Purification of calciferol-binding proteins from kidney: physicochemical and immunological properties.
The calciferol-binding system of rat kidney cytosol has been purified and is shown to consist of two proteins, each capable of binding either 25-hydroxy-vitamin D3 (25-OH-D3) or 1,25-dihydroxyvitamin D3  (1,25-(OH)2D3). The two proteins, designated A and B, have similar sedimentation coefficients (S20w) of 5.2 S. Component A binds 25-OH-D3 with a dissociation constant (Kd) of 10(-7) M while component B binds 1,25-(OH)2D3 with a Kd of 1.6 x 10(-8) M. The estimated molecular weights (Mr) of the two proteins are 105,000 for component A and 250,000 for component B. Amino acid analyses revealed that glutamic acid is the most abundant residue in both proteins, comprising 12% of the total number of amino acid residues. Immunodiffusion test using commercial anti-human serum group-specific protein antiserum gave a precipitin reaction when purified rat serum calciferol-binding protein was used as an antigen, but no reactions could be detected  with proteins A and B. This result significantly eliminated the possibility of the presence of the rat serum binding protein in either of the purified kidney proteins. In contrast, anti-rat serum calciferol-binding protein antiserum prepared in rabbits interacted with the rat serum and kidney proteins. This result suggests that the antigenic determinants recognized by the antiserum against the rat serum calciferol-binding protein appear to be similar to those recognized in the kidney proteins A and B. Immunoelectrophoresis of the three rat proteins demonstrated dissimilar electrophoretic mobilities with the serum protein showing the least mobility, a property consistent with its higher lysine content relative to proteins A and B.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000276:immunology / Q000302:isolation & purification*; D004100:Dihydroxycholecalciferols / Q000378:metabolism; D006887:Hydroxycholecalciferols / Q000378:metabolism*; D007122:Immunoelectrophoresis; D007668:Kidney / Q000378:metabolism*; D008970:Molecular Weight; D051381:Rats; D014461:Ultracentrifugation
D000596:Amino Acids; D002352:Carrier Proteins; D004100:Dihydroxycholecalciferols; D006887:Hydroxycholecalciferols