KINETICS, REGULATION, AND MECHANISMS OF BIOCHEMICAL REACTIONS

生化反应的动力学、调控和机制

• 批准号: 6109139
• 负责人: P. BOON Chock
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6109139
• 关键词: amyotrophic lateral sclerosis; biological signal transduction; calcium flux; chemical kinetics; electrochemistry; electron spin resonance spectroscopy; enzyme activity; enzyme mechanism; free radicals; hydrogen peroxide; isozymes; membrane activity; nitric oxide; oxidative stress; phospholipase C; phosphorylation; protein kinase; protein tyrosine kinase; superoxide dismutase; ubiquitin

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.

利用模型系统和EPR技术，我们有 结果显示，在糖化的葡萄糖中观察到的黄色荧光产物， 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。我们发现三种自由基， 交联自由基阳离子、甲基乙二醛自由基阴离子和 超氧阴离子自由基。后者仅在 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 发现蛋白质的交联席夫碱自由基阳离子 就像一种酶，催化自由的产生， 根的这些结果表明，糖化蛋白积累 可加速氧化损伤。蛋白酪氨酸 磷酸酶（PTP）在细胞内起着重要作用， 发信号。我们研究了PTP-1B的氧化还原调节， 发现PTP-1B的失活和恢复都比 有效与超氧阴离子自由基作为信号相对于 过氧化氢无法完全回收PTP-1B DTT与过氧化氢反应后， 这是因为，更多的蛋氨酸和半胱氨酸是 被氧化了使用我们自制的电转化系统， 磷脂囊泡，我们已经研究了诱导，分布， 和电场诱导的膜孔的寿命。我们也 探索了开发快速动力学方法的可能性， 装载的囊泡作为反应容器，因为它们可以在反应容器中破裂。 微秒时间范围内的电脉冲。但 由于囊泡制备及其破裂引起的异质性问题 需要解决。