title: Glutathione increases the binding affinity of a bovine B₁₂ trafficking chaperone bCblC for vitamin B₁₂.
abstract: Intracellular B(12) metabolism involves a B(12) trafficking chaperone CblC that is well conserved in mammals including human. The protein CblC is known to bind cyanocobalamin (CNCbl, vitamin B(12)) inducing the base-off transition and convert it into an intermediate that can be used in enzyme cofactor synthesis. The binding affinity of human CblC for CNCbl was determined to be K(d)=≈6-16 μM, which is relatively low considering sub-micromolar B(12) concentrations (0.03-0.7 μM) in normal cells. In the current study, we discovered that the base-off transition of CNCbl upon binding to bCblC, a bovine homolog of human CblC, is facilitated in the presence of reduced form of glutathione (GSH). In addition, GSH dramatically increases the binding affinity for CNCbl lowering the K(d) from 27.1 ± 0.2-0.24 ± 0.09 μM. The effect of GSH is due to conformational change of bCblC upon binding with GSH, which was indicated by limited proteolysis and urea-induced equilibrium denaturation of the protein. The results of this study suggest that GSH positively modulates bCblC by increasing the binding affinity for CNCbl, which would enhance functional efficiency of the protein.
authors.affiliation: School of Biotechnology, Yeungnam University, 214-1 Dae-dong, Gyeongsan-si, Gyeongsangbuk-do 712-749, Republic of Korea.
authors.forename: Jinju
authors.initials: J
authors.lastname: Jeong
authors.forename: Jihoe
authors.initials: J
authors.lastname: Kim
mesh_terms: D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle; D005978:Glutathione / Q000378:metabolism*; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D055550:Protein Stability; D014805:Vitamin B 12 / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D018832:Molecular Chaperones; D005978:Glutathione; D014805:Vitamin B 12