Effects of thiol-protecting reagents on the size of solubilized adenylate cyclase and on its ability to be stimulated by guanyl nucleotides and fluoride.

Abstract

The adenylate cyclase enzymes from pig kidney medulla, rat liver and rat brain membranes were solubilized with Triton X-100 into three different states: basal, NaF-activated and guanosine 5′-(β, γ-imino)triphosphate[GuoPP(NH)P]-activated. The effects of 2-mercaptoethanol and other thiol-protecting reagents on the response of solubilized adenylate cyclase to NaF and GuoPP(NH)P and on its apparent sedimentation constants were examined. In the absence of 2-mercaptoethanol, GuoPP(NH)P produced very little stimulation of the adenylate cyclase activity in 200000 ×g supernatant fractions of detergent-treated membranes. 2-Mercaptoethanol markedly enhanced the response to GuoPP(NH)P and to a lesser extent to NaF. When membranes were solubilized and ultracentrifuged without 2-mercaptoethanol, the adenylate cyclases its all of the three states studied had the same apparent sedimentation coefficient (7.4 S, 7.6 S and 7.9 S for the enzymes from pig kidney, rat liver and rat brain respectively). In the presence of 2-mercaptoethanol the apparent sedimentation constant of the adenylate cyclase solubilized in the basal activity state fell by about 1 S, that of the GuoPP(NH)P-preactivated state remained unchanged and that of the NaF-preactivated state dropped with a concomitant loss in activity. The reduction in size and activity were abolished when NaF was added to the sucrose density gradients. Stimulation by GuoPP(NH)P or NaF of the light form of adenylate cyclase (basal state solubilized in the presence of 2-mercaptoethanol) raised the apparent sedimentation constant, and the values obtained were identical to those found for the preactivated forms of the enzyme. The above results provide an explanation for the conflicting reports on the apparent size of solubilized adenylate cyclases and for their reactivity to guanyl nucteottae stimulation. They suggest that the presence of thiol-protecting reagents might be crucial in recomplementation experiments involving the solubilized components of the adenylate cyclase system.