Studies on the mode of action of calciferol: Comparison of the biochemical properties of an antiserum and the chick intestinal receptor both specific for 1,25-dihydroxyvitamin D3

The structural requirements for the interaction of 1,25-dihydroxyvitamin D₃ 1,25(OH)₂D₃] with an anti-1,25(OH)₂D₃ antiserum and with the natural cytosolic receptor for 1,25(OH)₂D₃ isolated from chick intestine have been evaluated quantitatively. The antiserum was raised in a rabbit against a 1,25(OH)₂D₃-hemisuccinate derivative which was linked to bovine serum albumin at the C-3 position of the steroid. For these cross-reaction studies structural analogs of 1,25(OH)₂D₃ were used in competitive protein binding assays; their ability to interact with the binding proteins was expressed as relative competitive index (RCI) values where the RCI of 1,25(OH)₂D₃ is defined to be 100. The results indicate that the 25-hydroxyl group is the most important hydroxyl for the interaction of 1,25(OH)₂D₃ with this antiserum. The absence of this hydroxyl group decreases the RCI value to 0.2. Lack of the hydroxyl at carbon-3 or carbon-1 decreases the RCI value to 33 or 25, respectively, indicating that the specificity of this antiserum for the A ring is much lower than for the side chain. The high specificity for the side chain is underlined by the fact that insertion of an additional hydroxyl group at C-24 or C-26 of 1,25(OH)₂D₃ decreases the binding affinity to the antiserum markedly. The chick intestinal mucosal receptor shows a comparable high specificity for the side chain of 1,25(OH)₂D₃, but an even higher specificity for the A ring in comparison to the antiserum. With the intestinal receptor, the 3-hydroxyl is only 1/ 10th as important as the 1-hydroxyl group and the 25-hydroxyl group for the binding process. Scatchard analysis showed a K_D value of 1.7 × 10^?10m for the antiserum and 2.3 × 10^?10m for the chick intestinal mucosal receptor for the equilibrium binding of 1,25(OH)₂D₃ at 2 °C. The association rate constant at 2 °C was determined to be 5.8 × 10⁷ M^?1 min^?1 for the antiserum and 0.55 × 10⁷ M^?1 min^?1 for the receptor, indicating a 10-fold more rapid association of 1,25(OH)₂D₃ to the antiserum in comparison to the receptor. Furthermore, the dissociation process was found to be slower for the chick intestinal receptor (dissociation rate constant 3.6 × 10^?5 min^?1 versus 21.0 × 10^?5 min^?1).