scholarly journals Proton translocation coupled to quinol oxidation in ox heart mitochondria

1973 ◽  
Vol 136 (3) ◽  
pp. 711-720 ◽  
Author(s):  
Hugh G. Lawford ◽  
Peter B. Garland
Keyword(s):  
Proton Translocation ◽  
Mitochondrial Protein ◽  
Glass Electrode ◽  
Measuring System ◽  
Heart Mitochondria ◽  
Analogue Computer ◽  
Back Flow ◽  
Ubiquinol Oxidase ◽  
True Value ◽  
Proton Generation

The suitability of ubiquinol1 and duroquinol as pulse reductants for initiating respirationdriven proton translocation by aerobic ox heart mitochondria was investigated. At 25°C the Vmax. for oxidation was close to 280nmol of quinol oxidized/min per mg of protein, and the Km values were 8μm for ubiquinol1 and 28μm for duroquinol. Pulses of ubiquinol1 and duroquinol were rapidly and completely oxidized by aerobic mitochondria with a simultaneous acidification of the suspending medium as detected with a glass electrode. The →H+/2e−ratios (Mitchell, 1966) calculated from the observed extent of acidification and the amount of quinol added were 3.62 for ubiquinol1 and 2.98 for duroquinol. These values are underestimates of the true value owing to proton back-flow across the membrane. An analogue computer model was used to correct the observed extent of respirationdriven acidification for proton back-flow. The corrected →H+/2e−values were 4.01 for ubiquinol and 3.86 for duroquinol oxidation. Attempts to measure the rate of proton translocation with a pH-measuring system with a response time of 0.4s were not entirely satisfactory, owing to the relative slowness of the electrode response. Nevertheless the maximal rate of proton generation during ubiquinol1 oxidation was about 1200ng-ions of H+/min per mg of mitochondrial protein. It is concluded, contrarily to Chance & Mela (1967), that mitochondria exhibit a proton-translocating ubiquinol oxidase activity with a →H+/2e−ratio of 4.0.

scholarly journals Identification of the protein responsible for pyruvate transport into rat liver and heart mitochondria by specific labelling with [3H]N-phenylmaleimide

1981 ◽  
Vol 196 (2) ◽  
pp. 471-479 ◽  
Author(s):  
A P Thomas ◽  
A P Halestrap
Keyword(s):  
Membrane Protein ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Mitochondrial Protein ◽  
Sodium Dodecyl ◽  
Liver Mitochondria ◽  
Heart Mitochondria ◽  
Approximate Amount ◽  
Specific Labelling

1. N-Phenylmaleimide irreversibly inhibits pyruvate transport into rat heart and liver mitochondria to a much greater extent than does N-ethylmaleimide, iodoacetate or bromopyruvate. alpha-Cyanocinnamate protects the pyruvate transporter from attack by this thiol-blocking reagent. 2. In both heart and liver mitochondria alpha-cyanocinnamate diminishes labelling by [3H]N-phenylmaleimide of a membrane protein of subunit mol.wt. 15000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 3. Exposure of mitochondrial to unlabelled N-phenylmaleimide in the presence of alpha-cyanocinnamate, followed by removal of alpha-cyanocinnamate and exposure to [3H]N-phenylmaleimide, produced specific labelling of the same protein. 4. Both labelling and kinetic experiments with inhibitors gave values for the approximate amount of carrier present in liver and heart mitochondria of 100 and 450 pmol/mg of mitochondrial protein respectively. 5. The turnover numbers for net pyruvate transport and pyruvate exchange at 0 degrees C were 6 and 200 min-1 respectively.

scholarly journals The effects of Mg2+ and adenine nucleotides on the sensitivity of the heart mitochondrial Na+-Ca2+ carrier to extramitochondrial Ca2+. A study using arsenazo III-loaded mitochondria

1987 ◽  
Vol 244 (3) ◽  
pp. 533-538 ◽  
Author(s):  
L H Hayat ◽  
M Crompton
Keyword(s):  
Adenine Nucleotides ◽  
Mitochondrial Protein ◽  
Arsenazo Iii ◽  
Heart Mitochondria ◽  
Small Decrease ◽  
Regulatory Site ◽  
Normal Heart ◽  
Partial Inhibition ◽  
Regulatory Sites

The technique of reversible Ca2+-induced permeabilization [Al Nasser & Crompton (1986) Biochem. J. 239, 19-29, 31-40] has been applied to the preparation of heart mitochondria loaded with the Ca2+ indicator arsenazo III (2 nmol of arsenazo III/mg of mitochondrial protein). The loaded mitochondria (‘mitosomes’) were used to study the control of the Na+-Ca2+ carrier by extramitochondrial Ca2+ mediated by putative regulatory sites. The Vmax. of the Na+-Ca2+ carrier and the degree of regulatory-site-mediated inhibition were similar to normal heart mitochondria. Ca2+ occupation of the sites in mitosomes yields partial inhibition, which is half-maximal with 0.8 microM external free Ca2+. The inhibition consists of a small decrease in Vmax. and a relatively large increase in apparent Km for internal Ca2+. Mg2+ also appears to interact with the sites, but this is largely abolished by ATP and ADP (but not AMP) under conditions in which the free [Mg2+] is maintained constant. The results indicate that the regulatory sites are effective in controlling the Na+-Ca2+ carrier at physiological concentrations of adenine nucleotides, Mg2+, intra- and extra-mitochondrial free Ca2+.

Preparation of oligomycin-sensitive ATPase enriched submitochondrial fraction from beef heart mitochondria

1979 ◽  
Vol 44 (9) ◽  
pp. 2854-2860 ◽  
Author(s):  
Petr Svoboda ◽  
Zdeněk Drahota
Keyword(s):  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Mitochondrial Protein ◽  
Sucrose Density Gradient ◽  
Heart Mitochondria ◽  
Beef Heart ◽  
Simple Method ◽  
Beef Heart Mitochondria ◽  
Relative Molecular Weight ◽  
Protein Components

A simple method for purification of oligomycin-sensitive ATPase from beef heart mitochondria is described. The isolation procedure is based on short term solubilization of mitochondrial membrane in deoxycholate and 1M-KCl followed by sequential precipitation of hydrofobic proteins and isopycnic centrifugation of crude particulate enzyme on sucrose density gradient. The oligomycin-sesitive ATPase preparation has a specific activity 15-20μmol P/min/mg protein and contains 5% of the total mitochondrial protein which can be separated by SDS-polyacrylamide gel electrophoresis into 13 protein components of relative molecular weight from 6 000 - 65 000 daltons, respectively.

Nitric oxide synthase in porcine heart mitochondria: evidence for low physiological activity

2001 ◽  
Vol 280 (6) ◽  
pp. H2863-H2867 ◽  
Author(s):  
Stephanie French ◽  
Cecilia Giulivi ◽  
R. S. Balaban
Keyword(s):  
Nitric Oxide ◽  
Physiological Activity ◽  
Mitochondrial Protein ◽  
Metabolic Effects ◽  
Heart Mitochondria ◽  
No Production ◽  
Porcine Heart ◽  
Nos Inhibitors ◽  
Oxide Synthase ◽  
Intact Heart

The capacity of isolated porcine heart mitochondria to produce nitric oxide (NO) via mitochondrial NO synthase (NOS) was evaluated. The mitochondrial NOS content and activity (0.2 nmol NO · mg mitochondrial protein−1 · min−1) were ∼10 times lower than previously reported for the rat liver. No evidence for mitochondrial NOS-generated NO was found in mitochondrial suspensions based on the lack of NO production and the lack of effect of eitherl-arginine or NOS inhibitors on the rate of respiration. The reason that even the low mitochondrial NOS activity did not result in net NO production and metabolic effects is because the mitochondrial metabolic breakdown of NO (1–4 nmol NO · mg mitochondrial protein−1 · min−1) was greater than the maximum rate of NO production measured in homogenates. These data suggest that NO production at the mitochondria via NOS is not a significant source of NO in the intact heart and does not regulate cardiac oxidative phosphorylation.

Redox-linked proton translocation in theb-c1complex from beef-heart mitochondria reconstituted into phospholipid vesicles

1983 ◽  
Vol 137 (3) ◽  
pp. 413-420 ◽  
Author(s):  
Michele LORUSSO ◽  
Domenico GATTI ◽  
Domenico BOFFOLI ◽  
Elisabetta BELLOMO ◽  
Sergio PAPA
Keyword(s):  
Proton Translocation ◽  
Heart Mitochondria ◽  
Phospholipid Vesicles ◽  
Beef Heart ◽  
Beef Heart Mitochondria

The errors involved in pH determination in soils.

1954 ◽  
Vol 5 (4) ◽  
pp. 716 ◽  
Author(s):  
M Raupach
Keyword(s):  
Reference Electrode ◽  
Glass Electrode ◽  
Measuring System ◽  
Ph Measurements ◽  
Soil Systems ◽  
Water Suspensions ◽  
Supernatant Liquid ◽  
Soil Variation ◽  
Single Determination ◽  
Suspension Effect

Errors of replication of pH values of 1: 5 soil-water suspensions are shown to differ significantly between routine observers and to be larger when duplicate determinations are made upon different days rather than on the same day. For the routine technique employed in these laboratories the 5 per cent. fiducial limits of a single determination do not rise above ±0.09 pH units due to the above causes. Errors due to soil variation over small distances in the field may show 5 per cent. limits as high as ±1.3 pH units. The causes of the errors which may arise within the measuring system are considered and details are given of errors in soil systems due to the suspension effect and to lack of equilibrium between the soil and aqueous phases. Absence of equilibrium may give differences as high as 1.0 unit when measurements are made upon sedimenting alkaline suspensions; no errors occur due to this cause below pH 5. The presence of salts does not modify the differences observed. The suspension effect is relatively small. It is recommended that where possible, pH measurements be made upon soil systems with the glass electrode in the suspension and the reference electrode in the dialysate or supernatant liquid. The description and use of a suitable electrode arrangement is given in an appendix. Generally pH measurements can be considered to no greater accuracy than ±0.1 unit and quite often circumstances do not justify this precision.

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