Euglena gracilis pellicle, separated by sucrose density gradient centrifugation, had a high cobalamin (Cbl) binding activity, Of the Cbl binding protein 65% was solubilized from E. gracilis pellicle by using 0.1 % SDS/2 M-urea and the residual 35 % by using 1 % (w/v) SDS. Both of the Cbl binding protein fractions showed a broad pH dependence for the binding of Cbl. No evidence for the involvement of SH-groups or metal ions in Cbl binding was obtained. The K, values for cyanocobalamin of the proteins solubilized with 0.1 % SDS/2 M-urea and with 1 % SDS were 0.22 and 0.19 nM, respectively. The M , of a Cbl binding polypeptide of each protein was estimated to be 38000 by SDS-PAGE. The Cbl binding protein solubilized with 0.1 % SDS/2 Murea was purified to homogeneity. Inhibition experiments on Cbl uptake in E. gracilis cells, using an antibody against the Cbl binding protein solubilized with 0-1 % SDS/2 M-urea, showed that the Cbl binding proteins solubilized with 0.1 % SDS/2 M-urea and 1 % SDS take part in the slower secondary phase and in the initial rapid phase of Cbl uptake in E. gracilis, respectively.
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