Glycyrrhetinic acid bound to 11β-hydroxysteriod dehydrogenase in rat liver microsomes

Abstract

A binding protein which exibits high affinity to [ 3H]glycyrrhetinic-acid in the rat liver microsomal fraction was solubilized with 0.2% Triton DF-18 and then purified to homogeneity. The equilibrium dissociation constant of the [ 3H]glycyrrhetinic-acid binding reaction and the maximal concentration for the binding of the purified protein, as determined by Scatchard plot analysis, were 27.6 nM and 7.79 nmol/mg protein, respectively. The molecular mass of the subunit (34 kDa) and 30 amino acids of N-terminal sequence of the purified protein were entirely the same as those of the reported 11β-hydroxysteriod dehydrogenase (11β-HSD). In each purification step, the recovery and purification (fold) of the glycyrrhetinic-acid binding activity corresponded to the values of 11β-HSD activity. These results show that the purified [ 3H]glycyrrhetinic-acid binding protein is 11β-HSD. From the molecular mass of 11β-HSD (135 kDa) and the maximal concentration of the binding site, it was calculated that one glycyrrhetinic acid molecule binds to one 11β-HSD molecule. The inhibitory effects of various glycyrrhetinic-acid derivatives on [ 3H]glycyrrhetinic acid binding and 11β-HSD activity indicate that the C 30-carboxyl and C 11-carbonyl groups of glycyrrhetinic acid are the principal structures for the 11β-HSD inhibition.