Immunochemical studies on the putative plasmalemmal receptor for 1,25-dihydroxyvitamin D 3: Ii. chick kidney and brain

Abstract

Chick kidney and brain were analyzed for the subcellular distribution (if any) of a putative plasma membrane receptor for 1,25-dihydroxyvitamin D 3 [1,25(OH) 2D 3]. Fractionation protocols were found to be based not only on differential centrifugation conditions, but also gentleness of resuspension procedures, and sufficiently dense Percoll gradients. The postnuclear pellets were resolved on 21.85% Percoll gradients overlayed on 2.4 M sucrose cushions. For both kidney and brain, fraction 1 (bottom of tube) was found to be enriched over whole homogenate 5.4- and 1.6-fold, respectively, in acid phosphatase activity, fractions 2 through 5 were enriched four- and eightfold, respectively, in succinate dehydrogenase activity, fraction 8 contained Golgi, as judged by a small peak of α-mannosidase activity, and fraction 9 was enriched sevenfold (for each tissue) in Na +,K +-ATPase activity. Western analyses, using a characterized antibody to the putative chick intestinal plasma membrane vitamin D receptor, revealed the highest levels of antigenicity in both chick kidney and brain in plasma membrane and Golgi fractions, followed by unidentified membranes in fractions 6 and 7 of Percoll gradients. Distribution of specific binding of [ 3H]1,25(OH) 2D 3 in Percoll gradient fractions paralleled that of antigenicity. Qualitatively, kidney plasma membrane contained more antigen than brain plasma membrane after Western blot analyses; these results were mirrored by differences in specific binding of the tritiated secosteroid (65 ± 14.5 and 34 ± 11.9 fmol/mg of protein, respectively).