Evaluation of Methods for the Determination of Mitochondrial Respiratory Chain Enzyme Activities in Human Skeletal Muscle Samples

Otto-von-Guericke-Universität Magdeburg, Magdeburg, Saxony-Anhalt, Germany
Analytical Biochemistry (Impact Factor: 2.22). 04/2000; 279(1):55-60. DOI: 10.1006/abio.1999.4434
Source: PubMed


The quantification of mitochondrial enzyme activities in skeletal muscle samples of patients suspected of having mitochondrial myopathies is problematic. Therefore, we have evaluated different methods for the determination of activities cytochrome c oxidase and NADH:CoQ oxidoreductase in human skeletal muscle samples. The measurement of cytochrome c oxidase activity in the presence of 200 microM ferrocytochrome c and the detection of NADH:CoQ oxidoreductase as rotenone-sensitive NADH:CoQ(1) reductase resulted in comparable citrate synthase-normalized respiratory chain enzyme activities of both isolated mitochondria and homogenates from control human skeletal muscle samples. These methods allowed the precise detection of deficiencies of respiratory chain enzymes in skeletal muscle of two patients harboring only 20 and 27% of deleted mitochondrial DNA, respectively. Therefore, citrate synthase-normalized respiratory chain activities can serve as stable reference values for the determination of a putative mitochondrial defect in human skeletal muscle.

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Available from: Wolfram S Kunz, Jan 16, 2015
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    • "The activity of citrate synthase, rotenone-sensitive NADH:CoQ 1 oxidoreductase, and COX was determined by standard methods (Wiedemann et al., 2000). The details on the performed biochemical assays are outlined in the Supplementary material. "
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    • "Enzymatic activities were measured using a dual wavelength spectrophotometer (Aminco DW 2000, SLM Instruments, Rochester, NY), as described before (Wiedemann et al., 2000). "
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    • "Then, the sample was sonicated for 15 s with the ultrasonic processor GEX-600. The activity of citrate synthase was determined by standard methods as described elsewhere (Wiedemann et al, 2000). The activity of rotenone-sensitive NADH : "
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    ABSTRACT: Genetic studies have implicated the evolutionary novel, anthropoid primate-specific gene locus G72/G30 in psychiatric diseases. This gene encodes the protein LG72 that has been discussed to function as a putative activator of the peroxisomal enzyme D-amino-acid-oxidase (DAO) and as a mitochondrial protein. We recently generated 'humanized' bacterial artificial chromosome transgenic mice (G72Tg) expressing G72 transcripts in cells throughout the brain. These mice exhibit several behavioral phenotypes related to psychiatric diseases. Here we show that G72Tg mice have a reduced activity of mitochondrial complex I, with a concomitantly increased production of reactive oxygen species. Affected neurons display deficits in short-term plasticity and an impaired capability to sustain synaptic activity. These deficits lead to an impairment in spatial memory, which can be rescued by pharmacological treatment with the glutathione precursor N-acetyl cysteine. Our results implicate LG72-induced mitochondrial and synaptic defects as a possible pathomechanism of psychiatric disorders.
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