Intracellular binding of radioactive hydroxocobalamin to cobalamin-dependent apoenzymes in rat liver.
We identified previously an intracellular cobalamin (Cbl) binding protein(s) in cultured human fibroblasts, distinct from known Cbl "R" binders and absent from mutant cells deficient in the synthesis of the two Cbl coenzymes. In order to further characterize this binding activity, we have investigated its homologue in rat liver. After being transported to the liver by the serum protein transcobalamin II, [57Co]Cbl was bound by at least two distinct proteins, one cytosolic, the other mitochondrial. Labeled Cbl bound to cytosolic protein faster than or prior to the mitochondrial protein. With time there was a decline in radioactivity associated with the cytosolic binder and a coordinate increase in that associated with the mitochondrial binder. Although both proteins cochromatographed on Sephadex G-150 and had apparent molecular weights of 120,000, they were separated into two discrete components by polyacrylamide gel electrophoresis and by DEAE-cellulose chromatography. The cytosolic binder cochromatographed with N5-methyltetrahydrofolate:homocysteine methyltransferase activity (5-methyltetrahydropteroyl-L-glutamate:L-homocysteine S-methyltransferase, EC 2.1.1.13); the mitochondrial one with methylmalonyl CoA mutase activity (methylmalonyl-CoA CoA-carbonylmutase, EC 5.4.99.2). These proteins were distinguished further by the chemical forms of [57Co]Cbl found with them, hydroxocobalamin and methylcobalamin with the cytosolic protein and adenosylcobalamin with the mitochondrial one. These results suggest that intracellular Cbl binding activity in rat liver can be accounted for by attachment of Cbl to the two known Cbl-dependent apoenzymes, methylmalonyl CoA mutase and methyltetrahydrofolate methyltransferase. The mechanism and significance of the observered binding protein deficiency in mutant human fibroblasts must, therefore, be re-evaluated.
D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase / Q000378:metabolism*; D000818:Animals; D001051:Apoenzymes / Q000378:metabolism*; D001059:Apoproteins / Q000378:metabolism*; D003038:Cobamides / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D007535:Isomerases / Q000378:metabolism*; D008099:Liver / Q000201:enzymology / Q000378:metabolism*; D008297:Male; D008765:Methylmalonyl-CoA Mutase / Q000378:metabolism*; D008780:Methyltransferases / Q000378:metabolism*; D008930:Mitochondria, Liver / Q000378:metabolism; D051381:Rats; D014155:Transcobalamins / Q000378:metabolism; D014805:Vitamin B 12 / Q000378:metabolism*
D001051:Apoenzymes; D001059:Apoproteins; D003038:Cobamides; D014155:Transcobalamins; D008780:Methyltransferases; D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase; D007535:Isomerases; D008765:Methylmalonyl-CoA Mutase; D014805:Vitamin B 12