Immunochemical studies on the putative plasmalemmal receptor for 1,25-dihydroxyvitamin D3 - II. Chick kidney and brain

Chick kidney and brain were analyzed for the subcellular distribution (if a ny) of a putative plasma membrane receptor for 1,25-dihydroxyvitamin D-3 [1 ,25(OH)(2)D-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 pell ets were resolved on 21.85% Percoll gradients overlayed on 2.4 M sucrose cu shions. 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 eightf old, respectively, in succinate dehydrogenase activity, fraction 8 containe d Golgi, as judged by a small peak of alpha-mannosidase activity, and fract ion 9 was enriched sevenfold (for each tissue) in Na+,K+-ATPase activity. W estern analyses, using a characterized antibody to the putative chick intes tinal plasma membrane vitamin D receptor, revealed the highest levels of an tigenicity in both chick kidney and brain in plasma membrane and Golgi frac tions, followed by unidentified membranes in fractions 6 and 7 of Percoll g radients. Distribution of specific binding of [H-3]1,25(OH)(2)D-3 in Percol l gradient fractions paralleled that of antigenicity. Qualitatively, kidney plasma membrane contained more antigen than brain plasma membrane after We stern 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). (C) 1999 Elsevier Science Inc. All rights reserv ed.