Purification and some properties of phospholipase C (alpha-toxin) of Clostridium perfringens.
1. Phospholipase C[EC 3.1.4.3] was purified from the culture filtrate of Clostridium perfringens by successive chromatographies on CM-Sephadex, DEAE-Sephadex, and Sephadex G-100. During the purification it was noted that, beside the monomer form of the enzyme which was purified, a part of the enzyme existed in active polymerized forms. 2. The purified preparation gave a single band on polyacrylamide gel electrophoresis and gave a single precipitin line in immunodiffusion with the National Standard gas gangrene (C. perfringens) antitoxin, indicating the homogeneity of the preparation. 3. The specific lecithin-hydrolyzing activity of the purified preparation was comparable to that of a preparation obtained by affinity chromatography, which had the highest specific activity previously reported. 4. The molecular weight of the purified enzyme was estimated to be 43,000 by SDS-polyacryl-amide gel electrophoresis, although the same preparation gave a molecular weight of 31,000 as determined by gel filtration on Sephadex G-150. From this and the above finding that a part of the enzyme exists in active polymerized forms, the discrepancy among reported values for the molecular weight of C. perfringens phospholipase C can be accounted for. 5. For maximum hydrolytic activity toward lecithin, the enzyme required sodium deoxycholate (SDC) and Ca2+ ions. In the presence of 6 mM Ca2+, the optimal molar ratio of SDC to lecithin for maximal hydrolytic activity was about 0.5 for dipalmitoyl lecithin and about 1.0 for egg lecithin. The effects of various divalent cations on the enzymatic hydrolysis were also investigated. 6. The effects of sodium deoxycholate and Ca2+ ions on the enzymatic hydrolysis are discussed, based on their possible roles in mixed micelle formation.
D000992:Antitoxins / Q000378:metabolism; D001427:Bacterial Toxins / Q000302:isolation & purification*; D002118:Calcium / Q000494:pharmacology; D002413:Cations, Divalent / Q000494:pharmacology; D003016:Clostridium perfringens / Q000201:enzymology*; D003840:Deoxycholic Acid / Q000494:pharmacology; D008970:Molecular Weight; D010713:Phosphatidylcholines / Q000378:metabolism; D010740:Phospholipases / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding
D000992:Antitoxins; D001427:Bacterial Toxins; D002413:Cations, Divalent; D010713:Phosphatidylcholines; D003840:Deoxycholic Acid; D010740:Phospholipases; D002118:Calcium