An improved spectrophotometric assay of pyruvate dehydrogenase in lactate dehydrogenase contaminated mitochondrial preparations from human skeletal muscle.
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.
Chretien
D
D
Unité de Recherches sur les Handicaps Génétiques de l'Enfant, INSERM U-393, Hôpital des Enfants-Malades, Paris, France.
Pourrier
M
M
Bourgeron
T
T
Séné
M
M
Rötig
A
A
Munnich
A
A
Rustin
P
P
D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000032:analysis* / Q000037:antagonists & inhibitors; D008931:Mitochondria, Muscle / Q000201:enzymology*; D018482:Muscle, Skeletal / Q000201:enzymology* / Q000648:ultrastructure; D009243:NAD / Q000032:analysis; D010072:Oxamic Acid / Q000494:pharmacology; D010084:Oxidation-Reduction; D011768:Pyruvate Dehydrogenase Complex / Q000032:analysis*; D011817:Rabbits; D013056:Spectrophotometry, Ultraviolet; D013552:Swine
D011768:Pyruvate Dehydrogenase Complex; D009243:NAD; D007770:L-Lactate Dehydrogenase; D010072:Oxamic Acid