Partial Purification and Characterization of Three 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 β form of NADH. The other two activities, peaks II and III, oxidize only β-NADH. All three peaks are insensitive to divalent cation chelators and a complex I inhibitor, rotenone. The major component to 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 II 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.     

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

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 (NH₄)₂SO₄ 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.

     

Partial Purification and Characterization of the Quinol Oxidase Activity of Arum maculatum Mitochondria

The menadiol oxidase activity of Arum maculatum mitochondria has been solubilized and fractionated. A preparation has been obtained which has an increased specific activity and a greatly decreased polypeptide composition when compared to the mitochondria. This preparation retains normal inhibitor sensitivities in that the oxidation of menadiol remains insensitive to cyanide and is inhibited by aromatic hydroxamates. Metal analyses of the preparation showed that only iron was closely correlated with the oxidase activity. No unusual lipid components were detected in the preparation. The results are discussed in relation to chemical quinol oxidation mechanisms and to several recent hypotheses concerning the nature of the higher plant alternative oxidase.     

Purification and Characterization of Two Distinct NAD(P)H Dehydrogenases from Onion (Allium cepa L.) Root Plasma Membrane

Highly purified plasma membrane fractions were obtained from onion (Allium cepa L.) roots and used as a source for purification of redox proteins. Plasma membranes solubilized with Triton X-100 contained two distinct polypeptides showing NAD(P)H-dependent dehydrogenase activities. Dehydrogenase I was purified by gel filtration in Sephacryl S-300 HR, ion-exchange chromatography in DEAE-Sepharose CL-6B, and dye-ligand affinity chromatography in Blue-Sepharose CL-6B after biospecific elution with NADH. Dehydrogenase I consisted of a single polypeptide of about 27 kD and an isoelectric point of about 6. Dehydrogenase II was purified from the DEAE-unbound fraction by chromatography in Blue-Sepharose CL-6B and affinity elution with NADH. Dehydrogenase II consisted of a single polypeptide of about 31 kD and an isoelectric point of about 8. Purified dehydrogenase I oxidized both NADPH and NADH, although higher rates of electron transfer were obtained with NADPH. Maximal activity was achieved with NADPH as donor and juglone or coenzyme Q as acceptor. Dehydrogenase II was specific for NADH and exhibited maximal activity with ferricyanide. Optimal pH for both dehydrogenases was about 6. Dehydrogenase I was moderately inhibited by dicumarol, thenoyltrifluoroacetone, and the thiol reagent N-ethyl-maleimide. A strong inhibition of dehydrogenase II was obtained with dicumarol, thenoyltrifluoroacetone, and the thiol reagent p-hydroxymercuribenzoate.     

Identification and Characterization of an Inducible NAD(P)H Dehydrogenase from Red Beetroot Mitochondria

Exogenous NADH oxidation of mitochondria isolated from red beetroots (Beta vulgaris L.) increased dramatically upon slicing and aging the tissue. Anion-exchange chromatography of soluble fractions derived by sonication from fresh and aged beetroot mitochondria yielded three NADH dehydrogenase activity peaks. The third peak from aged beetroot mitochondria was separated into two activities by blue-affinity chromatography. One of these (the unbound peak) readily oxidized dihydrolipoamide, whereas the other (the bound peak) did not. The latter was an NAD(P)H dehydrogenase with high quinone and ferricyanide reductase activity and was absent from fresh beet mitochondria. Further affinity chromatography of the NAD(P)H dehydrogenase indicated enrichment of a 58-kD polypeptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We propose that this 58-kD protein is the inducible, external NADH dehydrogenase.     

Characterization of cuckoo-pint (Arum maculatum) mitochondrial adenosine triphosphatases.

The catalytic properties of cuckoo-pint (Arum maculatum) mitochondrial adenosine triphosphatase have been analysed. The pH profile, effect of inhibitors, cold-stability and substrate specificity are characteristic of mitochondrial adenosine triphosphatases, although a high guanosine triphosphatase activity does appear to be restricted to plant mitochondrial adenosine triphosphatases. The kinetic properties of nucleoside 5'-triphosphate hydrolysis by membrane-bound and soluble enzymes have been studied by means of double-reciprocal plots. These plots were linear in the absence of an activating anion, which may indicate that the catalytic and/or regulatory mechanism of Arum maculatum adenosine triphosphatase is different from that of other enzyme preparations. It is suggested that the differences in subunit composition of plant and mammalian adenosine triphosphatases reported previously [Dunn, Slabas & Moore (1985) Biochem. J. 225, 821-824] are structurally, rather than functionally, significant.     

Purification and Characterization of an NAD(P)H:Quinone Oxidoreductase from Glycine Max Seedlings

Abstract

An NAD(P)H:(quinone acceptor) oxidoreductase (EC 1.6.99.2) was purified from Glycine max seedlings by means of chromatographic procedures. After 1371-fold purification, the enzyme showed a single band in IEF corresponding to an isoelectric point of 6.1. A single band was also found in native-PAGE both by activity staining and Coomassie brilliant blue staining. The molecular mass determined in SDS-PAGE was 21900 Da, while in HPLC gel-filtration it was 61000 Da. The NAD(P)H:quinone oxidoreductase was able to use NADH or NADPH as the electron donor. Among the artificial quinones which are reduced by this enzyme, 6-hydroxydopa- and 6-hydroxydopamine-quinone are of particular interest because of their neurotoxic effects.     

Partial purification and properties of the external NADH dehydrogenase from cuckoo-pint (Arum maculatum) mitochondria.

The external NADH dehydrogenase has been purified from Arum maculatum (cuckoo-pint) mitochondria by phosphate washing, extraction with deoxycholate, ion-exchange and gel-filtration chromatography. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis shows, when the gel is silver-stained, that the purified enzyme contains two major bands of Mr 78 000 and 65 000 and a minor one of Mr about 76 000. It is not possible at present to determine which of these, or which combination, constitutes the dehydrogenase. The enzyme contains non-covalently bound FAD and a small amount of FMN. Since the conditions of purification lead to considerable loss of flavin and possibly iron-sulphur centres, it is not possible to decide with certainty whether the enzyme is a flavo- or ferroflavo-protein. The enzyme has been distinguished from the other NADH dehydrogenases on the basis of its substrate specificity, its capability of reducing electron acceptors such as ubiquinone-1 and 2,6-dichlorophenol-indophenol and its sensitivity towards Ca2+, EGTA and dicoumarol.     

Lipid Peroxidation Induced by NAD(P)H and NAD+-Dependent Substrates in Soybean Mitochondria

Lipid peroxidation induced by Fe²⁺ADP in soybean mitochondriais stimulated by pyruvate, malate (in the presence of NAD²⁺)and by NAD(P)H. This lipid peroxidation is almost completelyinhibited by EDTA indicating that iron is essential. Also salicylhydroxamicacid, a specific inhibitor of the alternate oxidase, is a stronginhibitor of lipid peroxidation but its effect should be relatedto chelation or to a general antioxidant action. Rotenone doesnot show any effect on the malateNAD²⁺Fe²⁺ADP-induced lipidperoxidation. From the reported data, it is possible to concludethat, in soybean mitochondria, the peroxidation of unsaturatedlipids can be modulated through a balance between the systemssparking lipid peroxidation, like NAD(P)H Fe²⁺ADP, and the systemswhich protect against it, i.e. quinones, maintained at the reducedstate by the substrates. (Received April 20, 1987; Accepted July 21, 1987)     

Purification and Characterization of NAD(P)H:Nitrate Reductase and NADH:Nitrate Reductase from Corn Roots

The nitrate reductase activity of 5-day-old whole corn roots was isolated using phosphate buffer. The relatively stable nitrate reductase extract can be separated into three fractions using affinity chromatography on blue-Sepharose. The first fraction, eluted with NADPH, reduces nearly equal amounts of nitrate with either NADPH or NADH. A subsequent elution with NADH yields a nitrate reductase which is more active with NADH as electron donor. Further elution with salt gives a nitrate reductase fraction which is active with both NADH and NADPH, but is more active with NADH. All three nitrate reductase fractions have pH optima of 7.5 and Stokes radii of about 6.0 nanometers. The NADPH-eluted enzyme has a nitrate K_m of 0.3 millimolar in the presence of NADPH, whereas the NADH-eluted enzyme has a nitrate K_m of 0.07 millimolar in the presence of NADH. The NADPH-eluted fraction appears to be similar to the NAD(P)H:nitrate reductase isolated from corn scutellum and the NADH-eluted fraction is similar to the NADH:nitrate reductases isolated from corn leaf and scutellum. The salt-eluted fraction appears to be a mixture of NAD(P)H: and NADH:nitrate reductases.     

Purification,Characterization, and Submitochondrial Localization of a 58-Kilodalton NAD(P)H Dehydrogenase

An NADH dehydrogenase activity from red beet (Beta vulgaris L.) root mitochondria was purified to a 58-kD protein doublet. An immunologically related dehydrogenase was partially purified from maize (Zea mays L. B73) mitochondria to a 58-kD protein doublet, a 45-kD protein, and a few other less prevalent proteins. Polyclonal antibodies prepared against the 58-kD protein of red beet roots were found to immunoprecipitate the NAD(P)H dehydrogenase activity. The antibodies cross-reacted to similar proteins in mitochondria from a number of plant species but not to rat liver mitochondrial proteins. The polyclonal antibodies were used in conjunction with maize mitochondrial fractionation to show that the 58-kD protein was likely part of a protein complex loosely associated with the membrane fraction. A membrane-impermeable protein cross-linking agent was used to further show that the majority of the 58-kD protein was located on the outer surface of the inner mitochondrial membrane or in the intermembrane space. Analysis of the cross-linked 58-kD NAD(P)H dehydrogenase indicated that specific proteins of 64, 48, and 45 kD were cross-linked to the 58-kD protein doublet. The NAD(P)H dehydrogenase activity was not affected by ethyleneglycol-bis([beta]-aminoethyl ether)-N,N[prime] -tetraacetic acid or CaCl2, was stimulated somewhat (21%) by flavin mononucleotide, was inhibited by p-chloromercuribenzoic acid (49%) and mersalyl (40%), and was inhibited by a bud scale extract of Platanus occidentalis L. containing platanetin (61%).     

Purification and identification of an FMN-dependent NAD(P)H azoreductase from Enterococcus faecalis

Azoreductases reduce the azo bond (N=N) in azo dyes to produce colorless amine products. Crude cell extracts from Enterococcus faecalis have been shown to utilize both NADH and NADPH as electron donors for azo dye reduction. An azoreductase was purified from E. faecalis by hydrophobic, anion exchange and affinity chromatography. The azoreductase activity of the purified preparation was tested on a polyacrylamide gel after electrophoresis under native conditions and the protein that decolorized the azo dye (Methyl Red) with both NADH and NADPH was identified by mass spectrometry to be AzoA. Previously, the heterologously expressed and purified AzoA was shown to utilize NADH only for the reduction of Methyl Red. However, AzoA purified from the wild-type organism was shown to utilize both coenzymes but with more than 180-fold preference for NADH over NADPH as an electron donor to reduce Methyl Red. Also, its specific activity was more than 150-fold higher than the previous study on AzoAwhen NADH was used as the electron donor. The catalytic efficiency for Methyl Red reduction by AzoA from E. faecalis was several orders of magnitude higher than other azoreductases that were purified from a heterologous source.     

Purification and characterization of an oxygen-insensitive NAD(P)H nitroreductase from Enterobacter cloacae

The reductive products of several nitroaromatic compounds have been found to be toxic, mutagenic, and carcinogenic. The nitroreductases present in intestinal microflora have been implicated in the biotransformation of these compounds to their deleterious metabolites. A "classical" nitroreductase has been purified from Enterobacter cloacae 587-fold using a protocol which yields approximately 1 mg of purified nitroreductase from 10 liters of cell culture. An analysis of the physical properties of the nitroreductase indicates that the enzyme is active as a monomer with a calculated molecular mass of 27 kDa. FMN has been identified as a required flavin cofactor and is present at a stoichiometry of 0.88 mol of FMN bound/mol of active enzyme. The enzyme was found capable of reducing nitrofurazone under aerobic conditions indicating that the mechanism involves an obligatory two-electron transfer. Thus, this enzyme can be classified as an oxygen-insensitive nitroreductase. The purified nitroreductase can utilize either NADH or NADPH as a source of reducing equivalents and can reduce a variety of nitroaromatic compounds including nitrofurans and nitrobenzenes as well as quinones. Studies in which the rates of nitroreduction for a series of para substituted nitrobenzene derivatives were determined suggest that a linear free energy relationship exists between the rate and the redox midpoint potential of the substrate.     

Purification and Characterization of the Soluble F(1)-ATPase of Oat Root Mitochondria

The properties of the soluble moiety (F₁) of the mitochondrial H⁺-ATPase from oat roots were examined and compared to those of the native mitochondrial membrane-bound enzyme. The chloroform soluble preparation was purified by Sephadex G-200 and DEAE-cellulose chromatography. The purified F₁ preparation contained major polypeptides corresponding to α, β, γ, δ, and ε of apparent molecular mass 58, 55, 35, 22, and 14 kilodaltons, respectively. The purified F₁-ATPase, like the native enzyme, was inhibited by azide (I₅₀ = 10 micromolar), nitrate (I₅₀ = 7-10 millimolar), 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid (I₅₀ = 1-3 micromolar), and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (I₅₀ = 3 micromolar). F₁-ATPase activity was stimulated by bicarbonate but not by chloride. In both the native and the F₁-form of the ATPase, ATP was hydrolyzed in preference to GTP. The results indicate that these properties of the native membrane-bound mitochondrial ATPase have been conserved in the purified F₁. In contrast to the membrane-bound enzyme, the F₁-ATPase was not inhibited by oligomycin or by N,N′-dicyclohexylcarbodiimide. The mitochondrial F₁-ATPase from oat roots is analogous to other known F₁F₀-ATPases.     

Partial Purification and Characterization of the Phosphate Transporter from Bovine Heart Mitochondria

A highly active phosphate transporter was extracted with octylglucoside from bovine heart submitochondrial particles that were first partially depleted of other membrane components. It was then partially purified by ammonium sulfate fractionation. After reconstitution of the transporter into liposomes prepared with a crude mixture of soybean phospholipids, the P_i/OH exchange, but not the P_i/P_i exchange, was stimulated three- to fourfold by valinomycin and nigericin in the presence of K⁺. Both P_i/OH and P_i/P_i exchange activities were sensitive to mercurials and other SH reagents. The rutamycin-sensitive ATPase complex from mitochondria was reconstituted together with the phosphate transporter and adenine nucleotide transporter into liposomes. After inhibition of externally located ATPase, the hydrolysis of ATP was sensitive to atractyloside and mersalyl.     

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

An NAD(P)H dehydrogenase stimulated by quinone (P Pupillo, V Valenti, L de Luca, R Hertel 1986 Plant Physiol 80: 384-389) was solubilized from washed microsomes of zucchini squash hypocotyls (Cucurbita pepo L.) by use of 1% Triton X-100. The solubilized enzyme remained in solution in aqueous buffer and could be purified by a combination of Sepharose 6B chromatography and Blue Ultrogel chromatography. Of the three peaks of activity eluted from the latter column with a salt gradient, peak 3 had 50% or more of the activity and was almost pure enzyme. The preparation examined in SDS-gel electrophoresis consisted of two types of subunits, a (molecular weight 39,500) and b (37,000) in equal amounts. Peak 2 was less pure but had a similar polypeptide pattern. The active protein is proposed to be a heterotetramer (a₂b₂) having a molecular weight of about 150,000, as found by gel exclusion chromatography. The purified enzyme can reduce several quinones, DCPIP, cytochrome c, and with best efficiency ferricyanide, and is therefore a diaphorase. The kinetics for the substrates are negatively cooperative with Hill coefficients n_H = 0.55 ± 0.05 for NADPH and 0.22 ± 0.04 for duroquinone. A weak inhibition by p-hydroxymercuric benzoate and mersalyl (stronger with microsomal preparations) suggests the presence of essential sulfhydryl group(s). The possibility is discussed that the dehydrogenase is an NAD(P)H-P450 reductase or similar flavoprotein, and that it is responsible for the NADPH-cytochrome c reductase activity of plant microsomes.     

NAD(P)H dehydrogenase from rabbit and rat liver: Purification and some properties

• 1.1. NAD(P)H dehydrogenase from rabbit liver was purified to electrophoretic homogeneity using a procedure also found applicable for the rat liver enzyme.
• 2.2. Rabbit and rat liver enzymes showed different behaviour in isoelectric focusing and different K_m values and turnover numbers.
• 3.3. Both enzymes were inhibited to similar extents by warfarin.
• 4.4. The rabbit enzyme is composed of two subunits of mol. wt 27,000 and contained 1 FAD group per subunit.
• 5.5. Some absorption and circular dichroism properties of the rat enzyme are shown.

     

Functional Characterization and Partial Purification of the Ubiquinol-Cytochrome c Oxidoreductase from Higher Plant Mitochondria (Helianthus tuberosus)

The functional and thermodynamic characteristics of the ubiquinolcytochrome (Cyt) c oxidoreductase in a Cyt b/c₁-enriched fraction (defined S-1) isolated from Jerusalem artichoke mitochondria (JAM) (Helianthus tuberosus), have been analyzed. Fraction S-1, obtained through deoxycholate-KCl fractionation procedure, contained one Cyt of c type (formally c₁ with Em_7.0 of +240 millivolts), two b type Cyt with Em_7.0 values of +100 and −25 millivolts, ferredoxin-like centers presumably linked to succinic- and NADH-dehydrogenases, and a Rieske-type iron sulfur center (g_y = 1.89). The ubiquinol-dependent Cyt c reduction by fraction S-1 showed sensitivity to antimycin A, myxothiazol, and n-2-hepthyl-1-hydroxyquinoline N-oxide with I₅₀ of 12 nanomolar, 30 nanomolar, and 0.1 micromolar, respectively. Oxidation-induced extra b type reduction, a widespread phenomenon of bacterial and mitochondrial respiratory systems, has also been observed in both intact mitochondria and S-1 fraction. The data seem to blur previous experiments in which both spectral and functional differences between higher plant and mammalian mitochondria have been underlined.