Binding activity regulation of rabbit skeletal muscle adenosine 3'-5'-monophosphate-dependent protein kinases.

A cAMP-dependent protein kinase has been isolated from rabbit muscle and purified. The affinity constant of the enzyme for the nucleotide is Ka = 9.3 X 10(-9) M, with a Vmax = 0.013 X 10(12) moles bound cAMP/1 microgram protein. The influence exerted by different factors is studied: a) Inhibitor (I) of kinase activity: increases the binding capacity for cAMP, by percentages which depend on the amount of I. In the presence of inhibitor (120 microgram/100 microliter) the affinity constant is Ka = 4.1 X 10(-9) M, without change in Vmax. b) Effect of pH: it has a complex influence over binding, being also regulated by cAMP concentration. The positive effect on binding of ionic and bovine serum albumin concentrations, and the negative effect of enzyme preincubation before additions of (H3) cAMP, have also been studied. The importance of these effectors to obtain a high degree of sensitivity in the binding protein method has been assertained.