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)

Incubation of phospholipase C from Bacillus cereus with certain divalent metal cations caused enzyme inactivation with Cu(II) being particularly effective. The inactivation arose from the reversible exchange of Zn(II) in the enzyme with the metal cations. Both zinc atoms in the enzyme exchanged rapidly with Cu(II) whereas only one exchanged spontaneously with Co(II). With lecithin substrates, CoZn-phospholipase C had a specific activity of 3.6-11.3% of that of ZnZn-phospholipase C, whereas the CoCo-enzyme was less than 1% active relative to the native enzyme. The CoZn-enzyme had the same Km value for dihexanoyllecithin as had the native enzyme, but the Vm value was markedly lower. ZnZn-, CoZn- and CoCo-phospholipase C all had very low activities towards sphingomyelin micelles, although for the CoCo-enzyme, the sphingomyelinase activity was 4-7-fold greater than for the native enzyme.
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PMID:Effect of some divalent metal cations on phospholipase C from Bacillus cereus. 679 44

Phospholipid degradation by native phospholipase C from Bacillus cereus and enzyme forms where one or both of the Zn2+ prosthetic groups had been replaced with Co2+ was studied in human erythrocyte membranes (ghosts) and resuspended freeze-dried bovine brain myelin. The rate of total phospholipid degradation was 2-9-fold more rapid with erythrocytes than with myelin. With both membrane systems the activity decreased in the order ZnZn-enzyme greater than ZnCo-enzyme greater than CoCo-enzyme. For all three enzyme forms with either membrane system, phosphatidylethanolamine (or the ethanolamine-containing phosphoglycerides) and phosphatidylcholine were hydrolysed most rapidly and sphingomyelin least. The relative rate of sphingomyelin degradation was highest with the ZnCo-enzyme. In myelin at low ionic strength there seemed to be a core of phospholipid that was very resistant to degradation by native phospholipase C but which was much more accessible to the Co2+-substituted forms. It is suggested that ZnCo-phospholipase C has potential applications in membrane studies.
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PMID:Effect of Co2+-substitution on the substrate specificity of phospholipase C from Bacillus cereus during attack on two membrane systems. 681 48

A phosphatidylinositol-specific phospholipase C was purified from the culture broth of Bacillus thuringiensis to a homogeneous state as indicated by polyacrylamide gel electrophoresis. Specific activity of purified enzyme was 312 units/mg, and the recovery of the enzyme activity was 27.2%. The purified enzyme (molecular weight: 23 000 +/- 1000) was maximally active at pH 7.5 and not influenced by EDTA. The enzyme specifically hydrolyzed phosphatidylinositol, but did not act on phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol and sphingomyelin. The products from phosphatidylinositol of enzyme reaction were diacylglycerol and myoinositol 1,2-cyclic phosphate. The enzyme activity was stimulated by sodium deoxycholate or Triton X-100. Divalent cations such as Ca2+, Mg2+ and Zn2+ were inhibitory at concentrations above 10(-3) M. KCl and NaCl were inhibitory at the concentration higher than 10(-2) M. Alkaline phosphatase, an ecto-enzyme located on the surface of plasma membrane, was released from the slices of rat liver, kidney, pancreas and intestine by the treatment with this phospholipase.
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PMID:Purification and properties of phosphatidylinositol-specific phospholipase C of Bacillus thuringiensis. 741 68

An in vitro study examining the effects of zinc treatment on human PMN cell phagocytosis and killing of Staphylococcus aureus and Staphylococcus epidermidis and the cytoprotection of zinc against staphylococcal toxins. Phagocytosis was studied by transmission electron microscopy using different microbiological techniques, one of which was designed to follow the kinetics of bacterial killing. No effect was found on phagocytosis and bacterial killing. The cytotoxic effects of a crude toxin and an alpha-toxin extracted from Staphylococcus aureus preparations were studied on human PMN cells using the standard 51Cr release assay. Both toxins induced a dose-dependent leakage of 51Cr, indicating cell membrane damage. These results were confirmed by electron microscopy during the phagocytosis of S. aureus, where severe PMN cellular degeneration was observed. The addition of zinc to PMN cells strongly inhibited the release of 51Cr. In conclusion, our results show that zinc in higher than physiological concentrations does not inhibit PMN cell functions such as phagocytosis and intracellular killing of S. aureus and S. epidermidis. The addition of zinc may be beneficial in certain clinical situations, such as wound healing, zinc deficiency and infections involving toxin-producing bacteria, e.g. S. aureus.
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PMID:The effect of zinc on bacterial phagocytosis, killing and cytoprotection in human polymorphonuclear leucocytes. 748 84

The Bacillus thuringiensis CryIA(c) insecticidal delta-endotoxin binds to a 120-kDa glycoprotein receptor in the larval midgut epithelia of the susceptible insect Manduca sexta. This glycoprotein has recently been purified and identified as aminopeptidase N. We now report the cloning of aminopeptidase N from a M. sexta midgut cDNA library. Two overlapping clones were isolated, and their combined 3095-nucleotide sequence contains an open reading frame encoding a 990-residue pre-pro-protein. The N-terminal amino acid sequence derived from the glycoprotein is present in the open reading frame, immediately following a predicted cleavable signal peptide and a pro-peptide. There are four potential N-linked glycosylation sites. The C-terminal sequence contains a possible glycosylphosphatidylinositol (GPI) anchor signal peptide, which suggests that, unlike most other characterized aminopeptidases, the lepidopteran enzyme is anchored in the membrane by a GPI anchor. This was confirmed by partial release of aminopeptidase N activity from M. sexta midgut brush border membranes by phosphatidylinositol-specific phospholipase C. The deduced amino acid sequence shows significant similarity to the zinc-dependent aminopeptidase gene family, particularly in the region surrounding the consensus zinc-binding motif characteristic of these enzymes.
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PMID:Molecular cloning of an insect aminopeptidase N that serves as a receptor for Bacillus thuringiensis CryIA(c) toxin. 762 76

Serpulina hyodysenteriae produces an oxygen-stable heat-labile hemolysin that may be an important virulence factor in the pathogenesis of swine dysentery. We examined the effect of Ca2+, Co2+, Cu2+, Fe2+, Mg2+, Mn2+, Ni2+, and Zn2+ on the hemolytic activity of cell-free supernatant (CFS) from S. hyodysenteriae, isolate B204. Cells harvested from late logarithmic phase cultures were incubated in phosphate-buffered saline containing glucose and RNA-core (PBS-GR) with or without cations and the hemolytic activity of CFS obtained after successive 30 min incubation and washing cycles was determined. The addition of either ZnSO4 or CuSO4 to the PBS-GR caused complete inhibition of hemolytic activity after 3 cycles; other cations gave results similar to control extracts. Reduction in the concentration of Zn2+ in CFS by 60 to 80% after each incubation cycle and binding of Zn2+ by EDTA indicated that Zn2+ was associated with the cell fraction, and inhibition of hemolysin activity was specifically mediated by Zn2+. When the spirochetes were washed after incubation in the presence of ZnSO4 for 2 cycles and incubated in fresh PBS-GR without Zn2+, inhibition of hemolysin activity remained unchanged, indicating that the inhibitory effect of ZnSO4 was due to a direct action of ZnSO4 on the spirochetes. Since neither the viability of the spirochetes nor the activity of pre-formed hemolysin were affected by the presence of ZnSO4, the inhibitory effect of Zn2+ cations was attributed to reduced biosynthesis by viable S. hyodysenteriae cells rather than interference of Zn2+ cations with lysis of erythrocytes by the hemolysin. Transmission electron microscopic examination of spirochetes after incubation in PBS-GR containing ZnSO4 revealed clumping of ribosomes and clearing of cell cytoplasm.
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PMID:Effect of divalent cations on hemolysin synthesis by Serpulina (Treponema) hyodysenteriae: inhibition induced by zinc and copper. 780 26

Cd2+ provokes an immediate production of inositol trisphosphate and the release of Ca2+ from internal stores in human fibroblasts and some other mammalian cells. Ni2+, Co2+, Fe2+, and Mn2+ evoke the release of stored Ca2+, but are less potent than Cd2+ (apparent K0.5 = 40 nM). Zn2+ and Cu2+ competitively inhibit Ca2+ release evoked by Cd2+ without affecting Ca2+ release by hormones such as bradykinin. Zn2+ has the same apparent Ki value (80-90 nM) towards the five agonist metals, which suggests that the metals interact with the same site. Many other divalent cations neither released stored Ca2+ nor affected Cd(2+)-evoked Ca2+ release. The agonist metals appear to activate phospholipase C via a G protein rather than a tyrosine kinase. The production of reactive oxygen species is probably not involved in Ca2+ release by the metals. Cd2+ and other stimuli that raise cytosolic-free Ca2+ induce cyclic (AMP) production, apparently by activating a calmodulin-dependent adenylyl cyclase. We suggest that an orphan receptor mediates the hormonelike responses to Cd2+ and the other agonist metals. The receptor is referred to as an orphan because its physiological stimulus is unknown. Growth of the fibroblasts in high Zn2+ desensitizes them to the five agonist metals without affecting Ca2+ release by bradykinin or histamine. A several hour incubation in culture medium with normal Zn2+ fully restores responsiveness to the five active metals. Growth in high Zn2+ appears to repress the synthesis of the putative orphan receptor because inhibitors of RNA or protein synthesis, or asparagine-linked glycosylation, prevented the restoration of metal responsiveness.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Transmembrane signals and protooncogene induction evoked by carcinogenic metals and prevented by zinc. 784 95

Mutagenesis of H-68 or -148 in Clostridium perfringens alpha-toxin resulted in complete loss of hemolytic, phospholipase C, sphingomyelinase, and lethal activities of the toxin. These activities of the variant toxin at H-126 or -136 decreased by approximately 100-fold of the activities of the wild-type toxin. Mutation at H-46, -207, -212, or -241 showed no effect on the biological activities, indicating that these residues are not essential for these activities. The variant toxin at H-11 was not detected in culture supernatant and in cells of the transformant carrying the variant toxin gene. Wild-type toxin and the variant toxin at H-148 bound to erythrocytes in the presence of Ca2+; however, the variant toxins at H-68, -126, and -136 did not. Co2+ and Mn2+ ions stimulated binding of the variant toxin at H-68, -126, and -136 to membranes in the presence of Ca2+ and caused an increase in hemolytic activity. Wild-type toxin and the variant toxins at H-68, -126, and -136 contained two zinc atoms in the molecule. Wild-type toxin inactivated by EDTA contained two zinc atoms. These results suggest that wild-type toxin contains two tightly bound zinc atoms which are not coordinated to H-68, -126, and -136. The variant toxin at H-148 possessed only one zinc atom. Wild-type toxin and the variant toxin at H-148 showed [65Zn]2+ binding, but the variant toxins at H-68, -126, and -136 did not. Furthermore, [65Zn]2+ binding to wild-type toxin was competitively inhibited by unlabeled Zn2+, Co2+, and Mn2+. These results suggest that H-68, -126, and -136 residues bind an exchangeable and labile metal which is important for binding to membranes and that H-148 tightly binds one zinc atom which is essential for the active site of alpha-toxin.
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PMID:Site-directed mutagenesis of histidine residues in Clostridium perfringens alpha-toxin. 786 89

Phospholipase C produced by Pseudomonas fluorescens, isolated as a laboratory contaminant, has been purified to apparent homogeneity by ammonium sulphate fractionation, anion-exchange and size-exclusion chromatographies. The apparent molecular mass of the purified polypeptide was 39.5 kDa. Purified preparations of phospholipase C were used to characterize its enzymic properties and to obtain amino acid sequence of the N-terminus of the molecule. The P. fluorescens phospholipase C hydrolysed PtdEtn, PtdCho and PtdSer (PtdEtn > PtdCho >> PtdSer) and was relatively thermostable. The enzyme was inactivated in the presence of chelating agent o-phenanthroline and the activity restored after addition of zinc. Properties of this enzyme and in particular the requirement for zinc ions for the activity, revealed similarity with the well characterised Bacillus cereus phospholipase C. Similarities with other bacterial and mammalian enzymes reported to be related to the B. cereus type are discussed.
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PMID:Purification and properties of zinc-metallophospholipase C from Pseudomonas fluorescens. 792 9

Alkaline phosphatase activity was released up to 100% from the membrane by using 0.1 U of phosphatidylinositol-specific phospholipase C from B. thuringiensis. The M(r) of solubilized enzyme was 145,000 by Sephacryl S-300 gel filtration and 66,000 by SDS-PAGE, suggesting a dimeric structure. Solubilization of the membrane-bound enzyme with phospholipase C did not destroy its ability to hydrolyze p-nitrophenyl phosphate (PNPP) (264.3 mumol min-1 mg-1),ATP (42.0 mumol min-1 mg-1) and pyrophosphate (28.4 mumol min-1 mg-1). The hydrolysis of ATP and PNPP by solubilized 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 (Kd = 1.5 mM) but zinc ions were powerful non-competitive inhibitors (Kd = 6.2 microM) of solubilized enzyme. Treatment of solubilized alkaline phosphatase with Chellex 100 reduced the original PNPPase activity to 5%. Cobalt (K0.5 = 10.1 microM), magnesium (K0.5 = 29.5 microM) and manganese ions (K0.5 = 5 microM) restored the activity of the apoenzyme with positive cooperativity, suggesting that phosphatidylinositol-specific phospholipase C-solubilized alkaline phosphatase is a metalloenzyme. The stimulation of the apoenzyme by calcium ions (K0.5 = 653 microM) was lower than that observed for the other ions (26%) and exhibited site-site interactions (n = 0.7). Zinc ions had no effect on the apoenzyme of the solubilized enzyme.
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PMID:Osseous plate alkaline phosphatase is anchored by GPI. 808 Dec 65


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