EQUILIBRIUM AND KINETIC STUDY OF 1,25-DIHYDROXYVITAMIN-D3 BINDING TO CHICKEN INTESTINAL CYTOSOL EMPLOYING HIGH SPECIFIC ACTIVITY 1,25-DIHYDROXY[H-3-26, 27]VITAMIN-D3

A reexamination of the equilibrium and the kinetics of 1,25-dihydroxy vitamin D3 binding with its receptor in chick intestinal cytosol was performed because of the recent availability in our laboratory of high specific activity 1,25-dihydroxy[3H-26,27]vitamin D3 (160 Ci/mmol). Under saturating conditions at 25 °C, Scatchard analysis revealed an equilibrium dissociation constant (Kd) of 7.1 × 10-11 m which is several fold lower than previously reported for this binding reaction. Furthermore, an estimate of 1.8 × 103 receptor sites per cell was obtained from the intercept of the line with the abscissa of the Scatchard plot. From a kinetic analysis of 1,25-dihydroxy vitamin D3 binding with chick intestinal cytosol, association and dissociation rate constants were determined. Values that were obtained at 25 °C for these processes were 9.5 × 108 m- min- and 7.1 × 10-3 min-, respectively. Although these studies, such as for other steroid hormones, were carried out using a crude native cytosol preparation, we have been able to demonstrate unequivocally through the use of high specific activity 1,25-dihydroxy[3H-26,27] vitamin D3 a truly high affinity binding site. © 1979.