Modification of cardiac membrane adenylate cyclase activity and Gs alpha 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 microM GTP gamma S, 10 microM GppNHp or isoproterenol plus 2 nM GTP gamma S). Furthermore, the EC50 of isoproterenol to stimulate adenylate cyclase was reduced in the presence of NAD. In membranes incubated with [32P]-NAD, AlF4-, 10 microM GTP gamma S or isoproterenol plus 2 nM GTP gamma S produced a selective increase in the radiolabeling of a single 45-kDa protein which was identified as Gs alpha by immunoprecipitation. Cholera toxin catalysed radiolabeling of the same protein. Neutral hydroxylamine released [32P]-ADP-ribose from Gs alpha prelabeled in the presence of AlF4- and [32P]-NAD indicating that an arginine residue on Gs alpha was modified by an endogenous ADP-ribosyltransferase. ADP-ribosyltransferase inhibitors, novobiocin, vitamin K1 or 3-aminobenzamide, inhibited AlF4- stimulated ADP-ribosylation of Gs alpha and NAD potentiation of adenylate cyclase with similar efficacies. The activity responsible for NAD potentiation of adenylate cyclase and ADP-ribosylation of Gs alpha was not removed under hypotonic or hypertonic conditions and therefore appears to be tightly membrane bound. Collectively, these observations indicate that canine cardiac sarcolemma possess an ADP-ribosyltransferase which may constitutively catalyse transfer of an ADP-ribose to activated Gs alpha.
Quist
E E
EE
Department of Pharmacology, Texas College of Osteopathic Medicine, University of North Texas, Fort Worth 76107.
Coyle
D L
DL
Vasan
R
R
Satumtira
N
N
Jacobson
E L
EL
Jacobson
M K
MK
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D004285:Dogs; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006321:Heart / Q000187:drug effects; D066298:In Vitro Techniques; D007545:Isoproterenol / Q000494:pharmacology; D009206:Myocardium / Q000378:metabolism*; D009243:NAD / Q000378:metabolism / Q000494:pharmacology*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D012508:Sarcolemma / Q000187:drug effects / Q000378:metabolism
D009243:NAD; D011065:Poly(ADP-ribose) Polymerases; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D007545:Isoproterenol