KINETICS, REGULATION AND MECHANISMS OF BIOCHEMICAL REACTIONS

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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.