KINETICS, REGULATION, AND MECHANISMS OF BIOCHEMICAL REACTIONS

使用模型系统和 EPR 技术，我们有 表明在糖化中观察到黄色荧光产物 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。我们证明了三种自由基种类， 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、交联希夫碱和 发现蛋白质的交联希夫碱自由基阳离子 其功能类似于酶，催化游离态的产生 部首。这些结果表明糖化蛋白积累 在体内可加速氧化损伤。蛋白质酪氨酸 磷酸酶（PTP）被认为在细胞中发挥着重要作用 发信号。我们研究了PTP-1B的氧化还原调节 发现PTP-1B的失活及其恢复情况更 有效地以超氧阴离子自由基作为信号 过氧化氢。无法完全恢复 PTP-1B 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。使用我们自制的电穿孔器系统和 磷脂囊泡，我们研究了诱导、分布、 和电场感应膜孔的寿命。我们也 探索了开发快速动力学方法的可能性 负载囊泡作为反应容器，因为它们可以在 电脉冲的微秒时间范围。然而， 由于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.