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An improved spectrophotometric assay of pyruvate dehydrogenase in lactate dehydrogenase contaminated mitochondrial preparations from human skeletal muscle.
| Content Provider | Semantic Scholar |
|---|---|
| Author | Chrétien, Dominique Pourrier, Marc Bourgeron, Thomas Séne, Mariama Rötig, Agnès Munnich, Arnold Rustin, Pierre |
| Copyright Year | 1995 |
| Abstract | In mitochondria-enriched preparations of human skeletal muscle, the measurement of pyruvate dehydrogenase activity, as determined by conventional spectrophotometric assay of NADH accumulation, is underestimated due to the oxidizing activity of the contaminating lactate dehydrogenase. Using a model reaction system consisting of varying mixtures of purified lactate and pyruvate dehydrogenases, we found that the presence of oxamate, a competitive inhibitor of the lactate dehydrogenase, allowed the measurement of a linear rate of pyruvate dehydrogenase activity without interference from lactate dehydrogenase. In the presence of 25 mM oxamate, this holds true up to a ratio of 30:1 for lactate to pyruvate dehydrogenases, respectively. A similar result was obtained when using human skeletal muscle mitochondria contaminated by lactate dehydrogenase. Rates of pyruvate dehydrogenase activity ranging from 50 to 120 nmol/min/mg protein could be routinely measured in such mitochondrial fractions. We concluded that the use of oxamate allows a spectrophotometric assay for pyruvate dehydrogenase activity to be utilized when screening for pyruvate dehydrogenase deficiency in mitochondria-enriched preparations of human skeletal muscle. |
| File Format | PDF HTM / HTML |
| Alternate Webpage(s) | http://www.ufrgs.br/biofisica/Bio10003/PDHassay.pdf |
| PubMed reference number | 8548923v1 |
| Volume Number | 240 |
| Issue Number | 2 |
| Journal | Clinica chimica acta; international journal of clinical chemistry |
| Language | English |
| Access Restriction | Open |
| Subject Keyword | Centrifugation Kinetics Lactate Dehydrogenase Lactic acid Mitochondria, Muscle Mitochondrial Diseases Mitochondrial Inheritance Myalgia NADH Nanomole per Minute Oxidoreductase Pyruvate Dehydrogenase Complex Deficiency Disease Pyruvates Radioactivity Screening procedure Skeletal muscle structure pyruvate dehydrogenase activity |
| Content Type | Text |
| Resource Type | Article |
