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High‐Pressure Extraction of Membrane‐Associated Protein Kinase C from Rat Brain
Author(s) -
Lester David S.
Publication year - 1989
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.1471-4159.1989.tb07284.x
Subject(s) - cytosol , diacylglycerol kinase , membrane , protein kinase a , hydrostatic pressure , protein kinase c , endogeny , biochemistry , membrane protein , chemistry , kinase , phosphorylation , peripheral membrane protein , enzyme , integral membrane protein , microbiology and biotechnology , biology , biophysics , physics , thermodynamics
Extraction of rat brain membrane‐associated protein kinase C with high specific activity was obtained by applying benzyl alcohol (a membrane fluidizer), EDTA, and high hydrostatic pressures. Approximately 50% of total brain‐associated activity was extracted from membranes. The pressure‐extracted activity had an eightfold enrichment in the lipid/protein ratio when compared with the cytosolic fraction. This may explain the inability of exogenous diacylglycerol to stimulate endogenous phosphorylation in pressure‐extracted activity. The enzyme is extracted at > 1,300 atm, a result indicating it most likely has a portion inserted into the hydrophobic portion of the membrane bilayer. Perturbation of the native membrane induces a change in the membrane‐associated protein kinase C‐lipid interaction that permits extraction under conditions used for the cytosolic species. This is the first report of conversion of the endogenous membrane species to a cytosolic one and may be important in determining the role of protein kinase C in neuronal regulation.