Structural requirements for the interaction of 1 alpha, 25-(OH) 2- vitiamin D3 with its chick interestinal receptor system

Abstract

The mechanism by which 1 alpha,25-dihydroxycholecalciferol (1alpha,25-(OH)2D3), the biologically active metabolite of cholecalciferol (vitamin D3), stimulates intestinal calcium absorption has been shown to involve an interaction of the steroid with a specific cytosol-chromatin receptor system in this target organ. Thus, 1alpha,12(OH)2D3 binds to a specific cytoplasmic receptor protein and then, following a temperature-dependent step, becomes associated with a finite number of chromatin acceptor sites prior to the initiation of the physiological response. In this respect, 1alpha,25(OH) 2D3 is similar to a number of other steroid hormones. In this investigation, studies were performed to help define the essential structural features required for interaction of 1alpha,25-(OH)2D3 with its intestinal receptor system, and presumably, for biological activity. To this end, competition studies utilizing a series of closely related structural analogs of cholecalciferol were carried out by means of a competitive binding assay highly specific for 1alpha,25(OH)2D3. The competitive binding assay employed in this study is dependent upon the ability to duplicate, in vitro, the conditions which permit the saturable binding of 1alpha,25-(OH)2[3H]D3 to chick intestinal chromatin, in vivo. Optimal conditions for this assay were achieved by the incubation of a reconstituted intestinal receptor system consisting of separately isolated cytosol and Triton X-100 chromatin fractions at 25 degrees for 45 min with 2.0 X 10-8 M 1alpha,25-(OH)2[3H]D3. Maximal binding of about 21 to 24 pmol of radioactive steroid bound per chick intestinal chromatin occurred under these conditions. The ability of the various analogs to compete with the radioactive hormone was assessed by virtue of a decrease in the amount of radioactive steroid bound to the chromatin in the presence of increasing concentrations of nonradioactive analog.