Purification and Characterization Two NAD(P)H ...brane fractions by the two-phase partition method (Alberts- son et al., 1982) with a phase system composed of 6.4% dextran T 500 (Pharmacia,

$Page 1: Purification and Characterization Two NAD(P)H ...brane fractions by the two-phase partition method (Alberts- son et al., 1982) with a phase system composed of 6.4% dextran T 500 (Pharmacia,$
Plant Physiol. (1994) 106: 87-96

Purification and Characterization of Two Distinct NAD(P)H Dehydrogenases from Onion (Allium cepa 1.)

Root Plasma Membrane'

Antonio Serrano, Francisco Córdoba*, Jose Antonio Conzález-Reyes, Plácido Navas*, and Jose Manuel Villalba

Departamento de Biologia Celular, Facultad de Ciencias, Universidad de Córdoba, Avda. San Alberto Magno s/n, E-1 4004-CÓrdoba, Spain

~~~

Highly purified plasma membrane fractions were obtained from onion (Allium cepa 1.) 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-CB 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. Dehydro- genase I I 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-h ydroxymercuribenzoate.

Redox activities intrinsic to the plasma membrane have been demonstrated in all eukaryotic cells tested thus far (Crane et al., 1985). It has been proposed that part of these activities is due to the existence of redox systems able to catalyze the transmembrane transfer of electrons from reduced intracellular donors [NAD(P)H or ascorbate] to extemal impermeable acceptors (Askerlund and Larsson, 1991; Buck- hout and Luster, 1991). Transmembrane redox activity at the surface of plant cells has been associated with a variety of processes including light-mediated events, hormonal regula- tion of cell growth, maintenance of the reduced state of sulfhydryl groups of membrane proteins, iron uptake, H+

'This work was partially supported by the Spanish Dirección General de Investigación Científica y Tecnológica (Project No. PB 92-0714). J.M.V. was the recipient of a research contract from the Spanish Ministerio de EducaciÓn y Ciencia.

Present address: Departamento de Ciencias Agroforestales, Univ- ersidad de Huelva, Huelva, Spain.

* Corresponding author; fax 34-57-218634. 87

extrusion, and fluxes of other solutes (for reviews, see Mdller and Crane, 1990; Bottger et al., 1991; Buckhout and Luster, 1991; Rubinstein and Luster, 1993).

In addition to trans electron transport activity, the plant plasma membrane also contains cis activities exposed on both the cytoplasmic side and the external surface (Bottger et al., 1991). Activities such as NAD(P)H-Cyt c oxidoreductase, fatty acyl COA desaturase, and a portion of NADH-FeCN oxidoreductase fall into this category and are based on a Cyt b5 reductase-Cyt b5 system that is oriented cytoplasmically (Crane et al., 1991). Also, there exists an extemal NAD(P)H oxidase that likely corresponds to a plasma membrane-bound peroxidase (Mdller and Crane, 1990).

The purification and characterization of the protein components able to catalyze redox reactions at the plasma membrane are essential steps to clarify relevant aspects such as the actual composition of the redox systems, their substrate specificity, physiological significance, and regulatory mechanisms. For instance, although Bienfait (1985) proposed the existence of two different redox systems, one involved in the reduction of FeCN and Fe3+ chelates (turbo reductase) and another one unable to reduce Fe3+ chelates (standard reductase), the possibility that the standard reductase might be transformed into a turbo reductase under conditions of iron deficiency has not yet been ruled out (Bienfait, 1988; Holden et al., 1991).

In recent years, several attempts to purify redox proteins from plant microsomes (Guemni et al., 1987) or isolated plant plasma membranes (Brightman et al., 1988; Luster and Buckhout, 1988, 1989; Askerlund et al., 1991; Holden et al., 1991) have been reported. However, results obtained with microsomes are difficult to attribute unequivocally to the plasma membrane, and in most cases only partial purification of redox proteins from purified plasma membranes was achieved. Only Luster and Buckhout (1989) reported the purification to homogeneity of a 27-kD plasma membrane- associated electron transport protein from maize roots, although they acknowledged that the root plasma membrane should still contain more redox enzymes that were not recovered following their purification procedure.

Abbreviations: AFR, ascorbate free radical; DPIP, dichloropheno- lindophenol; FeCN, potassium ferricyanide; INT, p-iodonitrotetra- zolium violet; PHMB, p-hydroxymercuribenzoate; SHAM, salicylhy- droxamic acid; TTFA, thenoyltrifluoroacetone.

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88 Serrano et al. Plant Physiol. Vol. 106, 1994

Here we show the purification to homogeneity of two redox proteins isolated from highly enriched onion (Allium cepa L.) root plasma membrane fractions. The purified proteins show differential specificity for the electron donors NADH and NADPH and different sensitivity to inhibitors. Also, the purified proteins can be clearly distinguished on the basis of the rates of reduction of various electron acceptors and their physicochemical properties such as molecular mass and isoelectric point.

MATERIALS AND METHODS

Plant Material

Onion (Allium cepa L.) bulbs (approximately 35 g) were grown in the dark in tap water at a constant temperature (25OC) on plastic trays (30-36 bulbs/tray) in containers of about 3.5 L capacity in such a way that only their bases were in contact with water. The water was renewed every 24 h and aerated with an air pump. After 14 d the roots (about 14 cm long) were rinsed with distilled water and cut with scissors prior to homogenization for plasma membrane separation.

Isolation of Plasma Membrane Fractions

Unless otherwise stated all operations with roots, membranes, and purified fractions were carried out at O to 4OC. Washed and cut roots were homogenized in a Moulinex juice extractor as recommended by Serrano (1988), the extracted juice being collected directly over the homogenization buffer (20 mL/100 g fresh weight of roots). This buffer contained 30% (w/v) SUC, 25 m EDTA, 5 IT~M DTT, and 0.25 M Tris adjusted to pH 8 with solid Mes. Just before use, protease inhibitors (2.5 II~M PMSF and 125 rg/mL chymostatin) were added. Homogenates were filtered through cheese- cloth to remove fibrous residues and centrifuged for 10 min at 5,600g to remove debris. The supematant was then centrifuged for 30 min at 46,00Og, and the pellet containing the crude membrane fraction was washed again by centrifugation and resuspended with the aid of a hand-held homogenizer in 20 mL of 0.25 M SUC, 5 m potassium phosphate buffer, pH 7.8, containing 0.5 m PMSF and 25 r g mL-' chymostatin kg-' starting material.

Plasma membranes were then purified from crude membrane fractions by the two-phase partition method (Alberts- son et al., 1982) with a phase system composed of 6.4% dextran T 500 (Pharmacia, Uppsala, Sweden), 6.4% PEG 3350 (Fisher Scientific), 0.25 M SUC, and 5 m potassium phosphate buffer, pH 7.8. Briefly, crude membranes were combined with the phase systems, and the mixtures were shaken 40 times and centrifuged at lOOOg for 5 min to separate the phases. Upper phases, enriched in plasma membrane, were collected and added to unused lower phases; the process of mixing and separation was repeated three times (Albertsson et al., 1982). Final upper phases, containing highly purified plasma membrane, were diluted, washed twice to remove PEG, and resuspended in about 5 mL of 0.25 M SUC, 15 m Tris/Mes, pH 7.5, containing 0.5 ITLM PMSF and 25 r g mL-' chymostatin kg-' starting roots. Puri- fied plasma membranes were stored frozen in liquid nitrogen until needed.

Marker Enzymes

The fadlowing marker enzymes were used to check the purity of plasma membrane fractions: diethylstilbestrol-sensitive ATPase for plasma membrane (Serrano, 1988), cyanide- sensitive Cyt c oxidase for mitochondria, and NADPH-Cyt c oxidoreductase for ER (Lord et al., 1973; Stome and Madden, 1990), pyrophosphatase for tonoplast (Chanson, 1990), and latent IDPase for Golgi apparatus (Asard et al., 19 87).

EM and Morphometry

On-section staining with phosphotungstic acic! was used as an additional specific marker for plasma memtranes (Wi- dell and Larsson, 1990). Samples of crude membranes and purified plasma membranes were fixed in 2.5% glutaralde- hyde, postfixed in 1% osmium tetroxide, dehydrated, and embedded in Epon. Thin sections were cut, placell on nickel grids, and incubated for 30 min in 1% periodic x i d before staining with 1% phosphotungstic acid in 10% chromic acid. Purity of plasma membrane fractions was estimated by plan- imetry (VVeibel, 1979). Briefly, a line-test system was applied to obtain the number of intersections with plasma membrane vesicles (phosphotungstic acid stained) and with other possible membranes (unstained). The punty was calculated from the formiula P = 100 Ipm It-', where P = the estimated purity (in percentage), Ipm = the number of intersections with plasma membrane vesicles, and It = the total number of intersections in the test.

Solubilization of Plasma Membrane Proteins

Plasma membranes were extracted with 0.5 M KCl in 25 II~M Tris-HC1, pH 7.6, containing 1 m EDTA and 1 ITLM PMSF to remove peripheral proteins and adsort bed soluble proteins. The pellet obtained after centrifugation for 30 min at 105,OOOg was resuspended in buffer without KCI and solubilizttd with Triton X-100 at a protein concentration of 8 to 12 mg/mL and a detergent:protein ratio of 3:l. After incubation for 1 h on ice, solubilized proteins wen? separated by ultracentrifugation for 1 h at 105,OOOg. Solubilized fractions were pooled and stored in liquid nitrogen urttil needed.

Purification of Plasma Membrane Dehydrogenases

Step 1 . Size Exclusion Chromatography

Solubilized proteins were first fractionated in a L.6- X 100- an colunnn of Sephacryl 300 HR (Sigma). The gc?l was pre- equilibrated with column buffer (25 m Tris-HCl, pH 7.6, Containing 1 II~M EDTA, 0.5 mM PMSF, and 0.54; Triton X- loo), and, after application of the sample (10 mi, at 7 mg/ mL), fractions of 4.8 mL were collected. The flow rate was maintained at 20 mL/h.

Step 2. Ion-Exchange Chromatography

Fractions containing the peak of dehydrogenase activity from the preceding step were pooled and applied at 20 mL/ h to a column (1.6 X 14 cm) of DEAE-Sepharose CL-6B (Sigma) pre-equilibrated with column buffer. After the sample was washed with 10 volumes of column buffer, bound



NAD(P)H Dehydrogenases from Root Plasma Membrane 89

proteins were eluted with a linear KCl gradient (200 mL, 0-1 M).

Step 3. Dye-Ligand Affinity Chromatography

Final purification of dehydrogenases was achieved by chromatography in Blue-Sepharose CL-6B (Pharmacia). Dehydro- genase I was purified from the fraction adsorbed to the DEAE-Sepharose column. After salt elution, fractions containing dehydrogenase activity were desalted in small columns of Sephadex G-25 (PD-10 columns, Pharmacia) according to the manufacturer's recommendations. Dehydro- genase I1 was purified from the unbound DEAE fraction, which was used directly for further purification. Samples containing both dehydrogenases were applied separately at 8 mL/h to a 1.6- X 8-cm column of Blue-Sepharose that had been previously equilibrated with column buffer. The column was extensively washed with at least 10 column volumes of column buffer to remove unbound proteins (Luster and Buck- hout, 1989), and elution (1-mL fractions) was accomplished by including 30 PM or 1 mM NADH in the column buffer for dehydrogenases I and 11, respectively.

Oxidoreductase Activities

NAD(P)H-FeCN reductase activity was assayed spectro- photometrically at 3OoC by measuring the decrease in A340 in a medium containing 250 mM SUC, 0.2 mM FeCN, 50 m Tris-HC1, pH 7.6, 0.2 m NAD(P)H, 75 to 100 pg of plasma membrane protein or 15 to 200 pL of column eluate (depend- ing on the degree of purification), and f 0.025% (w/v) Triton X-100 with plasma membranes for latency determinations. The reaction was initiated with the addition of NAD(P)H, and the rates obtained were corrected for both nonenzymic reaction between NADH and FeCN and the rate of NAD(P)H oxidation in the absence of added acceptor. An extinction coefficient of 6.22 m-' cm-' was used in calculations of specific activities when pyridine nucleotides were recorded at 340 nm.

NAD(P)H-juglone (5-hydroxy-1,4-naphthoquinone), -duroquinone (tetramethyl-p-benzoquinone), -menadione (vitamin K,; 2-methyl-1,4-naphthoquinone), and -coenzyme Qo (2,3-dimethoxy-5-methyl-1,4-benzoquinone) reductase activities were assayed basically as described above, but 0.1 mM juglone, duroquinone, menadione, or coenzyme Qo was substituted for FeCN. Quinone stocks were made in absolute ethanol. A correction was made for the simultaneous decrease in A340 due to the reduction of quinones, assuming an NAD(P)H/quinone stoichiometry of 1. NAD(P)H-DPIP reductase was assayed in a final volume of 1 mL in the same reaction mixture containing 0.1 mM DPIP. Changes in A600 were recorded with an extinction coefficient of 20.6 m-' cm-'. NAD(P)H-INT reductase was assayed in a similar way with 0.2 mM INT. ASO0 was recorded with an extinction coefficient of 11.5 mM-' cm-'.

NAD(P)H-AFR reductase was assayed by measuring the decrease in ,4340 in 50 mM Tris-HC1, pH 7.6, and a final volume of 1 mL. To generate the free radical, 1 m ascorbate and 60 milliunits of ascorbate oxidase were added to the reaction mixture. After about 100 pg of protein were added, the reaction was started by the addition of 0.2 m NAD(P)H.

NAD(P)H-Cyt c reductase was assayed by measuring the increase in Asso (reduction of Cyt c) in a medium containing 0.2 m NAD(P)H, 80 PM Cyt c, 1 m KCN, and 75 to 100 pg of plasma membrane protein or 15 to 200 pL of column eluate in 50 mM Tris-HC1, pH 7.6. An extinction coefficient of 29.5 m-' cm-' was used in calculations of specific activities.

NAD(P)H-Fe3+ chelate reductase activity was measured essentially as described by Holden et al. (1991). Assays were in 1 mL of 15 m Mes-KOH buffer, pH 6.0, 250 p~ Fe3+- citrate (20:l citrate-KOH:FeC13, pH 6.0), 125 p~ bathophenantroline disulfonate, 40 Pg of plasma membrane protein or 50 to 100 pL of column eluate & 0.025% (w/v) Triton X-100 with plasma membranes, and the reaction was started by the addition of 0.2 m NAD(P)H. Change inA535 due to chelation of Fez+ by bathophenantroline disulfonate was recorded at 3OoC for 2 min. An extinction coefficient of 22 m-l cm-' was used in calculations of specific activities.

Nitrate reductase was assayed by measuring the decrease in in a medium containing 2 m KNO,, 0.2 m NAD(P)H, and 50 m Tris-HC1 buffer, pH 7.6.

SDS-PAG E

Samples were precipitated with deoxycholate-TCA. To avoid serious interference of Triton X-100, which is also precipitated by TCA, 2% 3-[(cholamidopropyl)dimethyl- ammoniol- 1 -propanesulfonate in water was added to the samples before precipitation. Concentrated protein was resuspended in SDS-DTT loading buffer and heated for 15 to 20 min at 42OC to avoid aggregation of membrane proteins (Findlay, 1987). Electrophoretic separation of proteins was then carried out in 10% polyacrylamide slab gels according to the method of Laemmli (1970). Gels were stained with Coomassie blue or silver (Merril et al., 1984) for protein visualization.

Nondenaturing IEF and Two-Dimensional Electrophoresis

Samples of solubilized plasma membranes and purified dehydrogenases I and I1 were analyzed by IEF in small rod gels (0.1 X 6 cm) under nondenaturing conditions. The composition of the gel was 5% (w/v) T (total monomer concentration, acrylamide plus bis-acrylamide) and 3% (w/w) C (cross-linking monomer, bis-acrylamide, with reference to total monomer) and contained 2% Ampholine (pH 3.5-10, LKB) and 0.5% Triton X-100. Samples (50 ng of protein for purified dehydrogenases) were diluted in 50 to 100 pL of 0.4% Ampholine, 10% Suc and then overlaid with the same volume of 0.4% Ampholine, 5% Suc to avoid contact with the cathode solution (0.5 M NaOH). The anode solution was 0.5 M orthophosphoric acid. Focusing was carried out according to the following scheme: 15 min at 20 V/cm, 30 min at 30 V/cm, 30 min at 40 V/cm, overnight at 50 V/cm, and 1 h at 75 V/cm. Temperature was kept constant at 4OC.

For two-dimensional electrophoresis, rod gels were equilibrated with denaturing SDS-DTT loading buffer for 10 min at room temperature and then placed on top of a flat stacking gel polymerized over a 10% separating gel. The second dimension was run as described above for SDS-PAGE. Gels




‘were silver stained according to the method of Meml et al. (1984).

Staining for Redox Activity

After nondenaturing- IEF, rod gels were pre-equilibrated with 10% glycerol, 50 mM sodium phosphate buffer, pH 7.4, and then stained for redox activity by incubation for 30 min at room temperature in the same buffer containing 0.4 m~ NADH and 0.5 m~ nitroblue tetrazolium as substrates. Con- trol gels were incubated in the absence of electron donor. After staining, gels were stored in 7.5% acetic acid.

Protein Quantification

Protein determinations were carried out by the dye-binding method of Bradford (1976) using bovine y-globulin as standard.

RESULTS

Isolation of Plasma Membranes

Fractions isolated by two-phase partition at a 6.4% polymer concentration were highly enriched in plasma membrane. The enzymic markers for mitochondria, ER, Golgi apparatus, and tonoplast were significantly decreased in plasma membrane fractions relative to the total homogenate. However, the specific marker for plasma membrane, diethylstilbestrol- sensitive ATPase activity, showed a 10-fold enrichment in plasma membrane fractions relative to the total homogenate (Table I). Plasma membrane purity was also checked by both on-section staining with phosphotungstic acid of Epon-embedded membrane fractions and morphometric analysis. Stained vesicles constituted about 95% of the total membrane vesicles, excluding the possibility that redox activities and purified enzymes might be derived from membranes other than the plasma membrane.

Purification of Plasma Membrane Dehydrogenases

Salt-extracted plasma membranes were solubilized with Triton X-100 at a detergent:protein ratio of 3:1, and the soluble fraction was separated by ultracentrifugation at 105,OOOg. Under these conditions NADH-FeCN and NADH- juglone oxidoreductase activities were effectively solubilized

Table 1. Marker enzyme analysis Specific activities are expressed as nmol mg-’ min-’. Values in

parentheses are the enrichment of specific activity in plasma membrane relative to the total homogenate. SDS were less than 7% of the mean values (n = 4).

Total Microsomal Plasma Homogenate Fraction Membrane Markers

Diethylstilbestrol-ATPase 3.5 12.7 36.1 (10.3) Cyt c oxidase 197.8 265.5 16 (0.08) NADPH-Cyt c oxidore- 20.1 10.1 2.4 (0.11)

Latent IDPase 62 91 25 (0.4) Pvrophosphatase 76.5 19.4 10 (0.13)

ductase

O 5 10 1 5 2 0 2 5 30 35 Fraction

Figure 1. Gel filtration. Membranes were solubilized with Triton X- 100 at a c1etergent:protein ratio of 3:1, and then solubilized samples were fractionated by chromatography in Sephacryl 300 HR. Both protein aind redox activities were measured in elution fractions. Discontinuous line, Protein profile. O, NADH-FeCN oxidoreductase; O, NADH-juglone oxidoreductase.

with a yield of more than 80%. Solubilized protl?ins (10 mL at 7 mg/”L) were first fractionated by molecular size with a column of Sephacryl 300 HR (Fig. 1). Protein and oxidoreductase activities were measured in eluted fractions. The elution profile showed that low amounts of NADH-juglone oxidoreductase activity collected at the void volume, thus showing that the enzyme was successfully soluhilized. This activity gave two main peaks, the first in fraction 23 and the second in fraction 26. NADH-FeCN oxidoreductase gave one major peak in fraction 24. An apparent shouldem* coincident with the second peak of NADH-juglone oxidoreductase was also observed. A plateau of activity collected at or near the void volume and likely corresponded to aggrege tes of non- fully solubilized enzyme. NADH-Fe3+ chelate oxidoreductase showed an elution profile similar to the first peak of NADH- juglone (oxidoreductase (not shown).

Active fractions (22-29) were pooled and al>plied to a DEAE-Sepharose CL-6B column (Fig. 2). The flow-through eluate contained significant oxidoreductase activiiy. The ratio of FeChl to juglone reductase activities was about 2 in the unbound fraction. Protein bound to the column was eluted with a linear gradient (0-1 M) of KCl, showing a major peak of protein and oxidoreductase activities that eluted immedi- ately after application of salt-containing buffer. The FeCN to juglone reductase activities were in a ratio of about 0.7 in eluted fractions.

Salt-eluted fractions that contained oxidoreductase activities were dialyzed in small columns of Sephader G-25 and applied to a column of Blue-Sepharose CL-6B. After the column was washed with buffer, the activity was eluted with 30 PM NADH (Fig. 3A). This fraction exhibited a FeCN to juglone reductase activities ratio of 0.7. In SDS gels, the purified fraction gave one polypeptide band of 27 kD (dehydrogenase I, Fig. 4A), as shown previously by Luster and Buckhout (1989). In nondenaturing IEF gels, the purified dehydrogenase gave one major band of oxidoreductase activity with an isoelectric point of about 6 (Fig. 4B).

The fraction that did not bind to the DEAE,-Sepharose




0 10 20 30 40 50 60 70 60 90 100

Bo.e

o.e

0.2

Figure 2. Ion-exchange chromatography. Active fractions from gelfiltration were pooled and loaded onto a column of DEAE-Sepha-rose CL-6B. Protein and redox activities were measured in unboundand salt-eluted fractions. Dotted line, KCI gradient; discontinuousline, protein. •, NADH-FeCN oxidoreductase; O, NADH-jugloneoxidoreductase.

column was applied directly to the column of Blue-SepharoseCL-6B. After the gel was washed, the activity could be elutedwith 1 rriM NADH (Fig. 3B). This fraction showed a ratio ofFeCN to juglone reductase activities of 2 to 2.5. Analysis ofpurified fractions by SDS-PAGE showed only one polypep-tide band corresponding to an apparent molecular mass of

s i MkD66- (B

45- •36- —.

29- W24- •

20- -

Pi9.3-8.3-

7.2-

•5.1- ;

3.6-

IIn

Figure 4. A, SDS electrophoresis of purified dehydrogenases. LaneS, Molecular mass standards; lanes I and II, 50 ng of purifieddehydrogenases I and II, respectively. After electrophoresis gelswere silver stained. B, Nondenaturing IEF. Rods I and II, 50 ng ofpurified dehydrogenases I and II, respectively. After focusing, rodgels were stained for redox activity (NADH-nitroblue tetrazoliumoxidoreductase). Positions of several isoelectric point (pi) markersare indicated on the left.

2SO

Figure 3. Dye-ligand affinity chromatography. A, Salt-eluted frac-tions from ion-exchange chromatography were dialyzed, bound toa column of Blue-Sepharose CL-6B, washed, and eluted specificallywith NADH. B, Samples that did not bind to the DEAE-Sepharosecolumn were applied directly to the Blue-Sepharose column. Afterthe column was washed, bound proteins were eluted specificallywith NADH. Discontinuous line, Protein profile. •, NADH-FeCNoxidoreductase; O, NADH-juglone oxidoreductase.

31 kD (dehydrogenase II, Fig. 4A). In nondenaturing IEF gelsthat had been stained for redox activity, the purified enzymeshowed one major stained band corresponding to an isoelec-tric point of about 8 (Fig. 4B).

Minor bands corresponding to different isoelectric pointswere also eventually observed with the two purified dehy-drogenases (Fig. 4B), but when samples were subjected toelectrophoresis in a second dimension, bands of enzymicactivity migrated to spots corresponding to 27 and 31 kD (notshown).

Specific activities and total recoveries during the differentsteps of purification are summarized in Table II.

Characteristics of Purified Dehydrogenases

Substrate Specificity and pH Profile

Dehydrogenase I catalyzed the electron transfer fromNADH or NADPH to a variety of electron acceptors, includ-ing FeCN and several quinones (Table HI). Maximal activitywas achieved when NADPH was used as a donor andquinones, such as juglone menadione or coenzyme Q0, wereused as acceptors. Dehydrogenase II was highly specific forNADH. Among different acceptors used, FeCN gave maximalactivity with this enzyme. Negligible activities were recordedwith NADPH.

Optimal pH was determined for activities that gave themaximal rate of electron transfer, i.e. NADPH-quinone oxi-doreductase for dehydrogenase I and NADH-FeCN oxido-reductase for dehydrogenase II. Maximal redox activities withthe two dehydrogenases were obtained at a pH of about 6(Fig. 5).




Table II. Purification of two different oxidoreductase enzymes from Triton X- 700-solubilized onion root plasma membranes

Assays for NADH-FeCN and NADH-juglone activities were carried out in 50 mM Tris-HCI buffer, pH 7.6, with samples of Triton X-100-treated plasma membrane, Triton X-100 solubilizates, and peak fractions obtained in successive steps of the purification procedure. DEAE (A), Flow-through eluate from ion-exchange chromatography. DEAE (B), Salt-eluted fraction from the DEAE-Sepha- rose column. Units used are: mg for protein, pmol min-' for total activity, and pmol min-' mg-' protein for specific activity. SDS were less than 9% of the mean values fn = 3).

Total Activity Specific Activity

FeCN Juglone FeCN Juglone Fraction Protein

Plasma membrane 321.3 98.6 54.6 0.307 0.1 7 Solubilizate 122.5 49 28.6 0.4 0.234 Gel filtration 57.2 27.1 16.7 0.473 0.292

DEAE (A) 36.7 9.3 3.6 0.253 0.099 Dye-ligand 0.0046 4.9 1.8 1066 389

DEAE (6) 19.9 5.4 7.0 0.272 0.352 Dye-ligand 0.0331 0.7 1.0 21.13 30.64

Inhibitor Studies and Kinetic Analysis

Sensitivity o f the t w o purif ied enzymes to a variety o f compounds known to affect the activity o f dehydrogenase enzymes was also tested (Table IV). Dehydrogenase I (NADPH-juglone) was moderately inhibited with the quinone antagonist dicumarol, the thiol reagent N-ethyl-maleimide, and the mitochondrial inhibitor at the Q site, TTFA. However, the enzyme was insensitive to carbonylcyanide-3- chlorophenylhydrazone and rotenone. Dehydrogenase I1 (NADH-FeCN) was strongly inhibited by PHMB, dicumarol, and TTFA. Rotenone and iodoacetate inhibited moderately, and a stimulatory effect was obtained with SHAM and adriamycin. K, values for pyridine nucleotides were calculated from reciprocal plots (Lineweaver-Burk) and were estimated

Table 111. Oxidoreductase activities with different electron donors and acceptors

Assays were carried out in 50 mM Tris-HCI buffer, pH 7.6. Specific activities are expressed as pmol min-' mg-' protein. ND, Not detected. SDS were less than 10% of the mean values (n = 3).

Acceptor

FeCN Juglone Duroquinone Menadione Coenzyme Qo AFR Cyt c DPlP I NT Fe3+ chelate Nitrate

Dehydrogenase I

NADH NADPH

21.1 47.9 30.6 75.3 10.4 16 32.3 65.6 37.9 77 ND ND 7.9 3.8 4.5 20.1 0.7 ND 3.0 ND

ND ND

Dehydrogenase II

NADH NADPH ~

1066 ND 389 ND ND ND ND ND 195 ND

13 ND 406 ND 266 ND

6.5 ND 320 ND ND ND

500 -

4w-

300 -

200 -

100 - b

7 + , , ~, , , , , ,

4 5 6 7 8 0 500

PH

Figure 5. pH curves of the purified dehydrogenases. 4, Dehydro- genase I. Activity measured was NADPH-juglone oxidoreductase. 6, Dehydrogenase I I . Activity measured was NADH-FeCN oxidoreductase. ., Forty millimolar citrate-phosphate buffer; O, 40 mM phosphate buffer; O, 40 mM Tris-HCI; O, 40 mM Gly-NaOH buffer.

to be 15 and 20 PM with dehydrogenase I (NADPH- and NADH-juglone, respectively) and 18 ~ L M with dehydrogenase I1 (NADH-FeCN).

DISCUSSION

Redox activities associated with the plant plasma membrane have received increased attention in recent years and

Table IV. Effect of various compounds on the activity oi' the two purified dehydrogenases

Assays were carried out at optimal pH (6.2) in 40 mM citrate- phosphate buffer containing 20 to 140 ng of purified enzyme. Activities measured were NADPH-juglone and NADH-FeCN oxidoreductase for dehydrogenases I and II, respectively. Values rep- resent differences between redox activities obtained in the pres- ence and absence of the listed compounds referred to activities obtained with no addition (in percentage). Negative values indicate inhibition, whereas positive values indicate activation. :DS were less than 10% of the mean values (n = 3).

Addition Dehvdronenase I Dehvdronenase I1

50 p~ Adriamycin 1 mM KCN 1 mM SHAM 100 p~ Dicumarol 1 p~ carbonylcyanide-3-

1 mM N-ethyl-maleimide 1 mM lodoacetate 5 p~ PHMB 67 p~ TTFA 1 O p~ Rotenone

chlorophenylhydrazone

O +13.96 -1 1.5 -6.8 -2 1 +43.8 -35.5 - 69

O -15.8

-30 -13.2 -3.5 - 30 -9.1 -95.2

-34 -77.2 O - 50.3




have been associated with processes at the cell surface such as light-mediated events, iron uptake, H+ efflux, and transport of solutes and nutrients (Mdler and Crane, 1990; Rubin- stein and Luster, 1993; Gonzilez-Reyes et al., 1994). Al- though physiological studies using several plant tissues and organs are abundant, the knowledge of molecular aspects of plasma membrane electron transport is surprisingly poor.

The aim of this work was the isolation of the enzymes able to catalyze NAD(P)H-dependent redox reactions at the plasma membrane. Onion root plasma membranes catalyzed the NADH-dependent reduction of electron acceptors such as FeCN, several quinones, dyes, and AFR. Much lower activities were achieved with NADPH as the electron donor (Luster and Buckhout, 1988; Serrano et al., 1994). However, microsomes obtained from Cucurbita hypocotyls contained more NADPH- than NADH-dependent activity, most likely because of an NAD(P)H-P450 reductase or similar flavoprotein (Guemni et al., 1987). Activities measured with microsomes might be derived from the ER and, therefore, should not be directly attributed to the plasma membrane.

Purification and Characterization of Redox Enzymes from Onion Root Plasma Membranes

NADH-FeCN and NADH-juglone oxidoreductase activities were resistant to salt extraction of membranes, thus demonstrating that the enzymes were integral membrane proteins. Gel filtration of Triton X-100-solubilized fractions in Sephacryl300 HR resolved two peaks of NADH-quinone oxidoreductase; however, the level of resolution achieved was relatively poor. Therefore, we pooled the fractions containing oxidoreductase activity and subjected them to the next chromatography step (ion-exchange in DEAE-resin), which successfully separated the two dehydrogenases. How- ever, starting with gel filtration was preferred because it eliminated significant amounts of protein that had not been fully solubilized (collecting at the void volume) and remained as aggregates that would impair the level of purification in further chromatography steps.

Chromatography in DEAE-Sepharose CL-6B resulted in a clear separation of two different NADH dehydrogenase activities. The fraction that did not bind to the column at pH 7.6 contained an enzyme that showed maximal activity when FeCN was used as the electron acceptor (dehydrogenase 11), On the other hand, the fraction bound to, and eluted from, the column contained a different enzyme that reduced juglone better than FeCN (dehydrogenase I). Final purification of both enzymes was accomplished by chromatography in Blue-Sepharose and affinity elution with NADH.

Dehydrogenase I consisted of a single polypeptide chain of about 27 kD with an isoelectric point of about 6. Dehydro- genase I1 also consisted of a single polypeptide chain, but the molecular mass was about 31 kD and the isoelectric point was about 8. Different isoelectric points in the two dehydrogenase enzymes explain why dehydrogenase I bound to the anion-exchange gel at pH 7.6 but dehydrogenase I1 did not.

Relationship to Other Purified Dehydrogenases

Holden et a1.- (1991) showed that the plasma membrane isolated from tomato roots contained redox proteins with

isoelectric points of approximately 6 that were induced under conditions of iron deficiency. Unfortunately, these proteins were not further characterized, and no data regarding their properties were available.

Luster and Buckhout (1989) purified a 27-kD plasma membrane-associated electron transport protein from maize roots with one-step chromatography in Blue-Sepharose. Dehydro- genase 1(27 kD) is likely equivalent to that enzyme. In fact, when Triton X-100-solubilized plasma membranes from onion roots were directly applied to the Blue-Sepharose column with no previous ion-exchange chromatography, NAD(P)H- dependent dehydrogenase activity was bound to the affinity gel, but only the 27 kD dehydrogenase was affinity eluted by NADH. In this case, an enzyme showing the highest activity with FeCN (most likely equivalent to dehydrogenase 11) could be eluted from the column nonspecifically with salt, although only a low level of purification was achieved (Ser- rano et al., 1994). The possibility exists that, upon solubilization with Triton X-100, dehydrogenase I is effectively isolated but dehydrogenase I1 forms a complex with another protein(s) that also binds to the Blue-Sepharose column but cannot be eluted by NADH. This (these) protein@), but not dehydrogenase I1 itself, might also bind to the DEAE-Sepharose column at pH 7.6 in such a way that the redox enzyme might be slowly separated during extensive washing with Triton X- 100-containing buffer. This scenario could explain why dehydrogenase I1 was recovered in the flow-through eluate of the DEAE-Sepharose column in a volume much larger than expected from the volume initially applied to the column (Fig. 2). This interpretation is also supported by the different recoveries of NADH-juglone and NADH-FeCN oxidoreductases collected at the void volume upon filtration in Sephacryl 300 HR (Fig. 1).

A 28-kD rotenone-insensitive dehydrogenase [NAD(P)H- ubiquinone oxidoreductase] has been isolated from inner mitochondrial membranes. Dehydrogenase I is clearly different, since the former enzyme can be purified from the soluble fraction and its activity is completely dependent on the addition of free Ca2+ (Mller et al., 1993).

The two purified enzymes were also different from the dehydrogenase purified by Guemni et al. (1987) from CUCUY- bita microsomes, which was apparently composed of two subunits, 37 and 39.5 kD, and exhibited maximal activity with NADPH.

Plasma membranes derived from sugar beet leaves appear to contain only one major NADH-FeCN reductase, presum- ably a 43-kD NADH-Cyt b5 reductase with both donor and acceptor sites located on the cytoplasmic surface, but not the 27- and 31-kD dehydrogenases (Askerlund et al., 1988, 1991). Cytochemical demonstration of NADH-FeCN reductase activity in hypocotyl segments and isolated plasma membranes also revealed that the site of FeCN reduction was oriented cytoplasmically (MorrC et al., 1987).

Substrate Specificity and pH Profile

Dehydrogenase I1 was highly specific for NADH as an electron donor, but dehydrogenase I used NADPH better than NADH. Most solubilized NADH-FeCN reductase activity from onion root plasma membranes can be attributed to



94 Serrano et al. Plant Physiol. Vcd. 106, 1994

dehydrogenase I1 (Serrano et al., 1994). Accordingly, whole plasma membranes utilize NADH much better than NADPH. Different NADPH:NADH activity ratios in whole plasma membrane and purified protein were explained by Luster and Buckhout (1988, 1989) on the basis of the existence of more than one FeCN reductase in maize root plasma membranes. Also, the low yield achieved in purification of FeCN reductase activity and the high yield in duroquinone reductase led the authors to assume that a significant portion of the activity present in whole and solubilized plasma membrane was not released from the affinity column.

Both dehydrogenases exhibited maximal activity at a pH of approximately 6 . This is in sharp contrast with optimal pH values obtained with whole membranes, which showed a plateau of maximal activity between pH 7 and 8.5 (Barr et al., 1986). Plasma membranes might contain still more redox proteins with different optimal pH values that could be either inactivated after detergent solubilization or not recovered from chromatography columns. Also, activities in which several redox camers participate could be lost after solubilization.

Sensitivity of Purified Dehydrogenases to Effectors

Dehydrogenase I1 was strongly inhibited by the thiol reagent PHMB. Involvement of thiol groups in plasma membrane redox activities from both plant and animal sources has been demonstrated (Barr et al., 1986; Luster and Buck- hout, 1989; Villalba et al., 1993). Interestingly, the enzyme was also inhibited about 90% by dicumarol, a vitamin K antagonist that inhibits the electron flow of the plasma membrane redox system and proton secretion induced by the electron transport (Doring et al., 1992a, 199213; Liithje et al., 1992). Since dehydrogenase I1 (activated by SHAM) was insensitive to cyanide, the enzyme is unlikely to be related to the plasma membrane-bound peroxidase activity (Mdler and Crane, 1990; Pene1 and Castillo, 1991). NADH-FeCN oxidoreductase measured in inside-out plasma membranes pre- pared by two-phase partition was also activated by SHAM (Barr et al., 1986; Barr and Crane, 1991).

Unlike dehydrogenase 11, dehydrogenase I was inhibited by SHAM. Inhibition of the NAD(P)H-dependent reduction of FeCN and Fe3' chelate by SHAM in plasma membranes isolated from peanut seedling hypocotyls has also been reported (Jiao et al., 1992).

Both dehydrogenases I and I1 were relatively insensitive to adriamycin (Luster and Buckhout, 1989; this work), which is reported to be an inhibitor of trans electron transport in plasma membranes from plant and animal sources (Sun et al., 1984; Morri. et al., 1988). The possibility exists that adriamycin is acting on a distinct NAD(P)H dehydrogenase or on a different site in a putative redox chain. Also, a possible reduction of adriamycin by purified enzymes has been proposed (Luster and Buckhout, 1989).

Relationship to Plasma Membrane Redox Activity and Physiological Implications

Whether or not either of the two purified dehydrogenases plays a role in transplasma membrane electron transport cannot be deduced from our data and remains for further

investigation. Topography studies have indicated that the 27- kD plasma membrane redox protein is likely located on the inner, cytoplasmic surface of the plasma menbrane, and possibly both donor and acceptor sites face thc cytoplasm. Howevler, the possibility that this protein parbcipates in a putative transmembrane redox chain, via the reduction of a quinone or Cyt, has not been discarded (Buckhout and Luster, 1991).

The biochemical study of transplasma membrane electron transport has gained greater advantage with the lise of sealed right-side-out plasma membrane vesicles loaded with different electron donors. For instance, electrogenic electron flow from intemal ascorbate to extemal FeCN, involving a high potential b-type Cyt, has been measured in plasma membranes (derived from hypocotyls and leaves (Hassidim et al., 1987; Askerlund and Larsson, 1991; Asard et al. 1992). Leaf plasma membranes loaded with an NADH-generating system are not (able to reduce extemal FeCN (Askerlund ,ind Larsson, 1991). However, Bottger et al. (1992) have demonstrated that bean hypocotyl plasma membranes indeed reduce extemal FeCN when loaded with NADH by electropor3tion. These results differ from those obtained by Askerlund and Larsson (1991), (and the use of different plant material and the loading procedures were discussed as possible explanations. As we have shown in this work, the composition of reliox proteins in plasrna membranes isolated from roots may be different from that found in leaf plasma membranes, and this would affect the results obtained in experiments with plasma membrane vesicles from different sources.

Some NADH-AFR reductase activity could be measured with dehydrogenase 11. AFR has been implicaíed in some plasma membrane redox processes related to cell elongation (Hidalgo et al., 1989, 1991), proliferation (De Cabo et al., 1993), and nutrient uptake (González-Reyes el al., 1994). However, from the data presented in this paper one cannot determine whether AFR reduction by dehydrogenase I1 is equivalent to the transmembrane reduction of AFR by intact tissues i hat results in membrane hyperpolarizatLon (Gonzá- lez-Reyes et al., 1992). Furthermore, a great part of plasma membrane NADH-AFR reductase activity is lost after solubilization with Triton X-100 (Serrano et al., 1994).

Since plasma membranes used for protein purification are derived from a variety of cell types, there is little chance to correlate biochemical activity of our purified proteins with a particular function. However, we point out the possibility that different plant organs could contain different redox systems determined according to their particular physiology. For instance, a redox system involved in reduction of femc chelates in the soil solution is expected to be specific for root tissues and redox systems associated with light-mediated events should be specific for aerial parts of the plant.

Received February 8, 1994; accepted April 19, 1994. Copyright Clearance Center: 0032-0889/94/106/0087,'10.

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