scholarly journals The oxidative activities of membrane vesicles from Bacillus caldolyticus. Energy-dependence of succinate oxidation

1978 ◽  
Vol 170 (2) ◽  
pp. 395-405 ◽  
Author(s):  
Anthony G. Dawson ◽  
J. B. Chappell
Keyword(s):  
Cytochrome C ◽  
Succinate Dehydrogenase ◽  
Cytochrome B ◽  
Atp Hydrolysis ◽  
Membrane Vesicles ◽  
Protonmotive Force ◽  
Anaerobic Conditions ◽  
Succinate Oxidation ◽  
Ample Supply ◽  
Energy Conserving

1. The properties of membrane vesicles from the extreme thermophile Bacillus caldolyticus were investigated. 2. Vesicles prepared by exposure of spheroplasts to ultrasound contained cytochromes a, b and c, and at 50°C they rapidly oxidized NADH and ascorbate in the presence of tetramethyl-p-phenylenediamine. Succinate and l-malate were oxidized more slowly, and dl-lactate, l-alanine and glycerol 1-phosphate were not oxidized. 3. In the absence of proton-conducting uncouplers the oxidation of NADH was accompanied by a net translocation of H+ into the vesicles. Hydrolysis of ATP by a dicyclohexylcarbodi-imide-sensitive adenosine triphosphatase was accompanied by a similarly directed net translocation of H+. 4. Uncouplers (carbonyl cyanide p-trifluoromethoxyphenylhydrazone or valinomycin plus NH4+) prevented net H+ translocation but stimulated ATP hydrolysis, NADH oxidation and ascorbate oxidation. The last result suggested an energy-conserving site in the respiratory chain between cytochrome c and oxygen. 5. Under anaerobic conditions the reduction of cytochrome b by ascorbate (with tetramethyl-p-phenylenediamine) was stimulated by ATP hydrolysis, indicating an energy-conserving site between cytochrome b and cytochrome c. However, no reduction of NAD+ supported by oxidation of succinate, malate or ascorbate occurred, neither did it with these substrates in the presence of ATP under anaerobic conditions, suggesting that there was no energy-conserving site between NADH and cytochrome b. 6. Succinate oxidation, in contrast with that of NADH and ascorbate, was strongly inhibited by uncouplers and stimulated by ATP hydrolysis. These effects were not observed when phenazine methosulphate, which transfers electrons from succinate dehydrogenase directly to oxygen, was present. It was concluded that in these vesicles the oxidation of succinate was energy-dependent and that the reoxidation of reduced succinate dehydrogenase was dependent on the outward movement of H+ by the protonmotive force. 7. In support of the foregoing conclusion it was shown that the reduction of fumarate by NADH was an energy-conserving process. 8. If the activities of vesicles accurately represent those of the intact organism it appears that in B. caldolyticus the reduction of fumarate to succinate at the expense of reducing equivalents from NADH is energetically favoured over succinate oxidation even under aerobic conditions. This may be related to the need for an ample supply of succinate for haem synthesis in order to provide cytochromes for the organism.

scholarly journals Biochemical heterogeneity of rat liver mitochondria separated by rate zonal centrifugation

1972 ◽  
Vol 129 (1) ◽  
pp. 209-218 ◽  
Author(s):  
M. A. Wilson ◽  
J. Cascarano
Keyword(s):  
Cytochrome C ◽  
Succinate Dehydrogenase ◽  
Dehydrogenase Activity ◽  
Cytochrome B ◽  
Rat Liver ◽  
Nadh Dehydrogenase ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Osmotic Gradient ◽  
Glycerophosphate Dehydrogenase

1. Rat liver mitochondria were separated on the basis of their sedimentation coefficients in an iso-osmotic gradient of Ficoll–sucrose by rate zonal centrifugation. The fractions (33, each of 40ml) were collected in order of decreasing density. Fractions were analysed by spectral analysis to determine any differences in the concentrations of the cytochromes and by enzyme analyses to ascertain any differences in the activities of NADH dehydrogenase, succinate dehydrogenase and α-glycerophosphate dehydrogenase. 2. When plotted as% of the highest specific concentration, the contents of cytochrome a+a3 and cytochrome c+c1 were constant in all fractions but cytochrome b was only 65% of its maximal concentration in fraction 7 and increased with subsequent fractions. As a result, the cytochrome b/cytochrome a+a3 ratio almost doubled between fractions 7 and 25 whereas the cytochrome c+c1/cytochrome a+a3 ratio was unchanged. 3. Expression of the dehydrogenase activities as% of highest specific activity showed the following for fractions 6–26: NADH dehydrogenase activity remained fairly constant in all fractions; succinate dehydrogenase activity was 62% in fraction 6 and increased steadily to its maximum in fraction 18 and then decreased; the activity of α-glycerophosphate dehydrogenase was only 53% in fraction 6 and increased slowly to its peak in fractions 22 and 24. 4. These differences did not result from damaged or fragmented mitochondria or from microsomal contamination. 5. These results demonstrate that isolated liver mitochondria are biochemically heterogeneous. The importance of using a system for separating biochemically different mitochondria in studies of mitochondrial biogenesis is discussed.

F0 Cysteine, bCys21, in the Escherichia coli ATP Synthase Is Involved in Regulation of Potassium Uptake and Molecular Hydrogen Production in Anaerobic Conditions

2002 ◽  
Vol 22 (3-4) ◽  
pp. 421-430 ◽  
Author(s):  
Nelli Mnatsakanyan ◽  
Karine Bagramyan ◽  
Anait Vassilian ◽  
Robert K. Nakamoto ◽  
Armen Trchounian
Keyword(s):  
Escherichia Coli ◽  
Hydrogen Production ◽  
Atp Synthase ◽  
Molecular Hydrogen ◽  
Atp Hydrolysis ◽  
Membrane Vesicles ◽  
Anaerobic Conditions ◽  
Whole Cells ◽  
B Subunit ◽  
Hydrolysis Of

The single cysteine in the b subunit of the membranous F0 sector and the 19 cysteines in extramembranous F1 sector of the Escherichia coli ATP synthase were replaced by alanine. When cells were grown under anaerobic conditions on glucose, the kcat for ATP hydrolysis of membrane vesicles containing the bCys21Ala mutant enzyme, but not enzymes with other cysteine replacements, was lower, while ATP-driven H+ pumping was unchanged. However, the ATP-dependent increase in the number of accessible thiol groups in membrane vesicles was negated. Furthermore, K+ uptake and molecular hydrogen production by whole cells and protoplasts was greatly decreased. These results indicate a role for the F0 subunit bCys21 in the functionality of F0F1 and coupling to other membranous activities under fermentative conditions.

scholarly journals The protonmotive force in phosphorylating membrane vesicles from Paracoccus denitrificans. Magnitude, sites of generation and comparison with the phosphorylation potential

1978 ◽  
Vol 174 (1) ◽  
pp. 257-266 ◽  
Author(s):  
Douglas B. Kell ◽  
Philip John ◽  
Stuart J. Ferguson
Keyword(s):  
Membrane Potential ◽  
Paracoccus Denitrificans ◽  
Atp Synthesis ◽  
Membrane Vesicles ◽  
Energy Coupling ◽  
Ph Gradient ◽  
Protonmotive Force ◽  
Electron Mediator ◽  
Succinate Oxidation ◽  
Phosphorylation Potential

1. The magnitude of the protonmotive force in phosphorylating membrane vesicles from Paracoccus denitrificans was estimated. The membrane potential component was determined from the uptake of S14CN−, and the transmembrane pH gradient component from the uptake of [14C]methylamine. In each case a flow-dialysis technique was used to monitor uptake. 2. With NADH as substrate, the membrane potential was about 145mV and the pH gradient was below 0.5 pH unit. The membrane potential was decreased by approx. 15mV during ATP synthesis, and was abolished on addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone. In the presence of KCl plus valinomycin the membrane potential was replaced by a pH gradient of 1.5 units. 3. Succinate oxidation generated a membrane potential of approx. 125mV and the pH gradient was below 0.5 pH unit. Oxidation of ascorbate (in the presence of antimycin) with either 2,3,5,6-tetramethyl-p-phenylenediamine or NNN′N′-tetramethyl-p-phenylenediamine as electron mediator usually generated a membrane potential of approx. 90mV. On occasion, ascorbate oxidation did not generate a membrane potential, suggesting that the presence of a third energy-coupling site in P. denitrificans vesicles is variable. 4. With NADH or succinate as substrate, the phosphorylation potential (ΔGp=ΔG0′+RTln[ATP]/ [ADP][Pi]) was approx. 53.6kJ/mol (12.8kcal/mol). Comparison of this value with the protonmotive force indicates that more than 3 protons need to be translocated via the adenosine triphosphatase of P. denitrificans for each molecule of ATP synthesized by a chemiosmotic mechanism. In the presence of 10mm-KNO3 the protonmotive force was not detectable (<60mV) but ΔGp was not altered. This result may indicate either that there is no relationship between the protonmotive force and ΔGp, or that for an unidentified reason the equilibration of SCN− or methylamine with the membrane potential and the pH gradient is prevented by NO3− in this system.

scholarly journals The probable site of action of thenolytrifluoracetone on the respiratory chain

1977 ◽  
Vol 164 (3) ◽  
pp. 617-620 ◽  
Author(s):  
W J Ingledew ◽  
T Ohnishi
Keyword(s):  
Succinate Dehydrogenase ◽  
Respiratory Chain ◽  
Close Association ◽  
Succinate Oxidation ◽  
Paramagnetic Resonance ◽  
Spin Interactions ◽  
Site Of Action ◽  
Transfer Inhibitor ◽  
Probable Site ◽  
Electron Paramagnetic

1. It is shown that the electron-transfer inhibitor thenoyltrifluoroacetone abolishes a respiratory-chain electron-paramagnetic-resonance absorbance due to spin-spin interactions of ubisemiquinones at concentrations similar to those required for inhibition of succinate oxidation. 2. A specific site of interaction of thenoyltrifluoroacetone with the respiratory chain is proposed to be on the ubisemiquinone with which succinate dehydrogenase reacts. 3. Our results further demonstrate the close association of the HiPIP (high-potential iron-sulphur) centre of succinate dehydrogenase with ubisemiquinone.

scholarly journals MITOCHONDRIAL BIOGENESIS DURING DIFFERENTIATION OF ARTEMIA SALINA CYSTS

1973 ◽  
Vol 58 (3) ◽  
pp. 643-649 ◽  
Author(s):  
H. Schmitt ◽  
H. Grossfeld ◽  
U. Z. Littauer
Keyword(s):  
Cytochrome C ◽  
Cytochrome Oxidase ◽  
Cytochrome B ◽  
Mitochondrial Biogenesis ◽  
Brine Shrimp ◽  
Morphological Changes ◽  
Artemia Salina ◽  
Mitochondrial Morphology ◽  
Second Stage ◽  
Two Stages

Mitochondria isolated from cysts of Artemia salina (brine shrimp) were found to be devoid of cristae and to possess a low respiratory capability. Hydration of the cysts induces marked biochemical and morphological changes in the mitochondria. Their biogenesis proceeds in two stages. The first stage is completed within 1 h and is characterized by a rapid increase in the respiratory capability of the mitochondria, their cytochrome oxidase, cytochrome b, cytochrome c and perhaps some morphological changes. In the second stage there is an increase in the protein-synthesizing capacity of the mitochondria as well as striking changes in mitochondrial morphology leading to the formation of cristae.

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.

Defibrillation depresses heart sarcoplasmic reticulum calcium pump: a mechanism of postshock dysfunction

1998 ◽  
Vol 274 (1) ◽  
pp. H98-H105 ◽  
Author(s):  
Douglas L. Jones ◽  
Njanoor Narayanan
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Atp Hydrolysis ◽  
Myocardial Dysfunction ◽  
Cardiac Contractility ◽  
Membrane Vesicles ◽  
Intracellular Ca ◽  
Current Densities ◽  
Langendorff Hearts ◽  
Pumping Function ◽  
Sarcoplasmic Reticulum Calcium

Presently, the only therapy for ventricular fibrillation is delivery of high-voltage shocks. Despite “successful defibrillation,” patients may have poor cardiac contractility, the mechanisms of which are unknown. Intracellular Ca2+ handling by the sarcoplasmic reticulum (SR) plays a major role in contractility. We tested the hypothesis that defibrillation shocks interfere with Ca2+ transport function of cardiac SR. Rats anesthetized with pentobarbital sodium had bilateral electrodes implanted subcutaneously for transthoracic shocks. A series of 10 shocks, 10 s apart, at 0–250 V was delivered from a trapezoidal defibrillator. The hearts were rapidly removed, SR-enriched membrane vesicles were isolated, and ATP-dependent Ca2+ uptake and Ca2+-stimulated ATP hydrolysis were determined. There was a marked, shock-related decline in Ca2+ uptake, whereas adenosinetriphosphatase activity remained unaltered. The polypeptide compositions were similar in control and shocked SR. In Langendorff hearts, shocks also decreased contractility and slowed relaxation. These data indicate that shocks with current densities similar to defibrillation depress Ca2+-pumping function of cardiac SR because of uncoupling of ATP hydrolysis and Ca2+ transport. Shock-induced impairment of Ca2+ pump function may underlie postshock myocardial dysfunction.

scholarly journals Multiple light-induced reactions of cytochromes b and c in Rhodopseudomonas spheroides

1969 ◽  
Vol 114 (4) ◽  
pp. 793-799 ◽  
Author(s):  
O. T. G. Jones
Keyword(s):  
Cytochrome C ◽  
Cytochrome B ◽  
Photochemical Reaction ◽  
Room Temperature ◽  
Close Association ◽  
Antimycin A ◽  
Reaction Centre ◽  
Difference Spectra ◽  
Cytochromes C ◽  
Rhodopseudomonas Spheroides

Illumination of chromatophore preparations from Rhodopseudomonas spheroides causes the oxidation of a cytochrome c and a slight oxidation of a cytochrome b with a maximum at 560nm. When illuminated in the presence of antimycin A the oxidation of cytochrome c was more pronounced and cytochrome b560 was reduced; the dark oxidation of cytochrome b560 was biphasic in the presence of succinate, but not in the presence of NADH, a less effective reductant. Split-beam spectroscopy showed that, in addition to the reduction of cytochrome b560, another pigment with maxima at 565 and 537nm. was reduced and was more rapidly oxidized in the dark than cytochrome b560. This pigment, tentatively identified as cytochrome b565, was also detected in spectra at 77°k, after brief illumination at room temperature; the maxima at 77°k were at 562 and 536nm. In the absence of antimycin A, light caused a transient reduction of cytochrome b565 and an oxidation of cytochrome b560. Dark oxidation of b565 was rapid, even in the presence of antimycin A and succinate. Difference spectra, at 77°k, of ascorbate-reduced minus succinate-reduced chromatophores or of anaerobic succinate-reduced minus aerobic succinate-reduced chromatophores suggested that two cytochromes c were present, with maxima at 547 and 549nm. When chromatophores frozen at 77°k were illuminated both these cytochromes c were oxidized, indicating a close association with the photochemical reaction centre. A scheme involving two reaction centres is proposed to explain these results.

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