KINETICS, REGULATION AND MECHANISMS OF BIOCHEMICAL REACTIONS

免疫细胞化学研究表明，泛素激活 酶E1在细胞分裂间期浓缩在细胞核酸酶中， 在有丝分裂中与染色体有关。 蛋白质印迹法， 多克隆抗El抗体显示El以两种同种型存在。 E1 可以被蛋白激酶C磷酸化，而 泛素载体酶E2可被酪氨酸激酶磷酸化。 在这两种情况下，磷酸化导致其活性加倍。 我们已经建立了一个仪器与实时荧光 成像能力，可产生多种波形， 在不同的场强下，频率可达1 MHz。 我们的研究 揭示了细胞膜的对称透化仅发生在 双极振荡电场，这种方法提供了 最有效的DNA转染。 从指标吸收率来看， 可以排除电渗作为该过程的驱动力。 电子顺磁共振谱和自旋俘获 方法被用来识别和监测地层， 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， 可以与金属结合的组氨酸残基反应， 酶失活，或与阴离子配体或清除剂反应， 与酶的带正电荷的活性通道结合， 次级自由基，或逃逸到本体介质中。 逸出的羟基 次级自由基可以破坏生物学上重要的分子。 这可能部分解释了某些疾病与升高的 这种酶的活性。 我们发明了一种方法来区分 配体置换反应的缔合机制。 分析 NADH在其复合物与 α-甘油-3-磷酸脱氢酶和乳酸脱氢酶 揭示了反应通过解离机制进行。 这 这与所提出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.