Transcobalamin from cow milk: isolation and physico-chemical properties.
The concentration of endogenous cobalamin (Cbl) in cow milk was 3.3 nM while the Cbl-binding capacity was 0.05 nM. Both endogenous and newly added Cbl showed similar quantitative distribution between a 280 kDa protein complex (45%) and a 43 kDa Cbl-binder (55%). Long time incubation, as well as urea treatment, was accompanied by a slow release of the 43 kDa Cbl-binder from the 280 kDa fraction. No other Cbl-binding proteins appeared after these procedures. The 43 kDa binder from cow milk, depleted of the ligand by urea treatment, reacted with Cbl even in the presence of a B12-analogue cobinamide (Cbi) at the ratio Cbl:Cbi = 1:40. The stokes radius of the binder changed from 2.7 nm for the Cbl-free protein to 2.5 nm for the Cbl-saturated form and the Cbl-saturated binder was able to displace human transcobalamin (TC) from the TC-receptor. The interaction between the protein and Cbl was significantly suppressed at pH 2.0. The N-terminal sequence of the purified 43 kDa Cbl-binder revealed homology with TC from human and rabbit plasma. In conclusion we have shown that TC is the main Cbl-binding protein in cow milk. This is surprising, since previous studies on human and rat milk have shown another Cbl-binder, apo-haptocorrin, to be the dominating Cbl-binding protein.
Department of Molecular Biology, University of Arrhus, Denmark.
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Fedosov
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Petersen
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Nexø
D000595:Amino Acid Sequence; D000818:Animals; D001059:Apoproteins / Q000032:analysis; D002417:Cattle; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008892:Milk / Q000737:chemistry*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011485:Protein Binding; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D014155:Transcobalamins / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D014508:Urea / Q000494:pharmacology; D014805:Vitamin B 12 / Q000378:metabolism*
D001059:Apoproteins; D011956:Receptors, Cell Surface; D014155:Transcobalamins; C028176:transcobalamin receptor; D014508:Urea; D014805:Vitamin B 12