Biomedical subjects

R A Wiklund

Publications and source records attributed to R A Wiklund.

8 recordsLinked to original sources

PubMed1976

Binding of (3H)prostaglandin E1 to putative receptors linked to adenylate cyclase of cultured cell clones.

A method for assessing the binding of 3H-labeled prostaglandin E1 ([3H]PGE1) to cell membranes has been developed and used to study the interaction of [3H]PGE1 with membranes from cultured mammalian cells. Receptor sites were identified by correlation of the potency of a series of compounds to compete for [3H]PGE1 binding sites and to stimulate adenylate cyclase activity, by correlation of rates of binding and change in enzyme activity, and by the correspondence of [3H]PGE1-binding activity with the presence or absence of PGE1-sensitive adenylate cyclase in several clones. In clone B82, a murine L-cell, [3H]PGE1 binds with an activation energy of 14 kcal/mol to a class of sites with an affinity of 0.5 X 10(8) M-1 and a capacity of 150 fmol/mg of protein. Concentration dependence of adenylate cyclase activation by PGE1 (KD =30 nM) and kinetic analysis of [3H]PGE1 binding (k1 = 4 X 10(6) liters/mol/min, k-1 0.15/min) verify this affinity. Concentration dependence and specificity of binding and activation of adenylate cyclases in neuroblastoma clone N4TG1 and N18TG2 substantiate the method. In several clones that lack PGE1-responsive adenylate cyclase, no specific [3H]PGE1 binding is detectable.

Adenylyl Cyclases↗

PubMed1976

Binding of (125I)iodohydroxybenzylpindolol to putative beta-adrenergic receptors of rat glioma cells and other cell clones.

[125I]Iodohydroxybenzylpindolol, an extremely potent beta-adrenergic antagonist, has been purified to theoretical specific activity (2200 Ci/mmol) and used as a ligand to characterize the beta-adrenergic receptors of cultured rat glioma cells and other cultured cell clones. Appropriate receptor sites were identified by stereoselective competition for binding by a number of adrenergic agonists and antagonists, by correlation between the potency of these compounds to inhibit binding and to affect adenylate cyclase activity, and by correlation of binding with the presence or absence of response to catecholamines (stimulation of adenylate cyclase) in various cell clones. In equilibrium experiments, the dissociation constant for binding of the iodinated ligand to the beta-adrenergic receptor of a clone of rat glioma cells (C6TG1A) was 250 pM; the corresponding value for a clone of human fibroblasts (VA2) was 15 pM. For C6TG1A, KD was verified by analysis of the kinetics of binding: k1 = 10(8) 1/mol/min: k-1 = 0.017/min. This rate of dissociation of ligand from the receptor was also established by study of the rate of activation of adenylate cyclase by isoproterenol after prior equilibration with iodohydroxybenzylpindolol. For VA2 cells, where affinity was higher, the rate of reversal of binding was only 0.0035/min. C6TG1A contained approximately 4000 receptor sites/cell (75 fmol/mg of protein), and these sites appeared to be coupled to adenylate cyclase in a stoichiometric manner. A second site with equal affinity for iodohydroxybenzylpindolol (KD = 250 pM) was also identified in C6TG1A by both kinetic analysis and equilibrium binding studies. While most compounds that interacted with the beta-adrenergic receptor also influenced binding to the second site, the latter did not distinguish between stereoisomers of propranolol, and its affinity for the other compounds tested was poorer.

Adenylyl Cyclases↗

PubMed1976