Determination of soluble CAMP-dependent protein kinase activity in guinea-pig tracheal smooth muscle Preferential use of kemptide as a phosphorylating substrate

A method is described for the determination of soluble (cytosolic) cyclic AMP (cAMP)-dependent protein kinase (A-kinase) activity in guinea-pig tracheal smooth muscle. The method relies upon the use of either histone or kemptide as a phosphorylating substrate. The relative merits of each of these substances were compared by studying the effects of a protein kinase inhibitor (PKI) and of Na+ on the phosphorylation of each substrate. PKI induced a concentration-dependent inhibition of basal and cAMP-stimulated phosphohistone formation but could not abolish it. Phosphokemptide formation was abolished by equivalent concentrations of PKI. Elevations in Na+ concentration in the reaction buffer inhibited cAMP-stimulated phosphohistone formation in a concentration-dependent manner with concomitant elevations in the enzyme activity ratio. Basal or cAMP-stimulated phosphokemptide formation was not inhibited by elevated Na+ concentrations. When tissues were homogenized in high Na+ concentration buffers, an increase in the basal A-kinase activity was observed using kemptide as the substrate. No apparent change in cAMP-stimulated activity was observed. Concomitant with this was an elevation in the enzyme activity ratio. However, a high Na+ concentration in the homogenizing buffer elevated basal phosphokemptide formation and the activity ratio. Separation of the isoenzymes of the enzyme yielded three peaks of activity upon assay of the fractions, which comprised free catalytic subunits (5% total activity), type I holoenzyme (5% total activity), and type II holoenzyme (90% total activity). Enzyme activity was increased upon pretreatment of tissues with isoprenaline and forskolin using both histone IIa and kemptide as phosphorylating substrates. The data support the preferential use of kemptide over histone IIa as a phosphorylating substrate during the determination of A-kinase activity in guinea-pig trachealis. The potential benefits of the use of kemptide are discussed.