PARTIAL-PURIFICATION AND CHARACTERIZATION OF 3 NAD(P)H DEHYDROGENASES FROM BETA-VULGARIS MITOCHONDRIA

Mitochondria isolated from the taproot of beet (Beta vulgaris) were used in an effort to identify and partially purify the proteins constituting the exogenous NADH dehydrogenase. Three NAD(P)H dehydrogenases are released from these mitochondria by sonication, and these enzymes were partially purified using fast protein liquid chromatography. One of the enzymes, designated peak I, is capable of oxidizing NADPH and the beta-form of NADH. The other two activities, peaks II and III, oxidize only beta-NADH. All three peaks are insensitive to divalent cation chelators and a complex I inhibitor, rotenone. The major component of peak I is a polypeptide with an apparent molecular mass of approximately 42 kilodaltons. Peak I activity was insensitive to platanetin, a specific inhibitor of the exogenous dehydrogenase, and insensitive to added Ca2+ or Mg2+. Peak I displayed a broad pH activity profile with an optimum between 7.5 and 8.0 for both NADPH and NADH. Purified peak 11 gave a single polypeptide of about 32 kilodaltons, had a pH optimum between 7.0 and 7.5, and was slightly stimulated by Ca2+ and Mg2+. As with peak I, platanetin had no effect on peak II activity. Peak III was not purified completely, but contained two major polypeptides with apparent molecular masses of 55 and 40 kilodaltons. This enzyme was not affected by Ca2+ and Mg2+, but was inhibited by platanetin. The peak III enzyme had a rather sharp pH optimum of approximately 6.5 to 6.6. The above data indicate that peak III activity is likely the exogenous NADH dehydrogenase.