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Purification, Properties, and Phosphorylation by Protein Kinase C of Two Phosphoinositidase C Isozymes from Rat Brain
Author(s) -
Blank Jonathan L.,
Foster Keith A.,
Hawthorne John N.
Publication year - 1991
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1991.tb02093.x
Subject(s) - biochemistry , phosphatidylinositol , enzyme , isozyme , phospholipase c , protein kinase c , inositol , substrate (aquarium) , polyacrylamide gel electrophoresis , phosphorylation , biology , kinase , microbiology and biotechnology , chemistry , chromatography , receptor , ecology
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 elec‐trophoresis gave apparent molecular masses of 151 kDa and 147 kDa. Western blotting with monoclonal antibodies showed that the isozymes corresponded to PLC‐β‐1 and PLC‐γ 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‐β‐1 and PLC‐γ on Mono Q. Purified rat brain protein kinase C phosphorylated PLO‐γ but not PLC‐β‐1. Incubation with the kinase increased the activity of both enzymes however, possibly by phosphory‐lation of another protein in the preparations.