KINETICS, REGULATION AND MECHANISMS OF BIOCHEMICAL REACTIONS

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Protein ubiquitination has been implicated in numerous intracellular processes. The enzymes involved are: the ubiquitin activating enzyme, E1, the ubiquitin carrier enzyme, E2, which delivers the activated ubiquitin to its ligase, E3, or directly ubiquitinates its target proteins. E1 was found to exist as two isoforms, designated as E1-117kDa and E1-110kDa. E1 in HeLa cells was found to be phosphorylated, suggesting protein ubiquitination may be regulated by the phosphorylation/dephosphorylation mechanism. This is in accord with the in vitro studies showing E1 is phosphorylated by protein kinase C, and two of the E2 isoforms, E2-32kDa and E2-20kDa are phosphorylated by a tyrosine kinase, and by a novel kinase in the cytosolic fraction of HeLa cells, respectively. The transcription factors, fos and jun, were found to undergo multiple ubiquitinations. When membrane of intact cells is subject to some critical electric field strength, they become transiently permeabilized. This transient time is inversely proportional to the ionic strength of the suspending medium. In addition, the ionic strength also affects the location of the permeabilization with respect to the electrodes, and the bulk-to-bulk membrane potential. Electron paramagnetic resonance spectroscopy and spin-trapping methods were used to identify and monitor the formation and the utilization of free radicals. Previously, we found that Cu,Zn-superoxide dismutase (Cu,Zn-SOD) catalyzes the conversion of hydrogen peroxide to hydroxyl radical which can generate secondary radicals from enzyme-bound anionic ligands or scavengers. Therefore, enhanced Cu,Zn-SOD activity can produced damaging effects to the cells. When NCB-20 cells, a mouse neuroblastoma-Chinese hamster brain hybrid cell line, is transfected with a human pSVneo Cu,Zn- SOD gene expression vector, the level of reduced glutathione is significantly reduced. This indicates that the Cu,Zn-SOD overexpressed cells maintain chronic pro-oxidant states due to free radical production catalyzed by the elevated level of Cu,Zn-SOD. Cytosolic Ca(II) oscillation in hepatocytes and mesangial cells has been investigated. It was found that vasopressin-induced production of inositol triphosphate proceeds with an initial spike and then reduces to an elevated steady-state level relative to that of basal. The data suggest that the frequency of Ca(II) oscillation, which is dependent on the concentration of agonist, is correlated with the steady-state level of inositol triphosphate.