Skip to main content
SpringerLink
Log in

Human quadricepts muscle mitochondria: A functional characterization

  • Published:
Molecular and Cellular Biochemistry Aims and scope Submit manuscript

Abstract

Human quadriceps mitochondria were isolated from ca. 80 mg tissue in ca. 45% yield. The preparation is described with respect to content of mitochondrial markers and nine different respiratory activities. The specific state 3 activities were high in comparison with literature data, indicating high integrity and purity of the preparation. Examples of state 3 rates, in µmol O min-1 g protein-1 (25°C): pyruvate + malate, 400; succinate, 514; malate + glutamate, 444. The notion of high integrity was also supported by the reproducibility of the preparation and the magnitude of the respiratory control ratios and the P/O ratios. The mitochondria most likely had lost ca. 30% of their cytochrome c upon isolation, but it was substantiated that this loss had not influenced the state 3 rates. Functional assays of single reactions or groups of reactions could be based on respiration experiments. The respiratory chain activity, for instance, was measured as respiration of NADH in freeze-permeabilized mitochondria (1263 μmol O min-1 g protein-1). Comparison of uncoupled rates of respiration and state 3 rates indicated that the ATP synthesis exerted major flux control over respiration of succinate + glutamate, malate + glutamate and pyruvate + malate. These reactions, showing very similar rates of ATP synthesis, could be used as a functional assay of ATP synthesis (1200 μmol ATP min-1 g protein-1). Respiration of succinate, palmitoyl-carnitine + malate, or glutamate could not support the maximal rate of ATP synthesis and the upstream reactions probably exerted major flux control in these cases. The specific activities appeared very constant in this group of young men, only the respiratory activity with glutamate might show biological variation.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Chinnery PF, Turnbull DM: Mitochondrial DNA and disease. Lancet 354: 17–21, 1999

    Google Scholar 

  2. Schapira AHV: Mitochondrial involvement in Parkinson's disease, Huntington's disease, hereditary spastic paraplegia and Friedreich's ataxia. Biochim Biophys Acta 1410: 159–170, 1999

    Google Scholar 

  3. Wallace DC: Mitochondrial diseases in man and mouse. Science 283: 1482–1488, 1999

    Google Scholar 

  4. Cortopassi GA, Wong A: Mitochondria in organismal aging and degeneration. Biochim Biophys Acta 1410: 183–193, 1999

    Google Scholar 

  5. Veksler VI, Kuznetsov AV, Sharov VG, Kapelko VI, Saks VA: Mitochondrial respiratory parameters in cardiac tissue: A novel method of assessment by using saponin-skinned fibers. Biochim Biophys Acta 892: 191–196, 1987

    Google Scholar 

  6. Kunz WS, Kuznetsov AV, Schulze W, Eichhorn K, Schild L, Striggow F, Bohnensack R, Neuhof S, Grasshoff H, Neumann HW, Gellerich FN: Functional characterization of mitochondrial oxidative phosphorylation in saponin-skinned human muscle fibers. Biochim Biophys Acta 1144: 46–53, 1993

    Google Scholar 

  7. Saks VA, Veksler VI, Kuznetsov AV, Kay L, Sikk P, Tiivel T, Tranqui L, Olivares J, Winkler K, Wiedemann F, Kunz WS: Permeabilized cell and skinned fiber techniques in studies of mitochondrial function in vivo. Mol Cell Biochem 184: 81–100, 1998

    Google Scholar 

  8. Williams RS: Regulation of mitochondrial biogenesis in striated muscles. In: G. Benzi (ed). Adv. Myochem, vol. 1. John Libbey Eurotext Ltd., 1987, 185–194

  9. Hoppeler H: The different relationship of VO2-max to muscle mitochondria in humans and quadrupedal animals. Resp Physiol 80: 137–146, 1990

    Google Scholar 

  10. Lee CP: Biochemical studies of isolated mitochondria from normal and diseased tissues. Biochim Biophys Acta 1271: 21–28, 1995

    Google Scholar 

  11. Bookelman H, Trijbels JMF, Sengers RCA, Janssen AJM, Veerkamp JH, Stadhouders AM: Pyruvate oxidation in rat and human skeletal muscle mitochondria. Biochem Med 20: 395–403, 1978

    Google Scholar 

  12. Fischer JC, Ruitenbeek W, Stadhouders AM, Trijbels JMF, Sengers RCA, Janssen AJM, Veerkamp JH: Investigation of mitochondrial metabolism in small human skeletal muscle biopsy specimens. Improvement of preparation procedure. Clin Chim Acta 145: 89–100, 1985

    Google Scholar 

  13. Scholte HR, Busch HFM, Luyt-Houwen IEM, Vaandrager-Verduin MHM, Przyrembel H, Arts WFM: Defects in oxidative phosphorylation. Biochemical investigations in skeletal muscle and expression of the lesion in other cells. J Inher Metab Dis 10(suppl 1): 81–97, 1987

    Google Scholar 

  14. Martens ME, Peterson PL, Lee C-P, Nigro MA, Hart Z, Glasberg M, Hatfeld JS, Chang CH: Kearns-Sayre syndrome: Biochemical studies of mitochondrial metabolism. Ann Neurol 24: 630–637, 1988

    Google Scholar 

  15. Chretien D, Bourgeron T, Rötig A, Munnich D, Rustin D: The measurement of the rotenone-sensitive NADH cytochrome c reductase activity in mitochondria isolated from minute amount of human skeletal muscle. Biochem Biophys Res Commun 173: 26–33, 1990

    Google Scholar 

  16. Wibom R, Hultman E: ATP production rate in mitochondria isolated from microsamples of human muscle. Am J Physiol 259: E204-E209, 1990

    Google Scholar 

  17. Tonkonogi M, Sahlin K: Rate of oxidative phosphorylation in isolated mitochondria from human skeletal muscle: Effect of training status. Acta Phys Scand 161: 345–353, 1997

    Google Scholar 

  18. Berthon PM, Howlett RA, Heigenhauser GJF, Spriet LL: Human skeletal muscle carnitine palmitoyltransferase I activity determined in isolated intact mitochondria. Am J Physiol 85: 148–153, 1998

    Google Scholar 

  19. Rasmussen HN, Rasmussen UF: Respiration measurements in small scale. Anal Biochem 208: 244–248, 1993

    Google Scholar 

  20. Rasmussen HN, Rasmussen UF: Small scale preparation of skeletal muscle mitochondria, criteria of integrity, and assays with reference to tissue function. Mol Cell Biochem 174: 55–60, 1997

    Google Scholar 

  21. Rasmussen HN, Andersen AJ, Rasmussen UF: Optimimization of preparation of mitochondria from 25–100 mg skeletal muscle. Anal Biochem 252: 153–159, 1997

    Google Scholar 

  22. Rasmussen UF, Rasmussen HN, Andersen AJ, Fogd Jørgensen P, Quistorff B: Characterization of mitochondria from pig muscle: Higher activity of exo-NADH oxidase in animals suffering from malignant hyperthermia. Biochem J 315: 659–663, 1996

    Google Scholar 

  23. Bergström J: Muscle electrolytes in man. Obtaining the muscle samples. Scan J Clin Lab Invest 14(suppl 68): 11–13, 1962

    Google Scholar 

  24. Shepherd D, Garland PG: Citrate synthase from rat liver. Meth Enzymol 13: 11–16, 1969

    Google Scholar 

  25. Hartree EF: Haematin compounds In: K. Paech, M.W. Tracey (eds). Modern Methods in Plant Analysis, vol. 4. Springer, Berlin, 1955, pp 208–210

  26. Schaffner W, Weissmann C: A rapid, sensitive, and specific method for the determination of protein in dilute solution. Anal Biochem 56: 502–514, 1973

    Google Scholar 

  27. Miller JC, Miller JN: In: Statistics for Analytical Chemistry, 3rd edn. Ellis Horwood PTR Prentice Hall, 1993, 108

  28. Scholte HR, Luyt-Houwen IEM, Busch HFM: Difficulties in assessing biochemical properties of abnormal muscle mitochondria. J Inher Metab Dis 8(suppl 2): 149–150, 1985

    Google Scholar 

  29. Scholte HR, Yihong Y, Ross JD, Oosterkamp II, Boonman AMC, Busch HFM: Rapid isolation of muscle and heart mitochondria, the lability of oxidative phosphorylation and attempts to stabilize the process in vitro by taurine, carnitine and other compounds. Mol Cell Biochem 174: 61–66, 1997

    Google Scholar 

  30. Federico A, Manneschi L, Paolini E: Biochemical differences between intermyofrillar and subsarcolemmal mitochondria from human muscle. J Inher Metab Dis 10(suppl 2): 242–246, 1987

    Google Scholar 

  31. Schmidt I, Herpin P: Postnatal changes in mitochondrial protein mass and respiration in skeletal muscle from newborn pig. Comp Biochem Physiol 118B: 639–647, 1997

    Google Scholar 

  32. Manneschi L, Federico A: Polarographic analyses of subsarcolemmal and intermyofibrillar mitochondria from rat skeletal and cardiac muscle. J Neurol Sci 128: 151–156, 1995

    Google Scholar 

  33. Byrne E, Trounce I: Oxygen electrode studies with human skeletal muscle mitochondria in vitro. J Neurol Sci 69: 319–333, 1985

    Google Scholar 

  34. Mitsumoto H, Aprille JR, Wray SH, Nemmi R, Bradly WG: Chronic progressive external ophthalmoplegia (CPEO): Clinical, morphologic, and biochemical studies. Neurology 33: 452–461, 1983

    Google Scholar 

  35. Elander A, Sjöström M, Lundgren F, Scherstén T, Bylund-Fellenius A-C: Biochemical and morphometric properties of mitochondrial populations in human muscle fibres. Clin Sci 69: 153–164, 1985

    Google Scholar 

  36. Cooper JM, Schapira AHV, Holt IJ, Toscano A, Harding AE, Morgan-Hughes JA, Clark JB: Biochemical and molecular aspects of human mitochondrial respiratory chain disorders. Biochem Soc Trans 18: 517–519, 1990

    Google Scholar 

  37. Bet L, Bresolin N, Moggio M, Meola G, Prelle A, Shapira AH, Binzoni T, Chomyn A, Fortunato F, Cerretelli P, Scarlato G: A case of mitochondrial myopathy, lactic acidosis and complex I deficiency. J Neurol 237: 399–404, 1990

    Google Scholar 

  38. Cardellac F, Martí MJ, Fernández-Solá J, Marín C, Hoek JB, Tolosa E, Urbano-Márquez A: Mitochondrial respiratory chain activity in skeletal muscle from patients with Parkinson's disease. Neurology 43: 2258–2262, 1993

    Google Scholar 

  39. Rasmussen UF, Rasmussen HN: The NADH oxidase system (external) of muscle mitochondria and its role in the oxidation of cytoplasmic NADH. Biochem J 229: 631–641, 1985

    Google Scholar 

  40. Fisher JC, Ruitenbeek W, Trijbels JMF, Veerkamp JH, Stadhouders AM, Sengers RCA, Janssen AJM: Estimation of NADH oxidation in human skeletal muscle mitochondria. Clin Chim Acta 155: 263–274, 1986

    Google Scholar 

  41. Pette D: Mitochondrial enzyme activities. Biochim Biophys Acta Library 7: 28–49, 1966

    Google Scholar 

  42. Hafner RP, Brown BC, Brand MD: Analysis of the control of respiration rate, phosphorylation rate, proton leak rate and proton motive force in isolated mitochondria using the 'top-down' approach of metabolic control theory. Eur J Biochem 188: 313–319, 1990

    Google Scholar 

  43. Rolfe DFS, Hulbert AJ, Brand MD: Characteristics of mitochondrial proton leak and control of oxidative phosphorylation in the major oxygenconsuming tissues of the rat. Biochim Biophys Acta 1118: 405–416, 1994

    Google Scholar 

  44. Moreno-Sánchez R, Devars S, López-Gómez F, Uribe A, Corona N: Distribution of control of oxidative phosphorylation in mitochondria oxidizing NAD-linked substrates. Biochim Biophys Acta 1060: 284–292, 1991

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biochemistry, August Korgh Institute, University of Copenhagen, Universitetsparken 13, DK-2100, Copenhagen, Denmark

    Ulla F. Rasmussen & Hans N. Rasmussen

Authors
  1. Ulla F. Rasmussen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hans N. Rasmussen
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Rasmussen, U.F., Rasmussen, H.N. Human quadricepts muscle mitochondria: A functional characterization. Mol Cell Biochem 208, 37–44 (2000). https://doi.org/10.1023/A:1007046028132

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1007046028132

Search

Navigation