Purification of cyclic AMP- and cyclic GMP-dependent protein kinases from rat skeletal muscle.
Cyclic AMP-dependent protein kinase (cAMP-PrK) regulatory subunits, RI and RII, and cyclic GMP-dependent protein kinase (cGMP-PrK) have been simultaneously purified from skeletal muscle, utilizing sequential affinity chromatography on cyclic AMP-Sepharose. Rat skeletal muscle extract was chromatographed over DEAE-cellulose. Appropriate fractions, enriched in RI, RII or cGMP-PrK were further purified by affinity chromatography on cAMP-Sepharose. The protein kinase units were specifically eluted with cAMP or cGMP. A novel procedure, using two affinity columns, differing in their linkage of cAMP via either N6 or C-8 bonds, was developed to obtain RII free of other cyclic nucleotide binding proteins. In all cases, affinity chromatography was followed by HPLC gel exclusion chromatography to remove residual contaminating proteins. Proteins were purified to essential homogeneity as judged by silver stained SDS polyacrylamide gels. This procedure yields protein kinase subunits of high purity, and may be applicable to the isolation of these proteins from other sources.
D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods; D002848:Chromatography, DEAE-Cellulose / Q000379:methods; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism; D046911:Macromolecular Substances; D008297:Male; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains
D046911:Macromolecular Substances; D011494:Protein Kinases; D019204:GTP-Binding Proteins