Abstract
A 43-kDa NAD(P)H dehydrogenase was purified from red beetroot mitochondria. An antibody against this dehydrogenase was used in conjunction with the membrane-impermeable protein cross-linker 3,3'-dithiobis(sulfosuccinimidylpropionate) to localize the dehydrogenase on the matrix side of the inner membrane. Immunoblotting showed that the dehydrogenase was found in mitochondria isolated from several plant species but not from rat livers. Antibodies against the purified dehydrogenase partially inhibited rotenoneinsensitive internal NADH oxidation by inside-out submitochondrial particles. The level of rotenone-insensitive respiration with NAD-linked substrates correlated with the amount of 43-kDa NAD(P)H dehydrogenase present in mitochondria isolated from different soybean tissues. Based on these results, we conclude that the 43-kDa NAD(P)H dehydrogenase is responsible for rotenone-insensitive internal NADH oxidation in plant mitochondria.
Highlights
It has been known for some time that plant mitochondria, unlike their mammalian counterparts, can oxidize NAD-linked substrates in the presence of the complex I inhibitor rotenone [1]
Rotenone lowered the ADP/O ratio for NADlinked substrates by one third [1], suggesting the presence of an internal NADH dehydrogenase that did not translocate protons and which was insensitive to rotenone. This was supported by the finding [2] that inside-out submitochondrial particles (SMP)1 from Jerusalem artichoke exhibited two different Km values for NADH, depending on whether rotenone was present or not. These results suggested that the rotenoneinsensitive bypass was mediated by a distinct NADH dehydrogenase that had a high Km for NADH relative to complex I
We report on the purification of a 43-kDa NAD(P)H dehydrogenase from red beetroot mitochondria and provide strong evidence that this enzyme is responsible for rotenone-insensitive internal NADH oxidation
Summary
Materials—Red beetroots (Beta vulgaris L.) and Potatoes (Solanum tuberosum L.) were purchased from local markets. Preparation and Fractionation of Mitochondria—Beetroot mitochondria were prepared by the method of Menz et al [17], rat liver mitochondria by the method of James et al [18], potato tuber mitochondria by the method of Liden and Akerland [19], and soybean mitochondria by the method of Day et al [20]. Anion exchange chromatography was performed on a Pharmacia resource Q (1 ml) column, while blue affinity chromatography was performed on a Pharmacia Hi-Trap blue column The buffer for these procedures was 20 mM Tris/HCl (pH 8.0), and proteins were eluted with gradients of NaCl (0 –350 mM) or NADPH (0 –10 M) as indicated. NADH:FeCN reductase activity was measured in a similar fashion except 0.5 mM FeCN replaced Q0, and the reduction of FeCN was monitored at 420 nm (extinction coefficient at 420 nm ϭ 1.05 mMϪ1 cmϪ1). Protein was estimated by the method of Bradford [25] using the Bio-Rad reagent and bovine serum albumin (fraction V) as a standard
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