Electrophoretic mobility shift assay identifies vitamin D binding protein (Gc-globulin) in human, rat, and mouse sera.
Serum vitamin D binding protein (DBP, also known as Gc-globulin) is a multifunctional protein capable of binding both vitamin D metabolites and actin. DBP can be visualized when analyzed by polyacrylamide gel electrophoresis followed by staining. Confirmation of its identity had previously required immunoprecipitation with specific anti-DBP antisera or occupancy of the protein with radioactive vitamin D sterols. We present studies showing that preincubation of G-actin with mammalian sera produced a discernible DBP protein band shift on native gel electrophoresis. Addition of DNaseI, a 33-kDa intracellular protein with an avid actin-binding site, to the incubations resulted in a supershift of DBP-actin complexes to an even more cathodal region of the gels. Following incubations with human, rat, and murine sera the same actin shift occurred as did the actin plus DNaseI supershift. The migrations of each complex were correlated with purified DBP migrations under identical conditions. It was confirmed that the supershifted bands contained DBP by Western blotting and detection of DBP by binding of 25-OH[3H]D3. After intravenous G-actin injections into living mice, a serum DBP-actin complex could be detected on native gels as the uncomplexed DBP band decreased in intensity. This simple, direct-staining technique appears to be suitable for identifying DBP/Gc phenotypes in human populations as well as for semiquantitatively monitoring the plasma actin-scavenger system in vivo in animal models or in human diseases.
Tang
W X
WX
Department of Medicine, University of Pennsylvania, School of Medicine, 415 Curie Boulevard, Philadelphia, Pennsylvania, 19104-6149, USA.
Bazaraa
H M
HM
Magiera
H
H
Cooke
N E
NE
Haddad
J G
JG
D000199:Actins / Q000302:isolation & purification; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005069:Evaluation Studies as Topic; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D013194:Staining and Labeling; D014809:Vitamin D-Binding Protein / Q000032:analysis* / Q000097:blood / Q000302:isolation & purification
D000199:Actins; D014809:Vitamin D-Binding Protein