Adenosine 3′,5′-Monophosphate-dependent Protein Kinase from Adipose Tissue

Abstract A procedure for partial purification of the adipose tissue adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase by substrate affinity chromatography is described. On the basis of specificity, apparent Km for ATP, pH dependence, metal ion requirement, and other properties, the enzyme showed a striking similarity to the protein kinases described in skeletal muscle and other tissues. Chromatography on casein-Sepharose columns in the presence of cAMP produced a cAMP-independent kinase. Gel filtration studies showed that this form had a molecular weight of approximately 42,000 as compared with 140,000 for the original cAMP-dependent enzyme. The adipose tissue cAMP-independent kinase was inhibited by a heat-stable muscle protein and by a preparation of muscle protein kinase regulatory subunit. The latter effect occurred only in the absence of cAMP. The cAMP-dependent and independent protein kinases were affected similarly by sodium chloride and potassium chloride and the direction of the effect was dependent on the substrate used. On the basis of the above findings and reports of results obtained with protein kinases from other tissues, it would appear that the adipose tissue cAMP-dependent protein kinase is composed of a regulatory (cAMP-binding) subunit and a cAMP-independent catalytic subunit. Lipolytic activity in adipose tissue homogenates is stimulated 2-fold in the absence of cAMP and in the presence of Mg2+ and ATP by the protein kinase catalytic subunit prepared from skeletal muscle. This stimulation is prevented if the muscle protein kinase regulatory subunit is also included in the incubation. The regulatory subunit added alone did not alter lipolytic activity significantly.