Cholera Toxin Action on Rabbit Corpus Luteum Membranes: Effects on Adenylyl Cyclase Activity and Adenosine Diphospho-ribosylation of the Stimulatory Guanine Nucleotide-binding Regulatory Component 1

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Abstract
Cholera toxin elicited 5- to 7-fold stimulation of adenylyl cyclase activity. Half-maximal activation was at 4.42 .mu.g/ml cholera toxin. Cholera toxin-mediated activation was time dependent. At 0.1 mM ATP, both GTP and NAD+ were required for cholera toxin activation of luteal adenylyl cyclase. The concentrations of GTP and NAD+ required for half-maximal activation were 1 and 200 .mu.M, respectively. The GTP requirement could be eliminated by increasing the ATP concentration to 1.0 mM. Guanosine-5''-O-(2-thiodiphosphate) [GDP.beta.S] did not support cholera toxin activation of the luteal enzyme. Cholera toxin treatment increased GTP-stimulated activity, did not significantly alter guanyl-5''-yl imidodiphosphate [GMP-P(NH)P]-stimulated activity, and depressed NaF-stimulated activity. Toxin treatment resulted in a 3.4-fold reduction in the Kact values for ovine luteinizing hormone (oLH) to activate adenylyl cyclase. A similar reduction in Kact values for oLH was obtained when concentration-effect curves performed in the presence of GMP-P(NH)P were compared to those performed in the presence of GTP. Luteal membranes treated with cholera toxin and [32P]NAD+ were subjected to autoradiographic analysis following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This treatment resulted in the [32P] ADP-ribosylation of a 45,000-dalton protein doublet, corresponding to the .alpha. subunit of the stimulatory guanine nucleotide-binding regulatory component (Ns). As with activation of adenylyl cyclase activity, cholera toxin-specific [32P]ADP-ribosylation was time dependent and increased with increasing concentrations of cholera toxin. GTP, GMP-P(NH)P and NaF, but not GDP.beta.S, were capable of supporting [32P]ADP-ribosylation of the protein doublet. oLH did not alter the ability of cholera toxin to ADP-ribosylate the protein activation of luteal adenylyl cyclase activity is due to the ADP-ribosylation of the .alpha.-subunit of Ns and the concomitant inhibition of a GTPase associated with adenylyl cyclase.

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