Trypsin cleavage of chick 1,25-dihydroxyvitamin D3 receptors. Generation of discrete polypeptides which retain hormone but are unreactive to DNA and monoclonal antibody.
Intestinal cytosol receptors for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) were subjected to limited trypsin digestion, and the properties of the resulting discrete polypeptide fragments were identified and contrasted with the native 1,25(OH)2D3 receptor. Physical characterization was achieved through sedimentation analysis, gel filtration chromatography, and DEAE anion exchange high performance liquid chromatography. Intactness of functional ligand-binding domains was evaluated by assessing macromolecular retention of 1,25(OH)2D3 as well as by determining reactivity to DNA and monoclonal antibody. While two differentially trypsin-sensitive effects on the 1,25(OH)2D3 receptor were noted, both produced a major polypeptide fragment which retained 1,25(OH)2D3. Action within region I (1 microgram of trypsin/A280-A310) had no effect on net charge but significantly decreased the Stokes radius of the 1,25(OH)2D3 receptor from 3.6 nm (60,000 daltons) to 3.2 nm, concomitant with a significant reduction in receptor aggregational capacity. This large hormone-bound fragment did not elicit detectable DNA-binding activity, and only a portion displayed reactivity to monoclonal antibody. Activity within region II (25 micrograms of trypsin/A280-A310) resulted in a less charged, more globular macromolecule with a Stokes radius of 2.9 nm which was completely unreactive to monoclonal antibody. Immunoblot methodology confirmed the protease-dependent loss of immunologic reactivity of the 60,000-dalton 1,25(OH)2D3 receptor and correspondingly identified receptor fragments of 50,000 and 20,000 daltons displaying positive immunologic reactivity. These studies provide the first evidence for the distinct nature of the molecular domains for 1,25(OH)2D3 and DNA on 1,25(OH)2D3 receptors while confirming the close spatial relationship between interactive sites for DNA and monoclonal antibody.
Allegretto
E A
EA
Pike
J W
JW
D000818:Animals; D000911:Antibodies, Monoclonal; D002117:Calcitriol / Q000378:metabolism*; D002645:Chickens; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D000939:Epitopes / Q000032:analysis; D007413:Intestinal Mucosa / Q000378:metabolism*; D007700:Kinetics; D008954:Models, Biological; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D014357:Trypsin / Q000494:pharmacology*; D014808:Vitamin D Deficiency / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid; D004247:DNA; D014357:Trypsin; D002117:Calcitriol