The apparent constants for the interaction of regulatory (R) and catalytic (C) subunits of CAMP-dependent protein kinase types I and 11 were estimated at 37 "C. Regulatory subunits I and II isolated from rabbit skeletal and bovine heart muscle, respectively, were eluted from CAMP-affinity columns by urea. The regulatory subunits were essentially free of CAMP and renatured after removal of the urea to a dimeric form. Holoenzymes (R2C2) of the respective isozymes were prepared from isolated

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... tory and catalytic subunits. The apparent constants for the interaction of R and C were estimated from activity measurements according to the following equation: An activity ratio (-cAMP/+cAMP value) of 0.5 was taken as indication that 50% of the subunits were present as free subunits and 50% as holoenzyme. Measurements were carried out either by using RaC2 at various concentrations or by adding R at various concentrations to C present at a fixed concentration (0.01 to 0.1 a). Catalytic activity was determined after apparent equilibrium between free and bound subunits was obtained. The concentration of R and C required for halfmaximal interaction was almost identical for the subunits from type I and II protein kinase (apparent K between 0.2 and 0.3 a) when 150 n w~ sodium chloride and 1 l l l~ magnesium was used. In the absence of sodium chloride, the respective constants were 0.15 and about 0.01 ILM. The effect of sodium chloride on the interaction of R and C was only seen when low concentrations of C (below 1.0 I~M ) were used. In the presence of 5 l l n~ magnesium and 150 n m sodium chloride, the apparent constant for type I subunits increased from 0.2 to 0.68 m, whereas that for the type II subunits was affected only marginally. CAMP-dependent protein kinases (EC 2.7.1.37; ATP: protein phosphotransferase) from several tissues are known to contain two types of subunits: catalytic subunits (C) which catalyze the transfer of the y-phosphate of ATP to proteins and regulatory subunits (R), which in the absence of CAMP inhibit the activity of the catalytic subunits (see Ref. 1). CAMP activates these enzymes by causing dissociation of the inactive holoenzyme (R2C.J to yield free C subunits and a CAMP. regulatory subunit complex (R2 -cAMP4) according to following equation established for the enzyme purified from skeletal muscle (2) or heart (3): ' The abbreviations used are: Mes, 2-(N-morpho1ino)ethannBsulfonic acid; EGTA, ethylene glycol his@-aminoethyl)N,N'-tetraacetic acid; PMSF, phenyhnethylsulfonyl fluoride; activity ratio, value obtained by dividing values measured in the absence of cAMP by those measured in the presence of cAMP (17).