STABILITY STUDY ON RENAL TYPE-I MINERALOCORTICOID RECEPTOR

The purpose of this work is to review stability and activation propert ies of type I receptor, in order to explain the reasons for its extrem e in vitro instability. We demonstrate that the treatment of rat kidne y cytosol with H2O2 prevents aldosterone binding, DNA/steroid-receptor complex interactions, and prevents the receptor thermal inactivation. In contrast, exogenous sulfhydryl reducing reagents are necessary to insure maximum binding of mineralocorticoid receptor and DNA/steroid-r eceptor interaction. However, the presence of beta-mercaptoethanol in thermal induced incubations reverts the H2O2 protection. We also demon strate that contaminations with free or sequestered iron are harmful f or both, receptor binding capacity (in a reversible form) and for horm one-receptor/DNA binding properties (in a partially reversible form). We propose a sulfhydryl oxidative mechanism for type I mineralocortico id receptor inactivation in which iron contaminants might accelerate t his process by oxidative catalysis. We also demonstrate that when thio l groups are blocked by specific reagents such as N-ethyl-maleimide or dithionitrobenzoic acid, type I sites loose binding capacity, but the protein is protected from oxidation as well as inactivation.