cytochrome-c-t and antimycin

Visible spectroscopy was used to measure real-time changes in the oxidation state of cytochrome c (cyt c) and the a-cytochromes (cyt aa(3)) of cytochrome oxidase during mitochondrial outer membrane permeabilization (MOMP) initiated by anisomycin in HL-60 cells. The oxidation state of mitochondrial cyt c was found to be approximately 62% oxidized before MOMP and became approximately 70% oxidized after MOMP. In contrast, the cytosolic pool of cyt c was found to be almost fully reduced. This oxidation change allows cyt c release to be continuously and quantitatively monitored in real time. Anoxia and antimycin were used to fully reduce and fully oxidize, respectively, the mitochondrial pool of cyt c and it was found that the release of cyt c was independent of it oxidation state consistent with a simple model of cyt c passively diffusing down a concentration gradient through a pore or tear in the outer membrane. After MOMP was complete, the flux of cyt c diffusing back into the mitochondria was measured from the residual mitochondrial oxygen consumption after complete inhibition of the bc(1) with antimycin and myxothiazol. The outer membrane was found to be highly permeable after MOMP implying that the reduction of cyt c in the cytosol must be very rapid. The permeability of the outer membrane measured in this study would result in the release of cyt c with a time constant of less than 1 s.

Topics: Anisomycin; Antifungal Agents; Antimycin A; Caspase 3; Caspase 9; Cytochromes c; Cytosol; Electron Transport; Enzyme Activation; HL-60 Cells; Humans; Methacrylates; Mitochondrial Membranes; Oxidation-Reduction; Oxygen; Oxygen Consumption; Permeability; Protein Synthesis Inhibitors; Rotenone; Spectrum Analysis; Thiazoles; Uncoupling Agents

Ultrasensitivity, hysteresis (a form of biochemical memory), and all-or-none (digital) responses are important signaling properties for the control of irreversible processes and are well characterized in the c-Jun N-terminal kinase (JNK) system using Xenopus oocytes. Our aim was to study these properties in the AMP-activated protein kinase (AMPK) signaling system under stress conditions that could engage a cell death program, and compare them to the JNK responses. After characterization of Xenopus AMPK, we show here that the response to antimycin (nonapoptotic) was slightly cooperative and graded (analog) in individual oocytes, whereas the response to sorbitol (which induced cytochrome c release and caspase activation) was ultrasensitive, digital in single cells, and without hysteresis, hallmarks of a monostable system. Moreover, initial graded responses of AMPK and JNK turned into digital during a critical period for the execution of the cell death program, although single cell analysis did not show complete correlation between AMPK or JNK activation and cytochrome c release. We propose a model where the life or death decision in the cell is made by integration of multiple digital signals from stress sensors.

Topics: AMP-Activated Protein Kinases; Animals; Antifungal Agents; Antimycin A; Caspases; Cell Death; Cytochromes c; Enzyme Activation; JNK Mitogen-Activated Protein Kinases; Models, Biological; Oocytes; Signal Transduction; Sorbitol; Stress, Physiological; Sweetening Agents; Xenopus laevis; Xenopus Proteins

IMS (intermembrane space) SOD1 (Cu/Zn-superoxide dismutase) is inactive in isolated intact rat liver mitochondria and is activated following oxidative modification of its critical thiol groups. The present study aimed to identify biochemical pathways implicated in the regulation of IMS SOD1 activity and to assess the impact of its functional state on key mitochondrial events. Exogenous H2O2 (5 microM) activated SOD1 in intact mitochondria. However, neither H2O2 alone nor H2O2 in the presence of mitochondrial peroxiredoxin III activated SOD1, which was purified from mitochondria and subsequently reduced by dithiothreitol to an inactive state. The reduced enzyme was activated following incubation with the superoxide generating system, xanthine and xanthine oxidase. In intact mitochondria, the extent and duration of SOD1 activation was inversely correlated with mitochondrial superoxide production. The presence of TxrR-1 (thioredoxin reductase-1) was demonstrated in the mitochondrial IMS by Western blotting. Inhibitors of TxrR-1, CDNB (1-chloro-2,4-dinitrobenzene) or auranofin, prolonged the duration of H2O2-induced SOD1 activity in intact mitochondria. TxrR-1 inactivated SOD1 purified from mitochondria in an active oxidized state. Activation of IMS SOD1 by exogenous H2O2 delayed CaCl2-induced loss of transmembrane potential, decreased cytochrome c release and markedly prevented superoxide-induced loss of aconitase activity in intact mitochondria respiring at state-3. These findings suggest that H2O2, superoxide and TxrR-1 regulate IMS SOD1 activity reversibly, and that the active enzyme is implicated in protecting vital mitochondrial functions.

Topics: Aconitate Hydratase; Animals; Antimycin A; Apoptosis; Cell Respiration; Cytochromes c; Electron Transport; Energy Metabolism; Enzyme Activation; Hydrogen Peroxide; Male; Membrane Potentials; Mitochondria, Liver; Oxidants; Rats; Rats, Wistar; Superoxide Dismutase; Superoxide Dismutase-1; Superoxides; Thioredoxin-Disulfide Reductase

Apart from energy generation, mitochondria perform a signalling function determining the life and death of a cell under stress exposure. In the present study we have explored patterns of heat-induced synthesis of Hsp101, Hsp70, Hsp17.6 (class I), Hsp17.6 (class II) and Hsp60, and the development of induced thermotolerance in Arabidopsis thaliana cell culture under conditions of mitochondrial dysfunction. It was shown that treatment by mitochondrial inhibitors and uncouplers at the time of mild heat shock downregulates HSP synthesis, which is important for induced thermotolerance in plants. The exposure to elevated temperature induced an increase in cell oxygen consumption and hyperpolarization of the inner mitochondrial membrane. Taken together, these facts suggest that mitochondrial functions are essential for heat-induced HSP synthesis and development of induced thermotolerance in A. thaliana cell culture, suggesting that mitochondrial-nuclear cross-talk is activated under stress conditions. Treatment of Arabidopsis cell culture at 50 degrees C initiates a programmed cell death determined by the time course of viability decrease, DNA fragmentation and cytochrome c release from mitochondria. As treatment at 37 degrees C protected Arabidopsis cells from heat-induced cell death, it may be suggested that Hsp101, Hsp70 and small heat-shock proteins, the synthesis of which is induced under these conditions, are playing an anti-apoptotic role in the plant cell. On the other hand, drastic heat shock upregulated mitochondrial Hsp60 synthesis and induced its release from mitochondria to the cytosol, indicating a pro-apoptotic role of plant Hsp60.

Topics: Antimycin A; Apoptosis; Arabidopsis; Arabidopsis Proteins; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Nucleus; Cell Survival; Cells, Cultured; Cytochromes c; Dinitrobenzenes; Heat-Shock Proteins; Hot Temperature; Membrane Potential, Mitochondrial; Mitochondria; Temperature

We have investigated the interaction between monomers of the dimeric yeast cytochrome bc(1) complex by analyzing the pre-steady and steady state activities of the isolated enzyme in the presence of antimycin under conditions that allow the first turnover of ubiquinol oxidation to be observable in cytochrome c(1) reduction. At pH 8.8, where the redox potential of the iron-sulfur protein is approximately 200 mV and in a bc(1) complex with a mutated iron-sulfur protein of equally low redox potential, the amount of cytochrome c(1) reduced by several equivalents of decyl-ubiquinol in the presence of antimycin corresponded to only half of that present in the bc(1) complex. Similar experiments in the presence of several equivalents of cytochrome c also showed only half of the bc(1) complex participating in quinol oxidation. The extent of cytochrome b reduced corresponded to two b(H) hemes undergoing reduction through one center P per dimer, indicating electron transfer between the two cytochrome b subunits. Antimycin stimulated the ubiquinol-cytochrome c reductase activity of the bc(1) complex at low inhibitor/enzyme ratios. This stimulation could only be fitted to a model in which half of the bc(1) dimer is inactive when both center N sites are free, becoming active upon binding of one center N inhibitor molecule per dimer, and there is electron transfer between the cytochrome b subunits of the dimer. These results are consistent with an alternating half-of-the-sites mechanism of ubiquinol oxidation in the bc(1) complex dimer.

Topics: Antimycin A; Cytochromes b; Cytochromes c; Dimerization; Electron Transport Complex III; Fungal Proteins; Heme; Hydrogen-Ion Concentration; Iron-Sulfur Proteins; Kinetics; Mutation; Oxidation-Reduction; Oxygen; Spectrophotometry; Time Factors; Ubiquinone; Ultraviolet Rays

Drosophila melanogaster is a key model organism for genetic investigation of the role of free radicals in aging, but biochemical understanding is lacking. Superoxide production by Drosophila mitochondria was measured fluorometrically as hydrogen peroxide, using its dependence on substrates, inhibitors, and added superoxide dismutase to determine sites of production and their topology. Glycerol 3-phosphate dehydrogenase and center o of complex III in the presence of antimycin had the greatest maximum capacities to generate superoxide on the cytosolic side of the inner membrane. Complex I had significant capacity on the matrix side. Center i of complex III, cytochrome c, and complex IV produced no superoxide. Native superoxide generation by isolated mitochondria was also measured without added inhibitors. There was a high rate of superoxide production with sn-glycerol 3-phosphate as substrate; two-thirds mostly from glycerol 3-phosphate dehydrogenase on the cytosolic side and one-third on the matrix side from complex I following reverse electron transport. There was little superoxide production from any site with NADH-linked substrate. Superoxide production by complex I following reverse electron flow from glycerol 3-phosphate was particularly sensitive to membrane potential, decreasing 70% when potential decreased 10 mV, showing that mild uncoupling lowers superoxide production in the matrix very effectively.

Topics: Animals; Antimycin A; Cytochromes c; Cytosol; Drosophila melanogaster; Electron Transport; Electron Transport Complex III; Fluorometry; Glycerolphosphate Dehydrogenase; Hydrogen Peroxide; Mitochondria; NAD; Superoxide Dismutase; Superoxides