[62] Characteristics and Purification of the Intestinal Receptor for 1,25-Dihydroxyvitamin D

This chapter describes the characteristics and purification of the intestinal receptor for 1,25-dihydroxyvitamin D. The primary metabolic pathway of vitamin D involves the hepatic conversion of the parent compound to 25-hydroxyvitamin D, and then subsequently an all important hormonally and ionically regulated hydroxylation of 25-OH-D in the kidney to 1,25-dihydroxyvitamin D [1,25-(OH)₂D]. Most studies have focused on receptors for 1,25-(OH)₂D in the intestines of rachitic (D-deficient) chicks 4–6 weeks old, although a similar cytoplasmic receptor has been identified in other tissues, such as the parathyroid glands, pancreas, bone, and kidney. Preparations derived from rachitic chicks benefit from the primary advantage that titers of endogenous 1,25-(OH)₂D are virtually nonexistent. The 1,25-(OH)₂D receptor can also be extracted from the tissue of normal chicks, rats, and pigs. Receptor extractions are most successfully made from freshly excised tissue, because preparations made from frozen whole tissue, although possible, suffer from relatively lower yields. The receptor for 1,25-(OH)₂D requires a theoretically calculated 200,000-fold enhancement of specific activity over cytosol to achieve homogeneity. Thus, the purification procedures as outlined in the chapter are capable of isolating the receptor from rachitic chick intestine to approximately 50% of homogeneity (86,000-fold).