STUDIES ON THE MODE OF ACTION OF CALCIFEROL .15. HYDROXYLAPATITE BATCH ASSAY FOR THE QUANTITATION OF 1-ALPHA,25-DIHYDROXYVITAMIN D3-RECEPTOR COMPLEXES

A versatile hydroxylapatite batch assay for 1α,25-dihydroxyvitamin D3-receptor complex from chick intestinal mucosa has been developed. The assay has been characterized with respect to time and temperature of incubations, protein concentration, amount of hydroxylapatite required to bind receptor-steroid complexes, pH, and effects of KCl and phosphate. Triton X-100 (0,5%, v v) was found to be essential for the removal of nonspecifically bound ligand. The hydroxylapatite was shown to bind the 1α,25-dihydroxy-vitamin D3 receptor as demonstrated by the specificity and high affinity for 1α,25-dihydroxy-vitamin D3 and the sedimentation properties of the phosphate-extracted hydroxylapatite-bound complex on sucrose density gradients. Binding appears to be nearly quantitative. The efficient separation of bound from free ligand utilizing this assay makes it possible to examine a number of aspects of the binding of this steroid hormone to its cytoplasmic receptor that has not previously been possible. © 1979.