Ammonium ions enhance proteolytic activation of adenylate cyclase and decrease its senszsitivity to inhibition by “P”-site agonists

Abstract

A detergent-dispersed adenylate cyclase from rat brain was used to study the effects of ammonium salts and polyamines on the proteolytic activation of the enzyme by a sperm protease and on the sensitivity of adenylate cyclase to inhibition via its “P”-site. A purified preparation of a trypsin-like, serine protease from bovine sperm was used to activate solubilized adenylate cyclase in the presence of guanosine 5′- O-(3-thiotriphosphate (GTPγS). The proteolytically activated form of adenylate cyclase was found to be particularly sensitive to further activation by ammonium bicarbonate. The activation by NH 4HCO 3 was found to be due to the NH 4 + cation and was characterized by an increased V max and by a decreased sensitivity of adenylate cyclase to inactivation by elevated concentrations of the sperm protease or by trypsin. NH 4Cl and (NH 4) 2SO 4 also caused biphasic effects on adenylate cyclase, which mimicked but were less effective than those caused by NH 4HCO 3. Consistent with observations of others, adenylate cyclase activity was enhanced by ammonium ions whether in the presence of reversible (Mn 2+) or irreversible (GTPγS) activators. Mn 2+- and GTPγS-stimulated activities were similarly optimally enhanced by 30 m m (NH 4) 2SO 4 and by 30 to 150 m m NH 4Cl or NH 4HCO 3. Ammonium ions did not increase the activity of the purified catalytic unit. Moreover, the effect of ammonium ions was not accompanied by an increased rate of activation by GTPγS, suggesting that the activation of G s (guanine nucleotide-dependent stimulatory component) may not be the primary cause of stimulation by ammonium salts. Several polyamines at millimolar concentrations blocked the stimulatory effect of NH 4 +. This was observed when adenylate cyclase was activated by Mn 2+, but not when it was activated by GTPγS or by the sperm protease + GTPγS. The inhibitory effect of polyamines was not due to the formation of a complex with ATP. Both the increase in V max of the Mn 2+-stimulated enzyme by NH 4 + and the decrease in V max caused by spermine were accompanied by an increase in the enzyme's K m MnATPapp. Spermine increased the IC 50 for inhibition of Mn 2+-activated adenylate cyclase by 2′,5′-dideoxyadenosine (2′,5′-ddAdo) from 0.75 to 4.6 μ m, consistent with the idea that increased sensitivity of P-site-mediated inhibition is associated with increased enzyme activity. In contrast, activation of Mn 2+-stimulated adenylate cyclase by 30 m m (NH 4) 2SO 4 also reduced sensitivity to inhibition by 2′,5′-ddAdo(IC 50 1.1 μ m). This decreased sensitivity to inhibition by 2′,5′-ddAdo induced by ammonium ion was also observed on GTPγS-activated or proteolytically activated adenylate cyclase. The data support the idea that activation of adenylate cyclase by ammonium salts may be due to an effect on the conformation and/or stability of the active form of the enzyme, leading to increased enzyme activity as well as to decreased proteolytic inactivation.