Studies on phospholipases from Streptomyces. III. Purification and properties of Streptomyces hachijoensis phospholipase C.
1. Phospholipase C [EC 3.1.4.3] found in the growth medium of Streptomyces hachijoensis was purified about sixty-fold by dialysis and column chromatography on Sephadex G-50. 2. The active fraction was separated by isoelectric focusing into two fractions, phospholipase C-I (pI 6.0) and phospholipase C-II (pI 5.6). 3. Both purified phospholipases C were homogeneous by immunodiffusion and were not differentiated as regards antigencity. 4. Phospholipase C-I had maximal activity at pH 8.0 and the optimal temperature was 50degree. Phospholipase C-I was stable at 50degrees for 30 min and was stable at neutral pH. 5. The activity of phospholipase C-I was inhibited by high concentrations of various detergents such as Triton X-100, sodium, cholate, SDS and was also inhibited by Ca2+, Ba2+, Al3+, and EDTA, but was stimulated by Mg2+, and ethyl ether. 6. The Km value of phospholipase C-I was 0.9 mM, using phosphatidylcholine as a substrate. 7. By the gel filtration procedure, the molecular weights of phospholipase C-I and -II were both determined to be 18,000. 8. Phosphatidylcholine, phosphatidylinositol, cardiolipin, sphingomyelin, and lysophosphatidylcholine were hydrolyzed by phospholipase C-I, but phosphatidylethanolamine and phosphatidylserine were hydrolyzed with difficulty under the same conditions, Phospholipase C-I also hydrolyzed phosphatidic acid.
D000535:Aluminum / Q000494:pharmacology; D002413:Cations, Divalent; D002414:Cations, Monovalent; D003902:Detergents / Q000494:pharmacology; D004355:Drug Stability; D004492:Edetic Acid / Q000494:pharmacology; D006461:Hemolysis / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D005779:Immunodiffusion; D007501:Iron / Q000494:pharmacology; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D010740:Phospholipases / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D013302:Streptomyces / Q000201:enzymology*; D013696:Temperature
D002413:Cations, Divalent; D002414:Cations, Monovalent; D003902:Detergents; D007527:Isoenzymes; D004492:Edetic Acid; D000535:Aluminum; D007501:Iron; D010740:Phospholipases