Molecular Pharmacology, Vol 15, 472-483, Copyright © 1979 by the American Society for Pharmacology and Experimental Therapeutics

Adenylate Cyclase Assembled In Vitro: Cholera Toxin Substrates Determine Different Patterns of Regulation by Isoproterenol and Guanosine 5'-triphosphate

¹ Division of Clinical Pharmacology, Departments of Medicine and Pharmacology and the Cardiovascular Research Institute, University of California, San Francisco, San Francisco, California 94143

Genetic and biochemical evidence indicates that hormone-sensitive adenylate cyclase consists of at least three separable components: hormone receptor (R), a catalytic unit (C) that synthesizes cAMP from ATP, and one or more regulatory factors, which we have termed N, that are required for functional coupling of R and C and also for cyclase stimulation by guanyl nucleotides, NaF, and cholera toxin. The functional absence of N in a S49 lymphoma variant clone, cyc^-, allows in vitro assembly of hormone-sensitive adenylate cyclase, using cyc^- membranes and N components in detergent extracts from membranes of other cells (Ross, E. M. and Gilman, A. G. Proc. Nat. Acad. Sci., USA 74,3715-3719, 1977). Such cyclase systems, assembled in vitro using N components from wild type S49 cells and turkey erythrocytes, are functionally distinguishable, each resembling—in responses to guanyl nucleotides and isoproterenol—the cyclase system from which the N component was derived. Thus, the N component determines certain functional characteristics of the response to guanine nucleotides and isoproterenol. Human erythrocyte membranes, which are virtually devoid of catalytic adenylate cyclase, also contain the functional N component. Donor extracts of human and turkey erythrocytes and S49 cells contain cholera toxin substrates, by two criteria: 1. The extracts transmit effects of toxin on donor membranes to adenylate cyclase assembled in vitro using cyc^- membranes; 2. Incubation with toxin plus [³²P]NAD⁺ specifically radiolabels substrates in each type of membrane, including a peptide of M_r = 42,000, common to all three. The results are consistent with the hypothesis that this peptide participates directly in multiple functions of N.

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ACKNOWLEDGMENT We thank Ms. Mary Gleason for expert technical assistance.

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