Binding of 5,5'-bis[8-(phenylamino)-1-naphthalenesulfonate] by the regulatory subunits of adenosine cyclic 3',5'-phosphate dependent protein kinase.
Binding to the regulatory subunits of types I and II adenosine cyclic 3',5'-phosphate (cAMP) dependent protein kinase (RI and RII, respectively) produces large distinctive increases in fluorescence and optical activity of 5,5'-bis[8-(phenylamino)-1-naphthalenesulfonate] [bis(ANS)]. Both specific and nonspecific interactions are involved. Association of the regulatory subunits with either the catalytic subunit or cAMP results in dissociation of a major portion of the bound bis(ANS) as detected by changes in fluorescence and circular dichroism. The results are consistent with the accepted cAMP binding properties of RI and RII, showing cooperativity in case of RI and two heterologous binding sites for RII. cGMP has the same overall effect on bis(ANS) binding as cAMP. However, very high concentrations are required for complete dissociation of bis(ANS) from RII, consistent with the observation that cGMP is inefficient in bringing about the dissociation of the type II holoenzyme. Magnesium binding to sites having dissociation constants of ca. 12 mM increases the interaction of bis(ANS) with both of the isolated regulatory subunits. Experiments involving the 37 000-dalton fragment of RII indicate that the limited proteolytic cleavage was heterogeneous, with only 24-39% of the resulting population interacting strongly with the catalytic subunit.
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism*; D055598:Chemical Phenomena; D002621:Chemistry; D002942:Circular Dichroism; D000242:Cyclic AMP / Q000378:metabolism*; D006152:Cyclic GMP / Q000378:metabolism; D046911:Macromolecular Substances; D008274:Magnesium / Q000378:metabolism; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D000817:Anilino Naphthalenesulfonates; D046911:Macromolecular Substances; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D000242:Cyclic AMP; D011494:Protein Kinases; D006152:Cyclic GMP; D008274:Magnesium