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 be phosphorylated in vivo and in vitro. Both E2-20kDa and E2-32kDa are also phosphorylated by a novel kinase purified from HeLa cells and by a tyrosine kinase, respectively. Together it implies that protein ubiquitination is regulated by a reversible phosphorylation mechanism. Transcription factors, fos and jun, were found to be multi-ubiquitinated with different pathways by the E2-20kDa, E2-32kDa and E2-14kDa/E3beta system, but only those ubiquitinated by the last system are degraded by the 26S protease. When membrane of intact cells is subjected to some critical electric field strength, it becomes transiently permeabilized. The susceptibility of membranes to this dilectric breakdown is found to be dependent not only the magnitude, the direction of the electric field, but also the resting membrane potential. Electron paramagnetic resonance spectroscopy and spin-trapping methods were used to identify and monitor the formation and the utilization of free radicals. It is believed that alterations in well regulated antioxidant enzyme reactions causes irreversible damage to cells and tissues. Recently we reported that Cu,Zn-superoxide dismutase behaves as an enzyme catalyzing the formation of free radicals using anionic scavengers and hydrogen peroxide as substrates. Now we show that glutathione and the thiol specific antioxidant enzyme function as scavengers of oxygen and thiyl radicals. In addition, glutathione functions as a first line of defense by scavenging the free radicals generated due to oxidative stress to form glutathionyl radicals in the cells. Cytosolic Ca(II) oscillation was studied using both mesangial cells and HeLa cells. With mesangial cells, CAMP-dependent protein kinase plays a role in regulating the vasopressin stimulated or electroporated inositol trisphosphate induced Ca(II) release. the Ca(II) oscillation in HeLa cells induced by histamine is regulated by calmodulin-dependent protein kinase II, at least via its capacity to activate the endoplasmic reticulum Ca-ATPase.