Quantification of muscle mitochondrial oxidative phosphorylation enzymes via histochemical staining of blue native polyacrylamide gels.
Blue native-polyacrylamide gel electrophoresis is a powerful technique that enables the separation of intact multi-subunit complexes. However, positive identification of particular enzymes generally requires further separation in a second dimension on a denaturing polyacrylamide gel. Histochemical staining is widely used to demonstrate enzyme activities in tissues, including oxidative phosphorylation enzymes. In this report, we demonstrate that the two techniques can be combined to quantify in situ mitochondrial enzymes, separated on nondenaturing polyacrylamide gels. The method gives quantitative results with human skeletal muscle as well as heart that contains higher mitochondrial numbers. Comparison of muscle from patients with oxidative phosphorylation enzyme deficiencies, such as those of two riboflavin-responsive patients, before and after vitamin treatment, gives results in agreement with those obtained by analyzing the activity of the mitochondrial enzymes in muscle homogenates.
Zerbetto
E
E
Dipartimento di Farmacologia, Universita' di Padova, Italy.
Vergani
L
L
Dabbeni-Sala
F
F
D000251:Adenosine Triphosphatases / Q000032:analysis; D000818:Animals; D002417:Cattle; D003902:Detergents; D003576:Electron Transport Complex IV / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006651:Histocytochemistry; D006801:Humans; D008928:Mitochondria / Q000201:enzymology*; D008929:Mitochondria, Heart / Q000201:enzymology; D018482:Muscle, Skeletal / Q000201:enzymology; D009132:Muscles / Q000648:ultrastructure*; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000032:analysis; D010085:Oxidative Phosphorylation; D051381:Rats; D013385:Succinate Dehydrogenase / Q000032:analysis
D003902:Detergents; D013385:Succinate Dehydrogenase; D016660:NAD(P)H Dehydrogenase (Quinone); D003576:Electron Transport Complex IV; D000251:Adenosine Triphosphatases