Adenylate cyclase system of human skeletal muscle (original) (raw)
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Bba Gen Subjects, 1978
Human skeletal muscle plasmalemmal adenylate cyclase activation by catecholamines and guanyl nucleotides was investigated. The enzyme was selectively stimulated by different fl-adrenergic catecholamines. Their order of potency was isoproterenol > epinephrine > norepinephrine. Phenylephrine, an a-adrenergic agonist, showed no effect. Stimulation of enzyme by fl-agonists was stereospecific as (--)-isomers of isoproterenol and epinephrine were at least two orders of magnitude more potent than their corresponding (+)-isomers. The fl-adrenergic blocking agents, propranolol and alprenolol, completely abolished the stimulation of adenylate cyclase by (--)-isoproterenol. Inhibition by flblockers was also stereospecific, (--)-isomers being more effective inhibitors than their corresponding (+)-isomers. This selectivity and stereospecificity suggests that the human plasmalemmal adenylate cyclase is closely associated with the fl-adrenergic receptors.
Archives of Biochemistry and Biophysics, 1978
Subcellular fractions of rat skeletal muscle enriched in plasma membrane (sarcolemma) were used to characterize properties of the fl-adrenergie receptor and adenylate cyelase. The equilibrium binding of (-)-[:~H]dihydroalprenolol to the sarcolemmal membrane was characterized by a high affinity, K = 3.6 • 10 ~ liters/tool and 1.2 • 10 ~ mol/g of membrane protein of homogeneous noninteracting sites. The binding was reversible, saturable, and stereospecific. Displacement of labeled dihydroalprenolol by adrenergic ligands revealed an order of potency corresponding to the fie-type response: (-)-isoproterenol > (-)-epinephrine > norepinephrine. The (+)-stereoisomers and the a-adrenergic antagonist phentolamine showed little ability to compete with (-)-[:~H]dihydroalprenolol for occupancy of the binding sites. In the membrane fractions, a substantial basal activity of adenylate cyclase activity was demonstrated (35-50 pmol of cyclic AMP/mg/5 min). This activity was stimulated 40-to 50-fold by sodium fluoride, 7-to 8-fold by isoproterenol, and 5-to 6-fold by guanylyl 5'-imidodiphosphate [Gpp(NH)p]. Maximum activation depended upon optimal Mg ~+ ion concentration (I0 mM). In the presence of 0.1 mM Gpp(NH)p, the isoproterenol activation of adenylate cyclase was increased twofold. In addition, the concentration of hormone required for half-maximal stimulation of enzyme activity was shifted from 2.2 • 10-~ M in the absence of Gpp(NH)p to 2.5 • I0 ~ M in the presence of Gpp(NH)p. Gpp(NH)p also stimulated epinephrine and norepinephrine activation without altering the fl2-type potency series for the enzyme. Gpp(NH)p had no effect on either the number or the affinity of (-)-[:JH]dihydroalprenolol binding sites, but it did increase the dissociation constant for isoproterenol by approximately fivefold. Deceased. This paper represents the last investigations carried out by Dr. Stuart P. Grefrath. His warmth, friendship, and critical acumen will be missed.
The Journal of biological chemistry, 1984
A hormone responsive adenylate cyclase has been reconstituted in phosphatidylcholine vesicles from its isolated protein components. The proteins used were the affinity chromatography purified (500-2000-fold) or pure Mr = 64,000 beta-adrenergic receptors (beta AR) isolated from hamster and guinea pig lung membranes, the pure heterotrimeric (Mr: alpha = 42,000; beta = 35,000; gamma approximately equal to 5,000) guanine nucleotide regulatory protein (Ns) isolated from human erythrocyte membranes; and the catalytic unit of the adenylate cyclase (C) solubilized from bovine brain caudate nucleus and resolved from beta AR and Ns by gel filtration. Adenylate cyclase activity in vesicles containing C alone was stimulated by forskolin but not by guanine nucleotides or by the beta-adrenergic agonist isoproterenol. Reconstitution of Ns and C interactions in the lipid vesicles resulted in guanine nucleotide but not beta-adrenergic agonist sensitivity. When beta AR was inserted together with Ns a...
Desensitization of β-adrenergic receptor-coupled adenylate cyclase activity
Biochemical Pharmacology, 1984
The effects on the Padrenergic receptors of intact L6 muscle cells of exposure to agonists were investigated. Treatment of cells with isoproterenol decreased GTP-, isoproterenol-, and zinterolstimulated adenylate cyclase activities, whereas exposure of cells to zinterol decreased only isoproterenol-
Proceedings of the National Academy of Sciences of the United States of America, 1980
Binding of the beta-adrenergic agonist [3H]hydroxybenzylisoproterenol to the beta-adrenergic receptor of rat reticulocyte membranes results in the coupling of the receptor to the guanine nucleotide regulatory protein associated with the adenylate cyclase system. This regulatory component, referred to as the G-protein, was identified by its specific [32P]-ADP-ribosylation catalyzed by cholera toxin. Incubation of [32P]ADP-ribosylated rat reticulocyte membranes with the [3H]hydroxybenzylisoproterenol agonist prior to membrane solubilization and gel exclusion chromatography resulted in the coelution of the 42,000 Mr [32P]ADP-ribosylated G-proteins with the agonist-occupied beta-adrenergic receptors. The receptor-G-protein complex was not formed when receptors were unoccupied or occupied with antagonists at the time of solubilization. Incubation of rat reticulocyte membranes with [3H]hydroxybenzylisoproterenol in the presence of guanine nucleotides reversed or prevented the formation of...
Proceedings of the National Academy of Sciences, 1984
Only part of the ,B-adrenergic receptors can undergo functional coupling to the adenylate cyclase regulatory unit. This receptor subpopulation shows an increased affinity for agonists in the presence of Mg2' and undergoes rapid "inactivation" (locking-in of the agonist) by the alkylating reagent N-ethylmaleimide in the presence of agonists. Several experimental conditions, known to modify the total receptor concentration without alteration of the other components of the acenylate cyclase system, do not affect the percentage of receptors that can undergo functional coupling: (i) homologous regulation of B13 receptors in rat brain by noradrenaline (through antidepressive drug or reserpine injections); (it) upand down-regulation of the 182 receptors in Friend erythroleukemia cells by, respectively, sodium butyrate and cinnarizine treatment; and (iiW) dithiothreitol-mediated inactivation of receptors in turkey erythrocytes, Friend erythroleukemia cells, and rat brain. Our findings argue against a stoichiometric limitation in the number of regulatory components, genetically different receptor subpopulations, bound guanine nucleotides, or reduced accessibility of part of the receptors to the agonists