Purification and reconstitution of the human platelet. cap alpha. /sub 2/-adrenergic receptor

Conference · Thu May 01 04:00:00 EDT 1986 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

OSTI ID:5179770

Regan, J W; Cerione, R A; Nakata, H; Benovic, J L; DeMarinis, R M; Caron, M G; Lefkowitz, R J

Human platelet ..cap alpha../sub 2/-adrenergic receptors have been purified approx.80,000 fold to apparent homogeneity by a five step chromatographic procedure. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radioiodinated protein from purified receptor preparations shows a single major band of M/sub r/ 64,000. The competitive binding of ligands to the purified receptor protein shows the proper ..cap alpha../sub 2/-adrenergic specificity. The ..cap alpha../sub 2/-adrenergic receptor contains an essential sulfhydryl residues. Thus, exposure of the purified receptor to the sulfhydryl specific reagent, phenylmercuric chloride (PMC), resulted in a 80% loss of binding activity. This loss of binding activity was prevented when exposure to PMC was done in the presence of ..cap alpha../sub 2/-adrenergic ligands and it was reversed by subsequent exposure to dithiothreitol. Partial proteolysis of purified ..cap alpha../sub 2/-adrenergic receptors was obtained with S. aureus V-8 protease, ..cap alpha..-chymotrypsin and papain. In a comparison with purified ..beta../sub 2/-adrenergic receptors no common partial proteolytic products were found. Partially purified preparations of the ..cap alpha../sub 2/-adrenergic receptor were successfully reconstituted into phospholipid vesicles with the inhibitory guanyl nucleotide-binding regulatory protein, N/sub i/. In these reconstituted preparations, epinephrine could stimulate, and phentolamine could block, the GTPase activity of N/sub i/.

• Research Organization: Duke Univ. Medical Center, Durham, NC
• OSTI ID: 5179770
• Report Number(s): CONF-8606151-
• Conference Information: Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Journal Volume: 45:6
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ADRENAL HORMONES
ADRENALINE
ANIMALS
AUTONOMIC NERVOUS SYSTEM AGENTS
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL MATERIALS
BLOOD
BLOOD CELLS
BLOOD PLATELETS
BODY FLUIDS
CARDIOTONICS
CARDIOVASCULAR AGENTS
CHEMICAL REACTIONS
CPB
DECOMPOSITION
DRUGS
ELECTROPHORESIS
ENZYMES
ESTERS
HORMONES
HYDROLASES
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LIGANDS
LIPIDS
MAMMALS
MAN
MATERIALS
MEMBRANE PROTEINS
NEUROREGULATORS
ORGANIC COMPOUNDS
ORGANIC PHOSPHORUS COMPOUNDS
PEPTIDE HYDROLASES
PHOSPHOLIPIDS
PRIMATES
PROTEINS
PROTEOLYSIS
PURIFICATION
REACTION KINETICS
REAGENTS
RECEPTORS
STEROID HORMONES
SYMPATHOMIMETICS
TRACER TECHNIQUES
VERTEBRATES