Purification and characterization of a soluble cyclic nucleotide-independent Ca2+-calmodulin-sensitive protein kinase from rat brain.
Following partial purification, the characteristics of a cytosol protein kinase were investigated. The protein kinase was purified by ammonium sulfate precipitation and diethylaminoethyl-cellulose, ATP-agarose, and hydroxyapatite chromatography. Analysis of the purified protein kinase preparation by polyacrylamide gel electrophoresis revealed three major protein bands. The cytosol protein kinase was purified approximately 442-fold, as calculated from the cyclic nucleotide independent protein kinase activity in the 40,000 g supernatant. The activity of the kinase was found to be independent of either cyclic AMP or cyclic GMP. Moreover, the kinase activity was unaffected by the addition of the endogenous protein kinase inhibitor, or the regulatory subunit from the type II cyclic AMP-dependent protein kinase from bovine heart. The molecular weight of the enzyme was determined to be 95,000 by Sephadex G-200 gel filtration. The activity of the kinase was increased approximately twofold in the presence of 10 microM Ca+2 and calmodulin. This increase was reversed by the addition of EGTA. The subcellular distribution of the protein kinase was also examined. The soluble fraction from nerve terminal was found to have the highest concentration of the kinase activity.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002364:Caseins / Q000378:metabolism; D002417:Cattle; D002848:Chromatography, DEAE-Cellulose; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008297:Male; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology; D011494:Protein Kinases / Q000302:isolation & purification*; D051381:Rats; D011919:Rats, Inbred Strains; D013347:Subcellular Fractions / Q000201:enzymology
D002147:Calmodulin; D002364:Caseins; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D011494:Protein Kinases; D006152:Cyclic GMP; D002118:Calcium