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Isolated potato (Solanum tuberosum) tuber mitochondria purified by isopycnic centrifugation in density gradients of Percoll were found to be highly intact, to be devoid of extramitochondrial contaminations and to retain a high rate of O2 consumption. When suspended in a medium that avoided rupture of the outer membrane, intact purified mitochondria progressively lost their NAD+ content by passive diffusion. This led to a slow decrease of oxoglutarate-dependent O2 consumption by isolated mitochondria. Addition of NAD+ to the medium restored the initial State-3 rate of oxoglutarate oxidation. The rate of NAD+ accumulation in the matrix space was concentration-dependent, exhibited Michaelis-Menten kinetics and was strongly inhibited by the analogue N-4-azido-2-nitrophenyl-4-aminobutyryl-NAD+.

Slow passive diffusion of NAD+ between intact isolated plant mitochondria and suspending medium