KINETICS, REGULATION AND MECHANISMS OF BIOCHEMICAL REACTIONS

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We showed that protein ubiquitination can be regulated by reversible phosphorylation. In addition to the kinases reported earlier, we purified a novel serine/threonine kinase, which is specific for E2-20kDa phosphorylation, from HeLa cells. Gel filtration indicates a MW of about 300 kDa and SDS-PAGE data suggest that the kinase may consist of 3 types of subunits. The stoichiometry is 0.45 mole phosphate incorporation per mole of E2 and phosphorylation enhances about 60% of the E2-20kDa activity to ubiquitinate histone H2A. Transcription factors fos and jun were found to be multi-ubiquitinated. E2-20kDa-catalyzed, multiubiquitination of jun proceeds via a stepwise dissociative mechanism, while polyubiquitination catalyzed by E2-14kDa and E3 proceeds via a processive mechanism in which ubiquitinated jun remains bound to E3. Cytosolic Ca(II) oscillations were studied using HeLa cells. The role of reversible phosphorylation in these oscillations was examined using various activators and inhibitors of protein kinases and phosphatases. Of the kinases examined, only CaMK II is essential. It phosphorylates inositol trisphosphatase receptor in vivo during oscillations. Our data suggest that CaMK II and a calyculin A-inhibitable phosphatase are involved in sustaining Ca(II) oscillations and in regulating the frequency in HeLa cells. Electron paramagnetic resonance spectroscopy and spin-trapping methods were used to identify and monitor the formation and utilization of free radicals. Glutathionyl radicals (GS) were formed (exist as DMPO-SG) inside the NCB-20 cells when these cells were subjected to oxidative stress. No DMPO-SG was observed when the cells were pretreated with N- acetyl-L-cysteine (NAC), which is known to protect the antioxidant enzymes from oxidative damage in the cells. The time course shows enhanced GS formation, observed only in the absence of NAC, after a lag phase consistent with the idea that the majority of GS was generated after the antioxidant enzymes failed to function. Using probe molecules, we found electroporation leads to the formation of asymmetric pores on both sides of the membrane-facing electrodes with small pores and higher population on the anode side and large pores and lower population on the cathode side. The asymmetric transport pattern is neither caused by electrophoresis nor is it due to one-sided membrane breakdown as previously believed.