Characterization of a cyclic AMP-binding protein from bakers' yeast. Identification as a regulatory subunit of cyclic AMP-dependent protein kinase.

Abstract

A cyclic AMP-binding protein with an szo,ur of 4.6 S and a Stokes radius of 50 di was purified to homogeneity from bakers' yeast.The native molecular weight and frictional ratio were calculated to be 95,000 and 1.66, respectively.The data imply that the protein has an axial ratio of 12.1, assuming a prolate elipsoid.The molecular weight determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 50,000.The findings suggest that the protein exists in solution as a dimer of identically sized subunits.The protein was judged to be a regulatory subunit (R) of cyclic AMP-dependent protein kinase, since it inhibited purified mammalian cyclic AMP-dependent protein kinase catalytic subunit in the absence but not in the presence of cyclic AMP; it also inhibited partially purified catalytic subunit from yeast.Furthermore, the ability of cyclic AMP to stimulate partially purified yeast cyclic AMP-dependent protein kinase was lost when the enzyme was passed through a cyclic AMP-affinity resin of the type used in the preparation of the binding protein.The NHz terminus of the yeast R subunit was not blocked as is the case with the corresponding mammalian proteins.The NHz-terminal sequence was Val-Ser-Ser-Leu-Pro-Lys-Glu-Ser-Gln-Ala-Glu-Leu-Gln-Leu -Phe -Gln-Asn-Gly -Ile -Asn -.Specificity studies showed that only 3':5'-cyclic nucleotides were effective in competing for cyclic AMP-binding to the subunit.The apparent dissociation constant for cyclic AMPbinding increased from 60 to 150 n~ when the protein concentration increased from 10 to 100 n ~.Antibody to yeast R subunit did not cross-react with types I or I1 mammalian R subunit.Cyclic AMP functions as a regulatory agent in controlling the rate of many physiological processes in both prokaryotic and eukaryotic organisms.In the bacterium Escherichia coli, cyclic AMP is necessary for transcription of specific genes repressed by catabolites.In eukaryotes, the cyclic nucleotide exerts many of its effects by activation of protein kinases, which have been found in a wide variety of vertebrate and invertebrate species (1-3).Cyclic AMP-dependent protein kinase or their subunit components have been purified to homogeneity from various mammalian sources (4-10).Several investigators (11)(12)(13)(14) have demonstrated the mechanism of enzyme activation by cyclic