Adenylate cyclase assembled in vitro: Cholera toxin substrates determine different patterns of regulation of isoproterenol and guanosine 5'-triphosphate

H. R. Kaslow, Z. Farfel, G. L. Johnson, H. R. Bourne

Research output: Contribution to journalArticlepeer-review

Abstract

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. 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 erythrocytes 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.

Original languageEnglish
Pages (from-to)472-483
Number of pages12
JournalMolecular Pharmacology
Volume15
Issue number3
StatePublished - 1979
Externally publishedYes

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