KINETICS, REGULATION, AND MECHANISMS OF BIOCHEMICAL REACTIONS

使用模型系统和EPR技术，我们有 显示黄色荧光产物，观察到糖化 蛋白质，是通过氨基酸之间的相互作用产生的 和甲基乙二醛。我们展示了三种自由基物种，即 交联自由基阳离子、甲基乙二醛自由基阴离子和 形成超氧阴离子自由基。后者仅形成于 氧气的存在。当氨基酸被替换为 牛血清白蛋白、交联席夫碱和 蛋白质的交联席夫碱自由基阳离子被发现 就像一种酶一样，催化产生游离 激进分子。这些结果表明糖化的蛋白质积累了 体内可能会加速氧化损伤。蛋白质酪氨酸 磷酸酶(PTP)被认为在细胞中起主要作用 发信号。我们研究了PTP-1B和PTP-1B的氧化还原调节 发现PTP-1B的失活及其恢复较多 使用超氧阴离子自由基作为相对于 过氧化氢。无法完全恢复PTP-1B 在与过氧化氢反应后，DTT的活性可以 归因于更多的蛋氨酸和半胱氨酸 被氧化了。使用我们自制的电穿孔系统和 磷脂囊泡，我们已经研究了诱导，分布， 和电场诱导的膜孔寿命。我们也 探索了开发一种快速动力学方法的可能性 加载的囊泡作为反应容器，因为它们可以在 以电脉冲表示的微秒时间范围。然而， 囊泡制备及其破裂引起的异质性问题 需要解决。

Using a model system and EPR techniques, we have shown that the yellow fluorescent products, observed in glycated proteins, were generated via the interaction between amino acids and methylglyoxal. We showed that three free radical species, the cross-linked radical cation, the methylglyoxal radical anion, and the superoxide anion radical, were formed. The latter is formed only in the presence of oxygen. When the amino acid was replaced with bovine serum albumin, the cross-linked Schiff base and the cross-linked Schiff base radical cation of the protein was found to function like an enzyme for catalyzing the generation of free radicals. These results suggest that glycated proteins accumulated in vivo could accelerate oxidative damage. Protein tyrosine phosphatase (PTP) has been implicated to play a major role in cell signaling. We have studied the redox regulation of PTP-1B and found that the inactivation of PTP-1B and its recovery are more efficient with superoxide anion radicals as a signal relative to hydrogen peroxide. The inability to fully recover the PTP-1B activity with DTT, after its reaction with hydrogen peroxide, could be attributed to the fact that more methionines and cysteines were oxidized. Using our home-build electroporator system and phospholipid vesicles, we have studied the induction, distribution, and lifetime of the electric field-induced membrane pores. We also explored the possibility of developing a rapid kinetic method using loaded vesicles as reaction vessels since they can be ruptured in the microsecond time range by electric pulses. However, the heterogeneity problems due to vesicle preparation and its rupture need to be resolved.