Tosyl-lysyl chloromethylketone detects conformational changes in the catalytic unit of adenylate cyclase induced by receptor and G-protein stimulation.

Incubation of striatal membranes with tosyl-lysyl chloromethylketone (TLCK) led to the irreversible inactivation of adenylate cyclase. However, under conditions where an interaction between the catalytic unit of adenylate cyclase and the alpha-subunit of the stimulatory G-protein GS were promoted, then the ability of TLCK to inhibit adenylate cyclase was markedly attenuated. The potency of stimulatory ligands, functioning through GS, to attenuate the sensitivity of adenylate cyclase to inactivation by TLCK was paralleled by their potency to activate adenylate cyclase. The local anaesthetic and membrane-fluidizing agent benzyl alcohol amplified GS-mediated stimulation of adenylate cyclase activity, whilst diminishing the ability of GS-mediated coupling to attenuate inactivation of adenylate cyclase by TLCK. In the absence of GS-mediated coupling, benzyl alcohol exerted only a small stimulatory effect on adenylate cyclase activity and had little effect on the ability of TLCK to inactivate this enzyme. We suggest that TLCK modifies a reactive group at or near the active site of adenylate cyclase which causes the functional inactivation of this enzyme. The reactivity of this group appears to be markedly affected by conformational changes elicited through coupling of adenylate cyclase to GS.