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

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+.