Purification and properties of phosphatidylinositol-specific phospholipase C from Streptomyces antibioticus.
A novel type of phosphatidylinositol-specific phospholipase C (PI-PLC) was purified from culture supernatant of a strain of Actinomycetales, Streptomyces antibioticus. The purified enzyme showed a single band on native polyacrylamide gel electrophoresis (native PAGE) with a molecular weight of 32 kDa, but showed two polypeptides, named alpha- and beta-peptides, on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) with molecular weights of 23 kDa and 15 kDa, respectively. From the results of both electrophoretic analysis and N-terminal amino acid sequencing, it was estimated that the enzyme was composed of alpha- and beta-peptides. The enzyme could hydrolyze phosphatidylinositol, but not any other glycerophospholipids. The enzyme had pH and temperature optima at around 7.0 and 30 degrees C, respectively, and was stable up to 50 degrees C when incubated at pH 8.0 for 30 min. The PI-PLC was strongly activated by SDS, sodium deoxycholate (SDC) and diethyl ether, but not by Triton X-100, and inhibited by cetylpyridinium chloride (CPC). The enzyme was activated a little by Ca2+ and was inhibited completely by a chelating agent such as ethylenediaminetetraacetic acid (EDTA) and glycoletherdiaminetetraacetic acid (EGTA). Their inhibitions were restored by the addition of Ca2+, suggesting that a certain amount of Ca2+ is essential for the enzymatic activity.
Iwasaki
Y
Y
Department of Applied Biological Sciences, School of Agriculture, Nagoya University, Japan.
Niwa
S
S
Nakano
H
H
Nagasawa
T
T
Yamane
T
T
D000595:Amino Acid Sequence; D002118:Calcium / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000302:isolation & purification*; D013303:Streptomyces antibioticus / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature
D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D002118:Calcium