Separation Procedure and Partial Characterization of Two NAD(P)H Dehydrogenases from Cauliflower Mitochondria

Abstract

A procedure was developed to separate and partially purify two NAD(P)H dehydrogenases from the inner membrane of cauliflower (Brassica oleracea L.) mitochondria. The procedure used Triton X-100 extraction followed by (NH(4))(2)SO(4) precipitation and gel filtration (Sepharose G-200 column) chromatography. The first dehydrogenase fraction (which eluted in the column void volume) was specific for NADH, was stimulated by KCl addition, and was inhibited by acidic pH, sulfhydryl reagents, and elevated temperature. This fraction contained two major polypeptides with molecular weights of about 57,600 and 32,600 daltons. The fraction exhibited electron paramagnetic resonance (EPR) signals associated with a reduced (ferredoxin-type) iron-sulfur center.A second dehydrogenase fraction was eluted from the column after removal of the first dehydrogenase. This fraction oxidized NADH and NADPH, was stable at high temperatures, and had a broad pH optima that ranged from 6.0 to 7.8. Although it was relatively insensitive to additions of monovalent and divalent cations, its activity was sensitive to incubation with sulfhydryl reagents. The second dehydrogenase fraction contained five major polypeptides and lacked the iron-sulfur protein EPR signals shown by the first dehydrogenase fraction.The dehydrogenase fractions represent three potential sites of entry to mitochondrial electron transport; two sites for NADH and a third site for NADPH.