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The binding of vitamin D3 analogues to the chick intestinal cytosol receptor was studied. In intestinal cytosol fraction, receptor proteins having the sedimentation constant of 2.5 S and 3.7 S to which 1 alpha,25-dihydroxyvitamin D3 binds were present, and the latter was specific for the compound. The binding of 1 alpha,24(R)-dihydroxyvitamin D3 and 1 alpha,24(S)-dihydroxyvitamin D3 to the receptor was also observed, while very weak binding was seen in the case of 24(R)25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3. The binding affinity of 1 alpha,24(R)-dihydroxyvitamin D3 to the 3.7 S receptor was 1.3 times as high as that of 1 alpha,25-dihydroxyvitamin D3, whereas those of 1 alpha,24(S)-dihydroxyvitamin D3, 1 alpha-hydroxyvitamin D3 and 25-hydroxyvitamin D3 were 10, 304 and 652 times lower than 1 alpha,25-dihydroxyvitamin D3, respectively. The dissociation constant of the receptor-1 alpha,25-dihydroxyvitamin D3 complex at 0 degrees C was 3.0 x 10(-11) M, and the dissociation constants were calculated to be 2.4 x 10(-11) M and 2.7 x 10(-10) M for the complexes with 1 alpha,24(R)-dihydroxyvitamin D3 and 1 alpha,24(S)-dihydroxyvitamin D3, respectively.

A heretofore unknown metabolite of vitamin D3 was isolated from the 1 alpha, 24, 25-trihydroxyvitamin D3 fraction of lipid extracts obtained from plasma of rats which were given intravenous or oral doses of 100 pmol/100 g of either 1 alpha-hydroxyvitamin D3 or 1 alpha, 23-dihydroxyvitamin D3. Doses of 25-250 pmoles of the new metabolite when given to a vitamin D deficient rat were completely inactive in terms of stimulating the classic vitamin D response of bone calcium mobilization. The nature of the metabolism of 1 alpha-hydroxyvitamin D3 or 1 alpha, 25-dihydroxyvitamin D3 to the metabolite is not clear at the present time, but it is probable that neither of these steroids undergo side-chain cleavage to yield the new metabolite.

Three protein fractions of the cytosol of the chick parathyroid glands, which had the sedimentation constants of 2.5 S, 3.7 S and 5.5 S, were found to bind with 1 alpha,25-dihydroxyvitamin D3. Among these proteins, the 3.7 S protein was assumed to be the specific receptor protein. The 3.7 S receptor protein was also capable of binding to 1 alpha,24-dihydroxyvitamin D3 but not 25-hydroxyvitamin D3. The binding affinity of 1 alpha,24(R)-dihydroxyvitamin D3 to the 3.7 S receptor protein was estimated to be 1.2 times greater than that of 1 alpha,25-dihydroxyvitamin D3, while 1 alpha,25-dihydroxyvitamin D3 bound to the receptor protein about 10 times stronger than 1 alpha,24(S)-dihydroxyvitamin D3. The dissociation constant for the receptor-1 alpha,25-dihydroxyvitamin D3 complex at 0 degrees C was 2.7 x 10(-11) M, the dissociation constants were calculated to be 2.2 x 10(-11) M and 2.6 x 10(-10) M for the complexes with 1 alpha,24(R)-dihydroxyvitamin D3 and 1 alpha,24(S)-dihydroxyvitamin D3.