Quantitation and Characterization of CytochromecOxidase in Complex Systems

Meunier, Brigitte; Rich, Peter R.

doi:10.1006/abio.1998.2704

Cited by 12 publications

(11 citation statements)

References 27 publications

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“…The kinetics of CO recombination were monophasic (for at least 80 % of the signal), which suggested that the mutations did not cause any significant structural distortions around the binuclear centre. An apparent rate constant (k obs ) of 60-65 s −" was observed for the wild type and for strains G317S, I280T, E243D and E243N, which was in agreement with values reported previously for wild-type enzymes (see [12] and references therein). With strain M273T, the rate of CO recombination was slightly higher (100 s −" ).…”

Section: Strainsupporting

confidence: 91%

“…Difference spectra (reduced minus oxidized, and reduced plus cyanide minus reduced) were then constructed. The reaction of cytochrome oxidase with CO was monitored as described in [11][12][13]. Roomtemperature photolysis of CO was achieved with short actinic light pulses (10 ns half peak width ; 532 nm ; 100 mJ\pulse) provided by a frequency-doubled Nd-YAG laser.…”

Section: Spectrophotometric Measurementsmentioning

confidence: 99%

Section: Quantification Of Cytochrome Oxidasementioning

confidence: 99%

“…Cytochrome oxidase content was estimated by using a molar absorption coefficient (ε) of 14 mM −" :cm −" at [604k(594j 614)\2] nm [12]. The amount of cyanide-binding cytochrome oxidase was estimated from the cyanide binding spectra by using ε l 18.7 mM −" :cm −" at (592-612) nm [12]. The amount of CObinding enzyme was estimated from the amplitude of the CO photolysis spectra by using ε l 113 mM −" :cm −" at (430k445) nm [11,12].…”

Section: Quantification Of Cytochrome Oxidasementioning

confidence: 99%

“…In strain I280T, the amount of cyanide-binding enzyme varied from 75 % to 90 %. Reactivity with CO was monitored by laser-flash photolysis [12,13]. Like cyanide, CO binds to reduced haem a $ and induces an optical change.…”

Section: Strainmentioning

confidence: 99%

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Site-directed mutations in the mitochondrially encoded subunits I and III of yeast cytochrome oxidase

Meunier

2001

Biochemical Journal

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Since yeast is amenable to mitochondrial transformation, designed mutations can be introduced in the mitochondrially encoded subunits of the respiratory complexes. In the present work, six mutations have been introduced by the biolistic method into yeast (Saccharomyces cerevisiae) cytochrome oxidase subunits I and III. The effects of these mutations on respiratory growth competence, cytochrome oxidase activity and optical properties were then characterized. Firstly, the conserved glutamate Glu-243 in the D-channel of subunit I was replaced by an asparagine or an aspartate residue. The effects of the mutations showed that Glu-243, which is essential for proton movement in bacterial oxidases, is also required for the activity of the eukaryotic enzyme. Secondly, four mutations associated with human disease were introduced in yeast, allowing detailed analysis of their deleterious effects on cytochrome oxidase function: Met-273 → Thr, Ile-280 → Thr and Gly-317 → Ser, affecting residues located in or near the K-channel in subunit I, and a short in-frame deletion comprising residues Phe-102 to Phe-106 in subunit III (∆F102–F106). The subunit III mutation was highly deleterious and abolished enzyme assembly. The change Gly-317 → Ser had no effect on respiratory function. However, mutations Met-273 → Thr and Ile-280 → Thr were mildly deleterious, decreased cytochrome oxidase activity and slightly perturbed the properties of the binuclear centre.

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Section: Strainsupporting

confidence: 91%

Section: Spectrophotometric Measurementsmentioning

confidence: 99%

Section: Quantification Of Cytochrome Oxidasementioning

confidence: 99%

Section: Quantification Of Cytochrome Oxidasementioning

confidence: 99%

Section: Strainmentioning

confidence: 99%

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Site-directed mutations in the mitochondrially encoded subunits I and III of yeast cytochrome oxidase

Meunier

2001

Biochemical Journal

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Direct chemiluminescent imaging detection of Cu/Zn-superoxidase dismutase, glutathione peroxidase, carbonic anhydrase-III, and catalase in rat liver cytosol separated by native porous gradient polyacrylamide gel electrophoresis

et al. 2005

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The cytosolic enzymes extracted from rat hepatocytes were separated by native porous gradient-PAGE (PG-PAGE) and were detected with a sensitive and fast chemiluminescence (CL) imaging method. Several peroxidases including glutathione peroxidase, Cu/Zn-superoxidase dismutase, and some other metallo-enzymes such as catalase, carbonic anhydrase-III (CA-III) present in the cytosol of rat hepatocytes have been selectively and sensitively detected by the direct CL imaging method using the luminol-H(2)O(2) chemiluminescent reagents. All detections after PG-PAGE were completed within 9 min. The linear range for the typical metallo-enzyme, e.g., CA-III is 0.75-4.9 microg/mL, with a detection limit of 0.25 microg/mL. In comparison with the traditional CBB-R250 staining method, the detection period decreased about 70 times and the detection sensitivity improved over ten times. Furthermore, two enzymes present in rat liver cytosol were identified employing MALDI-MS analysis of the tryptic digest after PG-PAGE.

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Cytochrome-c oxidase

Springer Handbook of Enzymes

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Quantitation and Characterization of CytochromecOxidase in Complex Systems

Cited by 12 publications

References 27 publications

Site-directed mutations in the mitochondrially encoded subunits I and III of yeast cytochrome oxidase

Site-directed mutations in the mitochondrially encoded subunits I and III of yeast cytochrome oxidase

Direct chemiluminescent imaging detection of Cu/Zn-superoxidase dismutase, glutathione peroxidase, carbonic anhydrase-III, and catalase in rat liver cytosol separated by native porous gradient polyacrylamide gel electrophoresis

Cytochrome-c oxidase

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