ascorbic-acid and malonic-acid

Ferricyanide reduction frequently is analyzed to determine the activity of membraneous reductases. An improved, highly sensitive, and rapid method for quantitative endpoint determination of ferrocyanide is presented. Ferrocyanide is oxidized by Fe(3+) in the presence of Ferene-S under acid conditions to form a chromogenic Ferene-S/Fe(2+) complex. The latter is quantitated at 593 nm with a sensitivity of 33.2 mM(-1) . cm(-1). The assay is 60% more sensitive to ferrocyanide (and with a 50% lower detection limit) than the prevailing method of Avron and Shavit, which employs sulfonated bathophenanthroline as the ferrous chromogen. Both pH dependence and potential sources of interference are discussed. Using the method, a sulfhydryl-sensitive, ascorbate-stimulated transplasma membrane ferricyanide reductase was assayed in human chronic myeloid (K562) leukemia cells. Furthermore, malonate-sensitive succinate dehydrogenase activity of heart mitochondria was easily assayed with ferricyanide as terminal electron acceptor. The current method will suit routine applications demanding high throughput, robustness, and sensitivity in a 96-well plate format.

Topics: Animals; Ascorbic Acid; Cations, Divalent; Cattle; Cell Membrane; Chelating Agents; Colorimetry; Ferricyanides; Ferrocyanides; Humans; Hydrogen-Ion Concentration; K562 Cells; Malonates; Mitochondria, Heart; Oxidation-Reduction; Succinates; Triazines

A decrease in total glutathione, and aberrant mitochondrial bioenergetics have been implicated in the pathogenesis of Parkinson's disease. Our previous work exemplified the importance of glutathione (GSH) in the protection of mesencephalic neurons exposed to malonate, a reversible inhibitor of mitochondrial succinate dehydrogenase/complex II. Additionally, reactive oxygen species (ROS) generation was an early, contributing event in malonate toxicity. Protection by ascorbate was found to correlate with a stimulated increase in protein-glutathione mixed disulfide (Pr-SSG) levels. The present study further examined ascorbate-glutathione interactions during mitochondrial impairment. Depletion of GSH in mesencephalic cells with buthionine sulfoximine potentiated both the malonate-induced toxicity and generation of ROS as monitored by dichlorofluorescein diacetate (DCF) fluorescence. Ascorbate completely ameliorated the increase in DCF fluorescence and toxicity in normal and GSH-depleted cultures, suggesting that protection by ascorbate was due in part to upstream removal of free radicals. Ascorbate stimulated Pr-SSG formation during mitochondrial impairment in normal and GSH-depleted cultures to a similar extent when expressed as a proportion of total GSH incorporated into mixed disulfides. Malonate increased the efflux of GSH and GSSG over time in cultures treated for 4, 6 or 8 h. The addition of ascorbate to malonate-treated cells prevented the efflux of GSH, attenuated the efflux of GSSG and regulated the intracellular GSSG/GSH ratio. Maintenance of GSSG/GSH with ascorbate plus malonate was accompanied by a stimulation of Pr-SSG formation. These findings indicate that ascorbate contributes to the maintenance of GSSG/GSH status during oxidative stress through scavenging of radical species, attenuation of GSH efflux and redistribution of GSSG to the formation of mixed disulfides. It is speculated that these events are linked by glutaredoxin, an enzyme shown to contain both dehydroascorbate reductase as well as glutathione thioltransferase activities.

Topics: Animals; Antioxidants; Ascorbic Acid; Buthionine Sulfoximine; Chromans; Disulfides; Fluorescent Dyes; Glutathione; Glutathione Disulfide; Malonates; Mesencephalon; Mitochondria; Oxidative Stress; Parkinson Disease; Rats; Rats, Sprague-Dawley

Compromised mitochondrial energy metabolism and oxidative stress have been associated with the pathophysiology of Parkinson's disease. Our previous experiments exemplified the importance of GSH in the protection of neurons exposed to malonate, a reversible inhibitor of mitochondrial succinate dehydrogenase/complex II. This study further defines the role of oxidative stress during energy inhibition and begins to unravel the mechanisms by which GSH and other antioxidants may contribute to cell survival. Treatment of mesencephalic cultures with 10 microM buthionine sulfoximine for 24 h depleted total GSH by 60%, whereas 3 h exposure to 5 mM 3-amino-1,2,4-triazole irreversibly inactivated catalase activity by 90%. Treatment of GSH-depleted cells with malonate (40 mM) for 6, 12 or 24 h both potentiated and accelerated the time course of malonate toxicity, however, inhibition of catalase had no effect. In contrast, concomitant treatment with buthionine sulfoximine plus 3-amino-1,2,4-triazole in the presence of malonate significantly potentiated toxicity over that observed with malonate plus either inhibitor alone. Consistent with these findings, GSH depletion enhanced malonate-induced reactive oxygen species generation prior to the onset of toxicity. These findings demonstrate that early generation of reactive oxygen species during mitochondrial inhibition contributes to cell damage and that GSH serves as a first line of defense in its removal. Pre-treatment of cultures with 400 microM ascorbate protected completely against malonate toxicity (50 mM, 12 h), whereas treatment with 1 mM Trolox provided partial protection. Protein-GSH mixed disulfide formation during oxidative stress has been suggested to either protect vulnerable protein thiols or conversely to contribute to toxicity. Malonate exposure (50 mM) for 12 h resulted in a modest increase in mixed disulfide formation. However, exposure to the protective combination of ascorbate plus malonate increased membrane bound protein-GSH mixed disulfides three-fold. Mixed disulfide levels returned to baseline by 72 h of recovery indicating the reversible nature of this formation. These results demonstrate an early role for oxidative events during mitochondrial impairment and stress the importance of the glutathione system for removal of reactive oxygen species. Catalase may serve as a secondary defense as the glutathione system becomes limiting. These findings also suggest that protein-GSH mixed disulfide formatio

Topics: Amitrole; Animals; Antioxidants; Ascorbic Acid; Catalase; Cells, Cultured; Chromans; Energy Metabolism; Enzyme Inhibitors; Glutathione Disulfide; Hydrogen Peroxide; Malonates; Mesencephalon; Neurons; Oxidative Stress; Parkinson Disease; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species

The sum of the flux control coefficients for group-transfer reactions such as electron transport has been proposed to be two when the coefficients are calculated from experiments in which the concentrations of the electron carriers are changed (CE) but one when they are calculated from changes in the rates of the electron-transfer processes (Cv). We tested this proposal using electron transport in uncoupled beef heart, potato tuber and rat liver mitochondria. First, with ascorbate plus N,N,N',N"-tetramethyl-p-phenylenediamine as substrate, the CE flux control coefficients of ascorbate, N,N,N',N"-tetramethyl-p-phenylenediamine, mitochondria and oxygen over electron-transport rate were measured by direct titration of the concentrations of these electron carriers. CE values were close to zero, one, one and zero, respectively, giving a sum of CE flux control coefficients of approximately two. At higher concentrations of N,N,N',N'-tetramethyl-p-phenylenediamine, its CE control decreased and the sum decreased towards one as predicted. Secondly, the Cv control coefficients of groups of electron-transfer processes with succinate or ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine as substrate were measured. This was achieved by measuring the effects of KCN (or malonate or N,N,N',N'-tetramethyl-p-phenylenediamine) on system flux when intermediates were allowed to relax and on local flux when intermediates were held constant. The Cv flux control coefficients were calculated as the ratio of the effects on system flux and on local flux. The sum of the Cv flux control coefficients was approximately one. Whether a sum of one or a sum of two was obtained depended entirely on the definition of control coefficients that was used, since either sum was obtained from the same set of data depending on the method of calculation. Both definitions are valid, but they give different information. It is important to be aware of which definition is being used when analysing control coefficients in electron-transport chains and other group-transfer systems.

Topics: Animals; Ascorbic Acid; Cattle; Cytochrome c Group; Electron Transport; Kinetics; Malonates; Mitochondria; Mitochondria, Heart; Mitochondria, Liver; Oxidation-Reduction; Oxygen Consumption; Potassium Cyanide; Rats; Solanum tuberosum; Succinates; Succinic Acid; Tetramethylphenylenediamine

A modified purification procedure has been developed for dopamine beta-hydroxylase isolated from bovine adrenal medulla. Catalase is included in the homogenization step starting with a suspension of either chromaffin granules or adrenal medulla tissue. With this precaution, the enzyme remains stable in the supernatant solution in preparation for the subsequent purification step involving concanavalin A-Sepharose chromatography. The homogeneous enzyme has a specific activity in the range of 60-70 mumol O2 consumed/min/mg. Using radiolabeled metal ion chelators, it was determined that several of the chelators remained tightly bound to the enzyme after removal of the copper leading to difficulties in establishing stoichiometry of enzyme-bound metal ions.

Topics: Adrenal Medulla; Animals; Ascorbic Acid; Catalase; Cattle; Chromaffin Granules; Chromaffin System; Chromatography; Copper; Dopamine beta-Hydroxylase; Edetic Acid; Histidine; Kinetics; Malonates; Oxygen