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Mechanisms of agonist and antagonist binding to. cap alpha. -/sub 2/ adrenergic receptors: evidence for a precoupled receptor-guanine nucleotide protein complex

Journal Article · · Biochemistry; (United States)
OSTI ID:5176188
; ;

The ..cap alpha../sub 2/ adrenergic receptor (AR) inhibits adenylate cyclase via an interaction with N/sub i/, a guanine nucleotide binding protein. The early steps involved in the activation of the ..cap alpha../sub 2/ AR by agonists and the subsequent interaction with N/sub i/ are poorly understood. In order to better characterize these processes, the authors have studied the kinetics of ligand binding to the ..cap alpha../sub 2/ AR in human platelet membranes on the second time scale. Binding of the ..cap alpha../sub 2/ antagonist (/sup 3/H)yohimbine was formally consistent with a simple bimolecular reaction mechanism with an association rate constant of 2.5 x 10/sup 5/ M/sup -1/ s/sup -1/ and a dissociation rate constant of 1.11 x 10/sup -3/ s/sup -1/. The low association rate constant suggests that this is not a diffusion-limited reaction. Equilibrium binding of the ..cap alpha../sub 2/ adrenergic full agonist (/sup 3/H)UK 14,304 was characterized by two binding affinities: K/sub d1/ = 0.3-0.6 nM and K/sub d2/ = 10 nM. The high-affinity binding corresponds to approximately 65% and the low-affinity binding to 35% of the total binding. The kinetics of binding of (/sup 3/H)UK 14,304 were complex and not consistent with a mass action interaction at one or more independent binding sites. The dependence of the kinetics on (/sup 3/H)UK 14,304 concentration revealed a fast phase with an apparent bimolecular reaction constant k..mu.. of 5 x 10/sup 6/ M/sup -1/ s/sup -1/. The rate constants and amplitudes of the slow phase of agonist binding were relatively independent of ligand concentration. These results were analyzed quantitatively according to several variants of the ternary complex binding mechanisms. The rates of interaction of liganded and unliganded receptor with N protein are estimated

Research Organization:
Univ. of Michigan, Ann Arbor (USA)
OSTI ID:
5176188
Journal Information:
Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 27:7; ISSN BICHA
Country of Publication:
United States
Language:
English

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Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINES
AROMATICS
AZAARENES
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL MATERIALS
BLOOD
BLOOD CELLS
BLOOD PLATELETS
BODY FLUIDS
CELL CONSTITUENTS
CELL MEMBRANES
CONFIGURATION INTERACTION
CYCLASES
ENZYME INDUCTION
ENZYMES
GENE REGULATION
GUANINE
HETEROCYCLIC COMPOUNDS
HYDROXY COMPOUNDS
INHIBITION
KINETICS
LABELLED COMPOUNDS
LIGANDS
LYASES
MATERIALS
MEMBRANE PROTEINS
MEMBRANES
NUCLEOTIDES
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PROTEINS
PURINES
REACTION KINETICS
RECEPTORS
TRITIUM COMPOUNDS