Receptors for 1,25-dihydroxyvitamin D 3 in chick pancreas: A partial physical and functional characterization

Abstract

1,25-Dihydroxyvitamin D 3(1,25-(OH) 2D 3) receptors from the rachitic chick pancreas have been partially characterized. Analyses of these receptors by isokinetic gradient centrifugation and analytical gel filtration reveal a sedimentation coefficient (S) of 3.3–3.7, a molecular weight ( M r ) of 58,500–68.000, and a calculated Stokes molecular radius ( R s ) of 34–36 Å. Polyethylenimine-ammonium sulfate precipitation of pancreatic cytosol partially purifies aporeceptor and reduces nonspecific binding (in part, 5.8S DBP), thus providing material more amenable to kinetic analyses. Binding studies incorporating this fractionated cytosol reveal an equilibrium dissociation constant ( K d ) of approximately 0.112 nM at 2°C for the 1,25-(OH) 2D 3-receptor interaction. Competition studies further demonstrate a particular preference for 1,25-(OH) 2D 3 over 1,24(R),25-trihydroxyvitamin D 3, 24(R),25-dihydroxyvitamin D 3, and 25-hydroxy-vitamin D 3. The pancreatic receptor also binds to immobilized group-selective affinity ligands such as DNA, cibacron blue, and heparin, and can be eluted as a single macromolecular species during standard linear KCl gradients. Its interaction with these ligands supports the premise that the 1,25-(OH) 2D 3 receptors' fundamental mode of action is at the level of the cellular genome. Salt-dependent nuclear uptake and chromatin localization studies with this receptor in vitro also support this potential site of action. Significantly, a physiologic dose of 1,25-(OH) 2[ 3H]D 3 to rachitic chicks leads to the in vivo formation of a receptor-hormone complex as identified by DNA-cellulose chromatography. These observations provide further evidence that the pancreatic protein is a biologically relevant component of the chick pancreas which functions to accumulate hormone intracellularly under physiologic situations.