Abstract
Mitochondrial function depends on the metabolite fluxes through the mitochondrial outer membrane. To directly measure the permeability of the outer membrane by using traditional methods is difficult due to the small volume of the intermembrane space and high permeability of the outer membrane. A method was developed to measure the permeability of the mitochondrial outer membrane by taking advantage of mitochondrial processes that consume the substrate being examined. A steady state concentration of substrate forms in the intermembrane space when the flux through the outer membrane equals the rate of substrate consumption. Thus, the rate of consumption of substrate depends on both processes and the extent of the contribution of each was sorted out by fitting to a steady-state theory. This report focuses on mitochondrial ATP phosphorylation and the determination of the permeability of the outer membrane to ADP. This approach also yields the intermembrane space [ADP] and shows how it differs from the extramitochondrial concentration.
Similar content being viewed by others
References
Werkheiser WC, Bartley W (1957). The study of the steady-state concentrations of internal solutes of mitochondria by rapid centrifugal transfer to a fixation medium. Biochem J 66: 79–91.
Pfaff E, Klingenberg M, Ritt E, Vogell W, et al. (1968). Korrelation des unspezifisch permeablen mitochondrialen raumes mit dem ‘Intermembranraum’. Eur J Biochem 5: 222–232.
Gellerich FN, Bohnensack R, Kunz W, et al. (1989). Role of the mitochondrial outer membrane in dynamic compartmentation of adenine nucleotides. In: Azzi A, Nałęcz KA, Nałęcz MJ, Wojtczak L (eds), Anion carriers of mitochondrial membranes. New York: Springer Verlag, pp 349–359.
Saks VA, Vasil'eva E, Belikova YuO, Kuznetsov AV, Lyapina S, Petrova L, Perov NA, et al. (1993). Retarded diffusion of ADP in cardiomyocytes: Possible role of mitochondrial outer membrane and creatine kinase in cellular regulation of oxidative phosphorylation. Biochim Biophys Acta 1144: 134–148.
Lee AC, Zizi M, Colombini M, et al. (1994). β-NADH decreases the permeability of the mitochondrial outer membrane to ADP by a factor of 6. J Biol Chem 269: 30974–30980.
Ahmadzadeh M, Horng A, Colombini M (1996). The control of mitochondrial respiration in yeast: A possible role of the outer mitochondrial membrane. Cell Biochem Funct 14: 210–208.
Brdiczka D, Kaldis P, Wallimann T (1994). In vitro complex formation between the octamer of mitochondrial creatine kinase and porin. J Biol Chem 269: 27640–27644.
Schwitzguebel JP, Siegenthaler PA (1984). Purification of peroxisomes and mitochondria from spinach leaf by Percoll gradient centrifugation. Plant Physiol 75: 670–674.
Tzagoloff A (1982). Mitochondria. New York: Plenum Press, pp 132–135.
Douce R, Bourguignon J, Brouquisse R, Neuburger M, et al. (1987). Isolation of plant mitochondria: general principles and criteria of integrity. Methods Enzymol 148: 403–412.
Zizi M, Forte M, Blachly-Dyson E, Colombini M, et al. (1994). NADH regulates the gating of VDAC, the mitochondrial outer membrane channel. J Biol Chem 269: 1614–1616.
Lee AC, Xu XF, Colombini M, et al. (1996). The role of pyridine dinucleotide in regulating the permeability of the mitochondrial outer membrane. J Biol Chem 271: 26724–26731.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Lee, AC., Colombini, M. An unique method for determining the permeability of the mitochondrial outer membrane. Methods Cell Sci 19, 71–81 (1997). https://doi.org/10.1023/A:1009761922055
Issue Date:
DOI: https://doi.org/10.1023/A:1009761922055