An evaluation of the effects of five buffers on respiratory parameters of isolated mitochondria

1968 ◽  
Vol 46 (1) ◽  
pp. 43-50 ◽  
Author(s):  
R. A. Stinson ◽  
Mary Spencer
Keyword(s):  
Cytochrome C ◽  
Respiration Rate ◽  
Mitochondrial Respiration ◽  
Phosphate Buffer ◽  
Respiratory Control ◽  
Isolated Mitochondria ◽  
Respiratory Parameters ◽  
Control Respiration ◽  
Ethanesulfonic Acid ◽  
Ph Range

Certain compounds, recently introduced as buffers for biological work in the pH range 6–8, were evaluated by measurement of their effects on mitochondrial respiration. Respiratory control, respiration rate, ADP/O ratio, and the effect of added cytochrome c were measured on bean cotyledon mitochondria, after isolation and incubation of the mitochondria in one of five buffers: phosphate, Tris, Tricine (N-tris(hydroxymethyl)methylglycine), TES (N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid), and HEPES (N-2-hydroxyethylpiperazine-N1-2-ethanesulfonic acid). In all parameters measured, except response to added cytochrome c, purified TES was found to be superior to all other buffers. HEPES was the best in prevention of leakage of cytochrome c from the mitochondria but in other respects was similar to Tricine. Phosphate buffer proved superior to Tricine only with respect to cytochrome c retention by the mitochondria. Tris was the poorest buffer in which to measure any of these parameters. Aging the mitochondria in the various buffers amplified the inadequacies of the buffers and served to show the superiority of purified TES.

Oxidative phosphorylation and respiratory control in mitochondria from Aspergillus oryzae

1969 ◽  
Vol 15 (8) ◽  
pp. 975-977 ◽  
Author(s):  
K. Watson ◽  
W. Paton ◽  
J. E. Smith
Keyword(s):  
Bovine Serum Albumin ◽  
Oxidative Phosphorylation ◽  
Aspergillus Oryzae ◽  
Reaction Medium ◽  
Respiratory Control ◽  
Adenosine Diphosphate ◽  
Yeast Mitochondria ◽  
Active Oxidation ◽  
Isolated Mitochondria ◽  
Ph Range

Mitochondria isolated from Aspergillus oryzae exhibited respiratory control with a range of substrates. Bovine serum albumin was required in the reaction medium to observe adenosine diphosphate (ADP) controlled respiration. The mitochondria carried out active oxidation and phosphorylation with citrate as substrate in the pH range 6–7 and showed a slight optimum for oxidative phosphorylation at pH 6.5. The respiratory properties of the isolated mitochondria were similar to those reported for A. niger and yeast mitochondria.

Control of proteoliposomal cytochrome c oxidase: the overall reaction

1990 ◽  
Vol 68 (9) ◽  
pp. 1128-1134 ◽  
Author(s):  
Peter Nicholls ◽  
Chris E. Cooper ◽  
John M. Wrigglesworth
Keyword(s):  
Steady State ◽  
Membrane Potential ◽  
Cytochrome C ◽  
Cytochrome Oxidase ◽  
Cytochrome C Oxidase ◽  
Respiration Rate ◽  
Respiratory Control ◽  
Ph Gradient ◽  
Activity Levels ◽  
Simulation Programme

The control of cytochrome c oxidase incorporated into proteoliposomes has been investigated as a function of membrane potential (ΔΨ) and pH gradient (ΔpH). The oxidase generates a pH gradient (alkaline inside) and a membrane potential (negative inside) when respiring on external cytochrome c. Low levels of valinomycin collapse ΔΨ and increase ΔpH; the respiration rate decreases. High levels of valinomycin, however, decrease ΔpH as valinomycin can also act as a protonophore. Nigericin (in the absence of valinomycin) increases ΔΨ and collapses ΔpH; the respiration rate increases. On a millivolt equivalent basis ΔpH is a more effective inhibitor of activity than is ΔΨ. In the absence of any ionophores the cytochrome oxidase proteoliposomes enter a steady state, in which there are both ΔpH and ΔΨ components of control. Present and previous data suggest that the respiration rate responds in a linear way ("ohmically") to increasing ΔpH but in a nonlinear way to ΔΨ ("non-ohmically"). High levels of both ΔΨ and ΔpH do not completely inhibit turnover (maximal respiratory control values lie between 6 and 10). The controlled steady state involves the electrophoretic entry and electroneutral exit of K+ from the vesicles. A model is presented in which the enzyme responds to both ΔpH and ΔΨ components of the proton-motive force, but is more sensitive to ΔpH than to ΔΨ at an equivalent ΔμH+. The steady state of the proteoliposome system can be represented for any set of permeabilities and enzyme activity levels using the computer simulation programme Stella™.Key words: cytochrome c, cytochrome oxidase, proteoliposomes, respiratory control, modelling, valinomycin, nigericin.

scholarly journals Changes in Alternative Pathway and Mitochondrial Respiration in Avocado in Response to Elevated Carbon Dioxide Levels

1997 ◽  
Vol 122 (2) ◽  
pp. 245-252 ◽  
Author(s):  
Diana L. Lange ◽  
Adel A. Kader
Keyword(s):  
Mitochondrial Respiration ◽  
Alternative Oxidase ◽  
Alternative Pathway ◽  
Respiratory Control ◽  
Persea Americana ◽  
Isolated Mitochondria ◽  
Harvest Dates ◽  
Avocado Fruit ◽  
Carbon Dioxide Levels

Partially ripened avocado [Persea americana (Mill.) cv. Hass] fruit harvested in either June or Aug. 1994 were kept at 10 °C in air (21% O2), 20% CO2 (17% O2, balance N2), or 40% CO2 (13% O2, balance N2) for 7 to 12 days and then were transferred to air at 10 °C for 2 to 3 days. Mitochondrial respiration was stimulated in response to elevated CO2 treatments at 10 °C. A shift to alternative pathway (Alt) respiration occurred on day 4 in experiments using avocados from both harvest dates, with a return to initial levels in only the 20% CO2-treated fruit (June-harvested fruit after return to air). Elevated CO2 at 20 °C decreased the in vitro O2 consumption of isolated mitochondria compared to mitochondria kept in air. The Alt pathway contributed less to the total O2 uptake of CO2-treated mitochondria compared to mitochondria kept in air. The respiratory control ratios of the CO2-treated fruit and mitochondria were higher and lower, respectively, than the air controls. Induction of 33 to 37 kD proteins (corresponding to the size of the alternative oxidase proteins) occurred in avocados after 4 days in 40% CO2. These results indicate that elevated CO2 has various effects depending on concentration, duration and temperature of exposure, and mitochondrial function of avocado fruit, such as increased and altered respiratory oxidation and up-regulation of alternative oxidase proteins.

scholarly journals Feeding effects on liver mitochondrial bioenergetics of Boa constrictor (Serpentes: Boidae)

2021 ◽  
Author(s):  
Helena Rachel da Mota Araujo ◽  
Marina Rincon Sartori ◽  
Claudia D. C. Navarro ◽  
José Eduardo de Carvalho ◽  
André Luis da Cruz
Keyword(s):  
Mitochondrial Respiration ◽  
Citrate Synthase ◽  
Mitochondrial Permeability Transition ◽  
Respiratory Control ◽  
Permeability Transition ◽  
Mitochondrial Bioenergetics ◽  
Retention Capacity ◽  
Isolated Mitochondria ◽  
Respiration Rates ◽  
Boa Constrictor

Snakes are interesting examples of overcoming energy metabolism challenges as many species can endure long periods without feeding, and their eventual meals are of reasonably large sizes, thus exhibiting dual extreme adaptations. Consequently, metabolic rate increases considerably to attend to the energetic demand of digestion, absorption, and protein synthesis. These animals should be adapted to transition from these two opposite states of energy fairly quickly, and therefore we investigated mitochondrial function plasticity in these states. Herein we compared liver mitochondrial bioenergetics of the boid snake Boa constrictor during fasting and after meal intake. We fasted the snakes for 60 days, and then we fed a subgroup with 30% of their body size and evaluated their maximum postprandial response. We measured liver respiration rates from permeabilized tissue and isolated mitochondria. From isolated mitochondria, we also measured Ca2+ retention capacity and redox status. Mitochondrial respiration rates were maximized after feeding, reaching approximately a 60% increase from fasting levels when energized with complex I-linked substrates. Interestingly, fasting and fed snakes exhibited similar respiratory control ratios and citrate synthase activity. Furthermore, we found no differences in Ca2+ retention capacity, indicating no increase in susceptibility to mitochondrial permeability transition (MPT), and no changes in mitochondrial redox state, although fed animals exhibited increases in the release of H2O2. Thus, we conclude that liver mitochondria from B. constrictor snakes increase mitochondrial respiration rates during the postprandial period and quickly improve the mitochondrial bioenergetics capacity without compromising redox balance.

Kinetics and mechanism of the reaction between oxidized cytochrome c and ascorbic acid

1991 ◽  
Vol 56 (2) ◽  
pp. 478-490 ◽  
Author(s):  
Joaquin F. Perez-Benito ◽  
Conchita Arias
Keyword(s):  
Ascorbic Acid ◽  
Cytochrome C ◽  
Redox Reactions ◽  
Arrhenius Equation ◽  
Horse Heart Cytochrome ◽  
First Order ◽  
Acidic Form ◽  
Ph Range ◽  
Horse Heart Cytochrome C ◽  
Mechanism Of The Reaction

The reaction between horse-heart cytochrome c and ascorbic acid has been investigated in the pH range 5.5 – 7.1 and at 10.0 – 25.0 °C. The rate shows a first-order dependence on the concentration of cytochrome c, it increases in a non-linear way as the concentration of ascorbic acid increases, it increases markedly with increasing pH and, provided that the ionic strength of the medium is high enough, it fulfills the Arrhenius equation. The apparent activation energy increases as the pH of the solution increases. The results have been explained by means of a mechanism that includes the existence of an equilibrium between two forms (acidic and basic) of oxidized cytochrome c: cyt-H+ -Fe3+ + OH- cyt -Fe3+ + H2O, whose equilibrium constant is (6.7 ± 1.4). 108 at 25.0 °C, the acidic form being more reducible than the basic one. It is suggested that there is a linkage of hydrogenascorbate ion to both forms of cytochrome c previous to the redox reactions. Two possibilities for the oxidant-reductant linkage (binding and adsorption) are discussed in detail.

Pyridine–Adenine Dinucleotide Transhydrogenase Activity in Cells Cultured from Rat Hepatoma

1972 ◽  
Vol 50 (5) ◽  
pp. 447-456 ◽  
Author(s):  
C. De Luca ◽  
R. P. Gioeli
Keyword(s):  
Phosphate Buffer ◽  
Pyridine Nucleotide ◽  
Maximum Activity ◽  
Ph Optimum ◽  
Apparent Lack ◽  
Minimal Deviation ◽  
Ph Range ◽  
Pyridine Nucleotide Transhydrogenase ◽  
Rat Hepatoma ◽  
Cells Cultured

Preparations from cells cultured from a minimal-deviation hepatoma in the rat exhibit pyridine nucleotide transhydrogenase (NAD(P)H: NAD(P) oxidoreductase, EC 1.6.1.1) activity. The pH optimum, its release by digitonin, and its apparent lack of dependence on steroids for activity tentatively classify it as a transhydrogenase of the type first described for animal tissue.Enzyme preparations from digitonin-treated homogenates were very unstable. The time necessary for the loss of one-half the activity was 16–18 h when the enzyme was stored at 5 °C; this was reduced to 4 h when storage was in polycarbonate tubes.The enzyme apparently transferred hydrogen directly and with equal ease from NADH to both the 3-acetyl-pyridine and thionicotinamide analogues of NAD. Half-saturation values for NAD and its acetylpyridine analogue were 0.99 × 10−5 M and 3.55 × 10−4 M, respectively. The enzyme exhibited its maximum activity in phosphate buffer at pH 5.8. It was inhibited by 50–60% over the pH range 7.0–8.5 in Tris buffer. This could be reversed by dithiothreitol; reversal was complete between pH 8.0 and 8.5.

Changes in oxygen tension affect cardiac mitochondrial respiration rate via changes in the rate of mitochondrial hydrogen peroxide production

2009 ◽  
Vol 47 (1) ◽  
pp. 49-56 ◽  
Author(s):  
Carla A. Di Maria ◽  
Marie A. Bogoyevitch ◽  
Douglas J. McKitrick ◽  
Leonard F. Arnolda ◽  
Livia C. Hool ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Oxygen Tension ◽  
Respiration Rate ◽  
Mitochondrial Respiration ◽  
Hydrogen Peroxide Production

Kinetics of the cytochrome c oxidase and reductase reactions in energized and de-energized mitochondria

1977 ◽  
Vol 55 (7) ◽  
pp. 706-713 ◽  
Author(s):  
Lars Chr. Petersen ◽  
Hans Degn ◽  
Peter Nicholls
Keyword(s):  
Steady State ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Oxygen Concentration ◽  
Respiration Rate ◽  
Redox State ◽  
Rat Liver Mitochondria ◽  
Excess Oxygen ◽  
Succinate Oxidation ◽  
State Reduction

1. Coupled, cytochrome-c-depleted ('stripped') rat liver mitochondria reducing oxygen in the presence of exogenous cytochrome c, with succinate or ascorbate as substrates, show marked declines in the steady-state reduction of cytochrome c in excess oxygen on addition of uncouplers. Calculated ratios of maximal turnover in the uncoupled state and in the energized state for the cytochrome c oxidase (EC 1.9.3.1) reaction lie between 3 and 6, as obtained with reconstituted oxidase-containing vesicles. The succinate-cytochrome c reductase activity in such mitochondria shows a smaller response to uncoupler than that of the oxidase.2. The respiration rates of uncoupled mitochondria oxidizing ascorbate in the presence of added cytochrome c follow a Michaelis–Menten relationship with respect to oxygen concentration, in accordance with the pattern found previously with the solubilized oxidase. But succinate oxidation tends to give nonlinear concave-upward double-reciprocal plots of respiration rate against oxygen concentration, in accordance with the pattern found previously with intact uncoupled mitochondria.3. From simultaneous measurements of cytochrome c steady-state reduction, respiration rate, and oxygen concentration during succinate oxidation under uncoupled conditions it is found that at full reduction of cytochrome c, apparent Km for oxygen is 0.9 μM and the maximal oxidase (aa3) turnover is 400 s−1 (pH 7.4, 30 °C).4. The redox state of cytochrome c in uncoupled systems reflects a simple steady state; the redox state of cytochrome c in energized systems tends towards an equilibrium condition with the terminal cytochrome a3, whose apparent potential under these conditions is more negative than that of cytochrome c.

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