptor kinase as well as inhibition of calmodulin-stimulated adenylyl cyclases, but had no effect on interactions between G beta gamma and G alpha o. Substitutions in this peptide identified a functionally important motif, Gln-X-X-Glu-Arg, that is also conserved in regions of potassium channels and beta-adrenergic receptor kinases that participate in G beta gamma interactions. Thus, the region defined by residues 956 to 982 of adenylyl cyclase 2 may contain determinants important for receiving signals from G beta gamma.
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PMID:A region of adenylyl cyclase 2 critical for regulation by G protein beta gamma subunits. 776 32

Several proteins that are important components of the calcium/phospholipid second messenger system (e.g. phospholipase C, protein kinase C, myristoylated alanine-rich C kinase substrate (MARCKS) and pp60src) contain clusters of basic residues that can interact with acidic lipids on the cytoplasmic surface of plasma membranes. We have studied the membrane binding of MARCKS and pp60src, peptides that mimic the basic regions of these proteins, and simple model peptides. Specifically, we determined how the binding of these model peptides depends on (1) the number of basic residues in the peptide (2) the fraction of acidic lipids in the membrane (3) the ionic strength of the solution (4) the chemical nature of the basic residues (Arg versus Lys) and the acidic phospholipids [phosphatidylglycerol (PG) versus phosphatidylserine (PS)] (5) the pressure and (6) the temperature. The results are consistent with a simple theoretical model: each basic residue in a peptide binds independently to an acidic lipid with an intrinsic microscopic association constant of 1-10 M-1 (binding energy congruent to 1 kcal/mol). The binding is described with a mass action formalism and the non-specific electrostatic accumulation of the peptides in the aqueous diffuse double layer is described with the Gouy-Chapman theory. This Gouy-Chapman/mass action model accounts surprisingly well for the sigmoidal dependence of binding on the percentage of acidic lipids in the membrane (apparent co-operativity or Hill coefficient > 1); the model assumes that the multivalent basic peptides bind > 1 acidic lipids and thus induce or stabilize domain formation.
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PMID:Does the binding of clusters of basic residues to acidic lipids induce domain formation in membranes? 776 86

Staphylococcal alpha-toxin is a primarily hydrophilic molecule that binds as a monomer to target membranes and then aggregates to form amphiphilic oligomers that represent water-filled transmembrane channels. Current evidence indicates that a region located in the center of the molecule inserts deeply into the bilayer. In the present study, we sought to determine whether membrane insertion was triggered by the oligomerization process, and whether insertion correlated with pore formation. Double mutants of alpha-toxin were prepared in which His-35 was replaced by Arg, and cysteine residues were introduced at positions 69, 130 and 186. Substitution of His-35 with Arg rendered the toxin molecules incapable of proper oligomerization, so that they remained in nonlytic form after binding to membranes. The sulfhydryl groups were labelled with the polarity-sensitive fluorescent dye acrylodan. Functionally intact, single mutant toxins containing only the cysteine residues were utilized as controls. Measurements of the fluorescence emission spectrum of acrylodan were performed for the active and inactive alpha-toxin mutants in free solution and in membrane-bound form. The collective results demonstrate that proper oligomerization is required for membrane insertion of the central region in the alpha-toxin molecule, and that lack of insertion correlates with absence of pore formation.
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PMID:Correct oligomerization is a prerequisite for insertion of the central molecular domain of staphylococcal alpha-toxin into the lipid bilayer. 779 60

Arginine-vasopressin (AVP) plays a determinant role in the normal ACTH response to stress in mammals. We cloned a human cDNA coding a 424 amino acid G-protein coupled receptor structurally related to the vasopressin/oxytocin receptor family. When expressed in COS cells, this receptor binds AVP with a high affinity (Kd = 0.55 +/- 0.13 nM) and is functionally coupled to phospholipase C. Competition studies with peptidic or non peptidic AVP analogues reveal that it is pharmacologically distinct from V1a and V2 AVP receptors and therefore it is designated V3. RT-PCR analysis shows that the human V3 receptor is expressed in normal pituitary and also in kidney, but is undetectable in liver, myometrium and adrenal gland. Northern blot analysis reveals a approximately 4.8 kb messenger in human corticotropic pituitary adenomas.
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PMID:Cloning and characterization of the human V3 pituitary vasopressin receptor. 780 41

The gene for Clostridium septicum alpha-toxin was cloned and expressed in Escherichia coli from C. septicum BX96. The toxin was determined to be 443 amino acids in length, with a 31-residue signal peptide that was removed from the toxin during secretion. No extended hydrophobic regions were observed in the mature toxin sequence. Expression of alpha-toxin in E. coli BL21 resulted in the production of ATpro, which was identical to native toxin from C. septicum with respect to activity and activation. The proteolytic activation site for alpha-toxin was determined to be on the carboxy-terminal side of arginine 398, which lies within the sequence KKRRGKR-398SVD. Previous work showing similarities in activation and mechanism between alpha-toxin and Aeromonas hydrophila aerolysin was extended to the primary structures of both toxins. The DNA-derived primary sequence of alpha-toxin exhibited 27% identity and 72% similarity over a 387-residue region with the primary structure of the A. hydrophila aerolysin toxin, a level of similarity heretofore unobserved between toxins produced by a gram-positive organism and a gram-negative organism.
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PMID:The primary structure of Clostridium septicum alpha-toxin exhibits similarity with that of Aeromonas hydrophila aerolysin. 780 74

The regulation of corticosteroid secretion of the adrenal cortex (interrenal tissue) of axolotl (Ambystoma mexicanum) was studied using in vitro preparations of kidney containing interrenal tissue. Normally, 0.3-0.65 ng/5 min corticosterone and 0.15-0.3 ng/5 min aldosterone were released from the tissue. Regulatory peptides were effective in the following range: ACTH = arginine vasotocin > urotensin II > angiotensin II. They stimulate an elevation of corticosterone (plus 0.2-1 ng/5 min) and of aldosterone (plus 0.05-0.15 ng/5 min). The three primary effector systems leading to second messengers, adenylate cyclase (forming cAMP), phospholipase C (forming InsP3 + DAG), and phospholipase A2 (liberating arachidonic acid) are involved in stimulation of biosynthesis. It can be suggested that the second messengers stimulate the biosynthesis at the level of the steps between pregnenolone and corticosterone ('3 beta-hydroxysteroid-dehydrogenase etc.), because the release of corticosterone is more stimulated than aldosterone. This is different than the regulation of anuran interrenal tissue. Ca++ ions are involved in corticosterone secretion. Verapamil inhibits immediately the secretion of corticosteroids and elevation of external Ca++ stimulates the release. It is suggested that Ca++ mediates the secretion process itself. Metamorphosis does not change the response of the interrenal gland compared with the neotenic animal.
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PMID:Regulation of interrenal secretion in the axolotl, Ambystoma mexicanum. 781 1

Bacterial toxin ADP-ribosyltransferases, e.g. diphtheria toxin (DT) and pertussis toxin, have in common consensus sequences involved in catalytic activity, which are localized to three regions. Region I is notable for a histidine or arginine; region II, approximately 50-75 amino acids downstream, is rich in aromatic/hydrophobic amino acids; and region III, further downstream, has a glutamate and other acidic amino acids. A similar motif was observed in the sequence of the glycosylphosphatidylinositol-linked muscle ADP-ribosyltransferase. Site-directed mutagenesis was performed to verify the role of this motif. Proteins were expressed in rat adenocarcinoma cells, released from the cell with phosphatidylinositol-specific phospholipase C, and quantified with polyclonal antibodies. Transferase His114 in region I aligned with His21 of DT; as with DT, the H114N mutant was active. Aromatic/hydrophobic amino acids (region II) were found approximately 30-50 amino acids downstream of this histidine. Although transferase has a Glu278-Tyr-Ile sequence characteristic of region III in DT, Glu278 was not critical for activity. In an alternative region III containing Glu238-Glu239-Glu240, Glu238 and Glu240 but not Glu239 were critical. Glu240 aligned with critical glutamates in DT, Pseudomonas exotoxin, and C3 transferase. Thus, the mammalian ADP-ribosyltransferases have motifs similar to toxin ADP-ribosyltransferases, suggesting that these sequences are important in ADP-ribose transfer reactions.
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PMID:Conservation of a common motif in enzymes catalyzing ADP-ribose transfer. Identification of domains in mammalian transferases. 782 77

Calcitonin (CT) is a peptide hormone that interacts with the cAMP-and phospholipase C-associated CT receptor subtypes. We investigated whether CT modulates the interaction of human tumoral osteoclast-like (GCT23) cells with a protein of the bone matrix, bone sialoprotein-II (BSP-II). Single GCT23 cells loaded with the intracellular Ca2+ indicator fura-2 were treated with the maximal active dose (300 micrograms/ml) of the 18-mer Arg-Gly-Asp (RGD)-containing BSP-IIA fragment, and the cytosolic free Ca2+ concentration ([Ca2+]i) was measured by dual wavelength microfluorometry. BSP-IIA stimulated an elevation in [Ca2+]i, consisting mainly of a peak, followed by a rapid return toward baseline. Pretreatment with CT induced a modest elevation of [Ca2+]i. However, CT significantly inhibited the response to BSP-IIA in a dose-dependent manner. Maximal inhibition (90% vs. untreated) was observed in the micromolar range. The intracellular mechanisms leading to this effect were investigated by pretreatment of GCT23 cells with the cAMP permeant analog, (Bu2)cAMP, and the protein kinase-C-activating agent, 12-O-tetradecanoylphorbol 13-acetate. Similar to CT, both agents inhibited the response to 300 micrograms/ml BSP-IIA. The effect induced by CT was specific, because an increase in the extracellular Ca2+ concentration, which is also known to inhibit bone resorption, failed to modify the ability of BSP-IIA to alter [Ca2+]i in GCT23 cells. To investigate whether the CT-induced alteration of BSP-IIA-dependent cell signals was due to a modification in the synthesis of cell surface receptors (integrins) for the extracellular matrix macromolecules, 1-h CT-treated [35S]methionine metabolically labeled GCT23 cell lysates were immunoprecipitated with anti-alpha 3-, -alpha v-, -beta 1-, and -beta 3-integrin subunit antibodies. Autoradiography demonstrated that 10(-7)-10(-6) M CT did not alter new synthesis of the alpha v beta 3 and the alpha 3 beta 1 receptors. Similarly, CT did not affect surface expression of these receptors, assessed by enzyme-linked immunosorbent assay. Finally, no alteration of the adhesion rate and spreading of GCT23 cells onto BSP-IIA-coated substrates was observed. This indicates that CT-induced down-regulation of immediate cell signals prompted by BSP-IIA in GCT23 cells is a postintegrin receptor event.
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PMID:Calcitonin down-regulates immediate cell signals induced in human osteoclast-like cells by the bone sialoprotein-IIA fragment through a postintegrin receptor mechanism. 786 71


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