The intracellular distribution of glutathine (GSH) in cultured hepatocytes hasbeen investigated by using the compound monochlorobimane (BmCI) Approximately 10-20% of total cellular GSH in rat liver is sequestered in the mitochondrial matrix (6, 7). The size ofthis pool depends on cytosolic GSH synthesis (8) and the active transport of GSH into mitochondria via a multicomponent system recently described (9).Conventional cell-fractionation studies have not provided evidence for the existence of functionally distinct pools of GSH in hepatocytes other than those in the cytosol and mitochondria. Despite the known functions of GSH in DNA synthesis (10) and protection from oxidative DNA damage (11), little is known about the nuclear localization ofGSH and the factors regulating the nuclear GSH level. Tirmenstein and Reed (12), using fractionation and centrifugation techniques in nonaqueous medium, measured the nuclear GSH content in rat kidney and found values similar to those in the cytosol. Other fractionation techniques (such as selective permeabilization of cell constituents with various detergents) provided equivocal results (13,14). However, the latter investigations have suggested that a nuclear pool of GSH may exist in intact cells.Recent advances in image-analysis technology, together with the development of additional, nontoxic fluorescent indicators that can be used in intact cells, have facilitated an enormous input into the study of various aspects of cell physiology (15,16 ITo whom reprint requests should be addressed.
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Cytoskeletal abnormalities occurring during oxidative stress generated by the metabolism of the redox cycling compound 2-methyl-1,4-naphtoquinone (menadione) have been investigated in different mammalian cells in culture. Extraction of the whole cytoskeleton as well as the intermediate filament- and the microtubule-enriched fractions from menadione-treated cells revealed a marked depletion of protein sulfhydryl groups. The analysis of the whole cytoskeletal fraction by PAGE showed a menadione-dependent and thiol-sensitive oxidation of actin, leading to the formation of high-molecular-weight aggregates. In addition, the extraction of this fraction with high concentrations of KCl entailed only a partial solubilization of actin. The comparative cytochemical analysis performed on treated cells showed a menadione-dependent clustering of actin microfilaments. The metabolism of menadione induced microtubule depolymerization and inhibition of GTP-induced microtubule assembly from soluble cytosolic components. The latter phenomenon was prevented by previously treating the cytosolic fraction with thiol reductants such as dithiothreitol. Menadione increased the protein content of the intermediate-size filament fraction, partially purified by one or more cycles of disassembly/assembly, and particularly enriched in polypeptides reacting with antikeratin antibodies. Furthermore, a reversible and oxidation-dependent change of the electrophoretic mobility of some polypeptides in this fraction was detected. The immunocytochemical investigation of intermediate-size filament distribution in menadione-treated cells, however, revealed only minor modifications mainly consisting of perinuclear condensation of cytokeratin structures. These findings suggest that cytoskeletal structures (actin microfilaments, microtubules, and intermediate-size filaments) are actually significant targets in quinone-induced oxidative stress.
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