We have established previously that the regulation of
adenylate cyclase is abnormal in adipose tissue membranes of ob/ob mice. To help establish the nature of the defect, we studied the time course of
guanine nucleotide activation and inhibition of
adenylate cyclase. The activation of
adenylate cyclase by
Gpp(NH)p in adipocyte membranes of normal (+/+) and ob/ob mice proceeds with a lag phase. In +/+ membranes, this lag could be shortened by increasing the concentration of Mg2+ in the incubation medium or by pretreatment of the membranes with
cholera toxin, and it could be abolished by
isoproterenol in combination with 4 mM
MgCl2. In contrast, in the ob/ob membranes, only pretreatment with
cholera toxin was effective in shortening the lag phase. These results indicate an impediment in the activation of
adenylate cyclase in ob/ob membranes. In the +/+ membranes,
Gpp(NH)p inhibited foreskolin-stimulated
adenylate cyclase, following a short lag phase, producing lower steady-state velocities than those seen with
forskolin alone. The inhibitory effect of
Gpp(NH)p on
forskolin-stimulated activity was abolished by
pertussis but not by
cholera toxin treatment. In the ob/ob membranes, neither
Gpp(NH)p nor
pertussis treatment had any effect on the steady-state velocity of the
forskolin-stimulated activity. These data have been interpreted as meaning that an anomaly in Ni rather than in Ns is likely to be responsible for the impairment of
adenylate cyclase activity in the membranes of the ob/ob mouse.