Visualization of the compartmentalization of glutathione and protein-glutathione mixed disulfides in cultured cells. 2003

Therese Söderdahl, and Mari Enoksson, and Mathias Lundberg, and Arne Holmgren, and Ole P Ottersen, and Sten Orrenius, and George Bolcsfoldi, and Ian A Cotgreave
Division of Biochemical Toxicology, Karolinska Institutet, Stockholm Sweden.

Fluorescence microscopy of A549 cells stained with a glutathione (L-gamma-glutamyl-L-cysteinylglycine, GSH)-specific polyclonal antibody displayed uniform staining of the peri-nuclear cytosol, with the nuclear region apparently lacking GSH staining. This discontinuous staining was confirmed in other cell types and also corroborated in A549 cells stained with the thiol-reactive dye mercury orange. The selectivity of antibody binding was confirmed by buthionine sulfoximine (BSO)-dependent inhibition of GSH synthesis. However, confocal visualization of antibody-stained A549 cells in the z-plane revealed the majority of the peri-nuclear staining intensity in the upper half of the cell to be associated with mitochondria, as confirmed by double staining for cytochrome oxidase. Integration of the confocal signals from the nuclear and cytosolic regions halfway down the z-plane showed that the GSH concentrations of these compartments are close to equilibrium. Confirmation of the relatively high levels of mitochondrial glutathione was provided in cells treated with BSO and visualized in z-section, revealing the mitochondrial GSH content of these cells to be well preserved in apposition to near-complete depletion of cytosolic/nuclear GSH. Localized gradients within the cytosolic compartment were also visible, particularly in the z-plane. The antibody also provided initial visualization of the compartmentalization of protein-GSH mixed disulfides formed in A549 cells exposed to diamide. Discontinuous staining was again evident, with heavy staining in membrane blebs and in the nuclear region. Using FACS analysis of anti-GSH antibody-stained Jurkat T lymphocytes, we also demonstrated population variations in the cellular compliment of GSH and protein-GSH mixed disulfides, formed in response to diamide. In addition, we showed cell-cycle variation in GSH content of the cells, with the highest levels of GSH associated with the G2/M mitotic phase of the cell cycle, using double staining with propidium iodide. Similar FACS analyses performed in isolated mitochondria presented a considerable variation in GSH content within mitochondria of uniform granularity from the same preparation.

UI MeSH Term Description Entries
D008928 Mitochondria Semiautonomous, self-reproducing organelles that occur in the cytoplasm of all cells of most, but not all, eukaryotes. Each mitochondrion is surrounded by a double limiting membrane. The inner membrane is highly invaginated, and its projections are called cristae. Mitochondria are the sites of the reactions of oxidative phosphorylation, which result in the formation of ATP. They contain distinctive RIBOSOMES, transfer RNAs (RNA, TRANSFER); AMINO ACYL T RNA SYNTHETASES; and elongation and termination factors. Mitochondria depend upon genes within the nucleus of the cells in which they reside for many essential messenger RNAs (RNA, MESSENGER). Mitochondria are believed to have arisen from aerobic bacteria that established a symbiotic relationship with primitive protoeukaryotes. (King & Stansfield, A Dictionary of Genetics, 4th ed) Mitochondrial Contraction,Mitochondrion,Contraction, Mitochondrial,Contractions, Mitochondrial,Mitochondrial Contractions
D008954 Models, Biological Theoretical representations that simulate the behavior or activity of biological processes or diseases. For disease models in living animals, DISEASE MODELS, ANIMAL is available. Biological models include the use of mathematical equations, computers, and other electronic equipment. Biological Model,Biological Models,Model, Biological,Models, Biologic,Biologic Model,Biologic Models,Model, Biologic
D011506 Proteins Linear POLYPEPTIDES that are synthesized on RIBOSOMES and may be further modified, crosslinked, cleaved, or assembled into complex proteins with several subunits. The specific sequence of AMINO ACIDS determines the shape the polypeptide will take, during PROTEIN FOLDING, and the function of the protein. Gene Products, Protein,Gene Proteins,Protein,Protein Gene Products,Proteins, Gene
D002451 Cell Compartmentation A partitioning within cells due to the selectively permeable membranes which enclose each of the separate parts, e.g., mitochondria, lysosomes, etc. Cell Compartmentations,Compartmentation, Cell,Compartmentations, Cell
D002453 Cell Cycle The complex series of phenomena, occurring between the end of one CELL DIVISION and the end of the next, by which cellular material is duplicated and then divided between two daughter cells. The cell cycle includes INTERPHASE, which includes G0 PHASE; G1 PHASE; S PHASE; and G2 PHASE, and CELL DIVISION PHASE. Cell Division Cycle,Cell Cycles,Cell Division Cycles,Cycle, Cell,Cycle, Cell Division,Cycles, Cell,Cycles, Cell Division,Division Cycle, Cell,Division Cycles, Cell
D002460 Cell Line Established cell cultures that have the potential to propagate indefinitely. Cell Lines,Line, Cell,Lines, Cell
D002478 Cells, Cultured Cells propagated in vitro in special media conducive to their growth. Cultured cells are used to study developmental, morphologic, metabolic, physiologic, and genetic processes, among others. Cultured Cells,Cell, Cultured,Cultured Cell
D003958 Diamide A sulfhydryl reagent which oxidizes sulfhydryl groups to the disulfide form. It is a radiation-sensitizing agent of anoxic bacterial and mammalian cells. Diazodicarboxylic Acid Bis(N,N-dimethyl)amide,Diazodicarboxylic Acid Bisdimethylamide,Dizene Dicarboxylic Acid Bis(N,N-dimethylamide),Dizenedicarboxylic Acid Bis(N,N-dimethylamide),Tetramethylazoformamide,Acid Bisdimethylamide, Diazodicarboxylic,Bisdimethylamide, Diazodicarboxylic Acid
D005434 Flow Cytometry Technique using an instrument system for making, processing, and displaying one or more measurements on individual cells obtained from a cell suspension. Cells are usually stained with one or more fluorescent dyes specific to cell components of interest, e.g., DNA, and fluorescence of each cell is measured as it rapidly transverses the excitation beam (laser or mercury arc lamp). Fluorescence provides a quantitative measure of various biochemical and biophysical properties of the cell, as well as a basis for cell sorting. Other measurable optical parameters include light absorption and light scattering, the latter being applicable to the measurement of cell size, shape, density, granularity, and stain uptake. Cytofluorometry, Flow,Cytometry, Flow,Flow Microfluorimetry,Fluorescence-Activated Cell Sorting,Microfluorometry, Flow,Cell Sorting, Fluorescence-Activated,Cell Sortings, Fluorescence-Activated,Cytofluorometries, Flow,Cytometries, Flow,Flow Cytofluorometries,Flow Cytofluorometry,Flow Cytometries,Flow Microfluorometries,Flow Microfluorometry,Fluorescence Activated Cell Sorting,Fluorescence-Activated Cell Sortings,Microfluorimetry, Flow,Microfluorometries, Flow,Sorting, Fluorescence-Activated Cell,Sortings, Fluorescence-Activated Cell
D005978 Glutathione A tripeptide with many roles in cells. It conjugates to drugs to make them more soluble for excretion, is a cofactor for some enzymes, is involved in protein disulfide bond rearrangement and reduces peroxides. Reduced Glutathione,gamma-L-Glu-L-Cys-Gly,gamma-L-Glutamyl-L-Cysteinylglycine,Glutathione, Reduced,gamma L Glu L Cys Gly,gamma L Glutamyl L Cysteinylglycine

Related Publications

Therese Söderdahl, and Mari Enoksson, and Mathias Lundberg, and Arne Holmgren, and Ole P Ottersen, and Sten Orrenius, and George Bolcsfoldi, and Ian A Cotgreave
September 1984, International journal of radiation oncology, biology, physics,
Therese Söderdahl, and Mari Enoksson, and Mathias Lundberg, and Arne Holmgren, and Ole P Ottersen, and Sten Orrenius, and George Bolcsfoldi, and Ian A Cotgreave
January 1981, Methods in enzymology,
Therese Söderdahl, and Mari Enoksson, and Mathias Lundberg, and Arne Holmgren, and Ole P Ottersen, and Sten Orrenius, and George Bolcsfoldi, and Ian A Cotgreave
January 1978, Doklady Akademii nauk SSSR,
Therese Söderdahl, and Mari Enoksson, and Mathias Lundberg, and Arne Holmgren, and Ole P Ottersen, and Sten Orrenius, and George Bolcsfoldi, and Ian A Cotgreave
September 1983, Biochemical pharmacology,
Therese Söderdahl, and Mari Enoksson, and Mathias Lundberg, and Arne Holmgren, and Ole P Ottersen, and Sten Orrenius, and George Bolcsfoldi, and Ian A Cotgreave
June 1983, Analytical biochemistry,
Therese Söderdahl, and Mari Enoksson, and Mathias Lundberg, and Arne Holmgren, and Ole P Ottersen, and Sten Orrenius, and George Bolcsfoldi, and Ian A Cotgreave
June 1994, Biochemistry,
Therese Söderdahl, and Mari Enoksson, and Mathias Lundberg, and Arne Holmgren, and Ole P Ottersen, and Sten Orrenius, and George Bolcsfoldi, and Ian A Cotgreave
August 1992, Comparative biochemistry and physiology. B, Comparative biochemistry,
Therese Söderdahl, and Mari Enoksson, and Mathias Lundberg, and Arne Holmgren, and Ole P Ottersen, and Sten Orrenius, and George Bolcsfoldi, and Ian A Cotgreave
January 1987, Methods in enzymology,
Therese Söderdahl, and Mari Enoksson, and Mathias Lundberg, and Arne Holmgren, and Ole P Ottersen, and Sten Orrenius, and George Bolcsfoldi, and Ian A Cotgreave
October 1995, Teratology,
Therese Söderdahl, and Mari Enoksson, and Mathias Lundberg, and Arne Holmgren, and Ole P Ottersen, and Sten Orrenius, and George Bolcsfoldi, and Ian A Cotgreave
February 1977, In vitro,
Copied contents to your clipboard!