Sulfhydryl group reactivity of adenosine 3',5'-monophosphate dependent protein kinase from bovine heart: a probe of holoenzyme structure.
The spectrophotometric titration of SH groups in adenosine 3',5'-monophosphate (cAMP) dependent protein kinase from bovine heart muscle with 5,5'-dithiobis(2-nitrobenzoic acid)(DTNB) is described. The holoenzyme (R2C2) contains 16 SH groups, 12 of which react with DTNB in the native enzyme. The SH groups are distributed 2 per catalytic (C) and 4 per regulatory (R) subunit. The binding of cAMP to the holoenzyme or isolated R subunit prevents the reaction of one SH group per R subunit. Modification of SH groups, however, has only a small effect on cAMP binding to R. Reaction of the C subunit with DTNB results in less than 95% loss of catalytic activity. The kinetics of the DTNB reaction and the reversal of the inactivation process by treatment with dithiothreitol suggest that the inactivation is associated with SH group modification. Inactivation studies with the holoenzyme show that: (1) the R subunit inhibits DTNG inactivation of the C subunit in the absence of cAMP; (2) the rate of inactivation of the dephosphoholoenzyme in the presence of cAMP is considerably faster than that of the free catalytic subunit; and (3) the rate of inactivation of the phosphoholoenzyme in the presence of cAMP is faster than that of the C subunit but slower than the dephosphoholoenzyme. The results are interpreted as evidence for a significant interaction of the R and C subunits in the presence of saturating concentrations of cAMP. This interaction is modulated by the state of phosphorylation of R. To account for the inactivation data, a short-lived ternary complex containing R, C, and cAMP is postulated to be in rapid equilibrium with the subunits.
D000818:Animals; D002417:Cattle; D000242:Cyclic AMP / Q000494:pharmacology; D004228:Dithionitrobenzoic Acid; D007700:Kinetics; D046911:Macromolecular Substances; D009206:Myocardium / Q000201:enzymology*; D011494:Protein Kinases / Q000378:metabolism; D013438:Sulfhydryl Compounds
D046911:Macromolecular Substances; D013438:Sulfhydryl Compounds; D004228:Dithionitrobenzoic Acid; D000242:Cyclic AMP; D011494:Protein Kinases