Abstract
Rhizopus stolonifer (Ehrenb.:Fr.) Vuill mitochondria contain the complete system for oxidative phosphorylation, formed by the classical components of the electron transport chain (complexes I, II, III, and IV) and the F1F0-ATP synthase (complex V). Using the native gel electrophoresis, we have shown the existence of supramolecular associations of the respiratory complexes. The composition and stoichiometry of the oxidative phosphorylation complexes were similar to those found in other organisms. Additionally, two alternative routes for the oxidation of cytosolic NADH were identified: the alternative NADH dehydrogenase and the glycerol-3-phosphate shuttles. Residual respiratory activity after inhibition of complex IV by cyanide was inhibited by low concentrations of n-octyl gallate, indicating the presence of an alternative oxidase. The K0.5 for the respiratory substrates NADH, succinate, and glycerol-3-phosphate in permeabilized cells was higher than in isolated mitochondria, suggesting that interactions of mitochondria with other cellular elements might be important for the function of this organelle.
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Acknowledgments
This work was supported by grants from Consejo Nacional de Ciencia y Tecnología (CONACyT 59855) and Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica (PAPIIT IN210311-3) from Universidad Nacional Autónoma de México, and Secretaría de Investigación y Posgrado (SIP), Instituto Politécnico Nacional, grants 20070822, 20080183, and 20080561. Leobarda Robles-Martínez is a Ph.D. student in the Quimicobiológicas Program from Escuela Nacional de Ciencias Biológicas of Instituto Politécnico Nacional and was supported by grants from CONACyT (206876) and SIP (Programa Institucional de Formación de Investigadores).
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Communicated by John Helman.
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203_2012_845_MOESM1_ESM.jpg
Solubilization of respiratory complexes by DDM. Increased concentrations of DDM were used to release the complexes from Rhizopus stolonifer mitochondria. (A) Blue native gel stained with Coomassie blue and (B) NADH dehydrogenase activity. (JPEG 100 kb)
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Solubilization of respiratory supercomplexes by digitonin. Increased concentrations of digitonin were used to release the supercomplexes from Rhizopus stolonifer mitochondria. (A) Blue native gel stained with Coomassie blue and (B) NADH dehydrogenase activity. (JPEG 159 kb)
203_2012_845_MOESM3_ESM.jpg
Respiratory activity and sensitivity by permeabilized Rhizopus stolonifer cells. Cells were permeabilized by digitonin (0.02%) in the absences of any substrate or inhibitor. Oxygen uptake was stimulated by exogenous 384 μM NADH in the (A) absence or (B) presence of 3 μM rotenone, respectively. The respiration was inhibited by 1 mM KCN. Additions are indicated by arrows. Number on the trace represents the rate of oxygen uptake in nmol O2 min-1 (mg cells)-1. Oximetric experiments were performed at 25 °C in buffer A. (JPEG 169 kb)
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Robles-Martínez, L., Guerra-Sánchez, M.G., Flores-Herrera, O. et al. The mitochondrial respiratory chain of Rhizopus stolonifer (Ehrenb.:Fr.) Vuill. Arch Microbiol 195, 51–61 (2013). https://doi.org/10.1007/s00203-012-0845-7
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DOI: https://doi.org/10.1007/s00203-012-0845-7