Dissociation and Activation of Adenosine 3′,5′-Monophosphate-dependent and Guanosine 3′,5′-Monophosphate-dependent Protein Kinases by Cyclic Nucleotides and by Substrate Proteins

Abstract

Abstract Two adenosine 3',5'-monophosphate (cyclic AMP)-dependent protein kinases, with sedimentation coefficients of 5.4 S (mol wt 84,000) and 7.7 S (mol wt 140,000), respectively, have been purified from bovine brain. Each enzyme migrated as a single band at three different pH values on polyacrylamide disc gel electrophoresis. The enzymes had similar properties. Catalytic subunits of cyclic AMP-dependent protein kinases from bovine brain, and of cyclic AMP-dependent and guanosine 3',5'-monophosphate (cyclic GMP)-dependent protein kinases from lobster muscle were prepared by means of Enzite CM-cellulose-protamine affinity chromatography, and their properties were studied. The catalytic subunits of the two bovine brain enzymes and of the lobster muscle cyclic GMP-dependent enzyme each had a sedimentation coefficient of 3.6 S (mol wt 40,000), whereas that of the lobster muscle cyclic AMP-dependent enzyme sedimented at 4.5 S (mol wt 60,000). These results agree with kinetic studies, indicating that cyclic GMP-dependent protein kinases of arthropods have certain similarities to cyclic AMP-dependent protein kinases of vertebrates. Histone and cyclic AMP caused the dissociation of the cyclic AMP-dependent protein kinase from lobster muscle (5.7 S; mol wt 90,000) and those from bovine brain into subunits; histone and cyclic GMP caused the dissociation of the lobster muscle cyclic GMP-dependent protein kinase (7.7 S; mol wt 140,000) into subunits. The dissociation of the enzymes by histone was accompanied by a concomitant increase in cyclic nucleotide-independent activity. Iso-electric points of catalytic and regulatory subunits of the 7.7 S component of the brain enzyme were about pH 7.8 and 3.8, respectively. Addition of isolated regulatory subunit, derived from bovine brain cyclic AMP-dependent enzyme, inhibited the activity of catalytic subunits obtained from bovine brain enzymes. Interestingly, the regulatory subunit of the cyclic AMP-dependent enzyme from mammalian brain also inhibited the activity of the catalytic subunit prepared from the lobster muscle cyclic GMP-dependent enzyme, converting it into a cyclic AMP-dependent form. Thus, an interaction between catalytic and regulatory subunits occurred, although the subunits were from different tissues, different phyla, and different classes (with respect to cyclic nucleotide specificity) of protein kinase.