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J Hanoune

Publications and source records attributed to J Hanoune.

At least 127 records · Page 7Linked to original sources
PubMed1979

Affinity chromatography of the beta-adrenergic receptor from turkey erythrocytes.

The beta 1-adrenergic receptors of turkey erythrocyte membranes have been identified by binding of the radioactively labeled antagonist (--)-[3H]dihydroalprenolol, solubilized by treatment of the membranes with the detergent digitonin, and purified by affinity chromatography. Binding of (--)-[3H]dihydroalprenolol to the membranes occurred to a single class of non-cooperative binding sites (0.2--0.3 pmol/mg protein) with a equilibrium dissociation constant (Kd) of 8 (+/- 2) nM. These sites were identified as the functional, adenylate-cyclase-linked beta 1-adrenergic receptors on the basis of: firstly, the fast association and dissociation binding kinetics at 30 degrees C; secondly, the stereospecific displacement of bound (--)-[3H]dihydroalprenolol by beta-adrenergic agonists and antagonists; and thirdly, the order of potencies for agonists to displace bound tracer (isoproterenol congruent to protokylol greater than norepinephrine congruent to epinephrine) similar to the one found for adenylate cyclase activation, and typical for beta 1-adrenergic receptors. Treatment of the membranes with the detergent digitonin solubilized 30% of the receptors in an active form. Digitonin solubilized also adenylate cyclase activity with a yield of 20 to 30%, provided the membranes were first treated with an effector known to produce a persistent active state of the enzyme: e.g. sodium fluoride. Binding sites for guanine nucleotides ([3H]p[NH]ppG) were solubilized as well. Their concentration (24 pmol/mg protein) was in large excess over the concentration of solubilized receptors (0.30--0.45 pmol/mg protein). Solubilized receptors were purified 500--2000-fold by affinity chromatography with a 25 to 35% yield, using an alprenolol-agarose affinity matrix. Affinity purified receptors were devoid of measurable adenylate cyclase activity and guanine nucleotide binding sites, thus showing that receptors and adenylate cyclase are distinct membrane constituents, and that guanine nucleotides apparently do not bind directly to the receptor molecules. Membrane-bound, solubilized and purified receptors were sensitive to inactivation by dithiothreitol, but not by N-ethylmaleimide, suggesting that receptors are at least partly constituted of protein molecules, with essential disulfide bonds.

Adenylyl Cyclases↗
PubMed1979

Activation of rat liver guanylate cyclase by proteolysis.

Guanylate cyclase activity (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2.), measured in purified rat liver plasma membranes, was markedly increased by treatment with various purified proteases. The effect was maximal with trypsin, alpha-chymotrypsin, papain, and thermolysin (6- to 8-fold increase with 5 to 20 microgram of protease/ml) and lower with subtilisin and elastase (3- to 4-fold increase). The activation was due to an increase in the maximal velocity of the cyclizing reaction. No modification was observed either in the apparent affinity for the substrate MnGTP or in the cooperative behavior of the enzyme kinetics which displayed Hill coefficients of 1.6 for both basal and activated states. The Triton X-100-dispersed guanylate cyclase remained sensitive to papain, which suggests that the action of proteases was not restricted to an indirect action upon the membranous environment of the guanylate cyclase. In contrast, the cytosolic soluble guanylate cyclase, assayed in the presence or absence of sodium azide, was absolutely insensitive to papain. Thus, proteolysis represents a previously undescribed mechanism for activating membranous guanylate cyclase systems, which might be of importance in the physiological regulation of this enzyme.

Animals↗
PubMed1979

The adenylate cyclase system in human liver: characterization, subcellular distribution and hormonal sensitivity in normal or cirrhotic adult, and in foetal liver.

1. Adenylate cyclase (EC 4.6.1.1) activity was characterized in human liver, and its subcellular distribution compared with that of three other potential enzyme markers of the pericellular membrane: leucine aminopeptidase (EC 3.4.11.1), gamma-glutamyltransferase (EC 2.3.2.2) and 5'-nucleotidase (EC 3.1.3.5). Although these three enzyme activities were detected in each of the subcellular fractions studied, 85% of the total adenylate cyclase activity was found in the 1000 g pellet ('nuclear' fraction) with a threefold increase in specific activity as compared with the homogenate. No adenylate cyclase activity existed in the 150 000 g supernatant fraction. 2. In the 'nuclear' fraction, adenylate cyclase activity was increased in a dose-dependent fashion by glucagon with a half-maximal stimulation at 10 nmol/l and a maximal four- to seven-fold increase at 1 mumol/l. Catecholamines activated adenylate cyclase 2.5- to three-fold, with an order of potency (protokylol greater than isoprenaline greater than adrenaline greater than noradrenaline) typical of a beta 2-adrenoreceptor. Prostaglandin E1 and NaF also stimulated cyclase two- and four-fold respectively. Insulin, serotonin, dopamine, thyroid-stimulating hormone and ACTH had no effect. Adenosine provoked a weak inhibition at 0.1 mmol/l. Finally guanosine triphosphate and 5'-guanylyl imidodiphosphate induced a marked increase in basal activity, four- and eight-fold respectively, but both reduced the relative increase in enzyme activity due to glucagon or adrenaline. 3. Cyclase from foetal liver (12--16 weeks old) and cirrhotic adult liver appeared to behave similarly to that from normal liver; however, foetal cyclase was more active, and cirrhotic enzyme less active than normal adult liver. Both systems responded to catecholamines via a beta 2-adrenoreceptor. 4. These results validate the use of rat liver adenylate cyclase as a tool for pharmacological and physiological studies.

Adenosine↗
PubMed1979

N-Aralkyl substitution increases the affinity of adrenergic drugs for the alpha-adrenoceptor in rat liver.

1 The alpha-adrenoceptor of rat liver plasma membranes was studied by use of the specific alpha-antagonist [3H]-dihydroergocryptine ([3H]-DHEC). Catecholamines and adrenergic compounds displayed an order of affinity that is typical of an alpha-receptor. Nevertheless, protokylol, a potent beta-adrenoceptor agonist, exhibited a higher affinity than that of adrenaline for alpha-sites. This result might be due to its bulky substituent on the amino group. 2 Further displacement experiments between [3H]-DHEC and four pairs of drugs differently substituted on the amino group (isoprenaline vs Cc-25, orciprenaline vs fenoterol, AH 3474 vs labetalol, pindolol vs hydroxybenzylpindolol) provided evidence that N-alkyl substitution decreased the affinity for alpha-sites (20 micromolar less than KD less than 200 micromolar), whereas an N-aralkyl one increased the affinity (0.17 micromloar less than KD less than 4.6 micromolar). 3 It is concluded that a substitution on the amino group by a bulky, hydrophobic moiety enhances the affinity of drugs for the alpha-adrenoceptors.

Alkylation↗
PubMed1978

The adenylate cyclase system of planaria Polycelis tenuis: activation by serotonin and guanine nucleotides.

The particulate fraction prepared after homogenization of planaria Polycelis tenuis in a buffer containing 3 mM EDTA and 15 mM 2-mercaptoethanol possesses an adenylate cyclase activity which was enhanced two-fold by serotonin and 20-fold by the nucleotide analog guanosine 5'-(beta-gamma-imino)triphosphate, Gpp(NH)p; when present together, the two activators exhibited a marked synergistic effect. The effect of serotonin was dose dependent, with a KA of 2 micrometer and a Hill coefficient of 0.4. In the presence of 10 micrometer Gpp(NH)p, these values became 45 nM and 1.5, respectively. The effect of serotonin was due to an increase in the maximal velocity of the enzyme and was specifically inhibited by methiotepin. The effect of methiotepin was half-maximal at 0.2 micrometer in the absence of Gpp(NH)p and at 5.0 micrometer in its presence. Planaria thus appear to be the lowest organisms in which guanine nucleotides are active upon adenylate cyclase. As serotonin is normally present in planaria, it is postulated that a serotonin-dependent regulation of adenylate cyclase activity plays a physiological role in this species.

Adenylyl Cyclases↗
PubMed1978

Biochemical evidence for the dual action of labetalol on alpha- and beta-adrenoceptors.

1 Labetalol (AH 5158A) inhibited the adrenaline-stimulated adenylate cyclase activity of rat liver and heart. This drug had no effect on basal or guanosine triphosphate (GTP)-activated adenylate cyclase activities. 2 Labetalol displaced the binding of the specific ligands [3H]-dihydroergocryptine and (-)-[3H]-dihydroalprenolol from their respective alpha and beta-adrenoceptors in rat heart and liver. The affinity of labetalol was 10 fold higher for the beta- than for the alpha-adrenoceptor. It appeared to be 10 to 100 times less potent than phentolamine in blocking alpha-adrenoceptors and 5 to 10 times less potent than propranolol in blocking beta-receptors. 3 It is concluded that labetalol exerts its dual alpha- and beta-antagonism by acting directly on the plasma membranes, where it binds competitively to alpha- and beta-adrenoceptors.

Adenylyl Cyclases↗