partia1 purification of the cyanide-resistant of … · for measurements of the ratio of o2 uptake...

Plant Physiol. (1993) 101: 113-119

Partia1 Purification of the Cyanide-Resistant Alternative Oxidase of Skunk Cabbage ( Symp/oca rpus foe tidus ) M i toc h o n d ri a'

Deborah A. Berthold' and James N. Siedow*

Department of Botany, Duke University, Box 90338, Durham, North Carolina 27708-0338

A partial purification of the cyanide-resistant, alternative oxidase from skunk cabbage (Symplocarpus foetidus L.) spadix mitochondria is described. Skunk cabbage mitochondria were solubilized in N,N-bis-(3-~-glucon-amido-propyl)deoxycholamide and the alternative oxidase was purified using a batch DEAE-cellulose treatment, followed by precipitation with Extracti-Cel and chro- matography on Sephadex C-200. Following pooling and concen- trating of the most active fractions from the gel filtration column, a 20- to 30-fold purification of the alternative oxidase was obtained, with no evidence of contamination by cytochrome c oxidase (complex IV) or cytochrome c reductase (complex 111). Polyacryl- amide gel electrophoresis of the partially purified oxidase showed major polypeptides at 36 and 29 kD, both of which react with monoclonal antibodies raised against the Sauromatum guttatum alternative oxidase. The purified oxidase fraction showed no absorbance in the visible spectral region, and addition of sodium borohydride induced no absorbance changes in the ultraviolet region. The purified alternative oxidase catalyzed the four-electron reduction of oxygen to water in the absence of citrate, but catalyzed an apparent two-electron reduction of oxygen to hydrogen per- oxide in the presence of 0.7 M citrate.

Cyanide-resistant respiration has been reported in a wide variety of plants and is associated with the presence of a second terminal oxidase found in the mitochondrial respiratory chain (Lance et al., 1985; Siedow and Berthold, 1986; Moore and Siedow, 1991). This "alternative" is believed to bring about a four-electron reduction of O2 to water, which, unlike Cyt c oxidase, is sensitive to inhibition by SHAM and completely insensitive to cyanide (Moore and Siedow, 199 1).

Following the initial characterization of the alternative oxidase as a ubiquinol oxidase by Rich (1978), there have been severa1 attempts to purify this enzyme utilizing soluble ubiquinol analogs, such as duroquinol and menadiol, as substrates for the enzyme (Huq and Palmer, 1978; Bonner and Rich, 1983; Bonner et al., 1986; Elthon and McIntosh, 1986, 1987). In spite of these efforts, the alternative oxidase has proved to be relatively refractory toward purification. Although an alternative oxidase associated polypeptide has

'During the course of this work, D.A.B. was supported by a National Institutes of Health Cell and Molecular Biology Training Grant GM07184-14. This work was supported by a grant from the National Science Foundation (DMB 85-16695) to J.N.S.

Present address: Department of Plant Biology, 111 GPBB, Uni- versity of California, Berkeley, CA 94720.

* Corresponding author; fax 1-919-684-5412.

been identified on SDS-polyacrylamide gels (Elthon and McIntosh, 1987), monoclonal antibodies have been raised against it (Elthon et al., 1989), and the sequences of cDNA clones associated with this protein have a11 been reported (Rhoads and McIntosh, 1991; Sakajo et al., 1991); to date, an active enzyme has not been purified to homogeneity. The alternative oxidase appears to be an integral membrane protein (Moore and Siedow, 1991), and attempts to purify it by solubilizing the mitochondrial membrane with detergent treatment have resulted in a large loss of activity. Therefore, the nature of its active site-what organic cofactors and/or metals might be present-remains unknown. In this article, we report the partial purification and characterization of the alternative oxidase from skunk cabbage spadix mitochondria.

MATERIALS A N D METHODS

Skunk cabbage (Symplocarpus foetidus L.) spadix tissue was collected in the wild, and mitochondria were isolated and stored at -8OOC as described previously (Berthold et al., 1988). Rates of cyanide-resistant, SHAM-sensitive, duroquinol-stimulated O2 consumption through the alternative pathway for these mitochondria ranged from 200 to 600 nmol O2 min-' mg-' protein.

Cyanide-resistant, SHAM-sensitive duroquinol oxidase activity in mitochondria and solubilized fractions was assayed polarographically at 25OC in a thermostatted cell. The mitochondrial reaction buffer was modified from that of Stegink and Siedow (1986) and consisted of 0.7 M sodium citrate in the standard mannitol reaction buffer at pH 6.8: 0.3 M mannitol, 10 mM KCI, 5 mM MgC12, 1 mM EDTA, 0.1% (w/v) BSA, 250 p~ KCN, and 10 mM K phosphate. O2 consumption was initiated by addition of 500 PM duroquinol. The nonenzymic 0% uptake obtained with the subsequent addition of 1 to 2 mM SHAM was subtracted from the total rate to yield a net rate of alternative oxidase O2 consumption. In the assay of activity following Extracti-Gel treatment, 1 mg mL-' asolectin and 3.33 mg mL-' deoxyBIGCHAP were included in the assay medium, unless otherwise noted.

Cyt c oxidase (EC 1.9.3.1) was assayed polarographically at 2 5 T in 50 mM K phosphate, pH 6.5, 0.1 mM EDTA, 0.1% (w/v) BSA, 8 mM sodium ascorbate, 0.5 mM N,N,N',N'-

Abbreviations: BCA, bicinchoninic acid; DCPIP, 2.6-dichloro- phenolindophenol; deoxyBIGCHAP, N,N-bis-(3-~-glucon-amido- propy1)deoxycholamide; E-64, ~-trans-epoxysuccinyl-leucylamido(4- guanidino)butane; SHAM, salicylhydroxamic acid.

113

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114 Berthold and Siedow Plant Physiol. Vol. 101, 1993

tetramethylphenylenediamine, 30 p~ horse heart Cyt c, and 1 mM lauryl maltoside (modified from Suarez et al., 1984). The enzymic activity was then inhibited with 250 p~ KCN to obtain the net rate of O, consumption.

Duroquino1:Cyt c reductase was assayed spectrophotomet- rically at 25OC in 50 mM Tes, pH 7.0, 1 mM EDTA, 1 mM KCN, 500 PM duroquinol, 157 PM lauryl maltoside, and 25 p~ Cyt c (modified from Soole et al., 1992). Cyt c reduction was monitored at 550 nm using an extinction coefficient of 19.8 mM-' cm-'. An identical sample was run in the presence of 5 p~ antimycin A to correct for the nonenzymic reduction

Succinate:DCPIP reductase was assayed spectrophotomet- rically at 25OC in 10 mM K phosphate, pH 7.4, 10 mM succinate, 1.6 mM phenazine methosulfate, 0.1 mM EDTA, and 70 p~ DCPIP (Hatefi, 1978a). The enzyme sample to be assayed was preincubated on ice for 10 min with 20 mM succinate, pH 7.4. Activity was monitored as DCPIP reduction at 600 nm using an extinction coefficient of 21 mM-' cm-'.

NADH:duroquinone reductase was assayed spectrophoto- metrically at 25OC in 0.3 M mannitol, 10 mM K phosphate, pH 7.2, 10 mM KCI, 10 mM Tes, 5 mM MgC12, 1 mM KCN, and 0.2 mM duroquinone (modified from Hatefi, 1978b). The reaction was initiated by addition of 200 PM NADH, and the consumption of NADH was monitored at 340 nm using an extinction coefficient of 6.22 mM-' cm-'.

NADH:FeCN reductase was assayed spectrophotometri- cally at 25OC in 0.3 M mannitol, 0.8 mM K3Fe(CN),, 10 mM K phosphate, 10 mM Tes, 10 mM KCI, and 5 mM MgCI,, pH 7.2 (Hatefi, 1978b). The reduction of ferricyanide in the presence of 5 ~ L M antimycin A was monitored at 420 nm using an extinction coefficient of 1.05 mM-' cm-'. The reaction was initiated by the addition of 200 p~ NADH.

of Cyt c.

Measurement of O2 Uptake

For assays of the ratio of 0, uptake to duroquinol oxidized by the solubilized alternative oxidase, the 0, electrode was calibrated prior to each run using distilled water at 25OC as the standard (=250 p~ 0, [Delieu and Walker, 19721). The concentration of the stock solution of duroquinol was calibrated using beef heart mitochondria in mitochondrial reaction buffer with or without 0.7 M citrate, which rapidly and completely oxidizes duroquinol with the concomitant reduction of O2 to water. Unlike Kay and Palmer (1985), we observed that the 0, concentration of our air-saturated mitochondrial reaction buffer in the presence of 0.7 M citrate did not differ appreciably from that obtained with air-saturated mitochondrial reaction buffer alone.

For measurements of the ratio of O2 uptake per duroquinol oxidized by the solubilized alternative oxidase, partially purified oxidase was added to the electrode chamber in the presence of the indicated reaction medium followed by a known concentration of duroquinol, and the reaction was allowed to proceed until no further O2 uptake occurred. Beef heart mitochondria were added after O2 uptake had ceased to verify that the duroquinol had been entirely oxidized at the end o€ the reaction. The ratio of duroquinol added to

moles of O2 consumed was then calculated from the amount of O2 taken up.

Protein Determination

Protein was determined using the BCA assay (Smith et al., 1985), modified as follows. Ten parts 20% SDS and 0.625 parts 5% CuS04.5H20 were added to the standard 1 part reagent B in 50 parts reagent A. This solution (2.4 mL) was added to 0.1 mL of protein sample. The samples were incubated for 30 min at 37OC, and the absorbance was read, versus a blank, at 562 nm. BSA served as a protein reference. This modification eliminated the cloudiness that resulted from the use of the unmodified BCA assay.

SDS-PAGE

SDS-PAGE was run on a discontinuous system (modified from Laemmli, 1970) containing 10% acrylamide, 0.1% SDS, 2.5 M urea, and 0.375 M Tris, pH 8.8, in the running gel, and 5% acrylamide, 0.1% SDS, 2.5 M urea, and 0.125 M Tris, pH 6.8, in the stacking gel. Protein samples (approximately 25 p g ) were denatured for 10 min at 25OC in sample buffer containing 75 mM Tris, 6.0 M urea, 5% glycerol, 1% SDS, 20 p~ E-64, and 0.001% broinphenol blue, pH 6.8. 2-Mercap- toethanol (1%) was added immediately before loading.

Spectrophotometry

A11 spectrophotometric enzyme assays and optical difference spectra were performed on a Shimadzu UV-265FW spectrophotometer. Ubiquinone was extracted from the purified oxidase using minor modifications of the procedure described by Kroger (1978). The resulting extract was subsequently separated on HPLC and quantified against standard ubiquinone-50 according to Schindler et al. (1984).

Reagents

DeoxyBIGCHAP and lauryl maltoside were purchased from Calbiochem. Extracti-Gel and the BCA protein assay were from Pierce. Taurodeoxycholate, taurocholate, Lubrol PX, E-64, and other standard reagents were purchased from Sigma Chemical Co. Cholic acid and deoxycholic acid were from Sigma and were charcoal-filtered and recrystallized from ethanol prior to use. Asolectin (Associated Concentrates, Woodside, NY) was purified before use by dissolving it in diethyl ether, followed by precipitation with acetone containing 1 mM DTT. This procedure was repeated once, and the final product was stored under nitrogen at -2OOC.

RESULTS

The presence of endogenous protease activity is a common problem associated with the isolation of organelles from plant tissues (Gray, 1982). A high leve1 of protease activity was initially discovered in skunk cabbage spadix mitochondrial preparations when an SDS-polyacrylamide gel of the preparation showed no clear polypeptide bands. To determine the class(es) of protease responsible for this degradation, mitochondria were incubated with a variety of different protease



Partia1 Purification of the Cyanide-Resistant Oxidase 115

inhibitors for 1 h at room temperature in SDS sample buffer containing 2-mercaptoethanol. Under these conditions, sev- era1 sulfhydryl protease inhibitors were found to inhibit the proteolytic activity, with iodoacetate and iodoacetamide being the most effective (data not shown). Later, the covalent inhibitor E-64 became commercially available and it was found to be much more effective (on a concentration basis) than iodoacetamide in blocking the protease activity in skunk cabbage mitochondria. Unlike iodoacetamide, E-64 does not react nonspecifically with sulfhydryl residues, but is specific to the active site of sulfhydryl proteases (Mehdi, 1991).

To purify the alternative oxidase, skunk cabbage mitochondria were diluted to 2.0 mg mL-' protein and solubilized with 24 mg mL-' deoxyBIGCHAP in 30 mM K phosphate, pH 6.8, containing 20 IM E-64. The solution was stirred for 10 min on ice and then centrifuged at 100,OOOg for 1 h (producing ultra sup-1, Table I). A variety of detergents were tested for their abilitjr to solubilize the alternative oxidase, including deoxycholate, Triton X-100, taurocholate, lauryl maltoside, and a combination of deoxycholate and octylthioglucoside. Similar to the results reported by Elthon and McIntosh (1986), deoxyBIGCHAP solubilized the alternative oxidase with the greatest recovery of activity in the subsequent supernatant fraction (data not shown). A typical solubilization using deoxyBIGCHAP is shown in Table 1.

A broad pH range from pH 6.5 to 7.5 was found to be optimal for solubilization of the alternative oxidase. Attempts to solubilize the alternative oxidase using a higher mitochondrial protein concentration were unsuccessful; increasing the protein concentration resulted in more activity remaining in the pellet. Increasing both the protein and the detergent concentration resulted in a greater loss of activity (data not shown). Upon addition of deoxyBIGCHAP to skunk cabbage mitochondria, there is an initial, immediate loss of altemative oxidase activity, the extent of which varies from preparation to preparation. Attempts to prevent this loss of activity with additions to the solubilization medium (including DTT, additional E-64, trimethylamine N-oxide [Mashino and Frido- vich, 19871, BSA, and asolectin) were a11 without success. After solubilization, the alternative oxidase activity remained stable and was relatively unaffected by freezing and thawing.

To purify the solubilized alternative oxidase activity, 0.5 volume of packed DEAE-cellulose, equilibrated with 30 mM K phosphate, pH 6.8, was added to the total volume of ultra

sup-1, and the slurry was swirled gently for 10 min at 4OC. The solubilized oxidase did not bind to the DEAE-cellulose at pH 6.8 and was separated from it either by filtration through a glass frit or by centrifugation. Likewise, the solubilized oxidase did not bind to carboxymethyl-cellulose at pH 6.8, which is in agreement with the calculated isoelectric point of 6.6 for the mature Sauromatum alternative oxidase, as determined from the nucleotide sequence (Rhoads and McIntosh, 1991). Typically, 50 to 60% of the contaminating protein bound to the DEAE, and 90 to 100% of the alternative oxidase activity was recovered in the filtrate (Table I). At this stage, the DEAE-treated filtrate was loaded on an Extracti- Gel column at a 1:l (v/v) ratio of filtrate to Extracti-Gel to remove the deoxyBIGCHAP. The Extracti-Gel was equilibrated before loading and subsequently eluted under gravity with 30 mM K phosphate, pH 6.8. An eluate volume approximately equivalent to the original DEAE filtrate volume, which contained the detergent-free alternative oxidase protein, was collected. This volume was subsequently centrifuged at 100,OOOg for 1 h to pellet the alternative oxidase activity. The recovery of alternative oxidase activity from what was loaded on the Extracti-Gel column was 90 to 100%. A number of detergents were tested for their ability to reso- lubilize the resulting pellet. Cholate derivatives proved to be most effective at solubilizing the protein and retaining activity. Taurodeoxycholate was superior to the other detergents because of its ability to separate the alternative oxidase activity from the major A280 peak on the subsequent Sephadex G- 200 gel filtration column.

The ultracentrifuge pellet (ultra pellet-2) was resolubilized in 0.5% taurodeoxycholate and 30 mM Hepes, pH 7.0, and centrifuged at 200,OOOg for 30 min, and the resulting yellow supernatant (ultra sup-3) was applied to a Sephadex G-200 column (Fig. 1). Active fractions from the G-200 column were pooled and concentrated by centrifugation in an Amicon Centricon-30 concentrator. Calibration of the column with mo1 wt standards revealed that the duroquinol oxidase activity ran in the range of 150 to 160 kD, whereas the large Azsa peak ran at 30 kD. The latter fractions were distinctly yellow in color, showing a typical carotenoid absorption spectrum with bands at 428,452, and 481 nm. Huq and Palmer (1978) reported an absorbance in this region in their partially purified alternative oxidase preparation from Arum maculatum, which they attributed to carotenoid. The later report of the

Table 1. Purification of the alternative oxidase from skunk cabbage mitochondria Alternative oxidase was purified as described in the text.

Specific Activitv

Sample Volume Activity Total Units Recovery Protein Purification

mL

Mitochondria 4.25 Ultra Sup-1 44.0 DEAE Sup 53.0 Extracti-Gel filtrate 56.0 Ultra Pellet-2 1.84 Ultra Sup-3 1.85 Pooled G-200 9.20

a Units of activity are nmol min-'.

units mL-'" % mg mL-' units mg-'

5404 22967 1 O0 22.5 240 215 9477 41 1.35b 159 O. 7 161 8582 37 0.42 383 1.6 144 8127 35 0.40 360 1.5

3905 7194 31 1.75 2231 9.3 3238 6004 26 1.79 1809 7.5

180 1656 7 0.037 4865 20.3

Protein was determined by subtracting the protein found in the pellet from the original protein.




to is ao » joFraction Number

Figure 1. Activity and absorbance profile of the skunk cabbagealternative oxidase fractionation on Sephadex G-200. A 43 x 1.0cm column was equilibrated in 50 HIM Hepes, pH 7.0, containing0.5% taurodeoxycholate and loaded with 1505 units (0.42 ml) ofthe taurocholate-solubilized Extracti-Gel precipitate. The columnwas run in the Hepes-taurodeoxy-cholate buffer, and fractions of0.92 ml each were collected. Shaded fractions were pooled forconcentration.

C\J

s (73 O

I §

r -66

-45

-36

-29

-24

1 2 3 4 5 6 7

Figure 2. SDS-PACE showing the sequential purification of theskunk cabbage alternative oxidase. The samples (25 M§ of proteinper lane) are as follows: lane 1, mitochondria; lane 2, ultracentrifugesupernate-1; lane 3, DEAE filtrate; lane 4, Extracti-Cel filtrate; lane5, ultracentrifuge pellet-2; lane 6, ultracentrifuge supernate-3; lane7, concentrated G-200 fractions. The gel was stained with Coo-massie blue. The scale on the right shows apparent molecular massX 10~3.

•66

'•;• -AS

- 36

-29

-24

Figure 3. Immunoblot showing the sequential purification of theskunk cabbage alternative oxidase. An SDS-polyacrylamide gel,similar to that of Figure 2, was blotted onto nitrocellulose andprobed with AOA mouse monoclonal antibody (Elthon et al., 1989)at a dilution of 1:1000. The blot was incubated with alkalinephosphatase-conjugated rabbit anti-mouse and developed (modi-fied from Pratt et al., 1986). The lanes are identical to those in thelegend of Figure 2. The scale on the right shows apparent molecularmass x 10~3.

partial purification of the Arum alternative oxidase by Bonneret al. (1986) also mentions the presence of carotenoid.

An analysis of the protein profile of each purification stepusing SDS-PAGE is shown in Figure 2. Lanes 1 and 2, theoriginal mitochondria and ultracentrifuge supernatant-1, arevery similar, indicating that most of the membrane-boundpolypeptides are solubilized along with the alternative oxi-dase. The final product of the purification (lane 7) shows twomajor diffuse bands around 29 and 36 kD and a pair of majorbands at 57 and 65 kD. A similar gel was blotted ontonitrocellulose and probed with the AOA monoclonal anti-body against the 36- to 37-kD alternative oxidase protein(Elthon et al., 1989) (Fig. 3). Close examination of lanes 1through 4 shows that, unlike the Sauromatum guttatum alter-native oxidase, which shows three distinct polypeptides clus-tering around 36 kD (Elthon et al., 1989), the skunk cabbagealternative oxidase shows only two polypeptides in this re-gion with apparent masses of 35 and 36 kD. The diffusenessof the bands shown in Figure 3 relative to the sharper bandsreported for immunoblots of Sauromatum mitochondria isprobably associated with our use of a simple 10% gel insteadof the gradient SDS gels used by other workers (Elthon et al.,1989). Some degradation of the 35- to 36-kD bands to a 29-kD product appears following collection by ultracentrifuga-tion and storage (—20°C) of the insoluble Extracti-Gel-treatedalternative oxidase (Fig. 3, lanes 5-7). Although the gel



Partiai Purification of the Cyanide-Resistant Oxidase 117

contains SDS and urea, there is also evidence of aggregation in the more purified fractions, with the appearance of a major, cross-reactive band at 65 kD and several minor bands at lower molecular masses (lanes 5-7). These bands probably represent mixed dimeric aggregates of the 29- to 36-kD polypeptides.

Table I1 shows the contaminating activities present in the final partially purified oxidase preparation. There is no meas- urable Cyt c reductase (complex 111) or Cyt c oxidase (complex IV) activity, but some NADH dehydrogenase and succinate dehydrogenase activities are present. Even so, there is a 20- to 40-fold enrichment of the alternative oxidase activity in the final product relative to these two activities. There was essentially no quinone ( 4 0 0 pmol ubiquinone. 10 mg-’ protein) present in the partially purified alternative oxidase, as determined by organic extraction and separation using HPLC. The purified oxidase also showed no absorption in the visible region of the spectrum (consistent with the lack of contamination by Cyts, carotenoids, or flavins). In addition, a borohydride-reduced minus air-oxidized redox difference spectrum of the partially purified oxidase showed no absorbance changes in the UV region between 250 and 400 nm (data not shown).

The partially purified alternative oxidase was assayed to determine the product of the reduction of O>. This was done by determining the amount of O2 consumed per mole of duroquinol substrate oxidized. The four-electron reduction of O2 to water would give a ratio of 0.5:1, but a two-electron reduction of O2 to H202 would give a ratio of O2 uptake to duroquinol oxidation of 1: 1. If endogenous catalase activity were present, the observed ratio would also be 0.5:1, even if HZ02 were the primary product, so the alternative oxidase samples were assayed for this activity. The solubilized alternative oxidase had no detectable catalase activity upon addition of 1 mM H202. In fact, when exogenous catalase was added as a control, the alternative oxidase preparation fully inhibited the catalase activity. The ratio of the moles of 0, consumed per mole of duroquinol added is shown in Table I11 for the alternative oxidase at two stages of purification. When assayed in mitochondrial reaction buffer, the alternative oxidase shows a ratio near 0.5, as expected if the product were H20. In mitochondrial reaction buffer containing 0.7 M citrate, the alternative oxidase shows a ratio approximating 1.0, suggesting that H202 is formed to a considerable extent.

Table 111. The ratio of the O, consumption per mole of duroquinol oxidized by the solubilized skunk cabbage alternative oxidase

Conditions are as described in the text. O2 Uptake/Duroquinol

Consumed

No citrate +0.7 M citrate Sample

Ultra Sup-3 0.60 1 .o2 Pooled, concentrated 0.51 0.85

G-200 fraction

DISCUSSION

This work reports an improved purification of the aroid alternative oxidase, resulting in a 20- to 30-fold increase in specific activity. There are a number of reasons why purification of the alternative oxidase has been difficult to achieve in spite of the efforts of several laboratories (Huqand Palmer, 1978; Bonner and Rich, 1983; Bonner et al., 1986; Elthon and McIntosh, 1986, 1987). First, ubiquinol and ubiquinol analogs donate electrons to the alternative oxidase of nonaroid spadix mitochondria only at very low rates. This precludes isolating the alternative oxidase from plants other than thermogenic aroids using quinol oxidase activity as an assay. Second, mitochondria from aroid spadices are only available either seasonally (A. maculatum; skunk cabbage) or in limited quan- tities (S. guttatum). Thus, the lack of a large and readily available supply of tissue from which to obtain the oxidase has hampered efforts to purify this enzyme. Another previously unrecognized difficulty in the purification of the aroid alternative oxidase was the presence of a highly active sulfhydryl protease activity. This protease may also be present in the spadix tissue of other aroids, and may account for.the observation of Bonner et al. (1986) that the alternative oxidase was “extraordinarily heat-labile after solubilization.” It was found that isolation of spadix mitochondria in the presence of E-64 inhibited this protease activity without any deleteri- ous effect on the alternative oxidase activity.

This purification of the alternative oxidase represents a significant advance over previous work. Bonner et al. (1986) obtained only a 4.8-fold purification of the alternative oxidase from A. maculatum. This preparation showed several major bands and a number of minor bands on a denaturing gel. Although they were able to separate the alternative oxidase

Table li. Mitochondríal activities in the partially purified skunk cabbage alternative oxidase Enzvme activities were assaved as described in ”Materials and Methods.”

Specific Activity

Mitochondria C-200 fraction

pnof min-’ mg-’

Activity Purification

Alternative oxidase 0.225 1.90 8.4 Cyt c oxidase 0.41 1 O Duroquino1:Cyt c reductase 0.141 O Succinate:DCPIP reductase 0.823 0.171 0.21 NADH:duroquinone reductase 2.92 0.062 0.02 NADH:FeCN reductase 4.26 1.65 0.39




activity from Cyt b and carotenoids, antibodies raised to this preparation were reactive toward an (unidentified) Cyt, succinate dehydrogenase, and two NADH dehydrogenases (Walsh and Moore, 1987). Huq and Palmer (1978), on the other hand, reported an 18-fold purification of the Arum alternative oxidase, with no detectable Cyt absorption, NADH dehydrogenase activity, or Cyt c oxidase activity. They did, however, report significant carotenoid contamination and a fluorescence emission spectrum consistent with a flavoprotein. Elthon and McIntosh (1987) reported that "if loss of activity is considered, a 166-fold purification was achieved" for their purification of the altemative oxidase from Sauromatum. However, they were unable to remove this fraction in the final phenyl-Sepharose step without using 2% SDS and losing a11 activity. The activity of their partially purified preparation prior to the phenyl-Sepharose column was 339 nmol O2 min-' mg-' protein, only a 3.4-fold purification.

In comparison to these earlier isolations, our purified product has no detectable Cyt, carotenoid, or flavin, as indicated by the lack of any visible absorbance, as well as essentially no extractable ubiquinone. There is no measureable complex I11 or complex IV activity in the purified oxidase, and contamination by activities associated with complexes I and I1 is limited .

The partially purified alternative oxidase activity runs on a gel filtration column with an apparent molecular mass of about 160 kD, but aggregation in aqueous medium is not atypical for solubilized, integral membrane proteins. An apparent aggregation of the oxidase polypeptides appears even under the conditions of SDS-2.5 M urea PAGE (Fig. 2), where the aggregates are believed to run at approximately the position expected of a dimer (approximately 60 kD).

We have noted some variation in the duroquinol oxidase activity from skunk cabbage mitochondria isolated from spadices harvested in different years from the same locale, together with a consistent pattern to the observed stimulation of the activity by citrate. When isolated skunk cabbage mitochondria show low levels of duroquinol oxidase activity (e.g. 70 nmol O, min-' mg-' protein), there is a large effect of added citrate (up to 400%). When the specific activity is intermediate (300-400 nmol 0 2 min-' mg-' protein), only a small citrate stimulation is observed (0-4096). With high levels of alternative oxidase activity (600 nmol O, min-' mg-' protein), there is no observed stimulation by citrate. We also note that the specific activity in the presence of citrate has never been observed to be higher than the specific activity for our most active skunk cabbage mitochondria in the absence of citrate. This pattern of stimulation by citrate has led us to believe that some modification (possibly proteolytic) of the native alternative oxidase occurs that both decreases the specific activity and causes it to become responsive to 0.7 M citrate. Rasmusson et al. (1990) have reported that in trypsin- digested submitochondrial particles from Arum, much of the lost duroquinol oxidase activity could be restored by including 0.7 M citrate in the reaction medium.

In contrast to the results of Kay and Palmer (1985), we found that the apparent stimulation of duroquinol oxidase activity by 0.7 M citrate is accompanied by a decrease in the amount of H 2 0 produced as the primary product, i.e. a

partially reduced product (H202 or superoxide) is produced. This means that some of the observed stimulation of duroquinol oxidase rate by citrate (at most, 2-fold) can be ac- counted for through the formation of H202 instead of H20 at the same rate of electron transfer. Additional stimulation in the rate of O, consumption may be due to the catalytic cycle leading to H,Oz formation by the altemative oxidase being kinetically faster than that for H20 formation. Al- though H202 formation during duroquinol oxidation by the skunk cabbage altemative oxidase apparently does occur in the presence of 0.7 M citrate, we find no evidence to suggest that H202 is a significant product of the alternative pathway under physiological conditions, in agreement with earlier studies (Moore and Siedow, 1991). However, the potential of citrate to induce H202 formation can be exploited in future studies to yield insights into the catalytic mechanism of the altemative oxidase.

ACKNOWLEDCMENTS

We would like to thank Dr. Lee McIntosh (Michigan State Uni- versity) for his gift of monoclonal antibody to the Sauromatum guttatum altemative oxidase, Dr. Nathalie Schmidt for doing the HPLC analyses of ubiquinone on the purified oxidase fraction, and Dr. Ann L. Umbach for helpful editorial suggestions.

Received June 23, 1992; accepted September 24, 1992. Copyright Clearance Center: 0032-0889/93/101/0113/07.

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partia1 purification of the cyanide-resistant of … · for measurements of the ratio of o2 uptake...

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