The isolation and characterization of the 25-hydroxyvitamin D-binding protein from chick serum.
The binding protein for 15-hydroxyvitamin D3 (25-OH-D3) was isolated from chick serum. The molecular weight was 60,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, without the existence of sub-units. Its sedimentation coefficient was 4.3 S. The isoelectric points of the major and minor components were 6.1 and 5.9, respectively. Addition of 25-hydroxyvitamin D3 causes an anodal shift in the isoelectric point of the two forms of the binding protein. Heating at 60 degrees C slowly destroys the binding protein but the addition of vitamin D metabolites increases its thermostability. The binding protein has probably a single binding site for all vitamin D metabolites and its association constant for 25-OH-D3 at 4 degrees C and pH 7.4 is 10(9) M-1. The same affinity is found for 24,25-dihydroxyvitamin D3 but 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 have a 3- and 10-fold lower affinity, respectively. The amino acid composition is very similar to that of the human and rat binding protein. The concentration of chick 25-hydroxyvitamin D3-binding protein was higher in egg-laying hens (10 microM) than in immature hens (4 microM) or immature or adult roosters (4 microM). Chick plasma contains virtually only a 4 S form of the 25-hydroxyvitamin D3-binding protein but partially hemolyzed chick serum also contains a 6 S form. Addition of pure actin to chick plasma or serum converts all 4 S form to the 6 S form. The serum 25-hydroxyvitamin D3-binding protein is thus heterogenous since it exists in two 4 S forms (with different pI) and a 6 S form (complexed with actin). No evidence was found for the existence of a separate vitamin D3-binding protein.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002645:Chickens; D007123:Immunoelectrophoresis, Two-Dimensional; D046911:Macromolecular Substances; D008970:Molecular Weight; D014807:Vitamin D / Q000302:isolation & purification / Q000378:metabolism*; D014809:Vitamin D-Binding Protein
D000596:Amino Acids; D002352:Carrier Proteins; D046911:Macromolecular Substances; D014809:Vitamin D-Binding Protein; D014807:Vitamin D