Kinetic Characterization of Reduced Pyridine Nucleotide Dehydrogenases (Duroquinone-Dependent) in Cucurbita Microsomes

Abstract

Some properties of microsomal electron transfer chains, dependent for oxidase activity on addition of NADH or NADPH, duroquinone, and oxygen (L. De Luca et al., 1984, Plant Sci Lett 36: 93-98) are described. Activity is characterized by negatively cooperative kinetics toward reduced pyridine nucleotides, with limiting K,, of 10 to 50 micromolar at pH 7.0 (increasing at lower pH), as well as toward duroquinone with limiting K, of 100 to 400 micromolar regardless of pH. Molecular oxygen is reduced by the enzyme complex with S_.s of about 30 micromolar and production of H20 and H202, without superoxide involvement. The ratio NAD(P)H:02 averages 1.35 in the presence of KCN and 1.85 in its absence. The pyridine nucleotide specificity of the dehydrogenases has been investigated by kinetic competition experiments. Some enzyme heterogeneity was established for all preparations. At least two enzymes are detectable in plasma membrane-enriched fractions: a major NAD(P)H dehydrogenase having an acid pH optimum, and an NADPH dehydrogenase active around neutrality. Addition of Triton X-100 strongly enhances the activity over most of the pH scale, but depresses it increasingly at pH values higher than 8.0, to the effect that pH profile shows, under these conditions, a major peak at about pH 5.8 for both NADH and NADPH oxidase. Results with endoplasmic reticulum preparations are similar, except that they suggest the presence of still more activities at and above pH 7. The results are interpreted in terms of different complexes catalyzing electron transfer from NAD(P)H to 02 without release of intermediates.Recent studies (1,3,7,21,22,25) indicate that reducing equivalents can be transferred from the inside of plant cells to the outer space through the plasma membrane, for control of the neighboring environment, uptake of nutrients, and intracellular regulation. An NADH oxidase ofroot cell surfaces, possibly coupled to a transport system (12,14), has been isolated and seems to contain quinone (15). Some typical redox chain components such as flavins and a light sensitive b-type Cyt are present in the plasmalemma (8,10,13,27 (16), and may not be related to the bulk of DQ-dependent oxidases of the plasma membrane.Because of the uncertainties, we have undertaken a detailed biochemical characterization of these oxidases. This paper deals mainly with the nature ofthe reactions. It also shows that among different components a pyridine nucleotide-nonspecific dehydrogenase is responsible for most of the activity.MATERIALS AND METHODS Plant Material and Tissue Fractionation (5, 9). Cucurbita pepo L. was grown from seeds (hybrid 'Senator', Asgrow Co.) for 5 d in the dark at 24°C on vermiculite. Hypocotyl segments 2 to 3 cm below the hook were harvested. All subsequent procedures were carried out on ice in daylight.Hypocotyl sections were chopped with razor blades in two volumes of extraction medium (0.25 M sucrose, 50 mM Tris-HCl pH 7.5, 1 mm Na2EDTA, 1 mm Na2EGTA), then ground in a mortar and squeezed through a nylon cloth. The...

NAD(P)H:(Quinone-Acceptor) Oxidoreductase of Tobacco Leaves Is a Flavin Mononucleotide-Containing Flavoenzyme

“…~ Plant NAD(P)H-QR (EC 1.6.99.2) has long been thought to be a peculiar type of DT-diaphorase (Pupillo et al, 1986;Valenti et al, 1989). Like the DT-diaphorase of animal tissues (Ernster, 1987), which does not seem to be present in higher plant cells (Trost et al, 1995), this plant enzyme can use either NADPH or NADH as an electron donor and hydrophilic quinones as acceptors.…”

Solubilization and Purification of NAD(P)H Dehydrogenase of Cucurbita Microsomes

“…fairly similar to that of the enzyme bound to the microsomes (19 were found to be sensitive to PHMB and especially mersalyl. With the purified enzyme the effect was small and was seen only at relatively high concentrations of PHMB and mersalyl (Table III).…”

“…The purified dehydrogenase (peak 3) exhibits a kinetic behavior towards its substrates similar to that observed with the enzyme in situ, i.e. convex curves in double reciprocal plots (19). In the high velocity-low affinity range (with 0.15 mM DQ) the response to increasing pyridine nucleotide concentrations is pseudolinear with either NADPH or NADH, with a common limiting Vm,, of 50 gmol min-' mg-' protein.…”

“…We have shown recently that the major, quinone-stimulated dehydrogenase of squash hypocotyl microsomes (7,18) can accept electrons from both NADPH and NADH, although it prefers NADPH, and can reduce molecular oxygen through duroquinone to H202 without detectable involvement of superoxide radicals (19). It was suggested that this pyridine-nucleotide nonspecific activity may bear a relationship to some redox systems reported for the plasmalemma of intact cells or organs, although some of these systems seem to be NADH-linked (12,15).…”

“…The major aim of our work was to solubilize and purify the microsomal enzyme which is responsible for quinone-stimulated NAD(P)H dehydrogenase of zucchini hypocotyls (7,19), in the attempt to understand more about the nature of the enzyme(s) concerned. full voltage).…”