Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1987 Sep 1;246(2):431–439. doi: 10.1042/bj2460431

Identification and purification from bovine brain of a guanine-nucleotide-binding protein distinct from Gs, Gi and Go.

G L Waldo 1, T Evans 1, E D Fraser 1, J K Northup 1, M W Martin 1, T K Harden 1
PMCID: PMC1148293  PMID: 3120696

Abstract

A guanine-nucleotide-binding protein (G-protein) was purified from cholate extracts of bovine brain membranes by sequential DEAE-Sephacel, Ultrogel AcA-34, heptylamine-Sepharose and Sephadex G-150 chromatography. Guanosine 5'-[gamma-[35S]thio]triphosphate (GTP[35S])-binding activity copurified with a 25,000 Da peptide and a 35,000-36,000 Da protein doublet. Neither pertussis toxin nor cholera toxin catalysed the ADP-ribosylation of a protein associated with the GTP[35S]-binding activity. Photoaffinity labelling of the purified protein with 8-azido[gamma-32P]GTP indicated that the GTP-binding site resides on the 25,000 Da protein. The 35,000-36,000 Da protein doublet was electrophoretically indistinguishable from the beta-subunits of other GTP-binding proteins, and the 36,000 Da protein was recognized by antiserum to oligomeric Gt. The purified protein specifically bound 17.2 nmol of GTP[35S]/mg of protein. The Kd of the binding site for radioligand was approx. 15 nM. The brain GTP-binding protein co-migrated during SDS/polyacrylamide-gel electrophoresis with a GTP-binding protein, named Gp, purified from human placenta [Evans, Brown, Fraser & Northup (1986) J. Biol. Chem. 261, 7052-7059], and cross-reacted with antiserum raised against the placental protein, but not with antiserum raised to brain Go. SDS/polyacrylamide-gel electrophoresis of the brain and placental GTP-binding proteins in the presence of Staphylococcus aureus V8 protease yielded identical peptide maps.

Full text

PDF
431

Images in this article

434Fig. 2.
on p.434
435Fig. 3.
on p.435
435Fig. 4.
on p.435
436Fig. 6.
on p.436
437Fig. 7.
on p.437
437Fig. 8.
on p.437

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Andrade R., Malenka R. C., Nicoll R. A. A G protein couples serotonin and GABAB receptors to the same channels in hippocampus. Science. 1986 Dec 5;234(4781):1261–1265. doi: 10.1126/science.2430334. [DOI] [PubMed] [Google Scholar]
  2. Bokoch G. M., Katada T., Northup J. K., Ui M., Gilman A. G. Purification and properties of the inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase. J Biol Chem. 1984 Mar 25;259(6):3560–3567. [PubMed] [Google Scholar]
  3. Breitwieser G. E., Szabo G. Uncoupling of cardiac muscarinic and beta-adrenergic receptors from ion channels by a guanine nucleotide analogue. Nature. 1985 Oct 10;317(6037):538–540. doi: 10.1038/317538a0. [DOI] [PubMed] [Google Scholar]
  4. Cassel D., Pfeuffer T. Mechanism of cholera toxin action: covalent modification of the guanyl nucleotide-binding protein of the adenylate cyclase system. Proc Natl Acad Sci U S A. 1978 Jun;75(6):2669–2673. doi: 10.1073/pnas.75.6.2669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cockcroft S., Gomperts B. D. Role of guanine nucleotide binding protein in the activation of polyphosphoinositide phosphodiesterase. Nature. 1985 Apr 11;314(6011):534–536. doi: 10.1038/314534a0. [DOI] [PubMed] [Google Scholar]
  6. Evans T., Brown M. L., Fraser E. D., Northup J. K. Purification of the major GTP-binding proteins from human placental membranes. J Biol Chem. 1986 May 25;261(15):7052–7059. [PubMed] [Google Scholar]
  7. FERGUSON K. A. STARCH-GEL ELECTROPHORESIS--APPLICATION TO THE CLASSIFICATION OF PITUITARY PROTEINS AND POLYPEPTIDES. Metabolism. 1964 Oct;13:SUPPL–SUPPL1002. doi: 10.1016/s0026-0495(64)80018-4. [DOI] [PubMed] [Google Scholar]
  8. Gilman A. G. G proteins and dual control of adenylate cyclase. Cell. 1984 Mar;36(3):577–579. doi: 10.1016/0092-8674(84)90336-2. [DOI] [PubMed] [Google Scholar]
  9. Hescheler J., Rosenthal W., Trautwein W., Schultz G. The GTP-binding protein, Go, regulates neuronal calcium channels. 1987 Jan 29-Feb 4Nature. 325(6103):445–447. doi: 10.1038/325445a0. [DOI] [PubMed] [Google Scholar]
  10. Hughes A. R., Martin M. W., Harden T. K. Pertussis toxin differentiates between two mechanisms of attenuation of cyclic AMP accumulation by muscarinic cholinergic receptors. Proc Natl Acad Sci U S A. 1984 Sep;81(18):5680–5684. doi: 10.1073/pnas.81.18.5680. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kahn R. A., Gilman A. G. Purification of a protein cofactor required for ADP-ribosylation of the stimulatory regulatory component of adenylate cyclase by cholera toxin. J Biol Chem. 1984 May 25;259(10):6228–6234. [PubMed] [Google Scholar]
  12. Kahn R. A., Gilman A. G. The protein cofactor necessary for ADP-ribosylation of Gs by cholera toxin is itself a GTP binding protein. J Biol Chem. 1986 Jun 15;261(17):7906–7911. [PubMed] [Google Scholar]
  13. Katada T., Oinuma M., Ui M. Two guanine nucleotide-binding proteins in rat brain serving as the specific substrate of islet-activating protein, pertussis toxin. Interaction of the alpha-subunits with beta gamma-subunits in development of their biological activities. J Biol Chem. 1986 Jun 25;261(18):8182–8191. [PubMed] [Google Scholar]
  14. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  15. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  16. Lam K. S., Kasper C. B. Sequence homology analysis of a heterogenous protein population by chemical and enzymic digestion using a two-dimensional sodium dodecyl sulfate-polyacrylamide gel system. Anal Biochem. 1980 Nov 1;108(2):220–226. doi: 10.1016/0003-2697(80)90572-2. [DOI] [PubMed] [Google Scholar]
  17. Litosch I., Wallis C., Fain J. N. 5-Hydroxytryptamine stimulates inositol phosphate production in a cell-free system from blowfly salivary glands. Evidence for a role of GTP in coupling receptor activation to phosphoinositide breakdown. J Biol Chem. 1985 May 10;260(9):5464–5471. [PubMed] [Google Scholar]
  18. Milligan G., Klee W. A. The inhibitory guanine nucleotide-binding protein (Ni) purified from bovine brain is a high affinity GTPase. J Biol Chem. 1985 Feb 25;260(4):2057–2063. [PubMed] [Google Scholar]
  19. Neer E. J., Lok J. M., Wolf L. G. Purification and properties of the inhibitory guanine nucleotide regulatory unit of brain adenylate cyclase. J Biol Chem. 1984 Nov 25;259(22):14222–14229. [PubMed] [Google Scholar]
  20. Neville D. M., Jr Molecular weight determination of protein-dodecyl sulfate complexes by gel electrophoresis in a discontinuous buffer system. J Biol Chem. 1971 Oct 25;246(20):6328–6334. [PubMed] [Google Scholar]
  21. Northup J. K., Smigel M. D., Gilman A. G. The guanine nucleotide activating site of the regulatory component of adenylate cyclase. Identification by ligand binding. J Biol Chem. 1982 Oct 10;257(19):11416–11423. [PubMed] [Google Scholar]
  22. Northup J. K., Sternweis P. C., Smigel M. D., Schleifer L. S., Ross E. M., Gilman A. G. Purification of the regulatory component of adenylate cyclase. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6516–6520. doi: 10.1073/pnas.77.11.6516. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Pfaffinger P. J., Martin J. M., Hunter D. D., Nathanson N. M., Hille B. GTP-binding proteins couple cardiac muscarinic receptors to a K channel. Nature. 1985 Oct 10;317(6037):536–538. doi: 10.1038/317536a0. [DOI] [PubMed] [Google Scholar]
  24. Ross E. M., Schatz G. Purification of subunit composition of cytochrome c1 from bakers' yeast Saccharomyces cerevisiae. Methods Enzymol. 1978;53:222–229. doi: 10.1016/s0076-6879(78)53027-9. [DOI] [PubMed] [Google Scholar]
  25. Sasaki K., Sato M. A single GTP-binding protein regulates K+-channels coupled with dopamine, histamine and acetylcholine receptors. Nature. 1987 Jan 15;325(6101):259–262. doi: 10.1038/325259a0. [DOI] [PubMed] [Google Scholar]
  26. Schaffner W., Weissmann C. A rapid, sensitive, and specific method for the determination of protein in dilute solution. Anal Biochem. 1973 Dec;56(2):502–514. doi: 10.1016/0003-2697(73)90217-0. [DOI] [PubMed] [Google Scholar]
  27. Shaltiel S. Hydrophobic chromatography. Methods Enzymol. 1974;34:126–140. doi: 10.1016/s0076-6879(74)34012-8. [DOI] [PubMed] [Google Scholar]
  28. Sternweis P. C., Robishaw J. D. Isolation of two proteins with high affinity for guanine nucleotides from membranes of bovine brain. J Biol Chem. 1984 Nov 25;259(22):13806–13813. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES