The purification and characterisation of the human-serum binding protein for the 25-hydroxycholecalciferol (transcalciferin). Identity with group-specific component.
The binding protein for 25-hydroxyvitamin D3 has been isolated from human serum by monitoring the recovery of 3H-labeled 25-hydroxyvitamin D3. After a 500-fold purification a pure protein was obtained as judged from the constant specific activity (ratio of absorbance versus radioactivity) on agarose and DEAE-Sephadex chromatography and on the presence of a single band on both cellulose acetate and polyacrylamide gel electrophoresis. The molecular weight of the purified protein was measured by gel filtration on agarose (56000), Sephadex G-75 (58000) and dodecylsulfate-polyacrylamide gel electrophoresis (56000). On sucrose gradient ultracentrifugation a sedimentation coefficient of 4.1 S was found. The isoelectric point was 4.89 S on isoelectric focusing. The stability of the protein at 60 degrees C was enhanced by the presence of excess 25-hydroxyvitamin D3. On tandem crossed immunoelectrophoresis the purified binding protein was found to be identical to the one present in whole serum. The activity of the isolated protein was demonstrated by a Ka at 4 degrees C of 1.2 X 10(10) l-mol-1. A binding capacity of 0.8 binding site/molecule was measured on a Sephadex G-25 column. During immunological studies with this protein it became evident that the binding protein is identical with another serum protein known as group-specific component (Gc). In analogy to other serum binding proteins we propose to call this group-specific component/25-hydroxyvitamin-D3-binding protein transcalciferin.
D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D001798:Blood Proteins / Q000302:isolation & purification / Q000378:metabolism; D004355:Drug Stability; D006358:Hot Temperature; D006801:Humans; D006887:Hydroxycholecalciferols / Q000097:blood*; D007123:Immunoelectrophoresis, Two-Dimensional; D007525:Isoelectric Focusing; D007700:Kinetics; D008970:Molecular Weight; D011485:Protein Binding
D000596:Amino Acids; D001798:Blood Proteins; D006887:Hydroxycholecalciferols