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

本工作的目的是研究钴胺素（Cbl）对大鼠肝脏和培养的COS-7细胞中L-甲基丙二酰辅酶A（MCM）活性和表达的影响。MCM全酶活性小于5%的总（全酶+脱辅基酶）的活性，在肝脏中，虽然大鼠喂养的饮食中含有足够的Cbl。当断乳大鼠维持在一个Cbl缺乏的饮食，holo-MCM活动变得几乎检测不到的年龄为10周。相反，在Cbl缺陷条件下，总MCM活性显著增加，并且在20周龄时，缺陷大鼠中的总MCM活性比对照大鼠高约3倍（108（SD 14.5）v.35（SD 8.5）nmol/mg蛋白/min（n5）; P<0.05）。Western blot分析证实，MCM蛋白水平显着增加，在CBL缺陷大鼠。然而，实时PCR测定的MCM mRNA水平却有所下降。当COS-7细胞在含有10%胎牛血清的培养基中培养时， 关于我们 CB 1的唯一来源，holo-MCM活性几乎未被检测到。添加Cbl后，细胞中的holo-MCM活性显著提高，但即使在10 μmol/l的Cbl存在下，其活性也不超过总MCM活性的30%。相反，补充Cbl显著降低总MCM活性，表明Cbl缺乏导致COS-7细胞以及大鼠肝脏中MCM蛋白水平增加。在缺乏的大鼠中，增殖细胞核抗原（PCNA）（细胞增殖的标志物）的表达水平显著增强，这表明在Cbl缺乏的条件下，肝中的细胞增殖被异常激活。此外，血浆丙氨酸氨基转移酶（ALT）活性，肝损伤的标志物，也显着升高，在缺陷大鼠。当临床上用于治疗具有甲基丙二酸尿症的Cbl缺陷患者的L-卡米汀通过腹膜内注射每天两次给予Cbl缺陷大鼠2周时，（各0.5mmol）时，排泄到尿中的甲基丙二酸的量显著减少，并且血浆ALT活性降低到正常水平，提示MCM全酶活性的降低导致Cbl缺陷大鼠的肝损伤。然而，PCNA在肝脏中的表达几乎没有影响的治疗与卡米汀。与此相反，当缺乏大鼠喂食L-蛋氨酸-补充的饮食（每公斤饮食4g L-蛋氨酸）2周时，增加的PCNA表达正常化，表明由于Cbl缺乏引起的蛋氨酸合成酶的减少诱导PCNA表达的异常增加。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