Purification, properties, and phosphorylation by protein kinase C of two phosphoinositidase C isozymes from rat brain.
Two forms of phosphoinositidase C have been purified from the soluble fraction of rat brain. The purification scheme included gel filtration followed by chromatography on cellulose phosphate, phenyl-Sepharose, and Mono Q. Gradient sodium dodecyl sulphate-polyacrylamide gel electrophoresis gave apparent molecular masses of 151 kDa and 147 kDa. Western blotting with monoclonal antibodies showed that the isozymes corresponded to PLC-beta-1 and PLC-gamma of bovine brain. With both enzymes phosphatidylinositol 4,5-bisphosphate was a better substrate than phosphatidylinositol at neutral pH and low calcium ion concentrations. Both enzymes produced a proportion of inositol 1:2-cyclic phosphates from each substrate, particularly at acid pH. Some GTPase activity was seen in the early stages of purification, but was separated from PLC-beta-1 and PLC-gamma on Mono Q. Purified rat brain protein kinase C phosphorylated PLC-gamma but not PLC-beta-1. Incubation with the kinase increased the activity of both enzymes however, possibly by phosphorylation of another protein in the preparations.
Blank
J L
JL
Department of Biochemistry, Medical School, Queen's Medical Centre, Nottingham, England.
Foster
K A
KA
Hawthorne
J N
JN
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007295:Inositol Phosphates / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008055:Lipids / Q000494:pharmacology; D008297:Male; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011506:Proteins / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains
D007295:Inositol Phosphates; D007527:Isoenzymes; D008055:Lipids; D011506:Proteins; D011493:Protein Kinase C; D010727:Phosphoric Diester Hydrolases; C021491:glycerophosphoinositol glycerophosphodiesterase; D002118:Calcium