Purification and some properties of cytosolic cobalamin-binding protein in Euglena gracilis.
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.
D000818:Animals; D003208:Concanavalin A / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D005056:Euglena gracilis / Q000378:metabolism*; D005779:Immunodiffusion; D007525:Isoelectric Focusing; D037102:Lectins / Q000494:pharmacology; D008970:Molecular Weight; D037121:Plant Lectins; D011485:Protein Binding / Q000187:drug effects; D014155:Transcobalamins / Q000302:isolation & purification / Q000378:metabolism*; D014805:Vitamin B 12 / Q000378:metabolism*
D037102:Lectins; D037121:Plant Lectins; D014155:Transcobalamins; C036554:dolichos biflorus agglutinin; D003208:Concanavalin A; D014805:Vitamin B 12