Catalytic Subunit of Adenosine Cyclic 3',5' Monophosphate- Dependent Protein Kinase from Rat Muscle: Basic Properties and Factors Influencing the Activity

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Abstract
The catalytic (C) subunit of isoenzyme II of cyclic[c]AMP-dependent protein kinase from rat muscle, purified to apparent homogeneity, was investigated for some of its basic properties and factors that influence its activity. The properties resembled those of C subunits of isoenzymes I and II from various other tissues of different species. This concerns the MW estimate (about 37 k), UV light absorption spectrum, isoelectric point (pH 9.0 .+-. 0.2), the apparent Km values for histone and protamine (for substrates .ltoreq. 10 .mu.M) and the apparent Km for co-substrate ATP (3-4 .mu.M). The enzyme is sensitive to the heat-stable inhibitory protein. The enzyme activity is stable up to 40.degree. C but is destroyed at higher temperatures; the half-life at 50.degree. C is slightly less than 1 min, and at .gtoreq. 60.degree. C activity is nullified within 1 min. Activity is lost on freezing and thawing. Substrate histone does not improve temperature stability; co-substrate ATP in absence of Mg2+ decreases, but in presence of Mg2+ increases stability. The optimum pH of enzyme activity is at pH 6.8 for histone and pH 7.8 for protamine phosphorylation. The enzyme becomes irreversibly inactivated at pH values below 5. For activity, the enzyme has absolute requirement for a divalent metal ion, highest activity is found in presence of Mg2+ or Co2+; the optima are at 5-10 mM (half maximum at 0.9 mM) and 0.5-0.8 mM (half maximum at 0.25 mM) for Mg2+ and Co2+, respectively. None of the various other metal ions tested were nearly as effective as these two. Ca2+ antagonized Mg2+. A considerable effect on activity by ionic strength was observed. In dependence on the substrate used, activity is influenced strongly by some anions and zwitterions, e.g., in presence of acetate, histone phosphorylation is by a factor of 3-4 higher than in presence of sulfate, whereas protamine phosphorylation is only half that in presence of sulfate. Roughly the following order of enzyme activity in presence of the respective buffer system was found: MOPS [N-2''-hydroxyethyl-piperazine-2-ethanesulfonic acid](100%) .gtoreq. TES [N-tris(hydroxymethyl)-methyl-2-aminoethanesulfonic acid](95%) .apprxeq. HEPES [N-2-hydroxyethylpiperazine-N''-2-ethanesulfonic acid] > MES [2-(N-morpholino) ethanesulfonic acid] (65%) .simeq. acetate .mchgt. ADA [N-(2-acetamido)-iminodiacetic acid] (20%) > phosphate (15%) > TRIS[tris(hydroxymethyl)-aminomethane].cntdot.maleate (7%).

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