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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)

A phosphatidylinositol phosphodiesterase from the culture broth of Bacillus cereus, was purified to a homogeneous state as indicated by polyacrylamide gel electrophoresis, by ammonium sulfate precipitation and chromatography with DEAE-cellulose and CM-Sephadex. The enzyme (molecular weight: 29000 +/- 1000) was maximally active at pH 7.2-7.5, AND NOT INFLUENCED BY EDTA, ophenanthroline, monoiodoacetate, p-chloromercuribenzoate or reduced glutathione. The enzyme specifically hydrolyzed phosphatidylinositol, but did not act on phosphatidylcholine, phosphatidylethanolamine and sphingomyelin, under the conditions examined. The products from phosphatidylinositol of enzyme reaction were diacylglycerols and a mixture of myoinositol 1- and 1, 2-cyclic phosphates, suggesting that the enzyme was a phosphatidylinositol-specific phospholipase C. The enzyme released alkaline phosphatase quantitatively from rat kidney slices. A kinetic analysis was made on the release of alkaline phosphatase. The results suggest that phosphatidylinositol-specific phospholipase C can specifically act on plasma membrane of rat kidney slices.
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PMID:Studies on phosphatidylinositol phosphodiesterase (phospholipase C type) of Bacillus cereus. I. purification, properties and phosphatase-releasing activity. 1 Sep 86

Phospholipase activity of 10 strains of Bacillus cereus was studied. The most active strain of Bac. cereus--phospholipase producer was selected. A cultivation mixture of Bac. cereus optimal for the phospholipase synthesis was found to include peptone, yeast extract, glucose, NaCl and Na2HPO4. Proper conditions for the synthesis of phospholipase in flasks, 20 l and 250 l fermenters were tested. The maximum increase of the phospholipase activity occurred by the 5-9th hour of microbial growth at pH 6.0-8.0. Further cultivation, foaming, strong aeration, pH increase (over 8.0) reduced the accumulated activity. By fractionation with (NH4)2SO4, ethanol precipitation, protamine sulphate treatment with subsequent Sephadex G-100 gel filtration phospholipase (EC 3.1.4.3) was purified 300-fold from the culture liquid of Bac. cereus str. 504. The preparation was examined electrophoretically in 7% polyacrylamide gel at alkaline pH. The effect of metal salts and EDTA on phospholipase activity was studied. Thermostability, substrate specificity and pH optimum of purified phospholipase were investigated.
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PMID:[Phospholipase of Bacillus cereus]. 1 9

The conditions necessary for the secretion of phospholipase C (phosphatidylcholine cholinephosphohydrolase) by Pseudomonas aeruginosa were studied. Enzyme secretion by washed cell suspensions required a carbon source and ammonium, potassium, and calcium ions. The calcium requirement could be substituted by magnesium and strontium but not by copper, manganese, cobalt, or zinc. During growth in liquid medium, cells secreted phospholipase C during late logarithmic and early stationary phases. Secretion was repressed by the addition of inorganic phosphate but not by organic phosphates, glucose, or sodium succinate. Studies with tetracycline indicated that de novo protein synthesis was necessary for the secretion of phospholipase C and that the exoenzyme was not released from a preformed periplasmic pool. Similarly, extraction of actively secreting cells with 0.2 M MgCl2 at pH 8.4 solubilized large quantities of the periplasmic enzyme alkaline phosphatase but insignificant amounts of phospholipase C. Bacteria continued to secrete enzyme for nearly 45 min after the addition of inorganic phosphate or rifampin.
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PMID:Secretion of phospholipase C by Pseudomonas aeruginosa. 11 87

The authors present a method of obtaining relatively homogeneous preparations of alpha-toxoid of Cl. perfringens, type A, including the primary conception of the alpha-toxin proteins, their chromatography on DEAE-cellulose, fractionation with (NH4)2SO4, detoxication, with the subsequent gel-filtration through sephadex and isoelectric focussing. Sedimentation coefficient of the preparation proved to be 3.8 S, isoelectric point-4.83 +/- 0.07. In studying the immunogenic properties of alpha-toxoid in experiments on guinea pigs and rabbits their high immunogenicity, exceeding that of the industrial toxoid 8- and 6-fold, respectively, was established. Homogeneous preparations of alpha-toxoid provided intense anti-microbial immunity. Interlinear differences in the levels of the immune response of inbred mice, highly-reactive (BALB/c) and low-reactive (C57BL/6) to alpha-toxoid, reached 20-fold; in combination with the high immunogenicity of this antigen for mice this permits to recommend it for immunogenic studies.
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PMID:[Homogeneous Cl. perfringens alpha-anatoxin: its physicochemical and immunogenic properties]. 20 30

The procedure for separation and purification of isoenzymes of phospholipase C from Clostridium perfringens (PLC) was developed. The procedure included primary concentration of culture liquid proteins and isoenzyme separtion on DEAE-cellulose during negative sorption of the major isoenzyme. Further purification of the isoenzymes was achieved by (NH4)2SO4 fractionation by Sephadex gel-filtration and isoelectric focusing. During polyacrylamide gel electrophoresis and precipitation reaction in the agar with the antiperfringens hyperimmune serum alpha1-PLC preparations were homogenous. Their coefficient of sedimentation was 3.8 S and isoelectric point was 5.50. The minor isoenzyme alpha2-PLC had the same coefficient of sedimentation and the isoelectric point of 5.35.
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PMID:[Separation and purification of isoenzymes of Clostridium perfringens phospholipase C]. 20 39

Phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) from Pseudomonas aureofaciens was purified 3600-fold from the culture filtrate with a recovery of 1.6%. Purification was performed with the useof (NH4)2SO4 precipitation, Sephadex G-100 gel filtration and by ion-exchange chromatography on DEAE-Sephadex A-50 and CM-Sephadex C-50. The purified enzyme appeared to be homogeneous as revealed by polyacrylamide disc gel electrophoresis at pH 9.3. The molecular weight was estimated to be 35 000 by gel filtration on Sephadex G-75. Under our experimental conditions, phosphatidylethanolamine was more rapidly hydrolysed than phosphatidylcholine. Lyso forms of these two phosphatides were poor substrates. Phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, cardiolipin and sphingomyelin were not hydrolysed. The enzyme activity with phosphatidylcholine as substrate was slightly stimulated by Ca2+, Mg2+, and Mn2+. However, these cations inhibited the activity with phosphatidylethanolamine as substrate. An anionic detergent, sodium deoxycholate, slightly enhanced the activity when phosphatidylcholine and phosphatidylethanolamine were used as substrates. A cationic detergent, cetyltrimethylammonium bromide, inhibited enzyme activity. EDTA and o-henanthroline inhibited the activity of the enzyme to a marked degree.
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PMID:Studies on phospholipase C from Pseudomonas aureofaciens. I. Purification and some properties of phospholipase C. 24 4

1. Phospholipase C (EC 3.1.4.3) from Clostridium novyi (oedematiens) type A was purified 2000-fold by (NH4)2SO4 precipitation, DEAE-Sephadex treatment in a batchwise system and Sephadex G-100 column chromatography. 2. The purified preparation had a specific activity of 95 mumol per min per mg protein toward phosphatidylcholine. This preparation was free from protease, lipase and oxygen-labile delta-hemolysin. 3. Phosphatidylcholine was hydrolyzed at the highest rate, while sphingomyelin and lysophosphatidylcholine were hydrolyzed at much lower rates. 4. Sodium deoxycholate and divalent cations such as Mg2+ and Ca2+ were extremely effective in stimulating phosphatidylcholine-hydrolyzing activity of this enzyme. 5. This enzyme hemolyzed horse red cells by hydrolyzing phosphatidylcholine, spingomyelin and phosphatidylethanolamine.
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PMID:Phospholipase C from Clostridium novyi type A. I. 24 23

rac-Phosphatidyl carnitine and rac-phosphatidyl beta-methylcholine were synthesized by direct condensation of phosphatidic acid and the appropriate alcohols in the presence of 2,4,6-triiso-propylbenzenesulphonylchloride and pyridine. Tetraphenylborates of the quarternary ammonium compounds beta-methylcholine and carnitine benzyl ester were shown to be particularly convenient for synthesis in homogeneous phase. Physical and chemical properties of the two phosphoglycerolipids and some intermediates were described. Phosphatidyl carnitine and phosphatidyl beta-methylcholine were hydrolyzed by phospholipase A2 (phosphatide acyl-hydrolase, EC 3.1.1.4), pancreatic lipase (triacylglycerol acyl-hydrolase, EC 3.1.1.3), and phospholipase C from Bacillus cereus (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3). Neither hydrolysis nor transphosphatidylation of phosphatidyl carnitine and phosphatidyl beta-methylcholine was achieved by phospholipase D (phosphatidylcholine phosphatidohydrolase, EC 3.1.4.4). The occurrence of phosphatidyl carnitine in embryonic chicken tissue was suggested by comparison with the synthesized compound. Phosphatidyl carnitine could not be detected in the tissue of rat embryos.
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PMID:Synthesis and properties of phosphatidyl carnitine and phosphatidyl beta-methylcholine. 80 79

The addition of ammonium sulfate to starved yeast cells leads to a 3- to 4-fold rapid increase of the second messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), the products of phosphoinositide-specific phospholipase C (PI-PLC). This response is reduced by dissecting the RAS-activating Cdc25 protein, and is completely abolished by the cdc25-1 mutation even at permissive temperature. Starved cdc25-1 mutant cells have a strongly reduced IP3 content, but an at least 10-fold increased DAG level compared to the isogenic wild-type strain. NH4 does not stimulate cAMP synthesis, and glucose does not induce IP3 and DAG. Our data suggest that the Cdc25 protein controls a nitrogen-specific signalling pathway involving the effector PI-PLC, in addition to the glucose-induced activation of adenylyl cyclase (AC).
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PMID:CDC25-dependent induction of inositol 1,4,5-trisphosphate and diacylglycerol in Saccharomyces cerevisiae by nitrogen. 132 32

We have purified to homogeneity the 33-kDa phosphatidylinositol-specific phospholipase C (PI-PLC) from the culture fluid of Listeria monocytogenes, a facultative intracellular pathogen. The protein was overexpressed, and secretion of PI-PLC was further enhanced by the addition of divalent cations to the culture medium. The basic protein (pI, approximately 9.4) was complexed with anionic proteins in the crude culture fluid. It bound to DEAE-Sepharose and was eluted from Sephacryl S-200 near the void volume in low-ionic-strength buffer, suggesting aggregates of greater than or equal to 150 kDa. Gel filtration chromatography on Sephacryl S-200 in the presence of 1 M ammonium sulfate resulted in disaggregation and complete separation of PI-PLC, which interacted with the column matrix. Amino-terminal sequencing of the pure protein gave results consistent with the previously deduced sequence and showed that the signal cleavage site was between alanine 29 and tyrosine 30. The enzyme was specific for PI and showed no activity with phosphatidylethanolamine, phosphatidylcholine, or phosphatidylserine. It did not cleave PI-4-phosphate or PI-4,5-bisphosphate, but it was active on the membrane form of the variable surface glycoprotein from Trypanosoma brucei, a PI-glycan-anchored protein. When assayed with deoxycholate-mixed micelles of PI, activity was highly dependent on added salt. Activation by salt was also observed with Triton X-100-mixed micelles. The optimal concentration of CaCl2 or MgCl2 was lower than that of KCl or (NH4)2SO4, but activity was not specifically dependent on divalent cations and was not inhibited by addition of EDTA. With deoxycholate, the optimum pH was 7.0. A broader pH optimum ranging from 5.5 to 6.5 was observed with Triton X-100-mixed micelles. These results are consistent with a postulated role for secreted PI-PLC in the acidified primary phagocytic vesicle of infected cells.
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PMID:Purification and characterization of Listeria monocytogenes phosphatidylinositol-specific phospholipase C. 139 18


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