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R J Lefkowitz

Publications and source records attributed to R J Lefkowitz.

At least 577 records · Page 32Linked to original sources
PubMed1975

Guanosine triphosphate binding sites in solubilized myocardium. Relation to adenylate cyclase activity.

Binding of (3H)GTP to solubilized preparations of myocardial adenylate cyclase, partially purified by DEAE-cellulose chromatography, as been studied in an attempt to gain further insight into the mechanisms by which guanine nucleotides regulate adenylate cyclase activity. Although several peaks of (3H)GTP-binding activity were present in crude preparations of solubilized myocardium, one peak was associated with the adenylate cyclase peak. Binding of (3H)GTP to this material was rapid (equilibrium within 3 min at 37 degrees) and reversible and not associated with nucleotide hydrolysis. Scatchard analysis revealed a single class of (3H)GTP binding sites with KA = 3 x 10-6 M-1 and total binding capacity of 50 pmol per mg of protein. The GTP analog Gpp(NH)p competed for the sites with an affinity somewhat lower than GTP, although its ability to activate the adenylate cyclase was far greater. GTP and other guanine nucleotides activated the soluble cyclase only weakly, although they antagonized competitively enzyme stimulation by Gpp(NH)p. Ability of GTP and other nucleotides to compete with (3H)GTP for binding sites and to antagonize competitively adenylate cyclase activation by Gpp(NH)p were directly parallel. The potency series was GTP = GDP = dGTP greater than GMP greater than ITP greater than UTP, CTP. Dissociation constants of nucleotides for the sites determined by inhibition of (3H)GTP binding and inhibition of Gpp(NH)p activation of cyclase agreed closely. Gpp(NH)p dose-response curves for activation of adenylate cyclase and inhibition of (3H)GTP binding were superimposable.

Adenylyl Cyclases↗
PubMed1975

Myocardial guanylate cyclase: properties of the enzyme and effects of cholinergic agonists in vitro.

The characteristics of myocardial guanylate cyclase (GTP pyrophosphatelyase, EC 4.6.1.2) were studied. Specific activity of the myocardial enzyme in five vertebrate species was guinea pig greater than man greater than cat greater than dog greater than rat. In the guinea pig, guanylate cyclase activity was uniformly distributed throughout the anatomical regions of the heart. The major portion of the enzyme activity was retrieved in the supernatant fraction after centrifugation at 12 000 times g. The Km for GTP was similar in supernatant (0.12 mM) and particulate (0.21 mM) preparations, although the Ka for Mn2+ in particulate preparations (0.3-0.6 mM) was less than that observed for guanylate cyclase in the supernatant fraction (0.8-2.0 mM). ATP competitively inhibited supernatant and particulate activity. Addition of 0.005-10.0 mM Ca2+ to assay incubations did not enhance guanylate cyclase activity. Suspension of 105 000 times g supernatant guanylate cyclase preparations with membrane lipids or phosphatidylserine stimulated activity 1.4-4.3 fold, whereas similar treatment of particulate preparations caused little alteration of enzyme activity. Addition of the cholinergic agonists acetylcholine, carbachol or methacholine (10-4-10-8 M) to homogenate, supernatant, particulate and disrupted tissue slice preparations in the presence of 0.0012-1.2 mM GTP, 0.3-10.0 mM Mn2+ and 0.005-10.0 mM Ca2+ or 0.0012-1.2 mM ATP did not stimulate guanylate cyclase activity. Similarly, further stimulation of guanylate cyclase activity was not elicited when enzyme-lipid suspensions were assayed in the presence of cholinergic agents.

Acetylcholine↗
PubMed1975

Identification of cardiac beta-adrenergic receptors by (minus) [3H]alprenolol binding.

(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 4.6.1.1]. 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.

Adenylyl Cyclases↗
PubMed1975

Catecholamine-induced subsensitivity of adenylate cyclase associated with loss of beta-adrenergic receptor binding sites.

Injection of frogs with beta-adrenergic catecholamines for 1-24 hr produces marked subsensitivity of the erythrocyte membrane adenylate cyclase [ATP pyrophosphate-lyase (cyclizing); EC 4.6.1.1.] to in vitro stimulation by isoproterenol. The subsensitization is specific for catecholamine stimulation, since basal and fluoride-stimulated enzyme activity are unaffected. Maximum isoproterenol-stimulated adenylate cyclase activity declines by 75% in the isoproterenol-treated animals (P less than 0.001). The concentration of isoproterenol causing one-half maximal activation of adenylate cyclase, however, is unaltered. (-)[3H]Alprenolol, a potent competitive beta-adrenergic antagonist, was used to study directly the beta-adrenergic receptor binding sites in the erythrocyte membranes from control and subsensitized animals. A highly significant (P less than 0.005) 60% fall in the number of the beta-adrenergic receptor binding sites ("specific"(-)[3H]alprenolol binding sites) in the treated animals was found. The binding affinity of the sites was not markedly altered. These data suggest that beta-adrenergic catecholamines are able to regulate catecholamine sensitivity of tissues in vivo, by regulating the properties of the beta-adrenergic receptor binding sites.

Adenylyl Cyclases↗