Table 2.

The NADH-supported generation of hydrogen peroxide by permeabilized rat heart mitochondria (pH 7.5, 30°C)^a

	Hydrogen peroxide production (nmol/min per mg protein)
	− NADH-OH		+ NADH-OHb
	− NH4Cl	+ NH4Clc	− NH4Cl	+ NH4Clc
NADH (50 μM)	5.1±0.4	19.2±2.6	2.4±0.2	15.1±0.6
NADH (50 μM) + NAD+ (50 μM)	1.0±0.2	3.1±0.6	0.7±0.1	3.9±1.0
NADH (2 mM)	5.3±0.2	33.3±2.3	3.9±0.5	32.9±0.9
NADH (2 mM) + NAD+ (2 mM)	1.2±0.1	2.2±0.3	0.9±0.1	1.7±0.2

^aAverage values from 4 experiments. Mitochondria (20 μg/ml) were incubated for 0.5 min in the standard reaction mixture containing alamethicin (40 μg/ml), 2.5 mM MgCl₂, and 5 μM rotenone. The reaction was started by the addition of NADH. Treatment with alamethicin increased the rate of externally added NADH (50 μM) oxidation by mitochondria in the absence of rotenone from 0.06 to 1.4 μmol/min per mg protein. The latter activity was more than 90% sensitive to NADH-OH.

^bNADH-OH (2.5 nmol/mg protein) was added after permeabilization and preincubation was continued for 1 min.

^c30 mM NH₄Cl was presented in the assay mixture.