Modification of Cardiac Membrane Adenylate Cyclase Activity and GSα by NAD and Endogenous ADP-ribosyltransferase

The mechanism by which NAD stimulates cardiac adenylate cyclase was investigated. In highly purified canine cardiac sarcolemma, NAD stimulated adenylate cyclase activity in the presence of agents which activate GS (i.e. 5 mM AlF4-, 10 μM GTPγS. 10 μM GppNHp or isoproterenol plus 2 nM GTPγS). Furthermore the EC50 of isoproterenol of stimulate adenylate cyclase was reduced in the presence of NAD. In membranes incubated with [32P]-NAD, AlF4-, 10 μM GTPγS or isoproterenol plus 2 nM GTPγS produced a selective increase in the radiolabeling of a single 45-kDA protein which was identified as GSα by immunoprecipitation. Cholera toxin catalysed radiolabeling of the same protein. Neutral hydroxylamine released [32P]-ADP-ribose from GSα prelabeled in the presence of AlF4- and [32P]-NAD indicating that an arginine residue on GSα was modified by an endogenous ADP-ribosyltransferase. ADP-ribosyltransferase inhibitors, novobiocin, vitamin K1 or 3-aminobenzamide, inhibited AlF4- stimulated ADP-ribosylation of GSα and NAD potentiation of adenylate cyclase with similar efficacies. The activity responsible for NAD potentiation of adenylate cyclase and ADP-ribosylation of GSα was not removed under hypotonic or hypotonic conditions and therefore appears to be tightly membrane bound. Collectively, these observations indicate that canine cardiac sarcolemma possess and ADP-ribosyltransferase which may constitutively catalyse transfer of an ADP-ribose to activated GSα.