Nechama S. Kosower

The Glutathione Status of Cells

Publisher Summary Glutathione (GSH) is the most important nonprotein thiol in living systems and is of widespread occurrence in the intracellular milieu of animals, plants, and microorganisms. GSH was isolated and named by the English biochemist Frederick Gowland Hopkins. This chapter discusses GSH status, the biologically relevant chemistry of GSH, the forms in which GSH can be present within the cell, along with the GSH content of cells and the methods for analysis of this substance. GSH-related biochemical reactions and the biological roles of GSH are discussed in the chapter. The use of perturbations in GSH status as a means for investigating GSH-related phenomena and an analysis of the consequences of perturbation are presented. A short summary of genetic lesions related to GSH is also included. Like chemically induced perturbations in GSH status, genetic lesions provide valuable insights into the role of GSH in normal functions and processes in cells. The chapter concludes with some brief comments about the future of the relationship of GSH status to cellular processes.

Diamide, a new reagent for the intracellular oxidation of glutathione to the disulfide

Abstract A new thiol-oxidizing agent, diamide, (CH3)2NCONNCON(CH3)2, is shown to stoichiometrically oxidize glutathione (GSH) within the human red blood cell to the disulfide (GSSG). Conversion of GSH to GSSG is very rapid, even at 1–40. No inter ference with cellular function is observed as shown by almost complete regeneration of GSH after incubation with glucose at 370, and no alteration in hemoglobin, osmotic fragility or cell density is found.

[11] Diamide: An oxidant probe for thiols

Publisher Summary The most convenient and chemically simple agent—diamide—is capable of producing a rapid diminution of the tripeptide thiol—glutathione (GSH)—within erythrocytes. An oxidant probe changes the oxidation state of the system; in the present case, diamide is an oxidant probe for thiols and changes the oxidation state of the thiols. Diamide perturbs the thiol status of a system. In most cases, the system can return to its original state by reduction. Information about the role of thiols in the biochemical, biophysical, and physiological economy of a biological system can be gained by the treatment of a system with an oxidant probe for thiols. The reaction of diamide with thiols can be followed spectrophotometrically between 300 and 325 nm and yields a second-order rate constant. The reduction of the diazene forms the diazane dicarboxylic acid bis( N,N -dimethylamide), a hydrazide that does not absorb down to 230 nm. The reaction of thiols with diazenecarbonyl derivatives—such as diamide—occurs in two observable stages, with thiolate anions (RS − ) as the reactive species. The reaction proceeds via addition and displacement steps. In the case of GSH, the GS − anion adds to the diazene double bond to form a sulfenylhydrazine, which, in a second step, reacts with a second GS − anion at sulfur to yield a disulfide and a hydrazine.

Thiol labeling with bromobimanes

Publisher Summary This chapter describes the use of three bromobimanes for fluorescent labeling of biological systems. The three bromobimanes, mBBr, bBBr, and qBBr, are commercially available. Bromobimanes are derived from a basic structure (two fused five-membered rings) with the requisite minimal size. In the course of preparing the bromo derivatives, it was found fortuitously that proteins were fluorescently labeled by bromobimanes. It was established that bromobimanes reacted preferentially with thiols and demonstrated the usefulness of such labeling for both small and large molecules in biological systems. Monobromobimane, mBBr, is the most frequently used in the series, not only as a labeling agent for thiols, but also for studies on the consequences of the alkylation of reactive thiols in biological systems. In a labeling reaction, involving multifunctional molecules like proteins, the first step involves displacement of the bromine of bBBr by a thiol, but the second bromine may react with a less reactive nucleophilic group if its local concentration is high, an expression of the neighboring group effect. This chapter discusses the labeling procedures including bromobimane solutions, reactions in solution, and reactions in cells and tissues.

Glutathione VII. Differentiation among substrates by the thiol-oxidizing agent, diamide

Abstract 1. 1. Glutathione reacts much more rapidly with the thiol-oxidizing agent, diamide, than do other small molecule cellular constituents like NADH, CoASH and lipoic acid. 2. 2. Combination of rate data and concentration data for important oxidizable cellular constituents leads to the conclusion that GSH is the major target for diamide within the cell. 3. 3. Conversion of GSH to GSSG is the most important immediate consequence of diamide treatment of a cell suspension. 4. 4. Consideration of the rate constants for thiol-disulfide interchange reactions leads to the idea that further chemical and physical changes may occur shortly after the initial GSH-GSSG equilibrium has been perturbed by diamide. The time scale of an experiment defines how extensive these sequellae might be. 5. 5. A new term, the thiol-disulphide status of the cell is defined. 6. 6. Perturbations of the thiol-disulfide status of cells and organisms by thiol-oxidizing agents have been demonstrated as important in many problems, including protein synthesis and neurotransmitter release.

Glutathione: VIII. The effects of glutathione disulfide on initiation of protein synthesis

Abstract 1. 1. Glutathione disulfide (GSSG) additions (1 · 10−4–2· 10−4 M or less) to rabbit reticulocyte lysates containing glutathione (GSH) in normal amounts (0.7 · 10−3–3 · 10−3 M) have no immediate effect on the rate of protein synthesis at 33°C but cause a rapid decrease in synthesis rate after 6–9 min (the lag period), followed by complete cessation of protein-synthesizing activity. 2. 2. The lag period varies with temperature; the length of the lag period and the time dependence of the decrease in synthesis provide important information for the interpretation of the GSSG effect on protein synthesis. 3. 3. Reduction of GSSG to GSH before or during the lag period prevents inhibition of protein synthesis. Such reduction has no effect on the behavior of the system if carried out after the lag period. 4. 4. Cessation of protein synthesis is accompanied by a conversion of polysomes to monosomes, with little dissociation into subunits. 5. 5. A ribosome-free supernatant is effective in reversing the inhibition of protein synthesis caused by GSSG. Fractionation of the supernatant leads to a protein (precipitated by 40–70 % (NH4)2SO4) which reverses the GSSG inhibition. Further fractionation yields a high molecular weight fraction with inhibition-reversing activity. 6. 6. Factor Q is the name given to the material with inhibition-reversing activity, the inhibition-reversal factor. A scheme for the behavior of Factor Q in protein synthesis is proposed. 7. 7. The effect of the antibiotic, cycloheximide, on initiation of protein synthesis is interpreted in light of the GSSG effect. 8. 8. A small rise in the usual GSSG concentration of a cell is recognized as a potentially important event, one requiring careful scrutiny.

[12] Bromobimane probes for thiols

Publisher Summary This chapter discusses bromobimane probes for thiols and describes the use of four bromobimanes for fluorescent labeling of biochemical and biological systems. The four bromobimanes are (1) mBBr, (2) bBBr, (3) qBBr, and (4) SBBr. The bromobimanes are essentially nonfluorescent and are relatively stable in the dry state when stored in the dark. Chromatography on thin-layer silica yields a yellow nonfluorescent spot that develops a blue fluorescence after several minutes of exposure to 360-nm light. The change is a convenient characteristic for the identification of the bromobimanes and suggests sensitivity to light (photolysis). Bromobimanes in solution react with small thiols (e.g., the tripeptide thiol glutathione [GSH]), and with reactive protein thiol groups (e.g., hemoglobin). The reactions of bromobimanes with thiols are second order and dependent on pH, the active nucleophile being the thiolate anion, such as GS − . The reaction of bromobimane with a thiolate converts the nonfluorescent agent into water-soluble fluorescent products. The chapter discusses the chemical and photophysical properties of bromobimanes, labeling procedures, labeling of thiols in tissues, and other applications of thiol labeling.

Formation of disulfides with diamide

Publisher Summary This chapter discusses the formation of disulfides with diamide. Several diazenecarbonyl derivatives were found to be active for the conversion of thiols to disulfides. The most convenient and chemically simple agent was diazenedicarboxylic acid bis(N,N-di-methylamide), now known by the trivial name, “diamide.” Diamide is a yellow, nonhygroscopic solid, easily soluble in both water and organic solvents, and rather stable toward hydrolysis. Diamide penetrates cell membranes within seconds and also reacts within the cell at a high rate (seconds to minutes) at physiological pH. Diamide is effective in the absence of oxygen, conditions under which metabolic activity is minimal. There is a simple stoichiometric relationship between the amount of agent added and the quantity of thiol reacted. Because the reaction of diamide with thiols has a low activation energy, thiol oxidation is fast at low temperatures. Reaction can be terminated through removal of diamide by washing of the cells or stopped instantaneously by the addition of acid. In most cases, diamide treatment does not cause any irreversible damage, and, after incubation of the cells with appropriate substrates at a suitable temperature, the original thiol status is recovered. Diamide treatment thus allows the study of cell functions altered by a perturbation in thiol status.

On the involvement of calpains in the degradation of the tumor suppressor protein p53

A crude fraction that contains ubiquitin–protein ligases contains also a proteolytic activity of ∼100 kDa that cleaves p53 to several fragments. The protease does not require ATP and is inhibited in the crude extract by an endogenous ∼250 kDa inhibitor. The proteinase can be inhibited by chelating the Ca2+ ions, by specific cysteine proteinase inhibitors and by peptide aldehyde derivatives that inhibit calpains. Purified calpain demonstrates an identical activity that can be inhibited by calpastatin, the specific protein inhibitor of the enzyme. Thus, it appears that the activity we have identified in the extract is catalyzed by calpain. The calpain in the extract degrades also N‐myc, c‐Fos and c‐Jun, but not lysozyme. In crude extract, the calpain activity can be demonstrated only when the molar ratio of the calpain exceeds that of its native inhibitor. Recent experimental evidence implicates both the ubiquitin proteasome pathway and calpain in the degradation of the tumor suppressor, and it was proposed that the two pathways may play a role in targeting the protein under various conditions. The potential role of the two systems in this important metabolic process is discussed.

Calpain and calpastatin in myoblast differentiation and fusion: effects of inhibitors.

Myoblast differentiation and fusion to multinucleated muscle cells can be studied in myoblasts grown in culture. Calpain (Ca(2+)-activated thiol protease) induced proteolysis has been suggested to play a role in myoblast fusion. We previously showed that calpastatin (the endogenous inhibitor of calpain) plays a role in cell membrane fusion. Using the red cell as a model, we found that red cell fusion required calpain activation and that fusibility depended on the ratio of cell calpain to calpastatin. We found recently that calpastatin diminishes markedly in myoblasts during myoblast differentiation just prior to the start of fusion, allowing calpain activation at that stage; calpastatin reappears at a later stage (myotube formation). In the present study, the myoblast fusion inhibitors TGF-beta, EGTA and calpeptin (an inhibitor of cysteine proteases) were used to probe the relation of calpastatin to myoblast fusion. Rat L8 myoblasts were induced to differentiate and fuse in serum-poor medium containing insulin. TGF-beta and EGTA prevented the diminution of calpastatin. Calpeptin inhibited fusion without preventing diminution of calpastatin, by inhibiting calpain activity directly. Protein levels of mu-calpain and m-calpain did not change significantly in fusing myoblasts, nor in the inhibited, non-fusing myoblasts. The results indicate that calpastatin level is modulated by certain growth and differentiation factors and that its continuous presence results in the inhibition of myoblast fusion.