Eur. J. Biochem. 68, 13- 19 (1Y76)
A Low-Molecular-WeightATPase from Wheat-Seedling Mitochondria Hans TUPPY and Giinther SPERK Institut fur Biochemie der Universitat Wien (Received February 2 / M a y 31, 1976)
An ATPase which strikingly differed from the mitochondrial ATPases of yeast and of animal tissues was obtained when wheat seedling mitochondria, or electron transport particles derived from them, were subjected to ultrasonication and treated with ammonium sulphate. The enzyme which was purified by chromatography on Sephadex G-100 and DEAE-Sephadex (A50) failed to be inactivated by keeping in the cold. The molecular weight was shown by gel filtration to be as low as 43000. The enzyme preparation was capable of hydrolysing ADP, in addition to ATP, and several other nucleoside diphosphates and triphosphates. In contrast to the ATPase of animal mitochondria, the activity of the wheat enzyme was almost as insensitive to oligomycin in intact mitochondria as it was after isolation from the organelles.
The ATPases isolated from mitochondria of baker's yeast, of heart muscle and liver tissue have molecular weights of approximately 360000 and are composed of several subunits (for a review, see [I]). The isolated subunits are devoid of ATPase activity. The active protein complex has been shown to lose its enzymic capacity upon keeping in the cold [2]. Attempts made in this laboratory to isolate ATPase from wheat seedling mitochondria failed to yield an enzyme resembling that of yeast and animal mitochondria with regard to high molecular weight and cold lability. Instead, a cold-stable and relatively low-molecular-weight ATPase was obtained. The isolation and properties of this enzyme will be discussed in this paper. MATERIALS AND METHODS Plant Material
Etiolated coleoptiles from winter wheat (Triticum aestivum L.) were obtained by layering seeds on moist paper towelling in plastic containers and germinating them in the dark for 3 days at 27 "C. Seeds were previously surface-sterilized by submersion in 1 % sodium hypochlorite for 15 min and rinsed thoroughly with distilled water. Abbreviations. AMP-(CHz)P, adenosine j'-(a,P-methylene)diphosphate; AMP-P(CHz)P, adenosine 5'-(fl,y-methylene)triphosphate. Enzymes. ATPase or adenosinetriphosphatase (EC 3.6.1.3) ; adenylate kinase (EC 2.7.4.3); j'-nucleotidase (EC 3.1.3.5); pyruvate kinase (EC 2.7.2.40); hexokinase (EC 2.7.1.1); glucose-6-phosphate dehydrogenase (EC 1.1.1.49); aldolase (EC 4.1.2.13); catalase (EC 1.11.1.6).
Preparation and Purification of Mitochondria
Mitochondria were prepared by the method described by Sarkissian and Srivastava [3], all steps being performed at 4 "C. When larger amounts of mitochondria were to be isolated for solubilization of ATPase, the method was slightly modified: 150250 g of fresh seedlings were homogenized for 15 s in a Waring blender with 2 1 of a solution containing 0.5 M sucrose, 50 mM Tris-chloride, pH 7.2, 2 mM EDTA and 0.75 mg albumin/ml. The homogenate was strained through a nylon fabric (mesh % 50 pm) and centrifuged in a Sorvall GS3 rotor 3 min at 3500 x g. Recentrifugation of the supernatant readjusted to pH 7.2 for 10 min at 13000 x g resulted in a bilayered sediment consisting of mitochondria on top of a small starch pellet. The mitochondria were carefully rinsed off with 11 of 0.5 M sucrose, 50 niM Tris-chloride, pH 7.2, without disturbing the starch layer. This suspension was recentrifuged for 10 min at 13000 x g . The resulting mitochondrial pellets were suspended in 0.5 M sucrose, 10 mM Trischloride, pH 7.2, 2 mM EDTA to give a protein concentration of 30 mg/ml. Portions of the suspension (0.8 ml) were layered separately on linear 20 - 70 "/, sucrose density gradients (37.5 ml) containing 10 mM Tris-chloride and 2 mM EDTA, pH 7.2. Upon centrifugation in a Beckman SW27 rotor for 2 h at 13000 x g the mitochondria formed a distinct band, which was removed by means of a syringe fitted with an L-shaped needle. The collected mitochondrial bands were diluted 5-fold and centrifuged for 20 min at 78000 x g in a Spinco 30 rotor. The sedimented mitochondria were suspended in 0.1 M sucrose, 50 mM Tris-chloride,
14
pH 7.2, at a protein concentration of 15 mg/ml and stored at - 20 "C. Purification of ATPase Preparation of Submitochondrial Particles. Electron transport particles were prepared from wheat seedling mitochondria according to the method of Senior and Brooks [4]. The frozen mitochondrial suspension was thawed and diluted 6-fold with 0.1 M sucrose, 10 mM Tris-chloride, pH 7.2. Fractions (50 ml) were placed in glass beakers submersed in an ice bath and sonicated at full power in a Branson €312 sonicator three times for 1 min, with intermissions of 1 min. The suspension was centrifuged at 105000 x g for 90 min in a Spinco 40 rotor. Sedimented submitochondrial particles were suspended in a solution of 0.1 M sucrose, 50 mM Tris-chloride, pH 7.2, at a protein concentration of 10 mgjml and stored at - 20 "C. The purity of the electron transport particles was checked by ultracentrifugation in a sucrose gradient (30 - 60 %>. All fractions were tested for ATPase and cytochrome oxidase activities. The peaks of these two enzymes were found to coincide, thus excluding the possibility of a significant contamination of the electron transport particles by other membrane or vesicle fractions. Release oj the Enzyme jrom Electron Transport Particles by Sonication. The frozen suspension of electron transport particles was thawed out, diluted 10-fold with 0.1 M sucrose, 50 mM Tris-chloride, pH 7.2, and supplemented with ammonium sulphate and Triplex 1V (cyclohexane-l,2-diaminetetraacetic acid) to give final concentrations of 0.25 M and 4 mM, respectively. Portions (50 ml) of this suspension (1 mg protein/ml) were immersed in an ice bath and sonicated for 8 min at full power. During this period the temperature of the sample rose from 4 "C to 30- 35 "C and thereupon was allowed to increase further up to 50 "C by sonicating without cooling. Denatured protein was removed by centrifuging 10 min at 38000 x g. The supernatant contained approximately 75 % of the ATPase activity of the electron transport particles. At least 70 of the ATPase activity of the supernatant could not be sedimented even upon ultracentrifugation at 250000 x g for 11 h. As a rule, all subsequent purification steps were performed at 4 "C. Ammonium Sulphate Fractionation. Solid amnionium sulphate was added slowly to the stirred solution and the pH maintained at 7.2 until 35% saturation was reached. After 10 min the precipitate was removed by centrifugation (10 min at 35000 xg). Such a precipitate did not form when this solution had been subjected to prolonged ultracentrifugation and the resulting sediment was removed prior to addition of ammonium sulphate. The supernatant was adjusted to 70% saturation of ammonium sulphate in the
ATPase from Wheat Seedling Mitochondria
same way and recentrifuged after 10 min. ATPase was found in the pellet and could be stored in a 75 % ammonium sulphate suspension or in aqueous solution at 4 "C with no detectable decrease of activity. Purification on Sephadex G-100. Aliquots of the 70 % ammonium sulphate precipitate containing about 10 mg protein were dissolved in 1 ml 0.25 M Trischloride, 0.25 M ammonium sulphate, 4 mM Titriplex IV, pH 7.2, and layered on a Sephadex G-100 column (1.7 x 94 cm) equilibrated with the same buffer. The elution was carried out at a flow rate of 50 ml/h. 2-ml fractions were collected in a LKB fraction collector and the absorbance at 280 nm was recorded by a LKB Uvicord spectrophotometer. Fractions 54- 60 were combined and the enzyme was salted out with 70 % ammonium sulphate saturation or concentrated by ultrafiltration in an Ainicon cell (50 nil) equipped with a UM2 filter. In order to achieve an optimum quality of the enzyme preparation, the gel filtration step was preferably repeated once. DEAE-Sephadex Chromatography. DEAE-Sephadex (A 50) was converted to the sulphate form and equilibrated with 40 mM Tris-sulphate, pH 7.2. The enzyme purified on Sephadex G-100 was dissolved in 0.5 ml 40 mM Tris-sulphate buffer, pH 7.2, layered on the column (2.1 x 27 cm) and eluted with 150 ml of the same buffer. Fractions (1 ml) were collected with simultaneous recording of the absorbance at 280 nm. Fractions 67 - 75 contained ATPase. Analytical Procedures ATPase Assay. The release of Pi from ATP was measured at 30 "C. The reaction was started by adding 20 pl of a solution of 6 pmol ATP in 0.05 M Trischloride, pH 7.2, to 1.0 ml incubation mixture containing enzyme, 50 pmol Tris-chloride, pH 7.2, and 4 pmol MgS04. When an ATP-regenerating system was to be included, the mixture was supplemented by 5 kmol phosphoenolpyruvate and 50 pg pyruvate kinase. When ATPase activity of mitochondria was determined, the incubation mixture contained from 0.1 M to 0.5 M sucrose in addition to 50 mM Trischloride, pH 7.2, and 4 mM MgS04, the ATP-regenerating system was included and the organelles were suspended at a protein concentration of 0.24 mg protein/ml. Oligomycin, rutamycin and N,N'-dicyclohexylcarbodiimide, when tested for their inhibitory action, were dissolved in ethanol and added to the mitochondrial preparations in portions of 10 pl. After 10-min incubation 1.0 ml 10 % trichloroacetic acid was added to stop the reaction and denatured protein was removed by centrifugation. Inorganic phosphate was estimated in 1.5-ml aliquots of the supernatant by the method of Fiske and SubbaRow [ 5 ] . The enzymic release of Pi from ADP, AMP and other nucleoside phosphates was measured using the
15
H. Tuppy and G. Sperk
same assay, 6 pmol of the respective nucleoside phosphate being added instead of ATP. Adenylute kinase was assayed using the spectrophotometric procedure of Sottocasa et al. [6]. Reaction mixtures (1.0 ml) contained 7.5 pmol ADP, 20 pmol glucose, 3 units hexokinase, 3 units glucose6-phosphate dehydrogenase, 20 pmol NADP', 2 pmol MgS04 and 100 pmol Tris-chloride, pH 8.0. The reaction was started by adding 50 1.11 of enzyme solution and followed on a Gilford spectrophotometer 240 equipped with an automatic recorder at 340 nm. In experiments assessing the effect of AMP-(CHz)P and AMP-P(CH2)P on adenylate kinase activity 7.5 pmol of the nucleotide analogue were added to the reaction mixture. Controls were carried out concerning a possible influence of these analogues on hexokinase and glucose-6-phosphate dehydrogenase (3 units each as well as 0.03 unit each), the same reaction mixture being used except adenylate kinase was omitted and ADP was replaced by 7.5 pmol of ATP. Cytochrome oxiduse was determined as described by Wharton and Tzagoloff [7]. Oxygen consumption of mitochondria and electron transport particles was recorded polarographically as described previously [8]. Protein was determined according to Lowry et al. [9]. Dodecylsukhate-polyucrylurnide gel electrophoresis was performed as described by Weber and Osborn [lo]. Materials
Seeds of winter wheat (Triticum aestivum L., line E.xtrem) were kindly donated by Prof. H. Hiinsel (Saatzuchtanstalt Probstdorf, Austria). Hydroxyapatite was prepared freshly by the method of Bernardi [ll]. Oligomycin was obtained from Sigma and rutamycin was a gift of Eli Lilly Comp. Adenosine 5'-(a,Bmethylene)diphosphate, AMP-(CHz)P, and adenosine 5'-(fl,y-tnethylene)triphosphate,AMP-P(CH2)P, were obtained from Miles Laboratories, Inc.
RESULTS Mitochondria prepared from wheat seedlings by the method of Sarkissian and Srivastava 131 showed respiratory control values of about 4 and ADP : 0 2 ratios of 3.7 with 2-oxoglutarate as substrate. Oligomycin did not impair their respiration in 'state 2' (i.e., in the presence of substrate and absence of ADP, cc Chance and Williams [12]), whereas the respiratory stimulation by ADP was totally inhibited by this antibiotic. The ATPase activity of these carefully prepared mitochondria was very low (0.08 pmol Pi
min-' mg protein-'). Activation of the enzyme could be achieved by subjecting the mitochondria either to lowered sucrose concentrations, or to freezing and thawing or to sonication. Specific activities of ATPase up to 1.2 pmol Pi min-' mg protein-' were found in mitochondria previously frozen and sonicated and subsequently assayed in a buffer lacking sucrose. The ATPase of native as well as that of damaged mitochondria was hardly alTected by oligomycin, rutamycin or N,N'-dicyclohexylcarbodiimide. Under conditions where the ATPase activity of beef heart mitochrondria, previously frozen and suspended in 0.1 M sucrose, was shown to be inhibited at least 901%;by oligomycin and rutamycin (20 pg/mg protein) and by N,N'-dicyclohexylcarbodiimide (10 pmol/mg protein), these agents reduced the ATPase activity of similarly treated wheat seedling mitochondria by less than 10 %. An increase in inhibitor concentration by a factor of 10 did not result in a significant increase of this inhibition. Soluhilization and Purification of Wheat Seedling ATPase
Several methods previously described for the solubilization of ATPase from animal and plant mitochondria were tested on wheat seedling mitochondria. These included shaking of the mitochondrial suspension with glass beads in a Merkenschlager homogenizer [13], extraction of phospholipids with acetone [14] and separation of ATPase from mitochondrial membrane fractions by incubation with ammonium sulphate and deoxycholate [15]. None of these procedures, however, yielded a cold-labile, high-molecularweight ATPase. In contrast, from wheat mitochondria a lower-molecular-weight and cold-stable ATPase could be isolated and purified by a procedure involving sonication in the presence of ammonium sulphate and an Mg2+-chelatingagent, precipitation by ammonium sulphate and chromatography on Sephadex G-100 and DEAE-Sephadex (A50) (Table 1). This kind of enzyme, rather than a cold-labile ATPase, was obtained irrespective of whether the isolation procedure was carried out at room temperature or at 4 "C. Gel filtration on Sephadex G-100 (Fig. 1) resulted in the separation of the ATPase from the bulk of other proteins including most of the adenylate kinase activity. 5'-Nucleotidase, which partly overlapped ATPase in the Sephadex G-100 chromatography, was subsequently removed completely by ion-exchange chromatography on DEAE-Sephadex (A50) (Fig. 2). The ATPase preparation resulting from chromatography on Sephadex G-100 and DEAE-Sephadex exhibited a residual adenylate kinase activity not exceeding 0.25 pmol of ATP formed min- ' mg protein-'. In the course of both purification steps ATPase and ADPase activities remained associated with each
16
ATPase from Wheat Seedling Mitochondria
Fraction number
Fig. 1. PuriJicalion of crude ATPasr by gelji//i,u//o/ioil Si;r,hadev G-100. Protein concentration (------)was determined by absorbance at 280 m i . ATPase (.---a) and 5'-nucleotidase (O-----O) activities have been expressed as rate of Pi release. There was a strict correlation between the capacities of effluent fractions to cleave ATP and ADP. The rate of ATP formation from ADP by adenylate kiiiase (A----A) was assayed according to the procedure of Sottocasa et al. [6] 8 .O
-
I-
c ._
E
E, %
0 c
x
m
8 m.
2 3
R
.s.
d4
2
,
j.
-... '1'..
.. .
.
...-,""... E . . . l .
Table 1. Pur(fication of ATPase fiorn wheat seedling mirochondria ATPase and nucleoside phosphate cleaving activities were assayed as described under Materials and Methods (10 min, 30 "C, in the presence of 6 pmol of substrate) Purification step
Protein ATPase Specific activity as P , yield released from AMP
Mitochondria Electron transport
ATP
mg
"/,
pmol min- mg protein-'
350
100
0.1
0.4
0.9
80 60
0.6 0.9
1.1 1.9
2.1 3.8
38
1.0
2.1
4.4
9
6.6
particles 120 Sonication supernatant 50 Ammonium sulphate fractionation 27.2 Sephadex G-100 chromatography DEAE-Sephadex chromatography
ADP
1.1
0.04
4
0
13 150
*' 3,0
'%._._
w
other. When chromatography on a hydroxyapatite column (1.3 x 48 cm) equilibrated with 0.05 M potassium borate buffer and elution with a linear gradient of potassium phosphate buffer (0-0.5 M, pH 7.2, 600 ml) was used instead of the DEAE-Sephadex (A50) chromatography, ATPase and ADPase did not separate, both activities emerging from the column at a potassium phosphate concentration of 0.89 M.
Characterization of ATPase
Gel filtration on a calibrated Sephadex G-100 column suggested that the ATPase from wheat mitochondria had a molecular weight of about 43000, the enzyme emerging from the column at an elution volume close to that of ovalbumin (Fig. 3). When the enzyme preparation was subjected to 7 % dodecylsulphate-polyacrylamide gel electrophoresis, two
H. Tuppy and G . Sperk
17 Table 2. Cleavage of various nucleoside diphosphaies and triphosphotrs h.y ~vlzeatmitochondria1 A TPase The purification of the enzyme has been described under Results and the assay of its activity under Materials and Methods
l 9o t
1 .o
2 .o
1.5
Substrate
Relative rate of P , release
ATP GTP CTP UTP ITP dGTP dCTP ADP GDP
1.0 1.2 2.0 1.7 0.4 0.3 0.5 0.5 0.5
2.5
ve 1 vo
Fig. 3. Determination of molecular weight o f A TPase by gelfiltration on Sephadev G-100. The column (1.7 x 54 cm) was equilibrated with 0.25 M Tris-sulphate, 0.25 M ammonium sulphate, pH 7.2. The reference proteins used were bovine serum albumin (molecular weight 67000). ovalbumin (45000), soybean trypsin inhibitor (21 500) and cytochrome c (12400). Abscissa: Elution volume of protein ( V , ) dividcd by the void volume of the column (V") as determined with the help of blue dextran
Table 3. Effect of nuckoside phosphate analogues on ATPuse and A D Past, ac I ivit its ATPase and ADPase activities of purified enzyme (6 pg protein) were determined as described under Materials and Methods, with the exception that 3 pmol of substrate (ATP o r ADP) were used instead of 6pmol. From AMP-(CHz)P and AMP-P(CH2)P no phosphate was released by the enzyme. n.d. = not determined ~
Added analogue
--
2.0
ATP
1
10
0
ADP
pmol/min None AMP-(CHz)P ( 3 p11101) AMP-(CHz)P (6 p11101) AMP-P(CH2)P (3 pmoi) AMP-P(CH1)P (6 pmol)
[EDTA] (rnM)
~
P, released from
10
20
30
40
50
[MgS041 (mM)
Fig. 4. DrJpundence qf ATPUSKactivity on M g z + concentration. ATPase purified on Sephadex G-100 was assayed in the presence of an ATP-regenerating system. The original enzyme solution was either supplemented with MgZt by addition of a small volume of MgS04 solution (0.1 -3.2 M) or, alternatively, EDTA (10 pM, p H 7.2) was added to give the final concentrations indicated on the abscissa
strong protein bands appeared. From their mobility relative to markers such as catalase and fumarase the molecular weight of the faster-moving protein was inferred to be about 40000, thus corresponding to the molecular weight of ATPase as found by gel filtration. The slower-moving protein having an apparent molecular weight of about 60000 is a contaminant which runs ahead but partially overlaps ATPase in Sephadex
0.18 0.20 n.d. 0.19 n.d.
0.09 0.16 0.22 0.10 0.10
G-100 chromatography. The ATPase activity of the enzyme preparation can be augmented by Mg2+ ions and is entirely abolished by EDTA in concentrations above 2 mM (Fig. 4). Dinitrophenol failed to activate the enzyme. Nucleoside triphosphates other than ATP, deoxynucleoside triphosphates and nucleoside diphosphates are cleaved by the purified enzyme at appreciable rates (Table 2). Neither AMP nor pyrophosphate were split. The ADPase activity was strongly increased by the ADP analogue, AMP-(CH2)P (Table 31, whereas the corresponding ATP analogue, AMP-P(CH2)P, did not have a comparable effect. The ATPase activity was not affected significantly by either analogue. By contrast, adenylate kinase present in wheat mitochondrial extracts and largely separated from ATPase by chromatography on Sephadex G-100 was inhibited 50% by AMP-(CHz)P, as well as by AMP-P(CH2)P when the analogues were used in concentrations equal to that of the substrate, ADP.
18
DISCUSSION The preparation of an ATPase from mitochondria of wheat seedlings as described in this paper combines the purification steps recommended by Senior and Brooks [4] including ultrasonication and heat treatment of electron transport particles with the dissociation of mitochondrial proteins by ammonium sulphate reported by Van de Stadt et al. [15]. When this preparative procedure was applied to mitochondria of beef heart as well as of several dicotylous plants such as castor bean and cauliflower [16] high-molecular-weight ATPases of the well-known type were obtained. By contrast, the ATPase prepared in this way from wheat mitochondria had markedly different properties. In the course of the preparation and purification of the wheat mitochondrial ATPase the ratio of the hydrolysing activities towards ATP and ADP did not change. This finding not only points to an identity of the ATP and ADP cleaving enzyme, but also to an identity of the original enzyme bound to wheat mitochondria with the solubilized and partially purified ATPase. Although adenylate kinase, large quantities of which were present in crude mitochondrial extracts, could be removed to a great extent in the course of purification, the final preparation of ATPase was still contaminated by a small amount of that enzyme. The activity of wheat mitochondrial adenylate kinase was inhibited markedly by the ADP and ATP analogues, AMP-(CH2)P and AMP-P(CH2)I’. These analogues, however, did not impair the hydrolysis of ADP by the ATPase preparation. This finding excludes the possibility of the ADP cleavage being due to conversion of ADP to AMP and ATP by adenylate kinase and subsequent hydrolysis of ATP by ATPase. On the contrary, AMP-(CH2)P significantly increased ADP hydrolysis by wheat seedling ATPase whereas the rate of ATP hydrolysis was hardly affected. The distinct behaviour of AMP-(CH2)P with respect to ADP and ATP hydrolysis may possibly be accounted for by separate binding sites of the ATPase for the two substances, ADP and ATP. Multiple nucleotide binding sites have previously been suggested by Hilborn and Hammes for beef heart mitochondrial ATPase [17]. Some further properties of the ATPase isolated from wheat seedling mitochondria are also significantly different from those of mitochondrial ATPases derived from other sources. Its molecular weight estimated by gel filtration was found to be approximately 43 000 as compared with molecular weights of about 360000 for beef heart, rat liver and yeast ATPases [l]. The relative mobility of wheat seedling ATPase on dodecylsulphate gel electrophoresis also pointed to a molecular weight around 40000, thus suggesting that this enzyme, in contrast with other
ATPase from Wheat Seedling Mitochondria
mitochondrial ATPases, failed to be dissociated by dodecylsulphate into subunits. In further contrast to such enzymes, the ATPase from wheat mitochondria proved itself stable to cold and was not affected by dinitrophenol. In view of these marked differences, the question arises whether this wheat ATPase might be comparable to the low-molecular-weight ATPases found by Le Deaut et a/. 1181 in the matrix of rat liver mitochondria rather than to those in the mitochondrial membrane involved in oxidative phosphorylation. In this paper, however, evidence has been presented that the wheat enzyme is membrane-bound: after freezing, thawing and mild sonication it was still associated with the electron transport particles, and drastic sonication in the presence of ammonium sulphate was required to solubilize it. The lowmolecuhr-weight enzyme appeared to be the only ATPase which could be obtained from wheat mitochondria or electron transport particles. Although several methods for solubilizing ATPase, such as disruption of the mitochondria in a Merkenschlager homogenizer, delipidation by acetone and disintegration of membranes by ammonium sulphate and detergents were tried, a cold-labile and high-molecular-weight enzyme comparable to the established mitochondrial membrane ATPases could never be obtained from wheat. In the course of the purification of the low-molecular-weight ATPase a high-molecular-weight ATPase having the expected properties was not encountered, irrespective of whether fractionations were carried out at room temperature or in the cold. There are strong indications that even in intact wheat seedling mitochondria the properties of the ATPase are different from those of the ATPase of animal or yeast mitochondria. In accordance with results reported by Jung and Hanson for corn mitochondria [19] we found previously that the ATPase activity of intact wheat mitochondria and electron transport particles derived from them is not inhibited by oligomycin, in contrast to the sensitivity of the animal and yeast organelles to this antibiotic [20]. This finding points to a significant difference between the ATPase or the membrane structure incorporating ATPase in wheat mitochondria compared with other mitochondria. Future investigations may show whether the low-molecular-weight ATPase of wheat mitochondria might correspond to one subunit of the ATPase complex found in other mitochondria. The distinctive difference could be that the subunit size molecule from wheat would be active whereas the ATPase complexes from other sources would lose their enzymic function when they are decomposed into subunits. This investigation was supported by the Hochschuljuhiluumsst$iung der Gemeinde Wien and by the Fonds iur Fiirdertang der wissenschaftlichen Forschung (Project 1210).
H. Tuppy and G. Sperk
REFERENCES
Weber, K.&Osborn, M. (1969) J . B i d Chem 244,4406-4412. Bernardi, G. (1971) Methods Enzymol. 22, 325-339. Chance, B. & Williams, G. R. (1956) A h . Enzymol. 17,56- 134. Penefsky, H. S., Pullman, M. E., Datta, A. & Racker, E. (1960) 4694-4702. J . Biol. Chem. 235, 3330- 3336. Sarkissian, I. V. & Srivastava, H. K. (1969) Proc,. Nut1 A C L J ~ . 14. M. J. (1967) Biochrm. J . 10.5, 279-288. Selwyn, Sci. U.S.A. 63, 302- 309. 15. Van dc Stadt, R . J., Kraaipoel, R. J. & Van Darn, K . (1972) Senior, A. E. & Brooks, J. G. (2970) Awh. Biochrm. Eiophyx. Eiochim. Siophys. A t t u , 276, 25- 36. 140, 257 - 266. 16. Sperk, G. & Tuppy, H. (1976) Planl Ph.vsiol. in press. Fiske, C. H. & SubbaRow, Y . (1925) J . B i d . Chem. 66, 37517. Hilborn, D . A. & Hammes, G . G. (1973) Biochemistry. 12, 400. 983 990. Sottocasa, G. L., Kuylenstierna, B., Ernster, L. & Bergstrand, 18. Le Deaut, J. Y., Ledig, M., Mandel, P. & Marfing, M. (1972) A. (1967) M e t h o h Enzymol. 10, 448-463. FEBS Lett. 20, 153- 156. Wharton, D. C. & Tzagoloff, A. (1967) Methods Enzymol. 10, 19. Jung, D. W. & Hanson. J . B. (1973) Arch. Biocltem. Biophys. 245 250. 158, 139-148. Zobl, R., Fischbeck, G., Kcydel, F., Latzko, E. & Sperk, G. 20. Sperk, G. & Tuppy, €3. (1973) Hoppe-Seylcr’s Z. Phy.c.iol. (1972) Plant Physiol. 50, 790-791. Chem. 350, 1244- 1245. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J .
1. Senior, A . E. (1973) Biochim. Biophys. Acta, 301, 249-277. 2. Penefsky, H. S. & Warner,C. R. (1965) J . B i d . Chrm. 240,
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H. Tuppy and G. Sperk. lnstitut fur Biochemie der Universitiit Wien, WahringerstraBe 17, A-1090 Wien, Austria
A Low-Molecular-WeightATPase from Wheat-Seedling Mitochondria Hans TUPPY and Giinther SPERK Institut fur Biochemie der Universitat Wien (Received February 2 / M a y 31, 1976)
An ATPase which strikingly differed from the mitochondrial ATPases of yeast and of animal tissues was obtained when wheat seedling mitochondria, or electron transport particles derived from them, were subjected to ultrasonication and treated with ammonium sulphate. The enzyme which was purified by chromatography on Sephadex G-100 and DEAE-Sephadex (A50) failed to be inactivated by keeping in the cold. The molecular weight was shown by gel filtration to be as low as 43000. The enzyme preparation was capable of hydrolysing ADP, in addition to ATP, and several other nucleoside diphosphates and triphosphates. In contrast to the ATPase of animal mitochondria, the activity of the wheat enzyme was almost as insensitive to oligomycin in intact mitochondria as it was after isolation from the organelles.
The ATPases isolated from mitochondria of baker's yeast, of heart muscle and liver tissue have molecular weights of approximately 360000 and are composed of several subunits (for a review, see [I]). The isolated subunits are devoid of ATPase activity. The active protein complex has been shown to lose its enzymic capacity upon keeping in the cold [2]. Attempts made in this laboratory to isolate ATPase from wheat seedling mitochondria failed to yield an enzyme resembling that of yeast and animal mitochondria with regard to high molecular weight and cold lability. Instead, a cold-stable and relatively low-molecular-weight ATPase was obtained. The isolation and properties of this enzyme will be discussed in this paper. MATERIALS AND METHODS Plant Material
Etiolated coleoptiles from winter wheat (Triticum aestivum L.) were obtained by layering seeds on moist paper towelling in plastic containers and germinating them in the dark for 3 days at 27 "C. Seeds were previously surface-sterilized by submersion in 1 % sodium hypochlorite for 15 min and rinsed thoroughly with distilled water. Abbreviations. AMP-(CHz)P, adenosine j'-(a,P-methylene)diphosphate; AMP-P(CHz)P, adenosine 5'-(fl,y-methylene)triphosphate. Enzymes. ATPase or adenosinetriphosphatase (EC 3.6.1.3) ; adenylate kinase (EC 2.7.4.3); j'-nucleotidase (EC 3.1.3.5); pyruvate kinase (EC 2.7.2.40); hexokinase (EC 2.7.1.1); glucose-6-phosphate dehydrogenase (EC 1.1.1.49); aldolase (EC 4.1.2.13); catalase (EC 1.11.1.6).
Preparation and Purification of Mitochondria
Mitochondria were prepared by the method described by Sarkissian and Srivastava [3], all steps being performed at 4 "C. When larger amounts of mitochondria were to be isolated for solubilization of ATPase, the method was slightly modified: 150250 g of fresh seedlings were homogenized for 15 s in a Waring blender with 2 1 of a solution containing 0.5 M sucrose, 50 mM Tris-chloride, pH 7.2, 2 mM EDTA and 0.75 mg albumin/ml. The homogenate was strained through a nylon fabric (mesh % 50 pm) and centrifuged in a Sorvall GS3 rotor 3 min at 3500 x g. Recentrifugation of the supernatant readjusted to pH 7.2 for 10 min at 13000 x g resulted in a bilayered sediment consisting of mitochondria on top of a small starch pellet. The mitochondria were carefully rinsed off with 11 of 0.5 M sucrose, 50 niM Tris-chloride, pH 7.2, without disturbing the starch layer. This suspension was recentrifuged for 10 min at 13000 x g . The resulting mitochondrial pellets were suspended in 0.5 M sucrose, 10 mM Trischloride, pH 7.2, 2 mM EDTA to give a protein concentration of 30 mg/ml. Portions of the suspension (0.8 ml) were layered separately on linear 20 - 70 "/, sucrose density gradients (37.5 ml) containing 10 mM Tris-chloride and 2 mM EDTA, pH 7.2. Upon centrifugation in a Beckman SW27 rotor for 2 h at 13000 x g the mitochondria formed a distinct band, which was removed by means of a syringe fitted with an L-shaped needle. The collected mitochondrial bands were diluted 5-fold and centrifuged for 20 min at 78000 x g in a Spinco 30 rotor. The sedimented mitochondria were suspended in 0.1 M sucrose, 50 mM Tris-chloride,
14
pH 7.2, at a protein concentration of 15 mg/ml and stored at - 20 "C. Purification of ATPase Preparation of Submitochondrial Particles. Electron transport particles were prepared from wheat seedling mitochondria according to the method of Senior and Brooks [4]. The frozen mitochondrial suspension was thawed and diluted 6-fold with 0.1 M sucrose, 10 mM Tris-chloride, pH 7.2. Fractions (50 ml) were placed in glass beakers submersed in an ice bath and sonicated at full power in a Branson €312 sonicator three times for 1 min, with intermissions of 1 min. The suspension was centrifuged at 105000 x g for 90 min in a Spinco 40 rotor. Sedimented submitochondrial particles were suspended in a solution of 0.1 M sucrose, 50 mM Tris-chloride, pH 7.2, at a protein concentration of 10 mgjml and stored at - 20 "C. The purity of the electron transport particles was checked by ultracentrifugation in a sucrose gradient (30 - 60 %>. All fractions were tested for ATPase and cytochrome oxidase activities. The peaks of these two enzymes were found to coincide, thus excluding the possibility of a significant contamination of the electron transport particles by other membrane or vesicle fractions. Release oj the Enzyme jrom Electron Transport Particles by Sonication. The frozen suspension of electron transport particles was thawed out, diluted 10-fold with 0.1 M sucrose, 50 mM Tris-chloride, pH 7.2, and supplemented with ammonium sulphate and Triplex 1V (cyclohexane-l,2-diaminetetraacetic acid) to give final concentrations of 0.25 M and 4 mM, respectively. Portions (50 ml) of this suspension (1 mg protein/ml) were immersed in an ice bath and sonicated for 8 min at full power. During this period the temperature of the sample rose from 4 "C to 30- 35 "C and thereupon was allowed to increase further up to 50 "C by sonicating without cooling. Denatured protein was removed by centrifuging 10 min at 38000 x g. The supernatant contained approximately 75 % of the ATPase activity of the electron transport particles. At least 70 of the ATPase activity of the supernatant could not be sedimented even upon ultracentrifugation at 250000 x g for 11 h. As a rule, all subsequent purification steps were performed at 4 "C. Ammonium Sulphate Fractionation. Solid amnionium sulphate was added slowly to the stirred solution and the pH maintained at 7.2 until 35% saturation was reached. After 10 min the precipitate was removed by centrifugation (10 min at 35000 xg). Such a precipitate did not form when this solution had been subjected to prolonged ultracentrifugation and the resulting sediment was removed prior to addition of ammonium sulphate. The supernatant was adjusted to 70% saturation of ammonium sulphate in the
ATPase from Wheat Seedling Mitochondria
same way and recentrifuged after 10 min. ATPase was found in the pellet and could be stored in a 75 % ammonium sulphate suspension or in aqueous solution at 4 "C with no detectable decrease of activity. Purification on Sephadex G-100. Aliquots of the 70 % ammonium sulphate precipitate containing about 10 mg protein were dissolved in 1 ml 0.25 M Trischloride, 0.25 M ammonium sulphate, 4 mM Titriplex IV, pH 7.2, and layered on a Sephadex G-100 column (1.7 x 94 cm) equilibrated with the same buffer. The elution was carried out at a flow rate of 50 ml/h. 2-ml fractions were collected in a LKB fraction collector and the absorbance at 280 nm was recorded by a LKB Uvicord spectrophotometer. Fractions 54- 60 were combined and the enzyme was salted out with 70 % ammonium sulphate saturation or concentrated by ultrafiltration in an Ainicon cell (50 nil) equipped with a UM2 filter. In order to achieve an optimum quality of the enzyme preparation, the gel filtration step was preferably repeated once. DEAE-Sephadex Chromatography. DEAE-Sephadex (A 50) was converted to the sulphate form and equilibrated with 40 mM Tris-sulphate, pH 7.2. The enzyme purified on Sephadex G-100 was dissolved in 0.5 ml 40 mM Tris-sulphate buffer, pH 7.2, layered on the column (2.1 x 27 cm) and eluted with 150 ml of the same buffer. Fractions (1 ml) were collected with simultaneous recording of the absorbance at 280 nm. Fractions 67 - 75 contained ATPase. Analytical Procedures ATPase Assay. The release of Pi from ATP was measured at 30 "C. The reaction was started by adding 20 pl of a solution of 6 pmol ATP in 0.05 M Trischloride, pH 7.2, to 1.0 ml incubation mixture containing enzyme, 50 pmol Tris-chloride, pH 7.2, and 4 pmol MgS04. When an ATP-regenerating system was to be included, the mixture was supplemented by 5 kmol phosphoenolpyruvate and 50 pg pyruvate kinase. When ATPase activity of mitochondria was determined, the incubation mixture contained from 0.1 M to 0.5 M sucrose in addition to 50 mM Trischloride, pH 7.2, and 4 mM MgS04, the ATP-regenerating system was included and the organelles were suspended at a protein concentration of 0.24 mg protein/ml. Oligomycin, rutamycin and N,N'-dicyclohexylcarbodiimide, when tested for their inhibitory action, were dissolved in ethanol and added to the mitochondrial preparations in portions of 10 pl. After 10-min incubation 1.0 ml 10 % trichloroacetic acid was added to stop the reaction and denatured protein was removed by centrifugation. Inorganic phosphate was estimated in 1.5-ml aliquots of the supernatant by the method of Fiske and SubbaRow [ 5 ] . The enzymic release of Pi from ADP, AMP and other nucleoside phosphates was measured using the
15
H. Tuppy and G. Sperk
same assay, 6 pmol of the respective nucleoside phosphate being added instead of ATP. Adenylute kinase was assayed using the spectrophotometric procedure of Sottocasa et al. [6]. Reaction mixtures (1.0 ml) contained 7.5 pmol ADP, 20 pmol glucose, 3 units hexokinase, 3 units glucose6-phosphate dehydrogenase, 20 pmol NADP', 2 pmol MgS04 and 100 pmol Tris-chloride, pH 8.0. The reaction was started by adding 50 1.11 of enzyme solution and followed on a Gilford spectrophotometer 240 equipped with an automatic recorder at 340 nm. In experiments assessing the effect of AMP-(CHz)P and AMP-P(CH2)P on adenylate kinase activity 7.5 pmol of the nucleotide analogue were added to the reaction mixture. Controls were carried out concerning a possible influence of these analogues on hexokinase and glucose-6-phosphate dehydrogenase (3 units each as well as 0.03 unit each), the same reaction mixture being used except adenylate kinase was omitted and ADP was replaced by 7.5 pmol of ATP. Cytochrome oxiduse was determined as described by Wharton and Tzagoloff [7]. Oxygen consumption of mitochondria and electron transport particles was recorded polarographically as described previously [8]. Protein was determined according to Lowry et al. [9]. Dodecylsukhate-polyucrylurnide gel electrophoresis was performed as described by Weber and Osborn [lo]. Materials
Seeds of winter wheat (Triticum aestivum L., line E.xtrem) were kindly donated by Prof. H. Hiinsel (Saatzuchtanstalt Probstdorf, Austria). Hydroxyapatite was prepared freshly by the method of Bernardi [ll]. Oligomycin was obtained from Sigma and rutamycin was a gift of Eli Lilly Comp. Adenosine 5'-(a,Bmethylene)diphosphate, AMP-(CHz)P, and adenosine 5'-(fl,y-tnethylene)triphosphate,AMP-P(CH2)P, were obtained from Miles Laboratories, Inc.
RESULTS Mitochondria prepared from wheat seedlings by the method of Sarkissian and Srivastava 131 showed respiratory control values of about 4 and ADP : 0 2 ratios of 3.7 with 2-oxoglutarate as substrate. Oligomycin did not impair their respiration in 'state 2' (i.e., in the presence of substrate and absence of ADP, cc Chance and Williams [12]), whereas the respiratory stimulation by ADP was totally inhibited by this antibiotic. The ATPase activity of these carefully prepared mitochondria was very low (0.08 pmol Pi
min-' mg protein-'). Activation of the enzyme could be achieved by subjecting the mitochondria either to lowered sucrose concentrations, or to freezing and thawing or to sonication. Specific activities of ATPase up to 1.2 pmol Pi min-' mg protein-' were found in mitochondria previously frozen and sonicated and subsequently assayed in a buffer lacking sucrose. The ATPase of native as well as that of damaged mitochondria was hardly alTected by oligomycin, rutamycin or N,N'-dicyclohexylcarbodiimide. Under conditions where the ATPase activity of beef heart mitochrondria, previously frozen and suspended in 0.1 M sucrose, was shown to be inhibited at least 901%;by oligomycin and rutamycin (20 pg/mg protein) and by N,N'-dicyclohexylcarbodiimide (10 pmol/mg protein), these agents reduced the ATPase activity of similarly treated wheat seedling mitochondria by less than 10 %. An increase in inhibitor concentration by a factor of 10 did not result in a significant increase of this inhibition. Soluhilization and Purification of Wheat Seedling ATPase
Several methods previously described for the solubilization of ATPase from animal and plant mitochondria were tested on wheat seedling mitochondria. These included shaking of the mitochondrial suspension with glass beads in a Merkenschlager homogenizer [13], extraction of phospholipids with acetone [14] and separation of ATPase from mitochondrial membrane fractions by incubation with ammonium sulphate and deoxycholate [15]. None of these procedures, however, yielded a cold-labile, high-molecularweight ATPase. In contrast, from wheat mitochondria a lower-molecular-weight and cold-stable ATPase could be isolated and purified by a procedure involving sonication in the presence of ammonium sulphate and an Mg2+-chelatingagent, precipitation by ammonium sulphate and chromatography on Sephadex G-100 and DEAE-Sephadex (A50) (Table 1). This kind of enzyme, rather than a cold-labile ATPase, was obtained irrespective of whether the isolation procedure was carried out at room temperature or at 4 "C. Gel filtration on Sephadex G-100 (Fig. 1) resulted in the separation of the ATPase from the bulk of other proteins including most of the adenylate kinase activity. 5'-Nucleotidase, which partly overlapped ATPase in the Sephadex G-100 chromatography, was subsequently removed completely by ion-exchange chromatography on DEAE-Sephadex (A50) (Fig. 2). The ATPase preparation resulting from chromatography on Sephadex G-100 and DEAE-Sephadex exhibited a residual adenylate kinase activity not exceeding 0.25 pmol of ATP formed min- ' mg protein-'. In the course of both purification steps ATPase and ADPase activities remained associated with each
16
ATPase from Wheat Seedling Mitochondria
Fraction number
Fig. 1. PuriJicalion of crude ATPasr by gelji//i,u//o/ioil Si;r,hadev G-100. Protein concentration (------)was determined by absorbance at 280 m i . ATPase (.---a) and 5'-nucleotidase (O-----O) activities have been expressed as rate of Pi release. There was a strict correlation between the capacities of effluent fractions to cleave ATP and ADP. The rate of ATP formation from ADP by adenylate kiiiase (A----A) was assayed according to the procedure of Sottocasa et al. [6] 8 .O
-
I-
c ._
E
E, %
0 c
x
m
8 m.
2 3
R
.s.
d4
2
,
j.
-... '1'..
.. .
.
...-,""... E . . . l .
Table 1. Pur(fication of ATPase fiorn wheat seedling mirochondria ATPase and nucleoside phosphate cleaving activities were assayed as described under Materials and Methods (10 min, 30 "C, in the presence of 6 pmol of substrate) Purification step
Protein ATPase Specific activity as P , yield released from AMP
Mitochondria Electron transport
ATP
mg
"/,
pmol min- mg protein-'
350
100
0.1
0.4
0.9
80 60
0.6 0.9
1.1 1.9
2.1 3.8
38
1.0
2.1
4.4
9
6.6
particles 120 Sonication supernatant 50 Ammonium sulphate fractionation 27.2 Sephadex G-100 chromatography DEAE-Sephadex chromatography
ADP
1.1
0.04
4
0
13 150
*' 3,0
'%._._
w
other. When chromatography on a hydroxyapatite column (1.3 x 48 cm) equilibrated with 0.05 M potassium borate buffer and elution with a linear gradient of potassium phosphate buffer (0-0.5 M, pH 7.2, 600 ml) was used instead of the DEAE-Sephadex (A50) chromatography, ATPase and ADPase did not separate, both activities emerging from the column at a potassium phosphate concentration of 0.89 M.
Characterization of ATPase
Gel filtration on a calibrated Sephadex G-100 column suggested that the ATPase from wheat mitochondria had a molecular weight of about 43000, the enzyme emerging from the column at an elution volume close to that of ovalbumin (Fig. 3). When the enzyme preparation was subjected to 7 % dodecylsulphate-polyacrylamide gel electrophoresis, two
H. Tuppy and G . Sperk
17 Table 2. Cleavage of various nucleoside diphosphaies and triphosphotrs h.y ~vlzeatmitochondria1 A TPase The purification of the enzyme has been described under Results and the assay of its activity under Materials and Methods
l 9o t
1 .o
2 .o
1.5
Substrate
Relative rate of P , release
ATP GTP CTP UTP ITP dGTP dCTP ADP GDP
1.0 1.2 2.0 1.7 0.4 0.3 0.5 0.5 0.5
2.5
ve 1 vo
Fig. 3. Determination of molecular weight o f A TPase by gelfiltration on Sephadev G-100. The column (1.7 x 54 cm) was equilibrated with 0.25 M Tris-sulphate, 0.25 M ammonium sulphate, pH 7.2. The reference proteins used were bovine serum albumin (molecular weight 67000). ovalbumin (45000), soybean trypsin inhibitor (21 500) and cytochrome c (12400). Abscissa: Elution volume of protein ( V , ) dividcd by the void volume of the column (V") as determined with the help of blue dextran
Table 3. Effect of nuckoside phosphate analogues on ATPuse and A D Past, ac I ivit its ATPase and ADPase activities of purified enzyme (6 pg protein) were determined as described under Materials and Methods, with the exception that 3 pmol of substrate (ATP o r ADP) were used instead of 6pmol. From AMP-(CHz)P and AMP-P(CH2)P no phosphate was released by the enzyme. n.d. = not determined ~
Added analogue
--
2.0
ATP
1
10
0
ADP
pmol/min None AMP-(CHz)P ( 3 p11101) AMP-(CHz)P (6 p11101) AMP-P(CH2)P (3 pmoi) AMP-P(CH1)P (6 pmol)
[EDTA] (rnM)
~
P, released from
10
20
30
40
50
[MgS041 (mM)
Fig. 4. DrJpundence qf ATPUSKactivity on M g z + concentration. ATPase purified on Sephadex G-100 was assayed in the presence of an ATP-regenerating system. The original enzyme solution was either supplemented with MgZt by addition of a small volume of MgS04 solution (0.1 -3.2 M) or, alternatively, EDTA (10 pM, p H 7.2) was added to give the final concentrations indicated on the abscissa
strong protein bands appeared. From their mobility relative to markers such as catalase and fumarase the molecular weight of the faster-moving protein was inferred to be about 40000, thus corresponding to the molecular weight of ATPase as found by gel filtration. The slower-moving protein having an apparent molecular weight of about 60000 is a contaminant which runs ahead but partially overlaps ATPase in Sephadex
0.18 0.20 n.d. 0.19 n.d.
0.09 0.16 0.22 0.10 0.10
G-100 chromatography. The ATPase activity of the enzyme preparation can be augmented by Mg2+ ions and is entirely abolished by EDTA in concentrations above 2 mM (Fig. 4). Dinitrophenol failed to activate the enzyme. Nucleoside triphosphates other than ATP, deoxynucleoside triphosphates and nucleoside diphosphates are cleaved by the purified enzyme at appreciable rates (Table 2). Neither AMP nor pyrophosphate were split. The ADPase activity was strongly increased by the ADP analogue, AMP-(CH2)P (Table 31, whereas the corresponding ATP analogue, AMP-P(CH2)P, did not have a comparable effect. The ATPase activity was not affected significantly by either analogue. By contrast, adenylate kinase present in wheat mitochondrial extracts and largely separated from ATPase by chromatography on Sephadex G-100 was inhibited 50% by AMP-(CHz)P, as well as by AMP-P(CH2)P when the analogues were used in concentrations equal to that of the substrate, ADP.
18
DISCUSSION The preparation of an ATPase from mitochondria of wheat seedlings as described in this paper combines the purification steps recommended by Senior and Brooks [4] including ultrasonication and heat treatment of electron transport particles with the dissociation of mitochondrial proteins by ammonium sulphate reported by Van de Stadt et al. [15]. When this preparative procedure was applied to mitochondria of beef heart as well as of several dicotylous plants such as castor bean and cauliflower [16] high-molecular-weight ATPases of the well-known type were obtained. By contrast, the ATPase prepared in this way from wheat mitochondria had markedly different properties. In the course of the preparation and purification of the wheat mitochondrial ATPase the ratio of the hydrolysing activities towards ATP and ADP did not change. This finding not only points to an identity of the ATP and ADP cleaving enzyme, but also to an identity of the original enzyme bound to wheat mitochondria with the solubilized and partially purified ATPase. Although adenylate kinase, large quantities of which were present in crude mitochondrial extracts, could be removed to a great extent in the course of purification, the final preparation of ATPase was still contaminated by a small amount of that enzyme. The activity of wheat mitochondrial adenylate kinase was inhibited markedly by the ADP and ATP analogues, AMP-(CH2)P and AMP-P(CH2)I’. These analogues, however, did not impair the hydrolysis of ADP by the ATPase preparation. This finding excludes the possibility of the ADP cleavage being due to conversion of ADP to AMP and ATP by adenylate kinase and subsequent hydrolysis of ATP by ATPase. On the contrary, AMP-(CH2)P significantly increased ADP hydrolysis by wheat seedling ATPase whereas the rate of ATP hydrolysis was hardly affected. The distinct behaviour of AMP-(CH2)P with respect to ADP and ATP hydrolysis may possibly be accounted for by separate binding sites of the ATPase for the two substances, ADP and ATP. Multiple nucleotide binding sites have previously been suggested by Hilborn and Hammes for beef heart mitochondrial ATPase [17]. Some further properties of the ATPase isolated from wheat seedling mitochondria are also significantly different from those of mitochondrial ATPases derived from other sources. Its molecular weight estimated by gel filtration was found to be approximately 43 000 as compared with molecular weights of about 360000 for beef heart, rat liver and yeast ATPases [l]. The relative mobility of wheat seedling ATPase on dodecylsulphate gel electrophoresis also pointed to a molecular weight around 40000, thus suggesting that this enzyme, in contrast with other
ATPase from Wheat Seedling Mitochondria
mitochondrial ATPases, failed to be dissociated by dodecylsulphate into subunits. In further contrast to such enzymes, the ATPase from wheat mitochondria proved itself stable to cold and was not affected by dinitrophenol. In view of these marked differences, the question arises whether this wheat ATPase might be comparable to the low-molecular-weight ATPases found by Le Deaut et a/. 1181 in the matrix of rat liver mitochondria rather than to those in the mitochondrial membrane involved in oxidative phosphorylation. In this paper, however, evidence has been presented that the wheat enzyme is membrane-bound: after freezing, thawing and mild sonication it was still associated with the electron transport particles, and drastic sonication in the presence of ammonium sulphate was required to solubilize it. The lowmolecuhr-weight enzyme appeared to be the only ATPase which could be obtained from wheat mitochondria or electron transport particles. Although several methods for solubilizing ATPase, such as disruption of the mitochondria in a Merkenschlager homogenizer, delipidation by acetone and disintegration of membranes by ammonium sulphate and detergents were tried, a cold-labile and high-molecular-weight enzyme comparable to the established mitochondrial membrane ATPases could never be obtained from wheat. In the course of the purification of the low-molecular-weight ATPase a high-molecular-weight ATPase having the expected properties was not encountered, irrespective of whether fractionations were carried out at room temperature or in the cold. There are strong indications that even in intact wheat seedling mitochondria the properties of the ATPase are different from those of the ATPase of animal or yeast mitochondria. In accordance with results reported by Jung and Hanson for corn mitochondria [19] we found previously that the ATPase activity of intact wheat mitochondria and electron transport particles derived from them is not inhibited by oligomycin, in contrast to the sensitivity of the animal and yeast organelles to this antibiotic [20]. This finding points to a significant difference between the ATPase or the membrane structure incorporating ATPase in wheat mitochondria compared with other mitochondria. Future investigations may show whether the low-molecular-weight ATPase of wheat mitochondria might correspond to one subunit of the ATPase complex found in other mitochondria. The distinctive difference could be that the subunit size molecule from wheat would be active whereas the ATPase complexes from other sources would lose their enzymic function when they are decomposed into subunits. This investigation was supported by the Hochschuljuhiluumsst$iung der Gemeinde Wien and by the Fonds iur Fiirdertang der wissenschaftlichen Forschung (Project 1210).
H. Tuppy and G. Sperk
REFERENCES
Weber, K.&Osborn, M. (1969) J . B i d Chem 244,4406-4412. Bernardi, G. (1971) Methods Enzymol. 22, 325-339. Chance, B. & Williams, G. R. (1956) A h . Enzymol. 17,56- 134. Penefsky, H. S., Pullman, M. E., Datta, A. & Racker, E. (1960) 4694-4702. J . Biol. Chem. 235, 3330- 3336. Sarkissian, I. V. & Srivastava, H. K. (1969) Proc,. Nut1 A C L J ~ . 14. M. J. (1967) Biochrm. J . 10.5, 279-288. Selwyn, Sci. U.S.A. 63, 302- 309. 15. Van dc Stadt, R . J., Kraaipoel, R. J. & Van Darn, K . (1972) Senior, A. E. & Brooks, J. G. (2970) Awh. Biochrm. Eiophyx. Eiochim. Siophys. A t t u , 276, 25- 36. 140, 257 - 266. 16. Sperk, G. & Tuppy, H. (1976) Planl Ph.vsiol. in press. Fiske, C. H. & SubbaRow, Y . (1925) J . B i d . Chem. 66, 37517. Hilborn, D . A. & Hammes, G . G. (1973) Biochemistry. 12, 400. 983 990. Sottocasa, G. L., Kuylenstierna, B., Ernster, L. & Bergstrand, 18. Le Deaut, J. Y., Ledig, M., Mandel, P. & Marfing, M. (1972) A. (1967) M e t h o h Enzymol. 10, 448-463. FEBS Lett. 20, 153- 156. Wharton, D. C. & Tzagoloff, A. (1967) Methods Enzymol. 10, 19. Jung, D. W. & Hanson. J . B. (1973) Arch. Biocltem. Biophys. 245 250. 158, 139-148. Zobl, R., Fischbeck, G., Kcydel, F., Latzko, E. & Sperk, G. 20. Sperk, G. & Tuppy, €3. (1973) Hoppe-Seylcr’s Z. Phy.c.iol. (1972) Plant Physiol. 50, 790-791. Chem. 350, 1244- 1245. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J .
1. Senior, A . E. (1973) Biochim. Biophys. Acta, 301, 249-277. 2. Penefsky, H. S. & Warner,C. R. (1965) J . B i d . Chrm. 240,
3. 4.
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H. Tuppy and G. Sperk. lnstitut fur Biochemie der Universitiit Wien, WahringerstraBe 17, A-1090 Wien, Austria
