KINETICS, REGULATION AND MECHANISMS OF BIOCHEMICAL REACTIONS

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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.