Properties and purification of the catalytic subunit of cyclic AMP-dependent protein kinase of adipose tissue.
The catalytic subunit of cAMP-dependent protein kinase from rat adipose tissue was purified to apparent homogeneity by making use of the differential binding of the holoenzyme and the free catalytic subunit to CM-Sephadex and by gel chromatography. Stability and yield was improved by inclusion of nonionic detergent in all steps after dissociation of the holoenzyme. Isoelectric focusing separated enzyme species with pI values of 7.8 and 8.6-8.8. The amino acid composition was similar to the enzyme purified from other tissues. Enzyme activity was markedly unstable in dilute solutions (less than 5 micrograms/ml). Additions of nonionic detergent, glycerol, bovine serum albumin and, especially, histones stabilized the enzyme. With protamine, the catalytic subunit had an apparent Km of 60 microM and Vmax of 20 mumol X min-1 X mg-1, corresponding values with mixed histones were 12 microM and 1.2 mumol X min-1 X mg-1. With both protein substrates the apparent Km for ATP was 11 microM. Concentrations of Mg2+ above 10 mM were inhibitory. Histone phosphorylation was inhibited by NaCl (50% at 0.5 M NaCl) while protamine phosphorylation was stimulated (4-fold at 1 M NaCl). Inorganic phosphate inhibited both substrates (histones: 50% at 0.3 M, and protamine: 50% at 0.5 M). pH optimum was around pH 9 with both substrates. The catalytic subunit contained 2.0 (range of three determinations, 1.7-2.3) mol phosphate/mol protein. It was autophosphorylated and incorporated 32Pi from [gamma-32P]ATP in a time-dependent process, reaching saturation when approx. 0.1 mol phosphate/mol catalytic subunit was incorporated.
D000273:Adipose Tissue / Q000201:enzymology*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D009994:Osmolar Concentration; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity
D000596:Amino Acids; D011494:Protein Kinases