Purification of stable protein kinase C from mouse brain cytosol by specific ligand elution using fast protein liquid chromatography.
The Ca2+- and phospholipid-dependent protein kinase (protein kinase C) has been purified to electrophoretic homogeneity from mouse brain cytosol. [20-3H]phorbol 12,13-dibutyrate binding activity was found to coelute quantitatively with the protein kinase activity throughout the purification procedure. The crude extract was first run over a DE52 column. Fractions containing peak activities were then chromatographed using a fast protein liquid chromatography system with a Mono Q column followed by chromatography on the same column run in the presence of 1 mM adenosine triphosphate. The adenosine triphosphate specifically shifted the elution position of the Ca2+- and phospholipid-dependent protein kinase, providing a powerful step in the purification procedure. The remaining minor contaminants were removed by hydrophobic chromatography on a TSK-phenyl-5-PW column. This purification procedure required less than 2 days after the initial large batch DE52 column chromatography. The molecular weight of the purified receptor was estimated to be 81,000 by its mobility on sodium dodecyl sulfate/polyacrylamide gels, in agreement with the published values. Optimal conditions for the storage of the purified receptor were sought. Both protein kinase and phorbol ester binding activities were stable for 2 mo when stored in the presence of 0.01% Triton X-100 at -70 degrees C. Polyclonal antibodies to the purified receptor have been prepared from rabbits. These antibodies recognized the purified receptor in electroblotting assays and were able to immunoprecipitate the purified receptor.
Jeng
A Y
AY
Sharkey
N A
NA
Blumberg
P M
PM
D000818:Animals; D001921:Brain / Q000201:enzymology*; D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D011232:Chemical Precipitation; D002853:Chromatography, Liquid; D003600:Cytosol / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D051379:Mice; D008815:Mice, Inbred Strains; D008970:Molecular Weight; D015240:Phorbol 12,13-Dibutyrate; D010703:Phorbol Esters / Q000378:metabolism; D011493:Protein Kinase C / Q000302:isolation & purification*; D011955:Receptors, Drug; D011971:Receptors, Immunologic / Q000276:immunology / Q000302:isolation & purification*
D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D010703:Phorbol Esters; D011955:Receptors, Drug; D011971:Receptors, Immunologic; C083141:phorbol ester binding protein; C027776:phorbol ester receptor; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C