Purification and properties of clostridium welchii phospholipase C.
TL;DR: Phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.4.3) from Clostridium welchii has been purified 32-fold over a commercial preparation.
About: This article is published in Biochimica et Biophysica Acta.The article was published on 1970-03-18. It has received 41 citations till now. The article focuses on the topics: Sephadex & Size-exclusion chromatography.
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TL;DR: Evidence is presented that the unique properties of the enzyme itself, rather than possible contaminations in the purified preparation, are responsible for the observed haemolytic effect.
340 citations
01 Jan 2002
TL;DR: In this article, the action of eight purified phospholipases on intact human erythrocytes has been investigated and it was shown that the unique properties of the enzyme itself, rather than possible contaminations in the purified preparation, are responsible for the observed haemolytic effect.
Abstract: 1 The action of eight purified phospholipases on intact human erythrocytes has been investigated Four enzymes, eg phospholipases A2 from pancreas and Crotalus adamanteus, phospholipase C from Bacillus cereus, and phospholipase D from cabbage produce neither haemolysis nor hydrolysis of phospholipids in intact cells On the other hand, both phospholipases A2 from bee venom and Naja naja cause a non-haemolytic breakdown of more than 50 ~ of the lecithin, while sphingomyelinase C from Staphylococcus aureus is able to produce a non-lytic degradation of more than 80 ~o of the sphingomyelin 2 Phospholipase C from Clostridium welchii appeared to be the only lipolytic enzyme tested, which produces haemolysis of human erythrocytes Evidence is presented that the unique properties of the enzyme itself, rather than possible contaminations in the purified preparation, are responsible for the observed haemolytic effect 3 With non-sealed ghosts, all phospholipases produce essentially complete breakdown of those phospholipids which can be considered as proper substrates for the enzymes involved 4 Due to its absolute requirement for Ca 2 ÷, pancreatic phospholipase A2 can be trapped inside resealed ghosts in the presence of EDTA, without producing phospholipid breakdown during the resealing procedure Subsequent addition of Ca 2 ÷ stimulates phospholipase A z activity at the inside of the resealed cell, eventually leading to lysis Before lysis occurs, however, 25 ~o of the lecithin, half of the phosphatidylethanolamine and some 65 ~ of the phosphatidylserine can be hydrolysed This observation is explained in relation to an asymmetric phospholipid distribution in red cell membranes
318 citations
TL;DR: Treatment of human red cell ghosts with pureospholipase C resulted in a nearly complete degradation of the main phospholipid classes (except for sphingomyelin) up to 70% of total phosphorus.
183 citations
TL;DR: This procedure produces acini which are morphologically and biochemically similar to the in situ pancreas and overcomes the poor response to secretagogues by isolated pancreatic acinar cells.
78 citations
TL;DR: The enzyme was found to be devoid of contaminating antigens as shown by immunodiffusion, immunoelectrophoresis and crossed electroimmunoassay against polyvalent antisera and no contaminating cytolytic toxins or enzymatic activities in the crude material were detected in the purified enzyme.
75 citations
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TL;DR: In this paper, the authors described a simplified version of the method and reported the results of a study of its application to different tissues, including the efficiency of the washing procedure in terms of the removal from tissue lipides of some non-lipide substances of special biochemical interest.
59,550 citations
TL;DR: The method is about seven times as sensitive as the Fiske–SubbaRow procedure and involves less pipetting, but it is not very satisfactory for determining inorganic phosphate if labile phosphate esters are present in large excess.
Abstract: Publisher Summary This chapter discusses the assay of inorganic phosphate, total phosphate, and phosphatases. The phosphomolybdate complex is reduced by ascorbic acid. The method is about seven times as sensitive as the Fiske–SubbaRow procedure and involves less pipetting. One can easily determine 0.01 micromole of phosphate. Pyrophosphate breaks down about 5% in the method and compounds such as glucose 1-phosphate also break down somewhat, so that the method is not very satisfactory for determining inorganic phosphate if labile phosphate esters are present in large excess. The sample of organic phosphate and a drop of magnesium nitrate solution in a small test tube are taken to dryness by shaking the tube in flame. The ashing procedure is rapid and is good for various biological materials and phosphate esters such as nucleic acid, carbohydrate phosphate esters, viruses, and phospholipids. The assay method of phosphatases for inorganic phosphate can be used as an assay for phosphatases hydrolyzing stable phosphate esters such as glucose-6-phosphate, ribose-5-phosphate, and histidinol phosphate. The enzyme incubation can be stopped with the one ascorbic-molybdate solution thus avoiding an extra pipetting.
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