Inhibitory regulation of adenylyl cyclase in the absence of stimulatory regulation. Requirements and kinetics of guanine nucleotide-induced inhibition of the cyc- S49 adenylyl cyclase.

cyc- S49 cell membranes contain an adenylyl cyclase activity which is stimulated by forskolin and inhibited by guanine nucleotides and NaF. These inhibitory effects are mediated by an inhibitory guanine nucleotide-binding regulatory component (Ni) affecting the adenylyl cyclase catalytic unit (Hildebrandt, J. D., Sekura, R. D., Codina, J., Iyengar, R., Manclark, C. R., and Birnbaumer, L. (1983) Nature (Lond.) 302, 706-709). Since cyc- S49 cells do not contain a stimulatory guanine nucleotide-binding regulatory component (Ns), these membranes were used to study the requirements and kinetics of activation of Ni in the absence of Ns. Activation of Ni by guanyl-5'-yl imidodiphosphate was time-dependent (i.e. hysteretic) and pseudo-irreversible. Although GTP and guanosine 5'-(beta-thio)diphosphate could prevent the inhibition caused by guanyl-5'-yl imidodiphosphate if added simultaneously with it, they could not reverse the inhibited state induced by previous exposure to guanyl-5'-yl imidodiphosphate. Activation of Ni had an absolute requirement for Mg2+. Unlike the activation of Ns, however, which requires millimolar concentrations of Mg2+ in the absence of hormonal stimulation, activation of Ni requires only micromolar concentrations of the divalent cation. These results support the contention that hormones which activate Ni or Ns do so by altering different parameters of a similar activation mechanism.