Purification and some properties of cytosolic cobalamin-binding protein in Euglena gracilis

Abstract

In Euglena gracilis SM-ZK, a bleached mutant of E. gracilis z, the cobalamin- (Cbl-)binding activity was distributed in cytosol (49.2%), mitochondria (20.3%) and microsomal fraction (20.4%). The cytosolic Cbl-binding activity gave two major peaks in isoelectric focusing. The Cbl-binding protein with pI 3.2 was purified 6500-fold in a yield of 19.9%, and that with pI 4.7 5800-fold in a yield of 11.9%. The monomeric Mr values of both Cbl-binding proteins were about 66,000. The Cbl-binding activity of both proteins showed a very low pH-dependency, and thiol groups and metal ions were not concerned with the Cbl-binding activities. The Ks values of the Cbl-binding proteins with pI 3.8 and 4.7 for CN-Cbl were 1.0 and 2.0 nM respectively. The Cbl-binding protein with pI 3.8 was shown to be immunologically identical with the protein with pI 4.7 by double-immunodiffusion experiments against antibody to the protein with pI 3.8. The two cytosolic Cbl-binding proteins did not show the activities of Cbl-dependent enzymes in E. gracilis, N5-methyltetrahydrofolate:homocysteine methyltransferase, methylmalonyl-CoA mutase and ribonucleotide reductase, suggesting that the two cytosolic Cbl-binding proteins play a physiological role as intracellular Cbl carriers.

Identification of the gene responsible for the cblA complementation group of vitamin B 12 -responsive methylmalonic acidemia based on analysis of prokaryotic gene arrangements

“…These findings are compatible with a block in mitochondrial transport, but no such transporter was identified (20,21). Instead, Watanabe and coworkers (22,23) identified an NADPH-dependent aquacobalamin reductase in rat liver and Euglena mitochondria, which was deficient in fibroblasts from one cblA cell line (14). They took this observation as evidence that the MMAA gene might code for a reductase.…”

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.…”

“…E. gracilis extracellular Cbl binding protein on the nitrocellulose sheet was detected immunologically with antibody against an E . gracilis cytosolic Cbl binding protein (Watanabe et al, 1987) and horseradish-peroxidase-anti-rabbit IgG goat serum conjugate as described by Towbin et al (1 979).…”

Biochemistry and Physiology of Vitamins in Euglena

“…gracilis Z contains numerous nonenzymatic B12-binding proteins, which are distributed in the cytosolic, mitochondrial, chloroplastic, and microsomal fractions (Isegawa et al 1984). Some of these B12-binding proteins have been purified and characterized (Watanabe et al 1987a(Watanabe et al , b, 1988b. Thus, Euglena has intracellular B12 transfer and/or accumulation systems (Watanabe et al 1989) which differ from the mammalian and bacterial systems (Watanabe and Nakano 1991).…”