Affinity chromatography of human platelet α ₂ -adrenergic receptors

Abstract

Catecholamines, such as epinephrine, inhibit the enzyme adenylate cyclase (EC 4.6.1.1) via a specific receptor mechanism involving a2-adrenergic receptors. In order to facilitate purification of these inhibitory receptors we have prepared a highly effective biospecific affinity adsorbent. The immobilized ligand SKF 101253 is a 3-benzazepine with a2-adrenergic antagonist activity. SKF 101253 is coupled to Sepharose CL-4B by using a bifunctional reagent (1,4-butanediol diglycidyl ether) which also provides a hydrophilic spacer moiety between the ligand and the gel matrix. Membranes from human platelets, containing a12-ad- To understand fully the coupling of stimulatory and inhibitory receptors to adenylate cyclase it will be important to purify these receptors. Progress in this regard has been made for the adenylate cyclase-stimulatory f3-adrenergic receptor (2); however, purification of the adenylate cyclase-inhibitory a2-adrenergic receptor has not been achieved. A formidable problem in the purification of membrane-bound receptors is their miniscule quantity relative to the complete chemical content of the cell. An additional difficulty is the need for solubilization of the receptor from the membrane. Solubilization of membranebound proteins is usually incomplete, and it may lead to receptor inactivation which exacerbates the problem of the low initial concentration of receptor. Furthermore, due to the similarity of physiochemical properties among detergent-solubilized proteins, solubilization vitiates the use of many of the traditional methods for the purification of macromolecules.Affinity chromatography, a technique that exploits the specificity and affinity of ligand-receptor interactions, can over-

.alpha.- and .beta.-Adrenoceptors: From the Gene to the Clinic. Part 1. Molecular Biology and Adrenoceptor Subclassification

“…These include denopamine (59)212 and xamoterol (60).213 However, as with Ro 363, both of these agents are partial agonists at the /81adrenoceptor, and some of the apparent in vivo ßadrenoceptor selectivity may be the result of their low intrinsic activities.214 Hence, when selective in vitro stimulation of /3i-adrenoceptors is desired, norepinephrine in the presence of an -adrenoceptor antagonist may still be the best pharmacological tool. of the /9-adrenoceptors, several antagonists having moderate selectivity for either /9-adrenoceptor subtype, such as practolol (61) (ß\) and butoxamine (62) (/Sa), were identified.215-217 However, these compounds had neither high potency nor high selectivity. Currently, the selective antagonists most commonly used are CGP 20712A, which has a dissociation constant below 1 nM at the /3i-adrenoceptor and a ß^-adrenoceptor selectivity ratio of 10 000.218'219 Another selective /^-antagonist commonly employed as a tool is bisoprolol (63), which has a dissociation constant of Careful analysis of agomst/antagonist interactions can be used to show that both ß -and /^-adrenoceptor activation contribute to many functional responses.…”

Role of catecholamines in platelet function: pathophysiological and clinical significance

“…The human platelet 2 -adrenergic receptor protein was purified as a single polypeptide chain of molecular weight 64 kD [104][105][106]; it contains large amino acid identities with the kidney 2 -adrenoceptor [66].…”

Development of an affinity ligand for purification of .alpha.2-adrenoceptors from human platelet membranes

“…Incubations were for 3 h at 4 °C, and bound ligand was separated from free ligand by Sephadex G-50 chromatography.19 Nonspecific binding was determined in parallel assays in the presence of 10 M phentolamine. The K¿ of [3M]yohimbine was 3 nM as determined from saturation isotherms (2-15 nM) in the same preparations.10 All data were analyzed by computer using nonlinear least-squares curve-fitting techniques.20 hKl± SE from three separate experiments using 10 inhibitor concentrations. CKÍ ± SE from four to five separate experiments using 12 inhibitor concentrations.…”

“…Affinity of columns were prepared from all of these agents (6,8, 9, and 13) by the methods described earlier. 10,16 Even though all of the uncoupled molecules had good affinity for the a2-receptor, only the column prepared from 13 was able to adsorb receptor. In the other cases, solubilized receptor was not bound to the column in any appreciable amount but passed through without adequate retention to allow sufficient purification.…”