Regulation in the expression of vitamin B12-dependent enzyme, methymalonyl-CoA mutase

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The aim of this work was to examine the effects of cobalamin (Cbl) on the activity and expression of L-methylmalonyl-CoA mutase (MCM) in rat liver and cultured COS-7 cells. The MCM holoenzyme activity was less than 5% of the total (holoenzyme+apoenzyme) activity in the liver although rats were fed a diet containing Cbl sufficiently. When weanling rats were maintained on a Cbl-deficient diet, the holo-MCM activity became almost undetectable at an age of 10 weeks. in contrast, a marked increase in the total-MCM activity occurred under the Cbl-deficient conditions, and at an age of 20 weeks it was about 3-fold higher in the deficient rats than in the controls (108 (SD 14.5) v.35 (SD 8.5) nmol/mg protein/min (n5); P<0.05). Western blot analysis confirmed that the MCM protein level increased significantly in the CBl-deficient rats. However, the MCM mRNA level, determination by realtime PCR, was rather decreased. When COS-7 cells were cultured in a medium in which 10% fetal bovine serum was … More the sole source of CBl, holo-MCM activity was barely detected. The supplementation of Cbl resulted in a great increase in the holo-MCM activity in the cells, but the activity did not exceed 30% of the total-MCM activity even in the presence of 10 μmol/l Cbl. In contrast, the total-MCM activity was significantly decrease by the Cbl supplementation, indicating that Cbl deficiency results in an increase in the MCM protein level in COS-7 cells as well as in rat liver.The expression level of proliferating cell nuclear antigen (PCNA), a marker for cell proliferation, in the liver was significantly enhanced in the deficient rats, suggesting that cell proliferation is abnormally activated in the liver under Cbl-deficient conditions. In addition, plasma alanine aminotransferase (ALT) activity, a marker for hepatic injury, was also significantly elevated in the deficient rats. When L-camitine, which is used clinically for the treatment of Cbl-deficient patients with methylmalonic aciduria, was administered to the Cbl-deficient rats by intraperitoneal injection twice per day for 2 weeks (each 0.5 mmol), the amount of methylmalonic add excreted into the urine was significantly reduced, and the plasma ALT activity was lowered to a normal level, suggesting that the decrease in the MCM holoenzyme activity results in hepatic injury in the Cbl-deficient rats. However; the PCNA expression in the liver was barely influenced by the treatment with camitine. In contrast, when the deficient rats were fed an L-methionine-supplemented diet (4 g of L-methionine per kg of the diet) for 2 weeks, the increased expression of PCNA was normalized, suggesting that a decrease in methionine synthase due to Cbl deficiency induces the abnormal increase in the expression of PCNA. Less