Methylglyoxal can modify GAPDH activity and structure
Methylglyoxal can modify GAPDH activity and structure
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DOI：
10.1196/annals.1333.017
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发表时间：
2005-01-01
期刊：
影响因子：
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通讯作者：
Beisswenger, PJ
中科院分区：
文献类型：
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作者：
Lee, HJ;Howell, SK;Beisswenger, PJ
甘油醛-3-磷酸脱氢酶（GAPDH）的活性在调节与糖尿病并发症发生相关的多个上游途径中发挥重要作用。GAPDH可以被许多代谢因素修饰，包括氧化和糖化产物。为了研究糖基化对GAPDH的影响，我们在将酶暴露于有效的α-二羰基糖甲基乙二醛（MG）后测量了GAPDH的结构和活性。将兔GAPDH与10-1000 μ M MG孵育96小时，并通过分光光度测定法间隔测量酶活性。用PROTEAN IEF系统对纯化的和细胞的GAPDH进行等电聚焦，并通过Western印迹法观察条带。糖化和天然GAPDH的质量通过MALDI用Applied Biosystems Voyager System 6235测定。GAPDH活性（在96小时）下降了20%，1.0微摩尔MG，并表现出越来越大的抑制活性与浓度增加到1 mM，其中活性下降了97%。GAPDH活性的降低在使用ImM MG两小时时迅速降低69.2%。IEF显示天然GAPDH的等电点（IEP）为8.5，而通过1 mM（IEP 7.5）和50 μ M（IEP 8.0）的MG水平的修饰观察到可测量的变化。用MALDI，GAPDH质量从36.012 kDa增加到37.071后，暴露于50 μ M MG和40.625后，1 mM MG。这表明分别向GAPDH添加了12.75和55.6 MG残基。GAPDH可以通过接近于先前在体内观察到的那些浓度的丙酮醛细胞内浓度进行修饰，其中等电点和质量具有可测量的变化。这些修饰可导致酶活性降低，表明与细胞内MG升高相关的条件可在体内修饰GAPDH活性。
The activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) can play an important role in regulating multiple upstream pathways relating to the development of diabetic complications. GAPDH can be modified by a number of metabolic factors, including oxidative and glycation products. To study the effect of glycation on GAPDH we have measured GAPDH structure and activity after exposure of the enzyme to the potent alpha dicarbonyl sugar methylglyoxal (MG). Rabbit GAPDH was incubated with 10-1000 mu M MG for 96 hours, and enzyme activity was measured at intervals by a spectrophotometric assay. Isoelectric focusing of purified and cellular GAPDH was performed with a PROTEAN IEF system and the bands visualized by Western blotting. The mass of glycated and native GAPDH was determined by MALDI with a Applied Biosystems Voyager System 6235. GAPDH activity (at 96 h) was decreased by 20% with 1.0 micromolar MG and showed progressively greater suppression of activity with increasing concentrations up to 1 mM, where activity was decreased by 97%. Reduction in GAPDH activity was rapidly decreasing by 69.2% by two hours with 1 mM MG. IEF showed an isoelectric point (IEP) of 8.5 for native GAPDH, while measurable changes were seen with modification by MG levels of 1 mM (IEP 7.5) and 50 mu M (IEP 8.0). With MALDI, GAPDH mass increased from 36.012 kDa to 37.071 after exposure to 50 mu M MG and to 40.625 following 1 mM MG. This indicates addition of 12.75 and 55.6 MG residues, respectively, to GAPDH. GAPDH can be modified by methylglyoxal intracellular concentrations close to those previously observed in vivo, with measurable changes in isoelectric point and mass. These modifications can lead to decreased enzyme activity, suggesting that conditions associated with elevated intracellular MG could modify GAPDH activity in vivo.