Isolation of Pellicular Cobalamin-binding Proteins of the Cobalamin Uptake System of Euglena gracilis

Abstract

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.

Direct exchange of vitamin B12 is demonstrated by modelling the growth dynamics of algal–bacterial cocultures

“…However, in evolutionary terms, the benefit of rendering an essential nutrient inaccessible seems questionable, and while B 12 -binding proteins have been characterised from different algal species these are much more likely to be involved in the uptake of the vitamin. Work with Euglena gracilis (Watanabe et al, 1988) and Thalassiosira pseudonana (Sahni et al, 2001) found high-affinity vitamin B 12 -binding factors in the growth medium of these two organisms. Similarly, a candidate B 12 -binding protein named CBA1 (for cobalamin acquisition) has recently been identified based on a transcriptomic and proteomic analysis of T. pseudonana and a second diatom Phaeodactylum tricornutum (Bertrand et al, 2012).…”

Purification, Some Properties and Possible Physiological Role of an Extracellular Cobalamin Binding Protein from Euglena gracilis

“…The inhibition of CN-Cbl binding activity by the Cbl analogues OH-Cbl, Me-Cbl and Ado-Cbl (45%, 35% and 46% inhibition, respectively) but not by 2-AMP-Cbl, suggests that the a-coordination moiety of the Cbl molecule is more important for formation of the protein-Cbl complex than the P-coordination moiety. These properties were similar to those of the cytosolic and pellicular Cbl binding proteins, but the extracellular Cbl binding protein was stable up to 30°C and therefore showed a lower thermal stability than these Cbl binding proteins (Watanabe et al, 1987(Watanabe et al, , 1988. The extracellular Cbl binding protein showed a lower affinity for CN-Cbl (K, = 1.1 nM) than did the pellicular binding proteins (Watanabe et al, 1988), but had a similar affinity to the cytosolic binding proteins (K, = 1-2nM) (Watanabe et al, 1987).…”

“…The extracellular Cbl binding protein showed a lower affinity for CN-Cbl (K, = 1.1 nM) than did the pellicular binding proteins (Watanabe et al, 1988), but had a similar affinity to the cytosolic binding proteins (K, = 1-2nM) (Watanabe et al, 1987). The M , of the polypeptide of the extracellular Cbl binding protein was estimated to be 56000 by SDS-PAGE, while those of the cytosolic and pellicular proteins were 66000 and 38 000, respectively (Watanabe et al, 1987(Watanabe et al, , 1988. The native extracellular Cbl binding protein was eluted in the void fraction on Sephacryl S-300 gel filtration, indicating that the protein has an M , of more than 1500000, unless nonspecific aggregation occurs.…”

Microalgal assimilation of vitamin B12toward the production of a superfood

“…Since, vitamin B 12 ‐binding proteins have been characterized in several microalgal species, the probability of having these proteins related with the uptake of the vitamin is favored, rather than the inaccessibility theory (Grant et al, ). Work with Euglena gracilis found high‐affinity vitamin B 12 ‐binding factors in the growth medium of this microorganism (Watanabe, Ito, Tabuchi, Nakano, & Kitaoka, ) and a candidate B 12 ‐binding protein named CBA1 (for cobalamin acquisition) has recently been identified based on a transcriptomic and proteomic analysis of Thalassiosira pseudonana and Phaeodactylum tricornutum (Bertrand et al, ). The protein was up to 160 times more abundant in vitamin B 12 ‐deficient medium, suggesting a role in acquiring this compound.…”

“…Since, vitamin B 12 -binding proteins have been characterized in several microalgal species, the probability of having these proteins related with the uptake of the vitamin is favored, rather than the inaccessibility theory (Grant et al, 2014). Work with Euglena gracilis found high-affinity vitamin B 12 -binding factors in the growth medium of this microorganism (Watanabe, Ito, Tabuchi, Nakano, & Kitaoka, 1988) and a candidate B 12 -binding protein named CBA1…”