Binding of 3H-estradiol-17 beta-(beta-D-glucuronide), a cholestatic organic anion, to rat liver plasma membranes. Evidence consonant with identification of organic anion carriers.
The binding of 3H-estradiol-17 beta(beta-D-glucuronide) (3H-E2 17G), a cholestatic organic anion, was examined in rat liver plasma membranes, and two saturable, specific binding sites were identified. The binding parameters are Kd1 = 3.9 X 10(-7) M, Bmax1 = 69 pmol/mg of protein; Kd2 = 4.90 X 10(-6) M, Bmax2 = 495 pmol/mg of protein according to Scatchard analysis of equilibrium experiments. Kinetic dissociation experiments showed that 3H-E2 17G binding was reversible and revealed two components. The dissociation rate constants did not vary with the method of dilution of radioligand, i.e., by "infinite" volume, or excess unlabeled ligand, ruling out the possibility of cooperativity. The rate of association of 3H-E2 17G binding was very rapid, so that maximal binding was reached within 15 sec at 4 degrees. Na+ was not required for binding and binding was not decreased in the presence of high osmolarity buffer (125 mM sucrose), indicating that transport into vesicles was not involved. The ability of a series of compounds to inhibit the binding of 3H-E2 17G was also examined. Taurocholate, cholate, taurodehydrocholate, and testosterone glucuronide were identified as ligands selective for the high affinity site (site 1). The A- and D-ring glucuronide conjugates of estradiol and estriol, bromosulfophthalein, dibromosulfophthalein, and the glucuronide conjugates of phenolphthalein, 4-methylumbelliferone, and menthol inhibited binding of 3H-E2 17G to both sites. Morphine glucuronide, estradiol, and glucuronic acid did not inhibit binding to either site. The substrate specificities of binding to the low affinity site (site 2) are consistent with data characterizing the transport of these substrates in hepatocytes and supports the postulate that site 2 represents a non-bile acid organic anion carrier. Site 1 is postulated to represent a carrier shared by the bile acids and non-bile acid organic anions.
Takacs
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AL
University of Kentucky College of Medicine, Department of Pharmacology, Lexington 40536.
Vore
M
M
D000818:Animals; D027321:Anion Transport Proteins; D001647:Bile Acids and Salts / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane; D002779:Cholestasis / Q000139:chemically induced*; D004958:Estradiol / Q000031:analogs & derivatives* / Q000378:metabolism; D005260:Female; D008099:Liver / Q000378:metabolism*; D010042:Ouabain / Q000494:pharmacology; D051381:Rats; D013329:Structure-Activity Relationship; D013347:Subcellular Fractions / Q000378:metabolism; D013696:Temperature; D013739:Testosterone / Q000031:analogs & derivatives / Q000378:metabolism
D027321:Anion Transport Proteins; D001647:Bile Acids and Salts; D002352:Carrier Proteins; C025483:estradiol-17 beta-glucuronide; D013739:Testosterone; D004958:Estradiol; D010042:Ouabain