KINETICS, REGULATION AND MECHANISMS OF BIOCHEMICAL REACTIONS

免疫细胞化学研究表明泛素激活 在间期和间期，酶E1集中在细胞核酸酶中。 在有丝分裂期间与染色体相关的。西方印迹使用 多克隆抗EL抗体表明EL以两种亚型存在。E1 可以被蛋白激酶C磷酸化，而蛋白激酶C的一个亚型 泛素载体酶E2可以被酪氨酸激酶磷酸化。 在这两种情况下，磷酸化导致它们的活性加倍。 我们已经构建了一台实时荧光仪器 成像功能，可通过以下功能生成多种类型的波形 在不同场强下，频率最高可达1 MHz。我们的研究 揭示了细胞膜的对称性通透性只发生在 具有双极振荡电场，该方法提供了 最有效的DNA转染法。从指标摄取率来看，1 可以排除电渗作用作为这一过程的驱动力。 电子顺磁共振波谱与自旋俘获 采用多种方法鉴定和监测细菌的形成和分布 自由基的利用。我们发现铜，锌-超氧化物歧化酶 催化过氧化氢转化为羟基自由基，从而 可以与金属结合的组氨酸残基反应并导致 酶失活，或与阴离子配体或清道夫反应 结合到酶的带正电的活性通道上产生 二次自由基，或逃逸到大块介质中。逃逸的羟基 而二次自由基可以摧毁生物上重要的分子。 这可能在一定程度上解释了某些与高血压相关的疾病 这种酶的活性。 我们已经开发出一种方法来区分离合词和 配基置换反应的缔合机制。分析 NADH在其络合物之间转移的动力学数据 α-甘油-3-磷酸脱氢酶和乳酸脱氢酶 揭示了反应是通过解离机制进行的。这 与提议的衬底沟道机制不一致 糖酵解途径。

Immunocytochemical studies revealed that ubiquitin activating enzyme, E1, is concentrated in the cell nuclease during interphase an associated with chromosomes during mitosis. Western blots using polyclonal anti-El antibodies showed that El exists in two isoforms. E1 can be phosphorylated by protein kinase C, while one of the isoforms of ubiquitin carrier enzyme E2, can be phosphorylated by tyrosine kinase. In both cases, phosphorylation resulted in doubling of their activities. We have constructed an instrument with real-time fluorescence imaging capability which can produce several types of waveforms with variable frequencies up to 1 MHz at different field strengths. Our study revealed that symmetrical permeabilization of cell membrane occurs only with bipolar oscillating electric fields, and this method provides the most efficient DNA transfection. From the rate of indicator uptake, one can exclude electro-osmosis as driving force for this process. Electron paramagnetic resonance spectroscopy and spin-trapping methods were used to identify and monitor the formation and the utilization of free radicals. We found that Cu,Zn-superoxide dismutase catalyzes the conversion of hydrogen peroxide to hydroxyl radicals which can either react with metal bound histidine residues and leads to the enzyme inactivation, or react with anionic ligands or scavengers which bind to the positively charged active channel of the enzyme to generate secondary radicals, or escape into the bulk medium. The escaped hydroxyl and secondary radicals can destroy biologically important molecules. This may, in part, explain certain diseases associated with elevated activity of this enzyme. We have developed a method to differentiate a dissociative from an associative mechanism for ligand displacement reactions. Analysis of the kinetic data for NADH transfers between its complexes with alpha-glycerol-3-phosphate dehydrogenase and with lactate dehydrogenase revealed that the reaction proceeds via a dissociative mechanism. This is inconsistent with a proposed substrate channeling mechanism for the glycolytic pathway.