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Mitochondrial function in normal and genetically altered cells and tissues
B Chance1, R A Waterland, A Tanaka
1Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia 19104.
Abstract:
The impact upon oxidative metabolism of normal and pathological variations of oxidative capability is just beginning to be understood, based upon the few examples of human and animal subject survivals and the relatively few cell systems in which the impact of molecular pathologies on function has been studied. On the one hand, difficulties of isolation of systems containing altered oxidases are significant because of ineffective assembly or small amounts of surviving isoenzymes, and on the other hand, unexpected fragilities of the oxidase system may lead to low yields when subjected to the preparative stresses appropriate to the wild types. To circumvent these problems, this paper describes the application, in vivo, of noninvasive, nondestructive techniques to study the function of cytochrome oxidase and other components of the respiratory chain, particularly cytochromes b-c1 in human subjects on the one hand, and in isolated cells on the other, principally mutants of Saccharomyces cerevisiae in which the subunit content is varied. Two principal spectroscopic approaches are employed: optical and phosphorus magnetic resonance spectroscopy (P MRS). Optical spectroscopy of the near red region of the spectrum provides effective analysis of brain and muscle, as does the surface coil of space-resolved phosphorus magnetic resonance. Both techniques are applicable to suspensions of single cells such as yeast. The optical method yields essential information on oxygen delivery to tissues by hemoglobin and myoglobin and oxygen utilization by cytochrome oxidase. P MRS affords essential information on the efficiency of ATP generation and the extent to which oxidative metabolism meets the needs of cell function in terms of the ratio of phosphocreatine to inorganic phosphate (PCr/Pi). This in turn enables the calculation of the velocity of oxidative metabolism, V, in relation to its maximum capability, Vm, according to a Michaelis-Menten relationship that involves control not only by ADP (Pi/PCr) and Pi, but also by oxygen and substrate deliveries. Thus, an overview of the functionality of mitochondria in cells and tissues is uniquely provided by this combined approach and thereby deficiencies of components of the respiratory chain are quantified.
Insights
This study introduces noninvasive optical and phosphorus magnetic resonance spectroscopy (P MRS) to assess oxidative metabolism. These methods quantify respiratory chain deficiencies in human tissues and yeast cells.
Area of Science:
- Biochemistry and Molecular Biology
- Cellular Respiration
- Spectroscopy
Background:
- Understanding oxidative metabolism's role in health and disease is limited due to challenges in studying altered oxidases.
- Isolating and analyzing systems with modified oxidases is difficult, often resulting in low yields or ineffective enzyme assembly.
Purpose of the Study:
- To apply noninvasive, nondestructive spectroscopic techniques to study cytochrome oxidase and respiratory chain function.
- To analyze oxidative metabolism in human subjects and isolated yeast cell mutants with varied subunit content.
Main Methods:
- Utilized near-infrared optical spectroscopy for analyzing oxygen delivery and utilization in tissues and cells.
- Employed phosphorus magnetic resonance spectroscopy (P MRS) for assessing ATP generation efficiency and metabolic rates.
- Applied both techniques to human subjects (brain, muscle) and yeast cell suspensions (Saccharomyces cerevisiae).
Main Results:
- Optical spectroscopy effectively measured hemoglobin, myoglobin, and cytochrome oxidase function.
- P MRS provided insights into phosphocreatine to inorganic phosphate ratios (PCr/Pi), indicating ATP generation efficiency.
- Combined methods allowed quantification of oxidative metabolism velocity (V) relative to maximum capacity (Vm), considering regulatory factors.
Conclusions:
- The combined spectroscopic approach offers a unique overview of mitochondrial functionality in cells and tissues.
- This methodology enables precise quantification of deficiencies within the respiratory chain components.
- The techniques are valuable for studying both human physiology and cellular models of metabolic dysfunction.