3 acetylpyridine adenine dinucleotide

Spectrophotometric assessment of NNT activity was performed essentially as described by others [49 (link), 50 (link)]. The reaction mixture was composed of 50 mM Tris (pH 8.0), 300 μM APAD (3-acetylpyridine adenine dinucleotide; Sigma-Aldrich), an analogue of NAD⁺, 300 μM β-NADPH and 10 μM rotenone. APAD was used instead of NAD⁺ because this compound has a different absorption spectrum from NADPH and NADH. The reaction was initiated by the addition of 100–150 μg homogeneized mitochondrial enriched-membrane proteins (20 μl) obtained by 3 cycles of freeze-thawing followed by a 5-s sonication with a Fisher 550 sonic dismembrator (intensity 2.5) [51 (link)]. Changes in absorbance were monitored at 375 nm for 3 min. Rates of NNT were converted to nmol APAD reduced/min using the extinction coefficient (5.1 mM⁻¹ × cm⁻¹) and normalized to protein, which was determined using the BCA assay. To provide experimental controls for NNT-mediated APAD reduction, homogenized mitochondria were incubated for 5 min with 1 mM palmitoylCoA, competitive inhibitor of NNT [52 (link)].

Tris, EDTA, HEPES, MOPS, NaCl, palmitoylCoA, rotenone, sucrose, 3-acetylpyridine adenine dinucleotide (APAD), and β-NADPH were all purchased from Sigma (St. Louis, MO). All reagents were of analytical grade or higher.