A novel effect of molybdate on the binding of [3H]aldosterone to gel-filtered type I receptors in brain cytosol.
Recently we reported that adding molybdate to crude steroid-free cytosol at 0 degree C results in a dose-dependent reduction in the binding of [3H]aldosterone ([3H]ALDO) to Type I adrenocorticosteroid receptors. In the experiments outlined here, we found that addition of molybdate to steroid-free brain cytosol produces a 30-50% increase in the subsequently measured maximal specific binding capacity (BMAX) of [3H]ALDO-Type I receptors if the cytosol is subjected to Sephadex G-25 gel filtration prior to steroid addition. These manipulations were found to have no effect on the equilibrium dissociation constant (Kd) of the receptors. In contrast, when gel filtration of steroid-free cytosol was performed in the absence of molybdate, there was a 2-fold increase in the Kd and over a 50% reduction in the subsequently measured BMAX of [3H]ALDO-Type I receptors. When molybdate was added to this steroid-free cytosol immediately following gel filtration, there was no reduction (or increase) in Type I receptor [3H]ALDO binding capacity compared with non-gel-filtered controls. The addition of as little as 2 mM molybdate to crude steroid-free cytosol was found to stabilize the binding capacity of Type I receptors during exposure to 22 degrees C incubations; however, when gel-filtered steroid-free cytosol was exposed to these conditions at least 10 mM molybdate was required to stabilize Type I receptor binding capacity. Adding the sulfhydryl reducing reagent, dithiothreitol, to the various steroid-free cytosols had little effect on [3H]ALDO-Type I receptor binding.(ABSTRACT TRUNCATED AT 250 WORDS)
Emadian
S M
SM
Department of Neuroscience, University of Florida College of Medicine, Gainesville 32610.
Luttge
W G
WG
D000450:Aldosterone / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000187:drug effects / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism*; D005260:Female; D007700:Kinetics; D051379:Mice; D008982:Molybdenum / Q000494:pharmacology*; D011965:Receptors, Glucocorticoid / Q000302:isolation & purification / Q000378:metabolism*; D018161:Receptors, Mineralocorticoid
D011965:Receptors, Glucocorticoid; D018161:Receptors, Mineralocorticoid; C044659:molybdate; D000450:Aldosterone; D008982:Molybdenum