Cholera-toxin-dependent ADP-ribosylation of the adenylate cyclase regulatory protein in turkey erythrocyte membranes

Robert W. Downs, Sharon A. Reen, Michael A. Levine, Gerald D. Aurbach, Allen M. Spiegel

Research output: Contribution to journalArticle

5 Scopus citations

Abstract

Incubation of turkey erythrocyte membranes with cholera toxin and [32P]NAD caused toxin-dependent incorporation of 32P into a 42,000 Mr peptide which could be distinguished from toxin-independent 32P incorporation into other membrane proteins. The radiolabeled 42,000 Mr peptide could be extracted from the membranes using Lubrol PX. When toxin-treated membranes were incubated with isoproterenol and GMP before detergent solubilization, the 42,000 Mr labeled peptide was adsorbed by GTP-γ-agarose which, with the same conditions, adsorbed the adenylate cyclase guanine nucleotide regulatory protein. The labeled peptide and guanine nucleotide regulatory protein activity were coeluted from the affinity matrix by guanylyl-β,γ-imidodiphosphate, GDP, and GMP. Guanosine 5′-O-(2-thiodiphosphate), an analog of GDP which blocks guanine nucleotide- and fluoride-stimulated adenylate cyclase activity, caused elution of labeled peptide which exhibited no regulatory protein activity. Our data support the view that the 42,000 Mr peptide is part of the adenylate cyclase guanine nucleotide regulatory protein. The labeled peptide allows identification of both active and inactive regulatory protein and should be useful in monitoring the purification of the regulatory protein from turkey erythrocytes.

Original languageEnglish (US)
Pages (from-to)284-290
Number of pages7
JournalArchives of Biochemistry and Biophysics
Volume209
Issue number1
DOIs
StatePublished - Jun 1981
Externally publishedYes

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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