Isolation and characterization of a gamma-type phosphoinositide-specific phospholipase C (PLC-gamma 2).
A novel bovine spleen phosphoinositide-specific phospholipase C (PLC) has been identified with respect to immunoreactivity with four independent antibodies against each of the PLC isoenzymes, and purified to near homogeneity by sequential column chromatography. Spleen contains three of the isoenzymes: two different gamma-types [gamma 1 and gamma 2, originally named as PLC-gamma [Rhee, Suh, Ryu & Lee (1989) Science 244, 546-550] and PLC-IV [Emori, Homma, Sorimachi, Kawasaki, Nakanishi, Suzuki & Takenawa (1989) J. Biol. Chem. 264, 21885-21890] respectively] and delta-type of the enzyme, but PLC-gamma 1 is separated from the PLC-gamma 2 pool by the first DEAE-cellulose column chromatography. Subsequently, PLC-delta is dissociated on the third heparin-Sepharose column chromatography. The purified enzyme has a molecular mass of 145 kDa on SDS/polyacrylamide-gel electrophoresis and a specific activity of 12.8 mumol/min per mg with phosphatidylinositol 4,5-bisphosphate as substrate. This enzyme activity is dependent on Ca2+ for hydrolysis of all these phosphoinositides. None of the other phospholipids examined could be its substrate at any concentration of Ca2+. The optimal pH of the enzyme is slightly acidic (pH 5.0-6.5).
Homma
Y
Y
Department of Pharmacology, Tokyo Metropolitan Institute of Gerontology, Japan.
Emori
Y
Y
Shibasaki
F
F
Suzuki
K
K
Takenawa
T
T
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols / Q000378:metabolism*; D013154:Spleen / Q000201:enzymology*; D013379:Substrate Specificity; D010738:Type C Phospholipases / Q000302:isolation & purification* / Q000378:metabolism
D007527:Isoenzymes; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D010738:Type C Phospholipases; D002118:Calcium