| 10702521 |
Brooks H, Krahenbuhl S: Development of a new assay for complex I of the respiratory chain. J Biol Chem. 2009 Jun 12;284(24):16236-45. Epub 2009 Apr 14.
BACKGROUND: Measurement of complex I activity has been hampered by the large amounts of tissue required and the resulting turbidity of the assay solution, which makes spectrophotometric analysis difficult. We have developed a new assay for measuring the activity of complex I in isolated mitochondria that is also applicable to skeletal muscle homogenate in patients with suspected mitochondrial diseases. METHODS: The method was a radioenzymatic assay based on the preferential oxidation of the 4B hydrogen of NADH by complex I. We prepared tritiated isoforms of NADH for both the respective 4A-(3) H and 4B-(3) H positions. Enzyme in the form of purified mitochondria or homogenate was prepared from rat or human skeletal muscle and incubated with the respective radioisotopes. The product ((3) H (2) O) was collected after charcoal adsorption of unreacted NADH and taken as an indicator of NADH oxidation. Sensitivity to rotenone was used as a measure of complex I specific activity. RESULTS: The assay was linear with time and protein for isolated mitochondria and tissue homogenates from rats and humans. The V (max) and K (m) values obtained for 4B-NADH with isolated rat skeletal muscle mitochondria were 35 micromol/L and 90 micromol. min (-1). mg protein (-1), respectively. The assay was reproducible and usable for routine measurements in human skeletal muscle. The sensitivity was > 10-fold higher than the sensitivities of spectrophotometric techniques. CONCLUSIONS: The results of our studies demonstrate the successful development of a new assay for complex I that is rapid, easy to perform, and that enables the processing of multiple samples at one time. |
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