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)

alpha-Toxin, a lethal hemolytic toxin secreted by Staphylococcus aureus, forms ionic channels of large size in lipid membranes. To investigate the mechanism of channel assembly we have studied the kinetics of pore formation on small unilamellar vesicles. We have used two assays of vesicle permeabilization: one is the release of a fluorescent molecule trapped in their inner compartment; the other is the dissipation of an imposed potential. Both methods indicate that the kinetics are complex consisting of an initial delay followed by a non-linear relaxation. The dependence of the pore formation rate and the extent of permeabilization on the toxin/vesicle ratio indicates that aggregation of 4-10 preinserted toxin monomers underlies channel assembly. The pH dependence of permeabilization suggests that protonation of an acidic group of the toxin is a prerequisite to channel formation. Inclusion of cholesterol in the target vesicles potentiates alpha-toxin effects, in a dose-dependent way, possibly by facilitating its protonation. The location of the proton-binding site on the two adjacent aspartic acid residues in positions 127 and 128 of the toxin monomer is proposed.
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PMID:Staphylococcal alpha-toxin increases the permeability of lipid vesicles by cholesterol- and pH-dependent assembly of oligomeric channels. 247 41

Placental alkaline phosphatase (PLAP) is initially synthesized as a precursor (proPLAP) with a C-terminal extension. We constructed a recombinant cDNA which encodes a chimeric protein (alpha GL-PLAP) comprising rat alpha 2u-globulin (alpha GL) and the C-terminal extension of PLAP. Two molecular species (25 kDa and 22 kDa) were expressed in the COS-1 cell transfected with the cDNA for alpha GL-PLAP. Only the 22 kDa form was labelled with both [3H]stearic acid and [3H]ethanolamine. Upon digestion with phosphatidylinositol-specific phospholipase C the 22 kDa form was released into the medium, indicating that this form is anchored on the cell surface via glycosylphosphatidylinositol (GPI). A specific IgG raised against a C-terminal nonapeptide of proPLAP precipitated the 25 kDa form but not the 22 kDa form, suggesting that the 25 kDa form is a precursor retaining the C-terminal propeptide. When a mutant alpha GL-PLAP, in which the aspartic acid residue is replaced with tryptophan at a putative cleavage/attachment site, was expressed in COS-1 cells, the 25 kDa precursor was the only form found inside the cell and retained in the endoplasmic reticulum, as judged by immunofluorescence microscopy. In vitro translation programmed with mRNAs coding for the wild-type and mutant forms of alpha GL-PLAP demonstrated that the C-terminal propeptide was cleaved from the wild-type chimeric protein, but not from the mutant one. This gave rise to the 22 kDa form attached with a GPI anchor, suggesting that GPI is covalently linked to the aspartic acid residue (Asp159) of alpha GL-PLAP. Taken together, these results indicate that the C-terminal propeptide of PLAP functions as a signal to render alpha GL a GPI-linked membrane protein in vitro and in vivo in cultured cells, and that the chimeric protein constructed in this study may be useful for elucidating the mechanism underlying the cleavage of the propeptide and attachment of GPI, which occur in the endoplasmic reticulum.
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PMID:Conversion of secretory proteins into membrane proteins by fusing with a glycosylphosphatidylinositol anchor signal of alkaline phosphatase. 751 12

A single point mutation that encodes an aspartic acid (Asp578) to glycine substitution in the LH/CG receptor (LH/CGR) gene, D578G, was recently found in American patients with familial male-limited precocious puberty and in a Japanese patient with a sporadic form of the disorder. Transfection of the mutant, compared to the wild-type, LH/CGR complementary DNA into COS-7 cells results in higher basal cAMP production, but a normal agonist-induced response; the mutation is, therefore, proposed to constitutively activate Leydig cells and elevate serum testosterone, despite low levels of gonadotropin. In the current study we examined two additional Japanese patients with male-limited precocious puberty without a family history of the disease. We describe a heterozygous cytosine (C) to thymine (T) transition at nucleotide 1715 in both; the mutation encodes an alanine to valine substitution in codon 572 of transmembrane helix 6, A572V. Transfected into COS-7 cells, the A572V mutant exhibited the same constitutively high basal cAMP levels and normal agonist-induced cAMP response as the D578G mutant. We conclude that the constitutively higher cAMP levels caused by the A572V mutation led to Leydig cell activation and male-limited precocious puberty, as in the previously described D578G mutation. As the mother of one of the two patients had the same heterozygous mutation, this patient represents the first recognized case of inherited male-limited precocious puberty in the Japanese population. The previously described D578G mutant did not increase basal or agonist-induced inositol phosphate production in transfected COS-7 cells, or the number of LH/CGRs or their affinity for LH/CG. In contrast, transfection of the A572V mutation in COS-7 cells exhibited significantly higher inositol phosphate levels basally and at 10(-11) mol/L hCG, but significantly lower inositol phosphate levels at 10(-7) mol/L hCG. These data suggest that the A572V mutation of the LH/CGR may have effects on the guanine nucleotide binding protein which activates phospholipase C (Gq) coupling and phospholipase-C activation in addition to its effects on Gs coupling and activation of adenylyl cyclase. A572V-transfected cells also exhibited a higher affinity, despite an apparent decrease in the number of binding sites, for [125I]hCG, compared to transfectants with the wild-type LH/CGR. We hypothesize that these differences between the A572V and D578G mutations reflect a greater impact of the A572V mutation on receptor conformation.
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PMID:A new constitutively activating point mutation in the luteinizing hormone/choriogonadotropin receptor gene in cases of male-limited precocious puberty. 771 85

The noradrenalin-evoked production of [3H]inositol phosphates in mouse striatal astrocytes in primary culture appeared to be the result of the combined stimulation of alpha 1- and alpha 2-adrenergic receptors. Indeed, the noradrenalin (100 microM) response was only partially reproduced by a maximally effective concentration of methoxamine (100 microM), a selective agonist of alpha 1-adrenergic receptors. In addition, the noradrenalin (100 microM)-induced production of [3H]inositol phosphates, which was completely suppressed by the alpha 1-adrenergic antagonist prazosin (1 microM), was also partially inhibited by yohimbine, a selective antagonist of alpha 2-adrenoceptors (maximum inhibition = -57 +/- 11%, measured in the presence of 10 microM yohimbine; six experiments). Finally, UK14.304, a selective alpha 2-adrenergic agonist that was ineffective alone, enhanced the methoxamine-evoked production of [3H] inositol phosphates (EC50 = 86 +/- 21 nM; three experiments). These results suggest that the stimulation of alpha 1-adrenergic receptors is required for the alpha 2-adrenergic receptor-mediated enhancement of phospholipase C activity. The increased production of [3H]inositol phosphates resulting from the stimulation of alpha 2-adrenergic receptors involved pertussis toxin-sensitive G proteins (Gi/o) and depended on extracellular calcium. As shown using the fluorescent dye indo-1, noradrenalin (100 microM) induced a long-lasting increase in cytosolic calcium in striatal astrocytes. Moreover, noradrenalin (100 microM) stimulated [3H]arachidonic acid release from these cells. These two latter responses may result from synergistic effects due to the combined stimulation of alpha 1- and alpha 2-adrenergic receptors, because they were inhibited by either prazosin (1 microM) or yohimbine (10 microM). Finally, the noradrenalin-evoked production of [3H]inositol phosphates seems to result partly from an inhibition by arachidonic acid of glutamate uptake into astrocytes, leading to the stimulation of glutamate metabotropic receptors coupled to phospholipase C. Indeed, the alpha 2-adrenergic component of the noradrenalin response was suppressed by either enzymatic removal of external glutamate or addition of 2-amino-3-phosphonopropionic acid (1 mM), an antagonist of glutamate metabotropic receptors that blocked the glutamate-evoked production of [3H]inositol phosphates in striatal astrocytes, and was reproduced by the direct application of either glutamate or an inhibitor of glutamate uptake, beta-methyl-DL-aspartic acid.
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PMID:Role of arachidonic acid and glutamate in the formation of inositol phosphates induced by noradrenalin in striatal astrocytes. 790 16

A plasma membrane rich fraction was prepared from olfactory rosettes of Atlantic salmon and used to study binding of L-glutamic acid and activation of phospholipase C (PLC). Glutamate binding was saturable, high affinity, and inhibited by aspartic acid and taurocholate but not by alanine and lysine. Binding of glutamate was potently inhibited by various ligands for rat brain metabotropic glutamate receptors (mGluR) and also by kainate and N-methyl-D-aspartate. Glutamate stimulated phosphatidylinositol 4,5-bisphosphate breakdown consistent with G protein-dependent activation of PLC. Northern blot analyses demonstrated the presence of olfactory rosette RNA that hybridizes with cDNA probes for mGluR1 and mGluR4 under low stringency conditions. The results indicate the salmon olfactory system includes a subtype of the metabotropic glutamate receptor family.
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PMID:A subtype of the metabotropic glutamate receptor family in the olfactory system of Atlantic salmon. 795 44

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

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

Eicosanoid receptors exhibit a highly conserved ERY(C)XXV(I)XXPL sequence in the second intracellular loop. The carboxyl end of this motif contains a bulky hydrophobic amino acid (L,I,V, or F). In human thromboxane A2 receptor (TXA(2)R), phenylalanine 138 is located at the carboxyl end of this highly conserved motif. This study examined the function of the F138 in G protein coupling. F138 was mutated to aspartic acid (D) and tyrosine (Y), respectively. Both mutants F138D and F138Y showed similar ligand binding activity to that of the wild type TXA(2)R. The Kd and Bmax values of either mutant were comparable to those of the wild type receptor. However, both mutants showed significant impairment of agonist induced Ca(2+) signaling and phospholipase C activation. These results suggest that the F138 plays a key role in G protein coupling.
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PMID:Phenylalanine 138 in the second intracellular loop of human thromboxane receptor is critical for receptor-G-protein coupling. 1052 59

Clostridium perfringens phospholipase C (PLC), also called alpha-toxin, is the major virulence factor in the pathogenesis of gas gangrene. The toxic activities of genetically engineered alpha-toxin variants harboring single amino-acid substitutions in three loops of its C-terminal domain were studied. The substitutions were made in aspartic acid residues which bind calcium, and tyrosine residues of the putative membrane-interacting region. The variants D269N and D336N had less than 20% of the hemolytic activity and displayed a cytotoxic potency 103-fold lower than that of the wild-type toxin. The variants in which Tyr275, Tyr307, and Tyr331 were substituted by Asn, Phe, or Leu had 11-73% of the hemolytic activity and exhibited a cytotoxic potency 102- to 105-fold lower than that of the wild-type toxin. The results demonstrated that the sphingomyelinase activity and the C-terminal domain are required for myotoxicity in vivo and that the variants D269N, D336N, Y275N, Y307F, and Y331L had less than 12% of the myotoxic activity displayed by the wild-type toxin. This work therefore identifies residues critical for the toxic activities of C. perfringens PLC and provides new insights toward understanding the mechanism of action of this toxin at a molecular level.
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PMID:Identification of residues critical for toxicity in Clostridium perfringens phospholipase C, the key toxin in gas gangrene. 1093 Dec 4

Tissue-non-specific alkaline phosphatase (TNSALP) is an ectoenzyme anchored to the plasma membrane via glycosylphosphatidylinositol (GPI). A TNSALP mutant with an Asn(153)-->Asp (N153D) substitution was reported in a foetus diagnosed with perinatal hypophosphatasia (Mornet, Taillandier, Peyramaure, Kaper, Muller, Brenner, Bussiere, Freisinger, Godard, Merrer et al. (1998) Eur. J. Hum. Genet. 6, 308-314). When expressed ectopically in COS-1 cells, the wild-type TNSALP formed active non-covalently associated dimers, whereas TNSALP (N153D) formed aberrant disulphide-bonded high-molecular-mass aggregates devoid of enzyme activity. Cell-surface biotinylation and digestion with phosphatidylinositol-specific phospholipase C showed that TNSALP (N153D) failed to reach the cell surface. Instead, double immunofluorescence demonstrated that TNSALP (N153D) partially co-localized with a cis-Golgi marker (GM-130) at the steady-state. Upon treatment with brefeldin A, TNSALP (N153D) was still co-localized with GM-130, further supporting the finding that this mutant is localized in the cis-Golgi. Consistent with morphological results, pulse-chase experiments showed that newly synthesized TNSALP (N153D) remained endo-beta-N-acetylglucosaminidase H-sensitive throughout the chase. Eventually, after a prolonged chase time, the mutant was found to be partly degraded in a proteasome-dependent manner. Since the mutant TNSALP was significantly labelled with [3H]ethanolamine, a component of GPI, comparable with the wild-type enzyme, it is unlikely that the abortive synthesis of the mutant is due to a defect in GPI-attachment. Interestingly, when asparagine was replaced by glutamine at position 153 (N153D), TNSALP (N153Q) was indistinguishable from the wild-type enzyme in terms of its molecular properties, suggesting the possible importance of amino acids with a polar amide group at position 153. Taken together, these findings indicate that replacing asparagine with aspartic acid at position 153 causes misfolding and incorrect assembly of TNSALP, which results in its retention at the cis-Golgi en route to the cell surface, followed by a delayed degradation, presumably as part of a quality-control process. We postulate that the molecular basis of the perinatal hypophosphatasia associated with TNSALP (N153D) is due to the absence of mature TNSALP at the cell surface.
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PMID:Retention at the cis-Golgi and delayed degradation of tissue-non-specific alkaline phosphatase with an Asn153-->Asp substitution, a cause of perinatal hypophosphatasia. 1180 76


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