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)

Membrane fragments rich in cholinergic (nicotinic) receptor sites, purified from homogenates of Torpedo marmorata electric organ, are dissolved in high concentrations of Na cholate and Tris buffer in the presence of Na ethylenediaminetetraacetate, without loss of the ability to bind Naja nigricollis [(3)H]alpha-toxin. After extensive dialysis to remove cholate, the solution is supplemented with crude lipids extracted from native membrane fragments and MgCl(2)-CaCl(2) added dropwise to the mixture as a concentrated solution. The reconstituted membranes obtained retain (22)Na(+). Release of Na from these microsacs increases in the presence of carbamylcholine and the alpha-toxin from N. nigricollis blocks this effect.
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PMID:Reconstitution of a chemically excitable membrane. 452 51

Rat kidney proximal tubule brush border membrane (BBM)-associated phosphatidylinositol-specific phospholipase C (PI-PLC) has been characterized previously in our laboratory. Here we report the effect of aminoglycosides on this enzyme. Enzyme activity is determined at 37 degrees C by increases in diacylglycerol or decreases in PI in the presence of Ca++, deoxycholate and [3H] arachidonate-labeled phospholipids in Tris buffer. Whereas activity of PI-PLC is inhibited 90% by gentamicin (1.5 mM) at pH 6 to 7, inhibition decreases to 72% at pH 7.4 and to zero at pH 7.8. As pH is raised from 7.8 to 9.5, gentamicin elicits a pH-dependent stimulation of activity. Alterations in Ca++ concentration (1-7.5 mM) have no effect on inhibition of PI-PLC by gentamicin, although the enzyme itself is critically dependent on divalent cations. Double reciprocal plots of activity vs. substrate concentration in the presence of 0, 1.0 and 1.5 mM gentamicin demonstrate uncompetitive interaction between enzyme and drug. Vmax of PI-PLC in the absence of gentamicin is 0.73 mumol/h/mg of protein and the Km is 7.05 microM (PI). Vmax and apparent Km decrease with increasing drug concentration. Comparative inhibition of PI-PLC by other aminoglycosides (at 1-2 mM concentrations) approximates their known nephrotoxic potential: streptomycin less than or equal to kanamycin less than or equal to amikacin less than tobramycin less than gentamicin less than neomycin. Kidney cortex cytosolic PI-PLC activity is also inhibited by gentamicin. However, BBM PI-PLC specific activity is 15 to 20-fold greater than for the cytosolic enzyme. Because marked gentamicin binding to BBM and damage/loss of BBM occurs early after administration of the drug, gentamicin-induced modulation of BBM PI-PLC may be an important factor in ensuing nephrotoxicity.
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PMID:Effects of aminoglycosides on proximal tubule brush border membrane phosphatidylinositol-specific phospholipase C. 609 5

The TSH receptor from human thyroid plasma membranes has been solubilized in 10 mM Tris/HCl, 50 mM NaCl, pH 7.4 containing 0.5% triton X-100. Binding of [125I]TSH to the soluble receptor showed rapid and reversible kinetics and reached a maximum within 30 min at 37 degrees C, by 1 h at 25 degrees C and by 24 h 4 degrees C. Optimal pH was 7.4. The soluble receptor retained specificity with cross-reactivity only to crude hCG (0.03%). Scatchard plots were curvilinear indicating the presence of at least two binding sites. The high affinity site showed an affinity content of 1.1 X 10(9) M-1 with binding capacity of 1.3 X 10(-10) M/mg protein. TSH-binding inhibitor immunoglobulins from patients with Graves' disease inhibited [125I]TSH binding to the soluble receptor in a dose-dependent manner. NaCl inhibited the TSh binding and this was ascribed to the decrease in the receptor capacity. Trypsin, neuraminidase and phospholipase C treatment of the solubilized receptor had no effect on TSH binding. The apparent molecular weight of the receptor, determined by gel filtration of Sepharose 6B, was approximately 300 000.
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PMID:Characterization of triton-solubilized TSH receptors from human thyroid plasma membranes. 626 43

The rate of phospholipid hydrolysis in erythrocyte ghosts by Bacillus cereus phospholipase C was markedly decreased by the presence of NaCl at concentrations between 25 and 200 mM. The inhibition seemed to be due to Cl- and was unaffected by the type of cation present. The larger univalent anions such as HCO3-, Br-, Cl-, NO3-, CNO- and I- seemed most effective, whereas the bivalent anion SO42- was relatively ineffective at 0.1 M, as were acetate and formate. Tris buffers at 0.1 M caused marked inhibition. With bovine brain myelin, phospholipid hydrolysis by phospholipase C was also much more strongly inhibited by I- and Cl- than by SO42- or acetate. NaCl inhibited the hydrolysis by the enzyme of the soluble substrate dihexanoylglycerophosphocholine, thereby suggesting that the inhibiton did not arise simply from substrate effects.
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PMID:Inhibition of Bacillus cereus phospholipase C by univalent anions. 681 Aug 75

The mechanism of increased tissue Ca2+ uptake during reoxygenation after hypoxia was studied in isolated, arterially perfused rabbit septum maintained at 27 degrees C or 37 degrees C. Tissue 47Ca2+, 85Sr2+, or 133Ba2+ uptake was measured by a juxtaposed gamma-probe and a counter. At 27 degrees C, Ba2+ flux across the sarcolemma is similar to that of Ca2+ and Sr2+, but Ba2+ is not taken up by sarcoplasmic reticulum or mitochondria. Therefore 133Ba2+ flux studies were used to delineate the effects of hypoxia and reoxygenation on sarcolemmal permeability to divalent cations. In muscles maintained at 27 degrees C, reoxygenation after 40 min of hypoxia caused significant increases in both 47Ca2+ and 85Sr2+ uptakes. In contrast, there was no change in tissue 133Ba2+ uptake, 133Ba2+ efflux, determined from 133Ba2+ washout studies, was also unchanged. When the sarcolemma was disrupted by perfusing the muscle with a solution containing phospholipase C, tissue 133Ba2+ uptake as well as 47Ca2+ and 85Sr2+ uptakes increased. Moreover an increase in 133Ba2+ efflux was observed after phospholipase infusion. Addition of an inhibitor or an uncoupler of mitochondrial respiration [sodium cyanide (5 X 10(-3) M) or dinitrophenol (5 X 10(-4) M), respectively] in the perfusate caused significant decreases in reoxygenation-induced tissue Ca2+ gain. In muscles perfused with a solution that did not contain permeant anions capable of proton donation (Tris buffer without HCO3(-) and H2PO4(-)), tissue CA2+ gain during reoxygenation was significantly reduced. Perfusion with Tris buffer also caused greater recovery of mechanical function and myocardial ATP concentration during reoxygenation. In muscles maintained at 37 degrees C, both tissue 47Ca2+ and 133Ba2+ uptakes increased during reoxygenation after 40 min of hypoxia. Isolated mitochondria accumulated both Ca2+ and Ba2+ at 37 degrees C. These data suggest that the reoxygenation-induced tissue Ca2+ uptake is primarily caused by an active uptake by mitochondria and that the increase in mitochondrial Ca2+ uptake can occur without any changes in sarcolemmal permeability to divalent cations (Ca2+, Sr2+, or Ba2+). The data also suggest that the increased mitochondrial Ca2+ uptake is responsible, at least in part, for the impaired recovery of myocardial mechanical and cellular function after hypoxia.
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PMID:Mechanism of tissue Ca2+ gain during reoxygenation after hypoxia in rabbit myocardium. 706 4

Amphiphilic monomers and dimers of acetylcholinesterase (AChE) and hydrophilic tetramers of butyrylcholinesterase (BuChE) were released by extracting human meningioma with Tris-saline and Tris-saline-Triton X-100 buffers. The amphiphilic or hydrophilic behavior of the AChE and BuChE forms was assessed by sedimentation analysis, hydrophobic chromatography and Triton X-114 phase-partitioning. A significant fraction of the amphiphilic AChE species was converted into hydrophilic components by incubation of the soluble enzyme with phosphatidylinositol-specific phospholipase C (PIPLC) from Bacillus thuringiensis, this fraction being increased by a double treatment with PIPLC and alkaline hydroxylamine. A significant amount of the membrane-bound AChE was released by incubation with PIPLC. These results demonstrate that AChE forms in meningioma are attached to the membrane via glycosylphosphatidylinositol, although part of the enzyme forms are resistant to PIPLC.
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PMID:Monomers and dimers of acetylcholinesterase in human meningioma are anchored to the membrane by glycosylphosphatidylinositol. 747 60

Listeria monocytogenes secretes a phospholipase C (PLC) which has 39% amino acid sequence identity with the broad-specificity PLC from Bacillus cereus. Recent work indicates that the L. monocytogenes enzyme plays a role during infections of mammalian cells (J.-A. Vazquez-Boland, C. Kocks, S. Dramsi, H. Ohayon, C. Geoffroy, J. Mengaud, and P. Cossart, Infect. Immun. 60:219-230, 1992). The homogeneous enzyme has a specific activity of 230 mumol/min/mg when phosphatidylcholine (PC) is dispersed in sodium deoxycholate. With phospholipid-Triton X-100 mixed micelles, the enzyme had a broad pH optimum between 5.5 and 8.0, and the rates of lipid hydrolysis were in the following order: PC > phosphatidylethanolamine (PE) > phosphatidylserine > sphingomyelin >> phosphatidylinositol (PI). Activity on PC was stimulated 35% by 0.5 M NaCl and 60% by 0.05 mM ZnSO4. When Escherichia coli phospholipids were dispersed in Triton X-100, PE and phosphatidylglycerol, but not cardiolipin, were hydrolyzed. The enzyme was active on all phospholipids of vesiculated human erythrocytes including PI, which was rapidly hydrolyzed at pH 7.0. PI was also hydrolyzed in PI-PC-cholesterol liposomes by the nonspecific PLC from L. monocytogenes and by the homologous enzyme from B. cereus. The water-soluble hydrolysis product was identified as inositol-1-phosphate. For the hydrolysis of human erythrocyte ghost phospholipids, a broad pH optimum was also observed. 32P-labelled Clostridium butyricum protoplasts, which are rich in ether lipids, were treated with PLC. The enzyme hydrolyzed the plasmalogen form of PE, its glycerol acetal, and cardiolipin, in addition to PE. I-, Cl- and F- stimulated activity on either PC- Triton X-100 mixed micelles or human erythrocyte ghosts, unlike the enzyme from B. cereus which is strongly inhibited by halides. Tris-HCl, phosphate, and calcium nitrate had similar inhibitory effects on the enzyme on the enzymes from L. monocytogenes and B. cereus.
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PMID:Nonspecific phospholipase C of Listeria monocytogenes: activity on phospholipids in Triton X-100-mixed micelles and in biological membranes. 833 Oct 63

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 mutant toxin (MT) that abolished almost 99% of the hemolytic activity of alpha-toxin was isolated by random polymerase chain reaction (PCR) mutagenesis of the gene for Clostridium perfringens alpha-toxin. In the mutant toxin, the amino acids at Tyr (Y)-62, Thr (T)-74 and Ile (I)-345 were substituted with His, Ile and Met, respectively. Replacement of T-74 with Ile by site-directed mutagenesis resulted in the loss of hemolytic, phospholipase C and sphingomyelinase activities by 1/250-fold of that of the wild-type. The replacement of Y-62 with Ile or I-345 with Met alone did not affect the activities of the toxin. T74I mutant bound to sheep erythrocyte membranes and specifically bound [65Zn]2+ in Tris-buffered saline, in the same manner as the wild-type, and contained 2 mol of zinc ions per mol of protein. These results suggest that the T-74 residue plays a key role in these biological activities of C. perfringens alpha-toxin.
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PMID:Threonine-74 is a key site for the activity of Clostridium perfringens alpha-toxin. 893 72

Bacillus cereus secretes a nonspecific phospholipase C (PLC) that catalyzes the hydrolysis of phospholipids to yield diacylglycerol and a phosphate monoester. B. cereus PLC has been overexpressed with its signal sequence in Escherichia coli using a T7 expression system. The expressed enzyme formed intracellular inclusion bodies which were solubilized in the presence of 8 M urea. Renaturation was initiated by gradual removal of urea and addition of zinc ions. The signal peptide was specifically cleaved by a protease, clostripain, added when the urea concentration was 1.5 M. Factors that led to protein reaggregation included rapid removal of urea, use of Tris instead of barbital buffer, and presence of the signal peptide when the urea concentration was below 1.5 M. The folded protein was purified by Q-Sepharose Fast flow chromatography to yield a preparation > 99% pure. The final yield of active enzyme was 30-40 mg per liter of culture. The recombinant PLC exhibited biochemical and kinetic properties identical to those of extracellularly produced PLC from B. cereus. Site-specific mutagenesis of Asn-134 was carried out as a test of the general effectiveness of the refolding procedure.
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PMID:Cloning, overexpression, refolding, and purification of the nonspecific phospholipase C from Bacillus cereus. 926 84


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