Alternative Pathway Respiration and Lipoxygenase Activity in ... - NCBI

Plant Physiol. (1982) 69, 1435-1438 0032-0889/82/69/1435/04/$00.50/0

Alternative Pathway Respiration and Lipoxygenase Activity in Aged Potato Slice Mitochondria' Received for publication October 9, 1981 and in revised form January 20, 1982

RICHARD M. SHINGLES2, GEOFFREY P. ARRON3, AND ROBERT D. HILL4 Department of Plant Science, University of Manitoba, Winnipeg, Canada R3T 2N2 ABSTRACT Mitochondrial preparations isolated from aged white potato (Solanum tuberosum L.) slices exhibited classical cyanide-insensitive 02 uptake which was inhibited by salicylhydroxamic acid and tetraethylthiuram disulfide (disulfiram). These mitochondria also possessed lipoxygenase activity, as determined by 02 uptake in the presence of 4 millimolar linoleic acid. Purification of the mitochondrial preparation on a continuous Percoll gradient resulted in a large decrease i lipoxygenase activity whereas cyanide-insensitive (disulfiram sensitive) 02 consumption was still observed. These data indicate that cyanide-insensitive 02 consumption in mitochondrial preparations isolated from aged white potato slices is of mitochondrial origin and not due to lipoxygenase contamination.

It has long been known that the aging of tissue slices from various tubers in liquid-aerated media induces metabolic changes within the tissue (7, 17). In potato slices, aging causes an increase in tissue respiration which becomes partially resistant to cyanide and carbon monoxide inhibition (5, 16). This resistance to cyanide has been attributed to the development of an alternative electron transfer pathway in the mitochondria (1, 6). It has been shown, however, that potato tubers contain high levels of lipoxygenase (11) and that, upon slicing of potatoes, there is a rapid loss of phospho- and galactolipid indicative of membrane lipid breakdown (15). Because of the possible contribution of lipoxygenase in the assessment of mitochondrial cyanide-insensitive respiration, we have attempted to determine whether cyanide-insensitive 02 consumption exhibited by mitochondrial preparations from aged potato slices was indeed attributable to a development of the alternative path or was due to lipoxygenase contamination.

MATERIALS AND METHODS Plant Tissue. White potato tubers (Solanum tuberosum L.) and red sweet potatoes (Ipomoea batatas Lam.) were purchased at local markets and stored at room temperature prior to use. Tubers of white potato were halved and slices (1.5-mm thick) were cut into ice water with a microtome blade fixed in an appropriate bed. Slices were rinsed with distilled H20 and trimmed with a knife to give squares (approximately 3 cm2). Square slices were aged for 24 h in 0.1 mm CaSO4 at 25°C in 2-L Erlenmeyer flasks on a shaker. The CaSO4 solution was changed at frequent intervals in the first few h and three times thereafter. Mung bean (Phaseolus aureus Roxb.) and soybean (Glycine max, Merr. cv. Maple Presto) seeds were surface sterilized in 2% HC104 for 2 min and then rinsed thoroughly in distilled H20. Seeds were germinated on wet paper towels for 3 d in the dark at 24°C. Isolation of Mitochondria. Mitochondria were prepared from white potato tubers and aged potato slices by differential centrifugation. Tissue was homogenized in a Waring Blendor in 250 ml of medium (0.25 M sucrose, 0.37 M mannitol, 5 mm cysteine, 5 mM EDTA, 0.2% BSA, 2% PVP-40, 25 mm Tes, with pH adjusted to 7.6 at 25°C with KOH). The resultant brei was filtered through two layers of cheesecloth and centrifuged at l,000g for 5 min. The supernatant layer was centrifuged at 20,000g for 5 min. The resulting pellets were resuspended in 40 ml of wash medium (0.4 M mannitol, 0.1% BSA, 25 mm Tes, with pH adjusted to 7.6 at 25°C with KOH) and centrifuged at 20,000g for 5 min. The ' Supported by grants from Natural Sciences and Engineering Research resulting pellet was resuspended in 2 ml of wash medium (washed Council and Canadian Department of Agriculture. mitochondrial preparation). Approximately 1.5 ml of this prepa2 Present address: Department of Horticultural Science, University of ration were layered onto a continuous Percoll gradient (10-60%o Guelph, Guelph, Canada NIG 2W1. [v/v] Percoll made up in 0.25 M sucrose). The gradient was 3 Present address: Chemical Research Division, Ontario Hydro, 800 centrifuged at 10,000g for 20 min in an angle rotor. The mitochondrial fraction was removed with a large bore syringe, susKipling Avenue, Toronto, Canada M8Z 5S4. 4 Send reprint requests to Dr. R. D. Hill. pended in 30 ml of wash medium and sedimented at 20,000g for 5 Abbreviation: SHAM, salicylhydroxamic acid. 5 min. The pellet of purified mitochondria was resuspended in 1

Goldstein et al. (3) have recently called for a reevaluation of the criteria necessary for the measurement of mitochondrial alternative oxidase-mediated, cyanide-insensitive respiration because of shortcomings inherent to electron transfer inhibitor studies. The presence of a functional alternative oxidase in plant mitochondria has been concluded from an insensitivity of 02 uptake to the presence of cyanide or antimycin A (inhibitors of the Cyt electron transfer path [6]) and specific inhibition by substituted hydroxamic acids, including SHAM5 (12). Hydroxamates also inhibit the activity of lipoxygenase (10), which catalyzes oxidation of polyunsaturated fatty acids. Crude mitochondrial preparations from wheat seedlings (3), mung bean (13), and soybean (9) have all been shown to be contaminated by lipoxygenase. Although purification of wheat seedling mitochondria on a continuous Percoll gradient resulted in a total loss of lipoxygenase activity as well as a complete loss of cyanide-insensitive respiration (3), other workers have been unsuccessful in attempts to totally remove lipoxygenase activity from soybean and mung bean mitochondria on Percoll and sucrose gradients, respectively (9, 13). However, Miller and Obendorf (9) reported that disulfiram, an inhibitor of cyanideinsensitive respiration in red sweet potato mitochondria (4), had no effect on lipoxygenase activity in soybean mitochondrial preparations and could be used to distinguish between residual lipoxygenase activity and cyanide-insensitive mitochondrial respiration.

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ml of wash medium. Mitochondria were prepared from mung Table II. Respiration, Cyanide Resistance, and Lipoxygenase Activity in Mitochondria Isolatedfrom Aged Potato Slices bean hypocotyls and soybean axes by homogenization of the plant tissue with a Polytron (PT 20 head) for 3 to 4 s in 250 ml of Conditions were the same as in Table I. Disulfiram (0.2 mM) was grinding medium (0.5 M mannitol, 0.2% BSA, 2 mm cysteine, 1 included where indicated. Numbers in parentheses refer to percentage of mm EDTA, 2% PVP-40, 10 mm K2HPO4 and pH adjusted to 7.5 state 3 rate that is cyanide-resistant. at 25°C with KOH). Red sweet potatoes were cut into small pieces Washed Purified Parameter and homogenized in the above medium in a Waring Blendor. The Substrate Mitochondria Mitochondria Measured rest of the mitochondrial isolation procedures were the same as Succinate 221 342 described for white potato. Mitochondrial protein was determined State 3 rate8 Succinate + CN 73(21%) 62(28%) by the method of Lowry (8) after solubilization with 5% deoxySuccinate + cholate. BSA was used as the standard. Percoll was obtained from 197 280 disulfiram Pharmacia Fine Chemicals. Succinate + Polarographic Measurements. Mitochondrial 02 consumption disulfiram + was measured with a Clark O2 electrode (Rank Bros., Bottisham, 0 0 CN Cambridge, U.K.) at 25°C in 3 ml of reaction medium (0.3 M 142 98 Malate mannitol, 0.2% BSA, 10 mm KCI, 5 mM MgCl2, 10 mm K2HPO4 33 (23%) 31 (31%) Malate + CN [pH 7.2]). When succinate was substrate, 0.1 mm ATP was included Malate + in the reaction medium to activate succinic dehydrogenase. 102 80 disulfiram ADP:O ratios were calculated by the method of Estabrook (2). Malate + SHAM and disulfiram were solubilized in ethylene glycol. The 02 disulfiram + concentration in air-saturated medium was taken as 240 iLM (2). 0 0 CN When disulfiram was used as an electron transfer inhibitor, it was added to the mitochondria 2 min before addition of substrate. Lipoxygenase Assay. Lipoxygenase activity in mitochondrial Respiratory 2.0 2.1 Succinate control ratio preparations was assayed polarographically in 1.5 ml of reaction Succinate + medium. The reaction was initiated by the addition of linoleic 2.6 2.7 disulfiram acid (final concentration 4 mM-stock solution of linoleic acid 2.1 2.0 Malate prepared in Tween 80 [14]). Malate + disulfiram

RESULTS Washed mitochondria isolated from white potato tubers and aged potato slices oxidized succinate at similar rates (Tables I and II), as shown by state 3 rates of oxidation (2). However, state 4 rates were higher in the mitochondria isolated from aged tissue, with the result that a decrease in respiratory control ratio was observed (Table II). A drop in the ADP:O ratios also indicated that oxidative phosphorylation was less efficient in these mitochondria. Similar results were obtained with malate as substrate. Cyanide totally inhibited electron transfer in mitochondria from fresh tissue with either succinate or malate as substrate (Table I). However, mitochondna isolated from aged potato slices were

Table I. Respiration, Cyanide Resistance, and Lipoxygenase Activity in Mitochondria Isolatedfrom Potato Tubers Oxygen consumption was measured as described under "Materials and Methods." State 3 rates were measured in the presence of 50 ,im ADP, with 10 mm succinate or 25 mm malate as substrate. NaCN (0.5 mM) was included where indicated. Purified Washed Parameter Mitochondria Mitochondria Substrate Measured 347 212 Succinate State 3 rate' 0 0 Succinate + CN 136 85 Malate 0 0 Malate + CN

Respiratory control ratio

Succinate Malate

3.0 3.3

3.4 4.9

ADP: 0 ratio

Succinate Malate

1.7 2.4

1.8 2.4

Lipoxygenase activity'

Linoleic acid

a

nmol O2/mg protein .

min.

475

40

ADP :0 ratio

Succinate Succinate + disulfiram Malate Malate + disulfiram

Lipoxygenase Linoleic acid activity' anmol O2/mg protein . min.

2.4

2.2

1.4

1.3

1.5 2.2

1.4 1.7

2.4

2.0

400

31

partially resistant to inhibition by cyanide with either substrate (Table II). In each case, the cyanide-insensitive respiration was fully inhibited by SHAM (data not shown). Similar results have been reported by Dizengremel and Lance (1). Purification of both fresh and aged slice mitochondrial preparations resulted in higher rates of substrate oxidation, with respiratory control and ADP:O ratios similar to those found in the washed mitochondrial preparations. Purification of the aged slice mitochondrial preparation did result in a slight loss of cyanide-insensitive path activity, although with both substrates the cyanide-insensitive rate was more than 20%o of the uninhibited rate (Table II). Disulfiram, an inhibitor of the cyanide-insensitive path in both red sweet potato mitochondria and soybean axes mitochondria (4, 9) totally inhibited cyanide-insensitive oxidation of malate or succinate by either washed or purified aged slice mitochondria (Table II). In the absence of cyanide, disulfiram caused a slight inhibition of malate or succinate oxidation, with an improvement in the respiratory control ratios and ADP:O values. Linoleic acid oxidation rates were higher in washed mitochondrial preparations from both fresh and aged potato slices than the oxidation rates of succinate or malate in the same preparation (Tables I and II). Purification of the washed mitochondrial preparations on Percoll gradients resulted in about 90% of the lipoxygenase activity being removed.

02 UPTAKE IN AGED POTATO SLICE MITOCHONDRIA

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Table III. Respiration, Cyanide Resistance, and Lipoxygenase Activity in Mitochondria Isolatedfrom Mung Bean Hypocotyls, Soybean Axes, and Sweet Potatoes Conditions were the same as in Table I. Disulfiram (0.2 mM) was included where indicated. Numbers in parentheses refer to percentage of state 3 rate that is cyanide-resistant. Mitochondrial Preparations Soybean

Mung bean Parameter Measured State 3 ratea

Substrate Succinate Succinate + tCN Succinate + disulfiram Succinate + disulfiram + CN Malate Malate+CN

Washed 258

Purified 320

Washed 547

Purified 624

Sweet potato, washed 118

43(17%)

55(17%)

62(12%)

100(16%)

73(61%) 105

264

505

566

11 188 41(22%)

0 337 51(15%)

0 265 64(24%)

4 50 18(35%)

Lipoxygenase

activitya a

nmol )2/mg

Linoleic acid

98

16

358

59

44

protein. min.

The linoleic acid oxidation rate observed in the purified aged slice by disulfiram. The results presented in this paper like those of Miller and mitochondrial preparation was lower than the cyanide-insensitive Obendorf (9) for soybean mitochondria, indicate that cyaniderespiration rates. Purification of mitochondria isolated from mung bean hypo- insensitive respiration in mitochondrial fractions prepared from cotyls and soybean axes on Percoll gradients gave similar results aged potato slices appears to be of mitochondrial origin. In to those obtained with aged potato slice mitochondria (Table III). contrast, upon purification of wheat seedling mitochondria there A large scale loss of lipoxygenase activity was accompanied by is a concomitant loss of lipoxygenase activity and cyanide-insenrespiration (3). little or no change in the rate of cyanide-insensitive respiration. sitive Our washed preparation of red sweet potato mitochondria Siedow and Girvin (13) have previously reported that purification exhibited a low level of lipoxygenase activity, while washed prepof mung bean mitochondria on a discontinuous sucrose gradient arations from white potatoes had high levels of contaminating also failed to remove all the lipoxygenase activity from the prep- lipoxygenase. This may be connected with the observation that aration, while soybean axes mitochondria purified on a discontin- there is little or no loss of lipid when red sweet potatoes are sliced uous Percoll gradient were found to retain high levels of lipoxy- (15), whereas there is a rapid loss of lipid observed when white genase activity (9). potato tubers are sliced. In contrast to the situation in washed preparations from fresh and aged potato slices, we observed that washed mitochondrial LITERATURE CITED preparations obtained from red sweet potatoes exhibited low levels of lipoxygenase activity, in fact the 02 consumption rate in the 1. DIZENGREMEL P, C LANCE 1976 Control of changes in mitochondrial activities during aging of potato slices. Plant Physiol 58: 147-151 presence of linoleic acid was lower than the rate of succinate 2. ESTABROOK RW 1967 Mitochondrial respiratory control and the polarographic oxidation in the presence of cyanide (Table III). measurement of ADP:O ratios. Methods Enzymol 10: 41-47

DISCUSSION There is lipoxygenase activity associated with mitochondria isolated from aged potato slices, but it does not appear to contribute significantly to the cyanide-insensitive 02 consumption observed in the presence of Krebs cycle substrates. Although purification of the mitochondrial preparation on a continuous Percoll gradient did not wholly remove lipoxygenase activity, the resultant activity was lower than the cyanide-insensitive rate of 02 consumption. This observation and the fact that disulfiram totally inhibited the cyanide-insensitive respiration meant that the latter was mitochondrial in origin and not due to lipoxygenase adhering to the organelles. Disulfiram did cause a slight inhibition of substrate oxidation in the absence of cyanide in all preparations examined, with an increase in respiratory control values and ADP:O ratios. This is consistent with disulfiram inhibition of the nonphosphorylating cyanide-insensitive path with the result that electron flow is directed through the phosphorylating Cyt chain. Grover and Laties (4) have previously reported a slight inhibitory effect of disulfiram on electron transfer via the Cyt chain of white potato mitochondria, and Miller and Obendorf (9) observed a slight inhibition of succinate oxidation in soybean mitochondria

3. GOLDSTEIN AH, JO ANDERSON, RG McDANIEL 1981 Cyanide-insensitive and cyanide-sensitive 02 uptake in wheat. II. Gradient-purified mitochondria lack cyanide-insensitive respiration. Plant Physiol 67: 594-596 4. GROVER SD, GG LATIES 1978 Characterization of the binding properties of disulfiram, an inhibitor of cyanide resistant respiration. In G Ducet, C Lance, eds, Plant Mitochondria. Elsevier/North Holland Biomedical Press, Amsterdam, pp 259-266 5. HACKETT, DP, DW HAAS, SK GRIFFITHs, DJ NIEDERPRUEM 1960 Studies on development of cyanide-resistant respiration in potato tuber slices. Plant Physiol 35: 8-19 6. HENRY M, E NYNS 1975 Cyanide-insensitive respiration. An alternative mitochondrial pathway. Subcell Biochem 4: 1-65 7. KAHL G 1974 Metabolism in plant storage tissue slices. Bot Rev 40: 263-314 8. LOWRY OH, NJ ROSEBROUGH, AL FARR, RJ RANDALL 1951 Protein measurements with the Folin phenol reagent. J Biol Chem 193: 265-275 9. MILLER MG, RG OBENDORF 1981 Use of tetraethylthiuram disulfide to discriminate between alternative respiration and lipoxygenase. Plant Physiol 67: 962-964 10. PARRISH DJ, AC LEOPOLD 1978 Confounding of alternate respiration by lipoxygenase activity. Plant Physiol 62: 470-472 11. PINSKY A, S GROSSMAN, M TROP 1971 Lipoxygenase content and antioxidant activity of some fruits and vegetables. J Food Sci 36: 571-572 12. SCHONBAUM GR, WD BONNER, BT STOREY, JT BAHR 1971 Specific inhibition of the cyanide-insensitive respiratory pathway in plant mitochondria by hydroxamic acids. Plant Physiol 47: 124-128 13. SIEDOW JN, ME GIRVIN 1980 Alternative respiratory pathway. Its role in seed

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respiration and its inhibition by propyl gallate. Plant Physiol 65: 669-674 14. SURREY K 1974 Spectrophotometric method for determination of lipoxidase activity. Plant Physiol 39: 65-70 15. THEOLOGIs A, GG LATIES 1980 Membrane lipid breakdown in relation to the wound-induced and cyanide-resistant respiration in tissue slices. Plant Physiol 66: 890-896

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16. THIMANN KV, CS YOCUM, DP HACKETT 1954 Terminal oxidases and growth in plant tissues. III. Terminal oxidation in potato tuber tissue. Arch Biochem Biophys 53: 239-257 17. VAN STEVENINCK RFM 1975 The "washing" or "aging" phenomenon in plant tissues. Annu Rev Plant Physiol 26: 237-258