Reconstitution of catecholamine-stimulated adenylate cyclase activity using three purified proteins

beta-Adrenergic receptors, the GTP-binding regulatory protein that stimulates adenylate cyclase (Gs), and adenylate cyclase were each purified and reconstituted into unilamellar vesicles composed of phosphatidylethanolamine and phosphatidylserine (3:2, w/w). The molar ratio of receptor:Gs:adenylate...

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Bibliographic Details
Published in:The Journal of biological chemistry 1985-12, Vol.260 (29), p.15829-15833
Main Authors: May, D C, Ross, E M, Gilman, A G, Smigel, M D
Format: Article
Language:English
Subjects:
Adenylyl Cyclases - metabolism
Animals
Biological and medical sciences
Catecholamines - pharmacology
Cattle
Cell physiology
Cyclic AMP - biosynthesis
Electrophoresis, Polyacrylamide Gel
Epinephrine - pharmacology
Freeze Fracturing
Fundamental and applied biological sciences. Psychology
GTP-Binding Proteins - metabolism
Hormonal regulation
In Vitro Techniques
Isoproterenol - pharmacology
Lipid Bilayers - metabolism
Microscopy, Electron
Molecular and cellular biology
Propranolol - pharmacology
Receptors, Adrenergic, beta - metabolism
Stereoisomerism
Terbutaline - pharmacology
Turkeys
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Summary:beta-Adrenergic receptors, the GTP-binding regulatory protein that stimulates adenylate cyclase (Gs), and adenylate cyclase were each purified and reconstituted into unilamellar vesicles composed of phosphatidylethanolamine and phosphatidylserine (3:2, w/w). The molar ratio of receptor:Gs:adenylate cyclase was estimated to be about 1:10:1. Adenylate cyclase activity in the vesicles was stimulated up to 2.6-fold by beta-adrenergic agonists. Stimulation was dependent on the presence of guanine nucleotide, displayed appropriate beta-adrenergic selectivity and stereoselectivity for agonists, and was blocked appropriately by beta-adrenergic antagonists. Therefore, while additional proteins may modulate adenylate cyclase activity in native membranes, these results show that these three proteins are sufficient for the expression of hormone-stimulated adenylate cyclase.
ISSN:0021-9258
1083-351X
DOI:10.1016/S0021-9258(17)36333-0