Adenosine 3':5'-cyclic monophosphate-binding proteins in bovine and rat tissues

Abstract

At least 2 classes of high-affinity cyclic[c]AMP-binding proteins were identified: those derived from cAMP-dependent protein kinases [EC 2.7.1.37] (regulatory subunits) and those that bind a wide range of adenine analogs (adenine analog-binding proteins). In fresh-tissue extracts regulatory subunits could be further subdivided into type I or type II, depending on whether they were derived from type I or type II protein kinase. The adenine analog-binding protein was detected in crude tissue supernatant fractions of bovine and rat liver. It differed from the regulatory subunit of cAMP-dependent protein kinase in many of its properties. Under the conditions of assay used, the protein accounted for about 45% of the binding of cAMP to bovine liver supernatants. The adenine analog-binding protein from bovine liver was partially purified by DEAE-cellulose and Sepharose 6B chromatography. It has a MW of 185,000 and was trypsin-sensitive. As shown by competition and direct binding experiments, it bound adenosine and AMP in addition to cAMP. At intracellular concentrations of adenine nucleotides, binding of cAMP was essentially completely inhibited in vitro. Adenosine binding was inhibited by only 30% under similar conditions. Rat tissues were examined for the presence of the adenine analog-binding protein, and of those examined (adipose tissue, heart, brain, testis, kidney and liver), significant amounts were only found in the liver. The possible physiological role of the adenine analog-binding protein is discussed. Because the adenine analog-binding protein or other cAMP-binding proteins in tissues may be products of partial proteolysis of the regulatory subunit of cAMP-dependent protein kinase, the effects of trypsin and aging on partially purified protein kinase and its regulatory subunit from bovine liver were investigated. In all studies the effects of trypsin and aging were similar. In fresh preparations the cAMP-dependent protein kinase had MW 150,000. Trypsin treatment converted it into a form of MW 79,500. The regulatory subunit of the protein kinase had MW 87,000. It would reassociate with and inhibit the catalytic subunit of the enzyme. Trypsin treatment of the regulatory subunit produced a species of MW 35,500 which bound cAMP but did not reassociate with the catalytic subunit. Trypsin treatment of the protein kinase and dissociation of the product by cAMP produced a regulatory subunit of MW 46,500 which reassociated with the catalytic subunit. These results may be explained by at least 2 trypsin-sensitive sites on the regulatory subunit. A model for the effects of trypsin is described.