The organo-specific external NADH dehydrogenase of mammal heart mitochondria has an artefactual origin.
The existence of an organo-specific (heart) external NADH dehydrogenase located on the outer face of the inner mitochondrial membrane has been recently proposed. We have studied the respiration on external NADH in rat and beef heart mitochondrial fractions: (i) by using different mitochondrial isolation procedures on the rat, we observed that the higher the criteria of quality toward classical substrate respiration of mitochondrial fractions, the lower the external NADH-linked respiration; (ii) by using an especially loosely fitting glass-Teflon homogenizer, we obtained rat heart mitochondrial fractions practically free from external NADH linked respiration and with the highest respiratory control ratio on glutamate plus malate respiration. In rat and beef heart mitochondrial fractions containing an external NADH respiration: (i) ethoxyformic anhydride used previously to distinguish internal and external NADH oxidation was shown not to be specific; (ii) external NADH-linked respiration (although associated to the normally functioning respiratory chain as was shown by the effects of classic respiratory inhibitors) did not lead to ADP phosphorylation while glutamate plus malate did; (iii) respiratory activity on glutamate plus malate and external NADH was totally additive and the oxidation corresponded to two separate cytochrome oxidase pools, indicating a total functional separation between the two respiratory systems; (iv) NAD+ addition stimulated states 3 and 4 glutamate plus malate respiration to the same extent, indicating the presence of an appreciable number of internal dehydrogenases accessible to external cofactors. These results show that external NADH-linked dehydrogenase activity, which is usually detectable in mammal heart mitochondrial fractions, is of artefactual origin.
Fraisse
L
L
Groupement de Recherche de Lacq, Artix, France.
Rey
E
E
Rigoulet
M
M
D000818:Animals; D002417:Cattle; D003576:Electron Transport Complex IV / Q000378:metabolism; D008297:Male; D008929:Mitochondria, Heart / Q000201:enzymology*; D009245:NADH Dehydrogenase / Q000737:chemistry*; D010766:Phosphorylation; D051381:Rats; D017208:Rats, Wistar
D009245:NADH Dehydrogenase; D003576:Electron Transport Complex IV