KINETICS, REGULATION AND MECHANISMS OF BIOCHEMICAL REACTIONS

我们发现蛋白质泛素化可以通过可逆的 磷酸化 除了先前报道的激酶，我们 纯化了一种新的丝氨酸/苏氨酸激酶，其特异于E2- 20 kDa， 磷酸化，来自HeLa细胞。 凝胶过滤表明MW为约 300 kDa和SDS-PAGE结果表明，该激酶可能由3种类型组成 的subunits。 化学计量为每摩尔磷酸盐掺入0.45摩尔磷酸盐。 摩尔的E2和磷酸化增强了约60%的E2- 20 kDa 泛素化组蛋白H2 A的活性。 转录因子fos和jun 被发现是多泛素化的。 E2- 20 kDa催化， Jun的多泛素化通过逐步解离 E2- 14 kDa和E3催化的多聚泛素化过程 通过一种进行性机制，在这种机制中，泛素化的jun仍然与 E3. 使用HeLa细胞研究了胞质Ca（II）振荡。 的作用 可逆磷酸化在这些振荡进行了检查，使用 蛋白激酶和磷酸酶的各种激活剂和抑制剂。 在检查的激酶中，只有CaMK II是必需的。 它磷酸化 肌醇三磷酸酶受体在体内振荡过程中。 我们的数据 表明CaMK II和calyculin A-可降解磷酸酶 参与维持Ca（II）振荡和调节 HeLa细胞中的频率。 电子顺磁共振波谱和自旋捕获方法 用于识别和监测自由基的形成和利用， 根的 形成谷胱甘肽自由基（GS）（以DMPO-SG形式存在） 在NCB-20细胞内，当这些细胞受到氧化作用时， 应力 当细胞用N-甲基吡咯烷酮预处理时，未观察到DMPO-SG。 乙酰-L-半胱氨酸（NAC），已知可保护抗氧化剂 酶的氧化损伤的细胞。 时间进程显示 增强的GS形成，仅在不存在NAC的情况下观察到， 相一致的想法，大部分GS产生 在抗氧化酶失效后 使用探针分子，我们发现电穿孔导致了 在面向膜的电极的两侧上具有不对称孔， 阳极侧上的小孔和较高的群体以及大孔， 在阴极侧上的更低的群体。 不对称运输模式 既不是由电泳引起的，也不是由于单面膜 正如之前所认为的那样。

We showed that protein ubiquitination can be regulated by reversible phosphorylation. In addition to the kinases reported earlier, we purified a novel serine/threonine kinase, which is specific for E2-20kDa phosphorylation, from HeLa cells. Gel filtration indicates a MW of about 300 kDa and SDS-PAGE data suggest that the kinase may consist of 3 types of subunits. The stoichiometry is 0.45 mole phosphate incorporation per mole of E2 and phosphorylation enhances about 60% of the E2-20kDa activity to ubiquitinate histone H2A. Transcription factors fos and jun were found to be multi-ubiquitinated. E2-20kDa-catalyzed, multiubiquitination of jun proceeds via a stepwise dissociative mechanism, while polyubiquitination catalyzed by E2-14kDa and E3 proceeds via a processive mechanism in which ubiquitinated jun remains bound to E3. Cytosolic Ca(II) oscillations were studied using HeLa cells. The role of reversible phosphorylation in these oscillations was examined using various activators and inhibitors of protein kinases and phosphatases. Of the kinases examined, only CaMK II is essential. It phosphorylates inositol trisphosphatase receptor in vivo during oscillations. Our data suggest that CaMK II and a calyculin A-inhibitable phosphatase are involved in sustaining Ca(II) oscillations and in regulating the frequency in HeLa cells. Electron paramagnetic resonance spectroscopy and spin-trapping methods were used to identify and monitor the formation and utilization of free radicals. Glutathionyl radicals (GS) were formed (exist as DMPO-SG) inside the NCB-20 cells when these cells were subjected to oxidative stress. No DMPO-SG was observed when the cells were pretreated with N- acetyl-L-cysteine (NAC), which is known to protect the antioxidant enzymes from oxidative damage in the cells. The time course shows enhanced GS formation, observed only in the absence of NAC, after a lag phase consistent with the idea that the majority of GS was generated after the antioxidant enzymes failed to function. Using probe molecules, we found electroporation leads to the formation of asymmetric pores on both sides of the membrane-facing electrodes with small pores and higher population on the anode side and large pores and lower population on the cathode side. The asymmetric transport pattern is neither caused by electrophoresis nor is it due to one-sided membrane breakdown as previously believed.