[Identification of the cardiac beta-adrenergic receptor protein: solubilization and purification by affinity chromatography].

Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 11/2007; 52(13 Suppl):1782-3. DOI: 10.1073/pnas.69.10.2828
Source: PubMed

ABSTRACT A protein that binds catecholamines with a specificity parallel to that of their in vivo effects on cardiac contractility (isoproterenol > epinephrine or norepinephrine > dopamine > dihydroxyphenylalanine) was solubilized from a microsomal fraction of canine ventricular myocardium. The binding protein was purified 500 to 800-fold by solubilization and subsequent affinity chromatography with conjugates of norepinephrine linked to agarose beads. Purified beta-adrenergic binding protein exists in two forms, corresponding to molecular weights of 40,000 and 160,000. The purified material has a single association constant, 2.3 x 10(5) liters/mol (as compared to two association constants, 10(7) and 10(6) liters/mol, for the binding protein in particulate form) but retains the identical binding specificity for beta-adrenergic drugs and antagonists.

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    ABSTRACT: (Minus) [3-H] alprenolol, a potent beta-adrenergic antagonist, was used to identify binding sites in a fraction of canine cyocardium. Beta adrenergic agonists and antagonists compete for these binding sites in a manner which directly parallels their known affinity for the cardiac beta-adrenergic receptor. Thus, binding was highly stereo-specific, with the (minus) isomers of beta-adrenergic agonists or antagonists being at least two orders of magnitude more potent than were the (plus) isomers in competing for these sites. The order of potency for inhibition of binding by beta-adrenergic agonists was (minus) isoproterenol greater than (minus) epinephrine greater than (minus) norepinephrine. The dissociation constant (KD) of (minus) alprenolol for the beta-adrenergic receptors was 7-11 nM as determined independently by direct binding studies or by inhibition of isoproterenol-stimulated adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC]. The beta-adrenergic antagonist (minus) propranolol also had high affinity for the binding sites (KD equals 12 nM). The physiologically inactive catechol-containing compounds pyrocatechol and (plus or minus) dihydroxymandelic acid, as well as the metabolite (plus or minus) normetanephrine, and the alpha-adrenergic antagonist phentolamine did not compete for the binding sites at a concentration of 160 muM. Binding was rapid (t1/2 less than 30 sec) and was rapidly reversible (t1/2 less than 15 sec). The binding sites were saturable and bound 0.35 pmol of (minus) [3-H] alprenolol per mg of membrane protein. These characteristics suggest that these binding sites represent the cardiac beta-adrenergic receptors.
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