Inactivation of [125I]-angiotensin II binding sites in rat renal cortex epithelial membranes by dithiothreitol.
Preincubation of renal epithelial membranes with DTT produced a dose-dependent inhibition of specific [125I]-angiotensin binding, with an IC50 of 1 mM and total loss of binding at 5-10 mM DTT. Inactivation of specific [125I]-angiotensin II binding by DTT was temperature sensitive; the t1/2 at 22 degrees was 6 min compared with 30 min at 4 degrees. A rapid inactivation rate was dependent on the presence of NaCl. In the presence of 120 mM NaCl the t1/2 for inactivation by DTT was 6 min and 33 min in the absence of NaCl. Protection against DTT inactivation was obtained by preincubating membranes with unlabelled angiotensin II greater than angiotensin I greater than renin substrate while the dipeptide, Ileu-His was only effective in protecting the binding site at high concentrations (10 mM). Preincubations with DTT (1 mM) caused a 43% decrease in Bmax from 217.0 +/- 39.5 to 123.7 +/- 30.9 fmol bound/mg protein while the KD was not significantly affected.
D000804:Angiotensin II / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D004229:Dithiothreitol / Q000494:pharmacology*; D004848:Epithelium / Q000378:metabolism; D006150:Guanine Nucleotides / Q000494:pharmacology; D066298:In Vitro Techniques; D007457:Iodine Radioisotopes; D007672:Kidney Cortex / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D012965:Sodium Chloride / Q000494:pharmacology
D006150:Guanine Nucleotides; D007457:Iodine Radioisotopes; D000804:Angiotensin II; D012965:Sodium Chloride; D004229:Dithiothreitol