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)

We report here the crystal structure of the complex formed between phospholipase C (PLC) from Bacillus cereus and the widely used biochemical buffer tris (hydroxymethyl)-methylamine (Tris). The structure has been determined at 1.9 A resolution and refined to R = 20.3%. Tris has metal-binding properties, especially to Zn2+, and has been reported to reduce the activity of PLC. The amine nitrogen atom in Tris is co-ordinated to one of the three Zn2+ ions in the active site of the enzyme, thus confirming its chelating properties and the involvement of the metal ions in the catalytic process. The occupancy of the Zn2+ ion in site 2 in native PLC is 0.6 which could imply the presence of Ca2+ rather than Zn2+. The fact that Tris binds to this metal ion, the nature of the site 2 co-ordination shell and comparison with several homologous Zn-metalloenzymes indicate that PLC is a 3-Zn metalloenzyme. This study is one of a series which explores the active site of PLC by complexing the enzyme with inhibitors and substrate analogues.
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PMID:The crystal structure of tris-inhibited phospholipase C from Bacillus cereus at 1.9 A resolution. The nature of the metal ion in site 2. 851 56

A new cytolytic toxin, designated as S-Hemolysin, was found in the culture filtrate of Streptomyces sp. strain No. A-6288, isolated from a soil sample. The molecular weight of S-Hemolysin was estimated to be 10,000 by SDS-polyacrylamide gel electrophoresis and to be 20,000 by Sephadex G-100. S-Hemolysin is a glycoprotein that is composed of 102 amino acid residues with 11.6% glucose, and the isoelectric point is around pH 5.8. The phospholipase C activity of S-Hemolysin was specific for the following substrates in this order: sphingomyelin > lysophosphatidylethanolamine > lysophosphatidylcholine > phosphatidylethanolamine > phosphatidylcholine. S-Hemolysin had hemolytic activity against rabbit, human, and sheep erythrocytes, but did not cause aggregation of human platelets. These activities were accelerated with Mg2+, Mn2+, and Co2+ ions and inhibited by the addition of Ca2+, Cu2+, and Zn2+ ions. This enzyme was shown to be different from the known bacterial phospholipase C.
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PMID:Purification and some properties of S-Hemolysin produced by Streptomyces sp. strain no. A-6288. 854 46

Metal selectivity of exocytosis was analyzed by comparing the effects of polyvalent metal cations Ca2+, Ba2+, Sr2+, Pb2+, La3+, Cd2+, Co2+, Tb3+, Mn2+, and Zn2+ on the release of norepinephrine (NE) from staphylococcal alpha-toxin-permeabilized bovine chromaffin cells. Pb2+, La3+, Cd2+, Sr2+, and Ba2+ activated NE secretion accompanied by the release of intragranular dopamine beta-hydroxylase but not cytosolic lactate dehydrogenase, indicating the activation of the mechanism of exocytosis. The release triggered by saturating concentrations of Pb2+, La3+, Cd2+, and Sr2+ was nonadditive with Ca2+, indicating a common site of action. In contrast, the Ba2(+)-evoked NE release was additive with Ca2+ and the Ca2+ agonists Pb2+, La3+, Cd2+, and Sr2+, suggesting that Ba2+ activates secretion at a site distinct from the Ca2+ receptor. In distinction to the NE release evoked by Pb2+, La3+, Cd2+, and Ba2+, the Sr(2+)-evoked NE release was associated with a significant elevation of Ca2+ concentration in the medium and abolished by Ca2+ chelation. This indicates that the secretagogue effect of Sr2+ was indirect and secondary to the displacement of bound Ca2+, Co2+ and Mn2+ inhibited the NE release evoked by Ca2+, Sr2+, Pb2+, La3+, and Cd2+ but had no effect on the Ba(2+)-dependent secretion. Tb3+ and Zn2+ were without effect on exocytosis.
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PMID:Metal selectivity of exocytosis in alpha-toxin-permeabilized bovine chromaffin cells. 859 35

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

Nonelectrolytes such as polyethylene glycols (PEG) and dextrans (i) promote the association of S. aureus alpha-toxin with liposomes (shown by Coomassie staining) and (ii) enhance the rate and extent of calcein leakage from calcein-loaded liposomes; such leakage is inhibited by H+, Zn2+ and Ca2+ to the same extent as that of nonPEG-treated liposomes. Incubation of liposomes treated with alpha-toxin in the presence of PEG with the hydrophobic photo-affinity probe 3-(trifluoromethyl)-3-m-[125I]iodophenyl) diazirine (125I-TID) labels monomeric and-predominantly-hexameric forms of liposome-associated alpha-toxin; in the absence of PEG little labeling is apparent. At high concentrations of H+ and Zn2+ but not of Ca(2+)-all of which inhibit calcein leakage-the distribution of label between hexamer and monomer is perturbed in favor of the latter. In alpha-toxin-treated planar lipid bilayers from which excess toxin has been washed away, PEGs and dextrans strongly promote the appearance of ion-conducting pores. The properties of such pores are similar in most regards to pores induced in the absence of nonelectrolytes; the differ only in being more sensitive to "closure" by voltage (as are pores induced in cells). In both systems, the stimulation by nonelectrolytes increase with concentration and with molecular mass up to a maximum around 2,000 Da. We conclude (i) that most of the alpha toxin that becomes associated with liposome or planar lipid bilayers does not form active pores and (ii) that the properties of alpha-toxin-induced pores in lipid bilayers can be modulated to resemble those in cells.
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PMID:Pore formation by S. aureus alpha-toxin in liposomes and planar lipid bilayers: effects of nonelectrolytes. 869 78

Clostridium perfringens KZ1340 isolated from Antarctic soil was first classified as Clostridium plagarum and later as a lecithinase-negative variant of C. perfringens. Although the strain produced no detectable lecithinase (phospholipase C, PLC) activity in the culture supernatant, it was shown by Southern blot hybridization to possess a PLC-encoding gene (plc). To determine the cause of the PLC deficiency, we cloned and sequenced the plc gene from KZ1340. The deduced amino acid sequence consists of 398 amino acid residues, coinciding with those of the plc genes previously determined. Tyrosine was substituted for histidine at amino acid position 148, which is thought to bind a zinc ion essential for PLC activity. Northern blot analysis revealed that KZ1340 expressed the plc gene at an extremely low level. Furthermore, the plc gene cloned from C. perfringens strain 13 into a plasmid was expressed weakly in KZ1340, compared to that in strain 13. This indicates that the former strain represses plc gene expression in trans. When a phylogenetic tree of plc genes was constructed, the KZ1340 plc gene formed a monophyletic branch along with those of various other C. perfringens strains, supporting the classification of the strain as a variant of C. perfringens.
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PMID:Analysis of the phospholipase C gene of Clostridium perfringens KZ1340 isolated from Antarctic soil. 870 60

Phospholipase C (phosphatidylcholine phosphohydrolase, EC 3.1.4.3) and lipase (EC 3.1.1.3) activities were detected in the supernatant fluid of Pseudomonas fluorescens strain D cultures. A combination of ultrafiltration and successive chromatography through columns of Sephadex G-75 and DEAE-cellulose was used to purify the phospholipase C over 700-fold from the culture medium, with 28.5% yield. The purified enzyme appeared as a single band after polyacrylamide gel electrophoresis. The apparent molecular mass of the phospholipase C was 36,000 daltons when estimated by gel permeation chromatography. The purified enzyme hydrolysed phosphatidylcholine more efficiently than phosphatidylethanolamine. The synthetic substrate p-nitrophenylphosphorylcholine, phosphatidylinositol or sphingomyelin were not hydrolysed. Hydrolysis of phosphatidylcholine was inhibited by EDTA (1mM) and stimulated by Zn2+, Mg2+ ions and detergents. These properties of the enzyme indicate that it is distinct from the previously reported Ps. fluorescens phospholipase C.
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PMID:Characterisation of a phospholipase C produced by Pseudomonas fluorescens. 872 7

Excitatory amino acids (EAA) became known as neurotransmitters of the central nervous system (CNS) in the last decade. The most studied EAA are glutamate and aspartate. Both are synthetized by the same mechanism as gamaaminobutyric acid. (Fig. 1). Glutamate is widely distributed in the CNS and the spinal cord, being the areas of higher concentration the cerebral cortex, the hypocampus and the cerebellum. There have been identified two type of receptors for glutamate: ionotropic and metabotropic. The former includes three different types: NMDA, AMPA and KA. NMDA receptor is coupled to a Na+ and Ca2+ channel being the second ion the most important one. This receptor has several sites of binding for various substances. Along with the site for N-methyl-D-aspartate, which binds glutamate and/or aspartate, there have been identified a site for the binding of glycine (which is different from the strychnine sensitive one), a site for poliamines such as spermine and spermidine, and a site for the binding of Zn2+ (Table 1). AMPA receptor is associated to a Ca(2+)-Na+ channel, being in this case the Na+ the most important ion. There are two metabotropic type receptors: L-AP4 and trans-ACPD. Both are coupled to a G protein and agonists exert their action increasing phospholipase C activity which in turn induces an increment of IP3 and diacyl-glicerol, and a consecutive releasing of Ca2+ from intracellular stores. EAA play a role in some physiological processes. One of them is long-term potentiation (LTP), an electrochemical phenomenon involved in memory consolidation. Antagonists of NMDA and AMPA receptor prevent the development of LTP, and conversely, the agonist of glycine site of NMDA receptor--D-cycloserine--facilitates memory consolidation. Since 1957, EAA are considered neurotoxic substances and there are many indirect evidences to support this statement. Pathogenesis of neuronal damage elicited by EAA involves the events shown in Fig. 3. Prevention of the cascade of events that provokes neurotoxicity may be achieved by NMDA antagonists, but once it has begun it may be only aborted subtracting the Ca2+ from the medium, using nifedipine or blocking AMPA receptor with an antagonist (CNQX). EAA have been shown to play a toxic role in neuronal damage induced by ischemia. Research using various experimental models demonstrated that NMDA receptor antagonists (i.e. MK 801) blocks postischemic damage. Interventions at various levels of the pathogenic cascade shown in Fig. 4 provoke the same results. There is enough evidence to suspect that NMDA and AMPA receptors are altered in epilepsy. NMDA antagonists (i.e. MK801 or AP5) prevent the development of epileptic seizures induced by kindling; CNQX, an AMPA antagonist, blocks the increase in electrical activity induced by K+ in slices of hypocampus; felbamate, an antiepileptic drug, blocks the glycine site (not strychnine sensitive) decreasing NMDA receptor activity. Several neurodegenerative disorders have been associated with exogenous administration or accidental intake of EAA. (i.e. neurolatirism, Guam disease). Similarities between these diseases and lateral aminotrophic sclerosis indicate that in the latter EAA may play a pathogenic role. Finally, the psychotomimetic effect of phencyclidine (an antagonist of NMDA receptor) suggests that in schizophrenia, together with dopaminergic neurotransmission impairment, some dysfunction of glutamate pathways may be present.
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PMID:[Role of excitatory amino acids in neuropathology]. 872 78

Alkaline phosphatase activity was released up to 100% from the membrane by incubating the rat osseous plate membrane-bound enzyme with phosphatidylinositol-specific phospholipase C. The molecular weight of the released enzyme was 145,000 on Sephacryl S-300 gel filtration and 66,000 on PAGE-SDS, suggesting a dimeric structure. Solubilization of the membrane-bound enzyme with phospholipase C did not destroy its ability to hydrolyse PNPP, ATP and pyrophosphate. The hydrolysis of ATP and PNPP by phosphatidylinositol-specific phospholipase C-released enzyme exhibited 'Michaelian' kinetics with K0.5 = 70 and 979 microM, respectively. For pyrophosphate, K0.5 was 128 microM and site-site interactions were observed (n = 1.4). Magnesium ions were stimulatory (K0.5 = 1.5 mM) and zinc ions were a powerful noncompetitive inhibitor (Ki = 6.2 microM) of phosphatidylinositol-specific phospholipase C-released enzyme. Phosphatidylinositol-specific phospholipase C-released alkaline phosphatase was relatively stable at 40 degrees C. However, with increasing temperature from 40-60 degrees C, the enzyme was inactivated rapidly following first order kinetics and thermal inactivation constants varied from 5.08 x 10(-4) min-1 to 0.684 min-1. Treatment of phosphatydilinositol-specific phospholipase C-released alkaline phosphatase with Chellex 100 depleted to 5% its original PNPPase activity. Magnesium (K0.5 = 29.5 microM), manganese (K0.5 = 5 microM) and cobalt ions (K0.5 = 10.1 microM) restored the activity of Chelex-treated enzyme, demonstrating its metalloenzyme nature. The stimulation of Chelex-treated enzyme by calcium ions (K0.5 = 653 microM) was less effective (only 26%) and occurred with site-site interactions (n = 0.7). Zinc ions had no stimulatory effects. The possibility that the soluble form of the enzyme, detected during endochondral ossification, would arise by the hydrolysis of the Pl-anchored form of osseous plate alkaline phosphatase is discussed.
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PMID:Characterization of the phosphatidylinositol-specific phospholipase C-released form of rat osseous plate alkaline phosphatase and its possible significance on endochondral ossification. 875 Nov 58

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


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