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

本工作的目的是观察钴胺(Cbl)对大鼠肝脏和培养的COS7细胞L甲基丙二酰辅酶A变位酶(MCM)活性和表达的影响。MCM全酶活性不到肝脏总(全酶+辅酶)活性的5%，而饲料中Cb1含量较高。当断奶的大鼠被维持在缺乏Cbl的饮食中时，在10周大时几乎检测不到全息MCM的活性。相反，在Cbl缺乏的条件下，总MCM活性显著增加，在20周龄时，缺陷大鼠的总MCM活性约为对照组的3倍(108(SD 14.5)比35(SD 8.5)nmol/mg Protein/min(N5)；P&lt；0.05)。Western印迹分析证实CBL缺陷大鼠的MCM蛋白水平显著升高。然而，实时定量聚合酶链式反应检测到的MCM基因表达水平却有相当程度的下降。COS7细胞在含10%胎牛血清…的培养液中培养作为CBL的唯一来源，几乎没有检测到HALO-MCM活性。补加Cbl后，细胞内MCM活性显著增加，但在10μ/L Cbl存在下，活性仍未超过总MCM活性的30%。与之相反，补充Cbl可显著降低COS-7细胞和大鼠肝脏中MCM的总活性，表明Cbl缺乏导致COS-7细胞和大鼠肝脏MCM蛋白水平升高；细胞增殖标志增殖细胞核抗原在肝脏中的表达水平显著增强，提示Cbl缺乏条件下肝脏细胞增殖被异常激活。此外，作为肝损伤标志的血浆丙氨酸氨基转移酶(ALT)活性在缺乏的大鼠中也显著升高。临床上用于治疗Cbl缺乏性甲基丙二酸尿症的L-卡米丁给Cbl缺乏症大鼠每日2次，连续2周(每次0.5 mm)，可显著减少尿中甲基丙二醛排泄量，并使血浆ALT活性降至正常水平，提示Cbl缺乏症大鼠肝损伤是由于MCM全酶活性降低所致。而山茶碱对小鼠肝脏中增殖细胞核抗原的表达影响不大。而添加L蛋氨酸的饲料(L蛋氨酸4g/kg)喂养2周后，增殖细胞核抗原表达的增加趋于正常，提示Cbl缺乏导致蛋氨酸合成酶表达降低，导致增殖细胞核抗原表达异常增加。较少

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