KINETICS, REGULATION AND MECHANISMS OF BIOCHEMICAL REACTIONS

研究泛素化/去泛素化的生理作用 Casade，我们主要关注泛素化途径中的第一种酶，即 泛素活化酶。从兔体内纯化该酶的过程中 网织红细胞，与泛素载体蛋白相互作用的酶 这表明它们是以复合体的形式存在的。免疫细胞化学数据显示 这种酶在细胞核中的显著区域化 大鼠脑、心、肝、肾和HeLa细胞。免疫化学研究 揭示了大脑中活化酶的水平略低 阿尔茨海默氏症患者的组织相对于正常人的组织。 氧自由基与蛋白质周转、衰老和 疾病。为了研究这些过程的基本机制，我们使用了EPR 和自旋陷阱方法来识别和监测形成和利用 自由基团。本研究揭示：(I)Mn(II)离子在 碳酸氢钠/二氧化碳缓冲液可催化歧化反应 过氧化氢，并产生超氧化物和羟基自由基。添加 一种氨基酸，如亮氨酸，会产生一个亮氨酸衍生的基团来代替 超氧自由基。使用各种同位素浓缩的Leu，这个自由基是 经鉴定为氢氧化亚氮-HOOCC(R)HNHO。(Ii)铜锌超氧化物 歧化酶能催化-OH自由基的产生 过氧化氢。这一发现意味着过度表达 铜，锌-超氧化物歧化酶基因，如唐氏综合症，将导致 体内产生的自由-OH自由基，可能是 他的病。 已提出糖酵解酶形成多酶复合体用于 代谢物的直接转移。对直接证据的重新审查 NADH与(α-甘油-3-磷酸)络合物之间的转移 脱氢酶与乳酸脱氢酶显示的数据一致 具有自由扩散机制。 我们已经开发了一种使用低幅度、双极的仪器 脉冲时间可变的正弦电场作为一种手段 将各种大分子引入细胞。我们的研究表明 使用双极交流电穿孔。菲尔德提供了最有效的DNA 用被测试的细胞进行转染，发生膜通透性 对称地位于面对电极的两个半球。

To study the physiological roles of the ubiquitination/deubiquitination cascade, we focused on the first enzyme in the ubiquitination pathway, the ubiquitin activating enzyme. During purification of this enzyme from rabbit reticulocytes, the enzyme copurified with the ubiquitin carrier proteins which suggests that they exist as a complex. Immunocytochemical data show significant compartmentalization of this enzyme in the cell nucleus from rat brain, heart, liver, and kidney and in HeLa cells. Immunochemical study revealed that the level of the activating enzyme is slightly lower in brain tissue from Alzheimer's patients relative to that of normal persons. Oxygen-free radicals have been implicated in protein turnover, aging, and disease. To study the fundamental mechanism of these processes, we used EPR and spin trap methods to identify and monitor the formation and utilization of free radicals. This study revealed: (i) Mn(II) ions in bicarbonate/carbon dioxide buffer can catalyze the disproportionation of hydrogen peroxide and generate superoxide and hydroxyl radicals. Addition of an amino acid such as Leu yields a Leu-derived radical in place of the superoxide radical. Using various isotope-enriched Leu, this radical was identified as a hydronitroxide -HOOCC(R)HNHO. (ii) Cu,Zn-superoxide dismutase can catalyze the generation of -OH radicals in the presence of hydrogen peroxide. This finding implies that overexpression of the Cu,Zn-superoxide dismutase gene, such as in Down's syndrome, will result in the free -OH radicals generation in vivo and may be, in part, a cause of the illness. It has been proposed that glycolytic enzymes form multienzyme complexes for direct transfer of metabolites. Reexamination of the evidence for direct transfer of NADH between its complexes with (alpha-glycerol-3-phosphate dehydrogenase and with lactate dehydrogenase show the data are consistent with a free-diffusion mechanism. We have developed an instrument which uses low amplitude, bipolar sinusoidal electric fields with variable pulse times as a means of introducing various macromolecules into cells. Our studies revealed that electroporation using a bipolar a.c. field provided the most efficient DNA transfection with the cells tested, and membrane permeabilization occurs symmetrically at the two hemispheres facing the electrodes.