An Mr = 52,000 peptide can mediate effects on cholera toxin on adenylate cyclase in intact cells.

Cholera toxin acts by ADP-ribosylating a membrane protein, variously termed N, G, or G/F, that is a guanine nucleotide-binding regulatory component of adenylate cyclase. Membranes of several mammalian cells contain two peptides, of Mr = 42,000 and 52,000, which are radiolabeled by incubation with cholera toxin and [32P]NAD. Toxin activation of N in avian and human erythrocytes leads to significant radiolabeling only of the Mr = 42,000 peptide. Thus ADP-ribosylation of the Mr = 42,000 peptide appears to suffice for stimulation of adenylate cyclase, and no biological function has yet been assigned to the Mr = 52,000 peptide. We have found that incubation with toxin and [32P]NAD significantly radiolabels an Mr = 52,000 peptide, but not an Mr = 42,000 peptide in particulate preparations of A9 mouse and 380-6 hamster transformed lung fibroblasts. Treatment of intact A9 or 380-6 cells with cholera toxin increases their cyclic AMP content and blocks subsequent toxin-catalyzed incorporation of 32P from NAD into the Mr = 52,000 peptide of particulate extracts from the toxin-treated cells. Treating A9 and 380-6 particulate extracts with toxin and NAD increases their N activity in the presence of GTP, assayed using N-deficient membranes of the S49 cyc- mutant. We conclude that (1) a functionally active form of the N protein can contain the Mr = 52,000 peptide and (2) ADP-ribosylation of the Mr = 52,000 peptide subunit of N can mediate the action of cholera toxin.