The regulatory component of adenylate cyclase. Purification and properties.

P. C. Sternweis, J. K. Northup, M. D. Smigel, A. G. Gilman

Research output: Contribution to journalArticle

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Abstract

The regulatory component (G/F) of adenylate cyclase, which has been purified previously, contains three putative subunits with molecular weights of 52,000, 45,000, and 35,000 (Northup, J. K., Sternweis, P. C., Smigel, M. D., Schleifer, L. S., Ross, E. M., and Gilman, A. G. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 6516-6520). The published procedure has been modified to reduce the time required for preparation and to increase the yield. Application of the improved procedure allows purification of .5 to 1.0 mg of purified G/F from 1.5 kg of frozen rabbit liver. Greater than 95% of the protein observed on sodium dodecyl sulfate polyacrylamide gels is found in the three bands mentioned above. Purified G/F has the following properties: 1. Hydrodynamic measurements in cholate indicate that purified hepatic G/F has a molecular weight of about 70,000. If G/F is activated with either fluoride or GTP analogs, its apparent molecular weight is reduced to 50,000. 2. The measurement of G/F by reconstitution with the catalytic moiety of adenylate cyclase is dependent on the concentrations of both G/F and catalytic moiety. This interaction is consistent with a model derived from a simple bimolecular binding equilibrium. 3. Purified G/F can be activated by fluoride and guanine nucleotide analogs in a Mg2+-dependent reaction. The rate of activation by guanine nucleotides is markedly stimulated by high concentrations of Mg2+, indicating a site of action of divalent metallic cations on G/F. 4. The 52,000- and 45,000-dalton polypeptides can be partially resolved by heptylamine-Sepharose chromatography. G/F fractions that are enriched in the 52,000-dalton protein reconstitute hormone-stimulated adenylate cyclase activity more efficiently and are activated by GTP analogs more rapidly than are fractions that are essentially free of this polypeptide. The 35,000-dalton protein is present in all cases.

Original languageEnglish (US)
Pages (from-to)11517-11526
Number of pages10
JournalJournal of Biological Chemistry
Volume256
Issue number22
StatePublished - Nov 25 1981

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Adenylyl Cyclases
Purification
Guanine Nucleotides
Molecular Weight
Molecular weight
Guanosine Triphosphate
Fluorides
Cholates
Agarose Chromatography
Peptides
Proteins
Liver
Divalent Cations
Hydrodynamics
Chromatography
Sodium Dodecyl Sulfate
Sepharose
Chemical activation
Hormones
Rabbits

ASJC Scopus subject areas

  • Biochemistry

Cite this

Sternweis, P. C., Northup, J. K., Smigel, M. D., & Gilman, A. G. (1981). The regulatory component of adenylate cyclase. Purification and properties. Journal of Biological Chemistry, 256(22), 11517-11526.

The regulatory component of adenylate cyclase. Purification and properties. / Sternweis, P. C.; Northup, J. K.; Smigel, M. D.; Gilman, A. G.

In: Journal of Biological Chemistry, Vol. 256, No. 22, 25.11.1981, p. 11517-11526.

Research output: Contribution to journalArticle

Sternweis, PC, Northup, JK, Smigel, MD & Gilman, AG 1981, 'The regulatory component of adenylate cyclase. Purification and properties.', Journal of Biological Chemistry, vol. 256, no. 22, pp. 11517-11526.
Sternweis, P. C. ; Northup, J. K. ; Smigel, M. D. ; Gilman, A. G. / The regulatory component of adenylate cyclase. Purification and properties. In: Journal of Biological Chemistry. 1981 ; Vol. 256, No. 22. pp. 11517-11526.
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