Action of mercurials on 3H-cAMP binding to the regulatory subunit-II of cAMP-dependent protein kinase.

Binding of the regulatory subunit type II (RII) of adenosine 3',5'-cyclic monophosphate (cAMP) dependent protein kinase was inhibited by Hg2+ with an IC50 value of 0.31 microM. Methyl mercury, p-chloromercuribenzoic acid (PCMB), and 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) also inhibited cAMP binding with IC50 values of 70-80 microM for organic mercurials and 130 microM for DTNB. Addition of 1 mM 2-mercaptoethanol and 1 M cysteine to the assay mixture reversed these inhibitions. N-ethylmaleimide (NEM) showed little effect on the binding. On the other hand, Hg2+ and methyl mercury markedly suppressed enzymatic activity of the catalytic subunit. The IC50 value was 0.13 microM for Hg2+ and 0.15 microM for organic mercurials. Scatchard plots of kinetic analysis data for the cAMP binding revealed a noncompetitive type of inhibition by mercurials and DTNB. It is suggested that blockade of sulfhydryl groups resulted in the inhibition of cAMP binding to the RII subunit, which might result in preserving the association of the RII subunit and the catalytic subunits and in preventing further inactivation of the catalytic subunit by Hg2+.