Size and charge of the functional 1,25-dihydroxyvitamin D receptor in porcine intestine.
The 1,25-dihydroxyvitamin D3 receptor from porcine intestine has been characterized by immunoprecipitation and immunoblotting experiments. Immunoprecipitation studies demonstrate that monoclonal antibodies to the receptor that recognize two different epitopes on the receptor protein quantitatively precipitate the 1,25-dihydroxyvitamin D3 binding activity from nuclear and whole cell extracts of intestinal mucosa. These antibodies were then used to study the porcine receptor by both one- and two-dimensional immunoblotting techniques. One-dimensional immunoblots of nuclear extract and whole cell extract show the receptor to be a single polypeptide of Mr approximately 55,000. The migration of the receptor on one-dimensional gels was unchanged by preassociation in vitro with 1,25-dihydroxyvitamin D3. In two-dimensional immunoblots two receptor proteins were detected, both of molecular weight 55,000. In addition, a form of the receptor did not enter the isoelectric focusing gel from either the acidic or basic direction. It did, however, migrate in the Mr 55,000 region in the molecular separation. The major receptor protein has a pI of 6.1; the minor protein has a pI of 5.9. These studies represent the first determinations of the size and charge of a mammalian 1,25-dihydroxyvitamin D3 receptor in crude tissue preparations, and they indicate that the pig receptor is a Mr 55,000 protein that exhibits charge heterogeneity in vivo. The nature of this apparent charge heterogeneity and its significance in receptor function are under investigation.
Pierce
E A
EA
Department of Biochemistry, University of Wisconsin-Madison 53706.
Dame
M C
MC
DeLuca
H F
HF
D000818:Animals; D000911:Antibodies, Monoclonal; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007163:Immunosorbent Techniques; D007422:Intestines / Q000032:analysis*; D008970:Molecular Weight; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid; D013552:Swine
D000911:Antibodies, Monoclonal; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid