INTESTINAL CYTOSOL BINDERS OF 1,25-DIHYDROXYVITAMIN D-3 AND 25-HYDROXYVITAMIN D-3

The binding of 25-hydroxy-[26,27-3H]vitamin D3 and 1,25-dihydroxy-[26,27-3H]vitamin D3 to the cytosol of intestinal mucosa of chicks and rats was studied by sucrose gradient analysis. The cytosol from chick mucosa showed variable binding of 1,25-dihydroxyvitamin D3 to a 3.0S macromolecule which has high affinity and low capacity for this metabolite. However, when the mucosa was washed extensively before homogenization, a 3.7S macromolecule was consistently observed which showed considerable specificity and affinity for 1,25-dihydroxyvitamin D3. Although 3.7S binders for 1,25-dihydroxyvitamin D3 could also be located in other organs, competition experiments with excess nonradioactive 1,25-dihydroxyvitamin D3 suggested that they were not identical to the 3.7S macromolecule from intestinal mucosal cytosol. As the 3.7S macromolecule was allowed to stand at 4.degree. C with bound 1,25-dihydroxy-[3H]vitamin D3, the 1,25-dihydroxy-[3H]vitamin D3 became increasingly resistant to displacement by nonradioactive 1,25-dihydroxyvitamin D3. The 1,25-dihydroxy-[3H]vitamin D3 remained unchanged and easily extractable with lipid solvents through this change, making unlikely the establishment of a covalent bond. Unlike the chick, mucosa from rats yielded cytosol in which no specific binding of 1,25-dihydroxy-[3H]vitamin D3 was detected. Instead, a 5-6S macromolecule which binds both 1,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 was found. This protein which was also found in chick mucosa shows preferential binding for 25-hydroxyvitamin D3. It could be removed by washing the mucosa with buffer prior to homogenization which suggests that it may not be a cytosolic protein. Although the 3.7S protein from chick mucosa has properties consistent with its possible role as a receptor, the 5-6S macromolecule does not appear to have receptor-like properties.