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Guanosine triphosphate binding sites in solubilized myocardium. Relation to adenylate cyclase activity.

Publication ,  Journal Article
Lefkowitz, RJ
Published in: J Biol Chem
February 10, 1975

Binding of (3H)GTP to solubilized preparations of myocardial adenylate cyclase, partially purified by DEAE-cellulose chromatography, as been studied in an attempt to gain further insight into the mechanisms by which guanine nucleotides regulate adenylate cyclase activity. Although several peaks of (3H)GTP-binding activity were present in crude preparations of solubilized myocardium, one peak was associated with the adenylate cyclase peak. Binding of (3H)GTP to this material was rapid (equilibrium within 3 min at 37 degrees) and reversible and not associated with nucleotide hydrolysis. Scatchard analysis revealed a single class of (3H)GTP binding sites with KA = 3 x 10-6 M-1 and total binding capacity of 50 pmol per mg of protein. The GTP analog Gpp(NH)p competed for the sites with an affinity somewhat lower than GTP, although its ability to activate the adenylate cyclase was far greater. GTP and other guanine nucleotides activated the soluble cyclase only weakly, although they antagonized competitively enzyme stimulation by Gpp(NH)p. Ability of GTP and other nucleotides to compete with (3H)GTP for binding sites and to antagonize competitively adenylate cyclase activation by Gpp(NH)p were directly parallel. The potency series was GTP = GDP = dGTP greater than GMP greater than ITP greater than UTP, CTP. Dissociation constants of nucleotides for the sites determined by inhibition of (3H)GTP binding and inhibition of Gpp(NH)p activation of cyclase agreed closely. Gpp(NH)p dose-response curves for activation of adenylate cyclase and inhibition of (3H)GTP binding were superimposable.

Duke Scholars

Published In

J Biol Chem

ISSN

0021-9258

Publication Date

February 10, 1975

Volume

250

Issue

3

Start / End Page

100 / 611

Location

United States

Related Subject Headings

  • Time Factors
  • Structure-Activity Relationship
  • Receptors, Drug
  • Protein Binding
  • Myocardium
  • Kinetics
  • Guanosine Triphosphate
  • Enzyme Activation
  • Dogs
  • Chromatography, DEAE-Cellulose
 

Citation

APA
Chicago
ICMJE
MLA
NLM

Published In

J Biol Chem

ISSN

0021-9258

Publication Date

February 10, 1975

Volume

250

Issue

3

Start / End Page

100 / 611

Location

United States

Related Subject Headings

  • Time Factors
  • Structure-Activity Relationship
  • Receptors, Drug
  • Protein Binding
  • Myocardium
  • Kinetics
  • Guanosine Triphosphate
  • Enzyme Activation
  • Dogs
  • Chromatography, DEAE-Cellulose