[7] Mitochondrial respiratory control and the polarographic measurement of ADP : O ratios☆
Publisher Summary
This chapter discusses the mitochondrial respiratory control and the polarographic measurement of ADP : O ratios. The polarographic oxygen electrode technique is used for measuring rapid changes in the rate of oxygen utilization by cellular and subcellular systems. Although the polarographic method measures changes in oxygen concentration of photosynthetic systems, yeast cells, and nerve, but the oxygen electrode technique is applied to a study the mitochondrial respiration and oxidative phosphorytation. The principle of the oxygen electrode has been summarized, and the design of the vibrating oxygen electrode for use with speetrophotometric studies is illustrated. The oxygen electrode apparatus can be calibrated in a number of ways. A more accurate calibration of oxygen content can be obtained by gas equilibration with various nitrogen-oxygen mixtures. When tightly coupled mitochondria are suspended in an isotonic buffer, a slow rate of oxygen uptake is measured in the presence of substrate and absence of ADP. Addition of ADP causes an immediate increase in the rate of oxygen utilization. The concentration of oxygen utilized is proportional to the amount of ADP phosphorylated to ATP. The type of oxygen electrode tracings is presented from which an ADP : O ratio (equivalent to a P : O ratio) can be directly calculated.
References (23)
- B. Hagihara
Biochim. Biophys. Acta
(1961) - D.O. Voss et al.
Anal. Biochem.
(1963) - H. Lardy et al.
J. Biol. Chem.
(1952) - P.W. Davies
- I.S. Longmuir
- W.W. Kielley et al.
J. Biol. Chem.
(1958) - J.S. Kahn
Anal. Biochem.
(1964) - R.W. Estabrook et al.
J. Biol. Chem.
(1957) - P.W. Davies et al.
Rev. Sci. Instr.
(1942) - L. Packer
Res. Rept. Walter Reed Army Inst. Res.
(1957)
Biochem. J.
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Spawning acts as a metabolic stressor enhanced by hypoxia and independent of sex in a broadcast marine spawner
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Dark-cutting beef mitochondrial proteomic signatures reveal increased biogenesis proteins and bioenergetics capabilities
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Morphological and physiological correlates of among- individual variation in basal metabolic rate in two passerine birds
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Mitochondrial physiology—Sturkie's book chapter
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See also W. W. Kielley, Vol. VI [33].