Characterization of the particulate and soluble acceptor for transcobalamin II from human placenta and rabbit liver.
We describe in both human placenta and rabbit liver membranes specific acceptors which bind the human transcobalamin II-vitamin B-12 (cobalamin) complex with an affinity of 2.3 . 10(9) (placenta) and 6.7 . 10(9) (liver) M-1 and which bind the rabbit transcobalamin II-cobalamin complex with an affinity of 1.1 . 10(9) (placenta) and 1.9 . 10(9) (liver) M-1, respectively. The binding requires Ca2+ and is sensitive to both 1 M NaCl and acid pH. A new ligand binding assay, based on the ability of the acceptor, but not transcobalamin II, to bind to concanavalin A, is described and is used to characterize the solubilized acceptors. The solubilized acceptors bind human transcobalamin II-cobalamin with high affinity (about 2-9 . 10(9) M-1) but do not bind free cobalamin; unsaturated transcobalamin II is bound with an affinity approximately one-third of that for transcobalamin II saturated with cobalamin. On gel filtration, the human acceptor saturated with transcobalamin II-cobalamin exhibits a Stokes radius of 6.7 nm, whereas the free acceptor has a Stokes radius of 5.1 nm. The rabbit liver acceptor either unsaturated or saturated with transcobalamin II-cobalamin exhibits a Stokes radius of 5.7 nm. Both acceptors bind to lectins such as concanavalin A, wheat germ agglutinin and phytohemagglutinin, indicating their glycoprotein nature, and both acceptors can be purified approximately 30-fold by affinity chromatography on wheat germ agglutinin-Sepharose columns. The concanavalin A assay, combined with lectin-Sepharose and transcobalamin II-cobalamin-Sepharose affinity chromatography will provide for the isolation and study of pure acceptors from a variety of tissue sources.
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D001798:Blood Proteins / Q000378:metabolism*; D002850:Chromatography, Gel; D005260:Female; D006801:Humans; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism*; D010920:Placenta / Q000378:metabolism*; D011247:Pregnancy; D011817:Rabbits; D012995:Solubility; D014155:Transcobalamins / Q000378:metabolism*; D014805:Vitamin B 12 / Q000378:metabolism
D001798:Blood Proteins; D014155:Transcobalamins; D014805:Vitamin B 12