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

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Abstract
A procedure was developed to separate and partially purify 2 NAD(P)H dehydrogenases from the inner membrane of cauliflower mitochondria. The procedure used Triton X-100 extraction followed by (NH4)2SO4 precipitation and gel filtration (Sepharose G-200 column) chromatography. The 1st 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 2 major polypeptides with MW of .apprx. 57,600 and 32,600 daltons. The fraction exhibited EPR signals associated with a reduced (ferredoxin-type) Fe-S center. A 2nd dehydrogenase fraction was eluted from the column after removal of the 1st dehydrogenase. This fraction oxidized NADH and NADPH, was stable at high temperatures, and had a broad pH optima that ranged from 6.0-7.8. Although it was relatively insensitive to additions of monovalent and divalent cations, its activity was sensitive to incubation with sulfhydryl reagents. The 2nd dehydrogenase fraction contained 5 major polypeptides and lacked the Fe-S protein EPR signals shown by the 1st dehydrogenase fraction. The dehydrogense fractions represent 3 potential sites of entry to mitochondrial electron transport; 2 sites for NADH and a 3rd site for NADPH.