Kolhouse J F, Allen R H
Proc Natl Acad Sci U S A. 1977 Mar;74(3):921-5. doi: 10.1073/pnas.74.3.921.
The granulocyte R-type cobalamin binding protein delivers cobalamin (Cbl) exclusively to hepatocytes, and transcobalamin II delivers Cbl to various mammalian cells. Both protein-Cbl complexes enter cells by pinocytosis, and the protein moieties are rapidly degraded in lysosomes. The liberated Cbl is subsequently bound to a high-molecular-weight intracellular cobalamin binding protein (ICB). The nature of ICB-Cbl is unknown but appears important because ICB-[57Co]Cbl is missing from cultured fibroblasts of a group of patients whose cells take up CN-[57Co]Cbl normally but do not convert it to either of its coenzyme forms. We have examined supernatants of sonicated rabbit livers and have found that 65% of the total endogenous Cbl elutes from Sephadex G-150 as ICB-Cbl and that this fraction also contains the two mammalian Cbl-dependent enzymes, methylmalonyl-CoA mutase (methylmalonyl-CoA CoA-carbonylmutase;EC 5.4.99.2) and methionine synthetase (tetrahydropteroylglutamate methyltransferase; 5-methyltetrahydropteroyl-L-glutamate:L-homocysteine-S-methyltransferase; EC 2.1.1.13). Gradient elution from DEAE-Sephadex reveals that 90--95% of the ICB--Cbl elutes with methylmalonyl-CoA mutase and 5--10% elutes with methionine synthetase. ICB--[57Co]Cbl first appears 2 hr after the intravenous injection of CN[57Co]Cbl bound to granulocyte R-type protein. This ICB-[57Co]Cbl is associated with either methylmalonyl-CoA mutase or methionine synthetase although the latter appears to be formed at a relatively faster rate. Our studies indicate that mammalian cells contain two ICBs, that these proteins are methylmalonyl-CoA mutase and methionine synthetase, and that the primary abnormality in the group of patients mentioned above lies at a step that is common to the formation of both Cbl coenzymes and that precedes the stable binding of Cbl to both methylmalonyl-CoA mutase and methionine synthetase.
粒细胞R型钴胺素结合蛋白仅将钴胺素（Cbl）传递给肝细胞，而转钴胺素II则将Cbl传递给各种哺乳动物细胞。两种蛋白质-Cbl复合物均通过胞饮作用进入细胞，蛋白质部分在溶酶体中迅速降解。随后释放的Cbl与一种高分子量的细胞内钴胺素结合蛋白（ICB）结合。ICB-Cbl的性质尚不清楚，但似乎很重要，因为在一组患者的培养成纤维细胞中缺少ICB-[57Co]Cbl，这些患者的细胞能正常摄取CN-[57Co]Cbl，但不能将其转化为任何一种辅酶形式。我们检测了超声处理的兔肝脏的上清液，发现65%的内源性总Cbl以ICB-Cbl的形式从Sephadex G-150上洗脱下来，并且该部分还含有两种依赖Cbl的哺乳动物酶，即甲基丙二酰辅酶A变位酶（甲基丙二酰辅酶A-CoA-羰基变位酶；EC 5.4.99.2）和甲硫氨酸合成酶（四氢蝶酰谷氨酸甲基转移酶；5-甲基四氢蝶酰-L-谷氨酸：L-高半胱氨酸-S-甲基转移酶；EC 2.1.1.13）。从DEAE-Sephadex上进行梯度洗脱显示，90%-95%的ICB-Cbl与甲基丙二酰辅酶A变位酶一起洗脱，5%-10%与甲硫氨酸合成酶一起洗脱。静脉注射与粒细胞R型蛋白结合的CN[57Co]Cbl后2小时，ICB-[57Co]Cbl首次出现。这种ICB-[57Co]Cbl与甲基丙二酰辅酶A变位酶或甲硫氨酸合成酶相关，尽管后者似乎以相对较快的速度形成。我们的研究表明，哺乳动物细胞含有两种ICB，这些蛋白质是甲基丙二酰辅酶A变位酶和甲硫氨酸合成酶，并且上述患者组中的主要异常发生在两种Cbl辅酶形成的共同步骤中，且该步骤先于Cbl与甲基丙二酰辅酶A变位酶和甲硫氨酸合成酶的稳定结合。
