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Biomedical subjects

L T Williams

Publications and source records attributed to L T Williams.

189 records · Page 11Linked to original sources
PubMed1976

Identification of alpha-adrenergic receptors in uterine smooth muscle membranes by [3H]dihydroergocryptine binding.

[3H]Dihydroergocryptine, a potent alpha-adrenergic antagonist, was used to label smooth muscle membrane binding sites which have the characteristics expected of alpha-adrenergic receptors. Binding of [3H]dihydroergocryptine to rabbit uterine membranes was rapid and reversible with rate constants of 1.26 X 10(7) M-1 min-1 and 0.034 min-1 for the forward and reverse reactions, respectively. [3H]Dihydroergocryptine binding was of high affinity, with an equilibrium dissociation constant (KD) of 8 to 10 nM. Binding was saturable with 0.14 to 0.17 pmol of [3H]dihydroergocryptine bound/mg of protein at maximal occupancy of the sites. No cooperative interactions among the sites were detected. The specificity of the binding sites for a large number of adrenergic agonists and antagonists was identical with the specificity of alpha-adrenergic responses to these agents. The alpha-adrenergic agonist (-)-epinephrine competed for binding with a KD of 0.23 muM. The order of potencies for several adrenergic agonists in competing for the binding sites was (-)-epinephrine greater than (-)-norepinephrine greater than (-)-phenylephrine greater than (-)-isoproterenol in agreement with their alpha-adrenergic potencies. A series of 19 phenylethylamine adrenergic agonists competed for binding in a manner paralleling their potencies as alpha-adrenergic agonists. alpha-Adrenergic antagonists such as phentolamine (KD = 15 nM) and phenoxybenzamine (KD = 18 nM) potently competed for the binding sites. In contrast, beta-adrenergic antagonists such as propranolol (KD = 27,000 nM) and practolol (KD greater than 10(6) nM) did not have high affinity for the binding sites. A series of ergot alkaloids competed for [3H]dihydroergocryptine binding in a manner which paralleled their potencies as alpha-adrenergic agents. Competition for binding sites by alpha-adrenergic agonists and antagonists was a stereospecific process. The (-)-stereoi somers of epinephrine, norepinephrine, and ergotamine were at least 20- to 50-fold more potent than the corresponding (+)-stereoisomers. Compounds devoid of significant alpha-adrenergic activity, such as pyrocatechol, 3,4-dihydroxymandelic acid, normetanephrine, and D-lysergic acid, did not effectively compete for [3H]dihydroergocryptine binding sites. These rabbit uterine binding sites for [3H]dihydroergocryptine appear to have characteristics indistinguishable from those of the physiologically active alpha-adrenergic receptors.

Animals↗
PubMed1976

Adipocyte beta-adrenergic receptors. Identification and subcellular localization by (-)-[3H]dihydroalprenolol binding.

(--)-[3H]Dihydroalprenolol, a potent beta-adrenergic antagonist, was used to identify binding sites which have the characteristics of beta-adrenergic receptors in membranes from rat adipocytes. The subcellular distribution of the (--)-[3H]Dihydroalprenolol binding sites was examined. The binding sites were predominantly in the plasma membrane fraction, consistent with the proposal that the physiologically significant beta-adrenergic receptors are localized in the adipocyte plasma membrane. Binding of (--)-[3H]dihydroalprenolol to unfractionated adipose membranes was saturable with 0.24 pmol bound/mg of protein at saturation. Half-maximal saturation occurred at 15 nM providing an estimate of the equilibrium dissociation constant, KD, for the interaction of (--)-[3H]dihydroalprenolol with its adipocyte receptor. Kinetic analysis of (--)-[3H]dihydroalprenolol binding provided a value of 2.4 X 10(7) M-1 min-1 for the forward bimolecular rate constant, k1. Dissociation of (--)-[3H]dihydroalprenolol was a first order reaction with a rate constant, k2, of 2.94 X 10(-1) min-1. The ratio k2/k1 = 12 nM provides an independent measurement of the KD for the interaction of (--)-[3H]dihydroalprenolol with its receptor which is in good agreement with the values obtained by steady state analysis (12 to 15 nM). Beta-Adrenergic agonists and antagonists competed for the binding sites in unfractionated adipocyte membranes with a typical beta1-adrenergic specificity. The order of potency of agonists was (--)-isoproterenol greater than (--)-norepinephrine congruent to (--)-epinephrine. The beta-adrenergic antagonist, (--)-propranolol, potently competed for the binding sites with a KD of 17 nM. Compounds such as dihydroxyphenylaline, dihydroxymandelic acid, normetanephrine, pyrocatechol, and phentolamine which are structurally related to beta-adrenergic agents, but are devoid of beta-adrenergic physiologicl effects in adipocytes, did not compete for the binding sites. Binding was highly stereospecific, the (+) isomers of adrenergic agonists and antagonists requiring 23- to 330-fold higher concentrations to half-maximally inhibit binding than the corresponding (--) stereoisomers. (--)-[3H]Dihydroalprenolol binding was examined highly enriched plasma membrane, mitochondrial, and microsomal (endoplasmic reticulum) fractions of adipocytes. In the presence of 12 nM (--)-[3H]dihydroalprenolol, the specific activity of binding in the plasma membrane fraction was 5-fold higher than that of the mitochondrial fraction and 8-fold higher than that of the microsomal (endoplasmic reticulum) fraction. The specificity and affinity characteristics of the plasma membrane binding sites were found to be virtually identical with those of the unfractionated adipocyte membranes. The observation that (--)-[3H]dihydroalprenolol binding sites are predominantly localized in the plasma membrane fraction suggests the potential usefulness of this ligand as a marker for adipocyte plasma membranes.

Adenylyl Cyclases↗
PubMed1976

Alpha-adrenergic receptor identification by (3H)dihydroergocryptine binding.

A radioactively labeled alpha-adrenergic antagonist, [3H]dihydroergocryptine, binds specifically to a site on rabbit uterine membranes. Binding is rapid, reaching equilibrium in less than 17 minutes at 25 degrees C. Adrenergic agonists compete for this binding site with an order of affinities identical to the pharmacological potency order of these agents as alpha-adrenergic agonists (epinephrine greater than norepinephrine greater than isoprotereonl). The (-) stereoisomers of epinephrine and norepinephrine are 30 times more potent in competing for the site than the corresponding (+) stereoisomers. alpha-Adrenergic antagonists, such as phentolamine and phenoxybenzamine, potently compete for the binding sites while the beta-adrenergic antagonist propranolol does not. Structural analogs of catecholamines that are devoid of alpha-adrenergic physiological activity do not compete for [3H]dihydroergocryptine binding sites. These data suggest that alpha-adrenergic receptors can be directly identified and studied by [3H]dihydroergocryptine binding.

Adrenergic alpha-Agonists↗
PubMed1976

Identification of beta-adrenergic receptors in human lymphocytes by (-) (3H) alprenolol binding.

Human lymphocytes are known to posessess a catecholamine-responsive adenylate cyclase which has typical beta-adrenergic specificity. To identify directly and to quantitate these beta-adenergic receptors in human lymphocytes, (-) [3H] alprenolol, a potent beta-adrenergic antagonist, was used to label binding sites in homogenates of human mononuclear leukocytes. Binding of (-) [3H] alprenolol to these sites demonstrated the kinetics, affinity, and stereospecificity expected of binding to adenylate cyclase-coupled beta-adrenergic receptors. Binding was rapid (t1/2 less than 30 s) and rapidly reversible (t1/2 less than 3 min) at 37 degrees C. Binding was a saturable process with 75 +/- 12 fmol (-) [3H] alprenolol bound/mg protein (mean +/- SEM) at saturation, corresponding to about 2,000 sites/cell. Half-maximal saturation occurred at 10 nM (-) [3H] alprenolol, which provides an estimate of the dissociation constant of (-) [3H] alprenolol for the beta-adrenergic receptor. The beta-adrenergic antagonist, (-) propranolol, potently competed for the binding sites, causing half-maximal inhibition of binding at 9 nM. beta-Adrenergic agonists also competed for the binding sites. The order of potency was (-) isoproterenol greater than (-) epinephrine greater than (-)-norepinephrine which agreed with the order of potency of these agents in stimulating leukocyte adenylate cyclase. Dissociation constants computed from binding experiments were virtually identical to those obtained from adenylate cyclase activation studies. Marked stereospecificity was observed for both binding and activation of adenylate cyclase. (-)Stereoisomers of beta-adrenergic agonists and antagonists were 9- to 300-fold more potent than their corresponding (+) stereoisomers. Structurally related compounds devoid of beta-adrenergic activity such as dopamine, dihydroxymandelic acid, normetanephrine, pyrocatechol, and phentolamine did not effectively compete for the binding sites. (-) [3H] alprenolol binding to human mononuclear leukocyte preparations was almost entirely accounted for by binding to small lymphocytes, the predominant cell type in the preparations. No binding was detectable to human erythrocytes. These results demonstrate the feasibility of using direct binding methods to study beta-adrenergic receptors in a human tissue. They also provide an experimental approach to the study of states of altered sensitivity to catecholamines at the receptor level in man.

Adenylyl Cyclases↗
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↗
PubMedDate not supplied

Identification of phosphotyrosine residues during protein sequence analysis.

Synthetic tyrosine-phosphorylated peptides were subjected to protein sequence analysis using a gas-phase sequencer and on-line phenylthiohydantoin (PTH) amino acid analysis. Our data show that phosphotyrosine is stable to the gas-phase sequencing chemistry and can be detected as its PTH-derivative during routine sequence analysis without the need of prior tyrosine radiolabeling.

Amino Acid Sequence↗