Critical micelle concentrations, molecular areas, coareas, and standard free energies of adsorption at an air-water interface have been determined for a series of bolaform electrolytes (R3N+-(CH2)n™+NR3 where R = methyl or n-butyl and = 4, 8, and 12). The parameters are compared with those of monoquaternary ammonium salts, CHsfCH^hi+NRs. It is found that the C12 bolaform electrolytes (but not the C4 and Cg homologs) form vertical loops or "wickets" at the air-water interface.

The interaction of cardiac adenylate cyclase [ATP pyrophosphate-lyase (cyclizing); EC 4.6.1.11 with a variety of nucleotide affinity resins was systematically investigated. None of these resins effectively bound the native, detergentsolubilized enzyme. However, after hydrophobic resolution on an uncharged resin consisting of long-chain alkyl groups linked to agarose via ether bonds, 40% of the adenylate cyclase activity biospecifically adsorbed to an ATP affinity resin. Gel filtration without detergent after hydrophobic chromatography demonstrated that the enzyme eluted in the identical position as the native enzyme chromatographed in the presence of detergent. This preparation almost completely biospecifically adsorbed to the same ATP-resin and was not eluted with 5 mM cyclic AMP, pyrophosphate, or GTP. If the GTP-washed immobilized enzyme was subsequently desorbed with ATP, then expected Gpp(NH)p (5'-guanylyliminodiphosphonate) sensitivity persisted. A preliminary purification scheme that resulted in an approximate 5000-fold increase in specific activity is presented. These observations indicate that a membrane-bound enzyme may appear to be intrinsically hydrophobic only by virtue of aggregation with other hydrophobic constituents and that prior separation of hydrophobic chromatography may permit such proteins to be fractionated subsequently by methods conventionally applied to hydrophilic proteins. Adenylate cyclase [ATP pyrophosphate-lyase (cyclizing); EC 4.6.1.1] is a membrane-bound enzyme present in almost all cell types which catalyzes the conversion of ATP to cyclic AMP. A number of polypeptide hormones as well as catecholamines, histamine, and prostaglandins exert all or part of their action through stimulation of this enzyme (1, 2). Cyclic AMP also may exert direct effects on a variety of cellular regulatory mechanisms, including microtubule assembly (3, 4), mobility, and chemotaxis (5), secretion (6, 7), and neoplastic transformation '(8, 9). Despite the critical role of this enzyme, minimal progress has been made in its purification. This difficulty arises for several reasons. First, the enzyme probably constitutes less than 0.005% of the total membrane protein (10). Second, the enzyme is notoriously labile, with a half-life at room temperature of only minutes (11). Third, nonionic detergents are required to solubilize the enzyme. Following such a procedure, several investigators have achieved only minimal success using classical procedures including gel filtration and ion-exchange chromatography (12, 13). We have recently investigated the interaction of the solubilized enzyme with a variety of long-chain alkyl resins linked to uncharged matrices via ether bonds. These gels have been shown to demonstrate all of the properties expected for hydrophobic interaction chromatography (14). Such resins effect an apparent separation of cyclase from more hydrophobic proteins, as demonstrated by the behavior of the enzyme on subsequent gel filtration and ion-exchange chromatography.These results suggest...

An azide derivative of the ,-adrenergic antagonist acebutolol has been synthesized and its effect examined on the isoproterenol-stimulated adenylate cyclase [ATP pyrophosphate-lyase (cyclizing); EC 4.6.1.1] activity of rat reticulocytes. It behaved as an effective competitive antagonist (Kd = 2 X 10-7 M) prior to photolysis. However, when the reticulocyte preparation pretreated with acebutolol azide was photolyzed, a noncompetitive inhibition of isoproterenol-stimulated adenylate cyclase was obtained. Photolysis-of the azide derivative in buffer alone did not convert it to a product of higher affinity.Labeling of the P-adrenergic receptor appeared to be irreversible; multiple washings could not reverse the inhibition produced during photolysis with the label whereas washing would completely reverse the antagonism produced by the same concentration of label prior to photolysis. The effect appears to be specific for the ,-adrenergic receptor because the inhibition could be blocked stereoselectively by propranolol and there was no inhibition of fluoride-or GMP-P(NH)P-stimulated adenylate cyclase. Furthermore, no effect was observed on the glucagonmediated stimulation of adenylate cyclase of liver membranes, whereas the catecholamine response in the same membranes was inhibited.In this communication, we report on the synthesis of an azide derivative of the f3-adrenergic antagonist acebutolol and its characterization as a photoaffinity label for the ,B-adrenergic receptor by its effect on isoproterenol-stimulated adenylate cyclase activity of rat reticulocytes. Although there are previous reports in which affinity labels for this receptor have been described (1-4), this study demonstrates both the irreversible nature and specificity of such inhibition after light-induced chemical activation. Furthermore, the structure of this ligand is unique in that the reactive functional site is located on the phenyl ring rather than on the ethanolamine side chain which is the structure, common to both agonists and antagonists, that is required for ligand binding to the receptor.Photoaffinity labels offer the advantage over affinity labels of being completely reversible until photoactivation occurs. Thus, reliable affinity constants can be obtained and the labels can be characterized prior to their photolysis and concomitant irreversible binding. Furthermore, the specificity of this ligand is demonstrated by its lack of effect on a second adenylate cyclase-linked receptor system in the same membrane preparation in which covalent binding to the f3-adrenergic receptor had been achieved. Stereoselective protection of the receptor from irreversible labeling was also shown with the stereoisomers of propranolol.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 4449MATERIALS AND METHODS Materials. Acebutolol amine, RS-1-(2-acetyl-4-amino)-2-hydroxy-3-isopropylaminopropan...

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