Purification and properties of the inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase

G. M. Bokoch, T. Katada, J. K. Northup, M. Ui, A. G. Gilman

Research output: Contribution to journalArticlepeer-review

310 Scopus citations

Abstract

Attenuation of GTP-dependent inhibition of adenylate cyclase by islet-activating protein (pertussis toxin) is due to the ability of the toxin to catalyze the ADP-ribosylation of a 41,000/35,000-Da membrane-bound protein, which is thought to be the inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase (G1). We describe and document the purification of this protein from rabbit liver, and, in conjunction with evidence presented in the subsequent papers of the series, identify it as G(i). Purified G(i) serves as an excellent substrate for islet-activating protein and can be ADP-ribosylated to the extent of 1 mol of ADP-ribose/mol of protein. The extent of ADP-ribosylation of G(i) correlates with the amount of guanine nucleotide that can be bound to the protein. Studies of nucleotide binding site on the 41,000-Da subunit of G(i) reveal a high affinity site that is specific for guanine nucleotides, Ran. order of affinities for various nucleotides in GTPγS > Gpp(NH)p = GTP = GDP > GMP >> App(NH)p, ATP. High affinity binding of guanine nucleotides is dependent on Mg2+ and is essentially irreversible in the presence of divalent cation. Bound nucleotide readily dissociates from its site on the 41,000-Da subunit of G(i) in the absence of Mg2+. This reversal of binding is markedly enhanced by the presence of the 35,000-Da subunit of G(i). The physical characteristics of G(i) are important determinants of its role as the inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase.

Original languageEnglish (US)
Pages (from-to)3560-3567
Number of pages8
JournalJournal of Biological Chemistry
Volume259
Issue number6
StatePublished - 1984

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint

Dive into the research topics of 'Purification and properties of the inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase'. Together they form a unique fingerprint.

Cite this