With mitochondria from Saccharomyces carlsbergensis the functional relationship of the exogenous and endogenous NAD system has been investigated with respect to the membrane barrier, the localisation of NADH dehydrogenase and other NAD-and flavine-linked dehydrogenases.
- 1Added NAD and NADH do not permeate through the inner membrane into the matrix space of mitochondria from S. carlsbergensis despite the high rate of oxidation. There is no exchange between exogenous and endogenous NADH and NAD.
- 2A transhydrogenation between external and internal NADH and NAD across the inner membrane is not observed.
- 3By applying the “ferricyanide method” two separate dehydrogenases can be identified: one for the oxidation of exogenous NADH, located towards the outer surface of the inner mitochondrial membrane; and a second for the oxidation of endogenous NADH directed towards the inner surface. Both dehydrogenases are connected to the cytochrome chain through the ubiquinone pool.
- 4About 50% of the total NADH dehydrogenase activity of yeast mitochondria is solubilized by sonication. The solubilized NADH dehydrogenase is tentatively identified with the external enzyme. This agrees with the finding that on opening the membrane by sonication the total activity of NADH-ferricyanide reduction increases by 65%.
- 5Applying both [4B-3H]NADH and [4A-3H]NADH the oxidation of both exogenous and endogenous NADH by ferricyanide and by oxygen are found to be B-specific.
- 6By application of the “ferricyanide method” to other substrates it is concluded that the dehydrogenases for glycerolphosphate and possibly lactate are localized before the barrier for ferricyanide while the dehydrogenases for ethanol, isocitrate and succinate are localized behind it. This barrier is identical with the inner mitochondrial membrane.
- 7The existence of a mitochondrial alcohol dehydrogenase, accounting for about 6% of the total cellular activity and located in the matrix space, is established. The dual localization of alcohol dehydrogenase is considered to facilitate the equilibration between the intra- and extra-mitochondrial NAD systems via an ethanol-acetaldehyde shuttle.
ethylene glycol bis(β-aminoethyl)-N, N′-tetraacetic acid
NADH dehydrogenase or reduced-NAD: (acceptor) oxidoreductase (EC 126.96.36.199)
succinate dehydrogenase or succinate: (acceptor) oxidoreductase (EC 188.8.131.52)
lipoamide dehydrogenase or reduced-NAD: lipoamide oxidoreductase (EC 184.108.40.206): malate dehydrogense or l-malate:NAD oxidoreductase (EC 220.127.116.11)
alcohol dehydrogenase or alcohol:NAD oxidoreductase (EC 18.104.22.168)
isocitrate dehydrogenase or threo-ds-isocitrate:NAD oxidoreductase (EC 22.214.171.124)