PURIFICATION, CHARACTERIZATION, AND SUBMITOCHONDRIAL LOCALIZATION OF THE 32-KILODALTON NADH DEHYDROGENASE FROM MAIZE

Plant mitochondria have the unique ability to directly oxidize exogeno us NAD(P)H. We recently separated two NAD(P)H dehydrogenase activities from maize (Zea mays L.) mitochondria using anion-exchange (Mono Q) c hromatography. The first peak of activity oxidized only NADH, whereas the second oxidized both NADH and NADPH. In this paper we describe the purification of the first peak of activity to a 32-kD protein. Polycl onal antibodies to the 32-kD protein were used to show that it was pre sent in mitochondria from several plant species. Two-dimensional gel a nalysis of the 32-kD NADH dehydrogenase indicated that it consisted of two major and one minor isoelectric forms. Immunoblot analysis of sub mitochondrial fractions indicated that the 32-kD protein was enriched in the soluble protein fraction after mitochondrial disruption and fra ctionation; however, some association with the membrane fraction was o bserved. The membrane-impermeable protein cross-linking agent 3,3'-dit hiobis-(sulfosuccinimidylpropionate) was used to further investigate t he submitochondrial location of the 32-kD NADH dehydrogenase. The 32-k D protein was localized to the outer surface of the inner mitochondria l membrane or to the intermembrane space. The pH optimum for the enzym e was 7.0. The activity was found to be severely inhibited by p-chloro mercuribenzoic acid, mersalyl, and dicumarol, and stimulated somewhat by flavin mononucleotide.