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The β-subunit of pea stem mitochondrial ATP synthase exhibits PPiase activity
Mitochondrion 3 (2003) 111–118 www.elsevier.com/locate/mito
The b-subunit of pea stem mitochondrial ATP synthase exhibits PPiase activity Marco Zancania, Valentino Casoloa, Carlo Peressona, Giorgio Federicib, Andrea Urbanib, Francesco Macrı`a, Angelo Vianelloa,* a
Sezione di Biologia Vegetale, Dipartimento di Biologia ed Economia Agro-Ind., Universita` di Udine, via Cotonificio 108, Udine 33100, Italy Laboratorio di Biochimica Clinica, Ospedale Pediatrico del Bambino Gesu`—IRCCS, Piazza S. Onofrio 4, Rome 00166, Vatican City State
b
Received 12 February 2003; received in revised form 8 July 2003; accepted 29 July 2003
Abstract A soluble protein with a molecular mass of 55 kDa has been purified from etiolated pea stem mitochondria. The protein exhibits a Mg2þ-requiring PPiase activity, with an optimum at pH 9.0, which is not stimulated by monovalent cations, but inhibited by F2, Ca2þ, aminomethylenediphosphate and imidodiphosphate. The protein does not cross-react with polyclonal antibodies raised against vacuolar, mitochondrial or soluble PPiases, respectively. Conversely, it cross-reacts with an antibody for the a/b-subunit of the ATP synthase from beef heart mitochondria. The purified protein has been analyzed by MALDI-TOF mass spectrometry and the results, covering the 30% of assigned sequence, indicate that it corresponds to the b-subunit of the ATP synthase of pea mitochondria. It is suggested that this enzymatic protein may perform a dual function as soluble PPiase or as subunit of the more complex ATP synthase. q 2003 Elsevier B.V. and Mitochondria Research Society. All rights reserved. Keywords: ATP synthase; b-Subunit; MALDI-TOF; Mitochondria; Pisum sativum; PPiase
1. Introduction
Abbreviations: BTP, 1,3-bis[tris(hydroxymethyl)methyl amino]propane; EDTA, ethylene-diamminetetraacetic acid; EGTA, ethylene glycol-bis(2-aminoethylether)-N,N,N0 ,N0 -tetra acetic acid; ELFE, electro-endosmotic preparative electrophoresis; MALDI, matrix-assisted laser desorption-ionization; PAGE, polyacrylamide gel electrophoresis; PSD, post-source decay; SDS, sodium dodecyl sulphate; TOF, time-of-flight. * Corresponding author. Tel.: þ39-432-558-781; fax: þ 39-432558-784. E-mail address: [email protected] (A. Vianello).
Inorganic pyrophosphatases (PPiases) (EC 3.6.1.1) are ubiquitous enzymes that catalyze the hydrolysis of pyrophosphate (diphosphate, PPi) to orthophosphate (Pi). The known PPiases can be subdivided into three main families: (1) tightly membrane-bound Hþpumping PPiases; (2) earlier known family of soluble PPiases; (3) recently found family of soluble PPiases (Baltscheffsky et al., 1999). Their presence in both prokaryotic and eukaryotic organisms raises the question whether PPi could be a direct ancestor of
1567-7249/$20.00 q 2003 Elsevier B.V. and Mitochondria Research Society. All rights reserved. doi:10.1016/S1567-7249(03)00105-3
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ATP, as an energy source, in the origin and evolution of life. In mammalian cells, PPi is considered a simple byproduct of a number of biosynthetic reactions (e.g. DNA, RNA synthesis, amino acid activation, activation of fatty acids, etc.), which has to be continuously removed by cytoplasmic soluble PPiases to drive forward these reactions (Kornberg, 1957). In higher plant cells, PPi is always produced by the same type of reactions, but the cytosol of these cells does not contain soluble PPiases, thus permitting an accumulation of a significant amount of PPi (0.2 – 0.3 mM) (Weiner et al., 1987). This cytosolic PPi can then act as an energy donor for at least three reactions, namely PPi-dependent proton pumping at the level of tonoplast (Maeshima, 2000; Rea et al., 1992), sucrose degradation via sucrose synthase, UDP-glucose phosphorylase and phosphofructokinase (Stitt, 1990; Kruger, 1997), and entry into glycolysis via the pyrophosphate fructose-6phosphate phosphotransferase (Stitt, 1990; Davies, 1997). These PPi-consuming reactions are ubiquitous in higher plants and operate in parallel with the corresponding ATP-dependent enzymes. Therefore, PPi is now considered an energetic alternative to ATP in plant cells (Stitt, 1998). This situation could be related to the sessile nature of plants and, consequently, to the need of having a more flexible metabolism. Higher plant mitochondria have a matrix concentration of PPi (0.2 mM) which appears to be in equilibrium with that of the cytoplasm by an exchange mediated by the adenine nucleotide translocase (Casolo et al., 2002). The mitochondrial PPi can be produced/utilized by a Hþ-PPiase, which seems to be loosely bound to the inner surface of the inner membrane (Vianello and Macrı`, 1999). This enzyme has a Hþ/PPi stoichiometry of two (Zancani et al., 1998) and a catalytic subunit of 35 kDa, which is probably part of a more complex multimeric enzyme (Zancani et al., 1995). On the basis of radiation inactivation analysis, the functional masses of enzymatic and proton translocation of mitochondrial Hþ-PPiase are 170 and 156 kDa, respectively (Jiang et al., 2000). In the light of the above considerations, it appears clear that higher plant mitochondria have an appreciable PPi metabolism. In this framework, we have now
identified a soluble (matrix) PPiase, which presents a high degree of homology with the b-subunit of the ATP synthase.
2. Materials and methods 2.1. Isolation of purified mitochondria and matrix proteins Pea stem Percoll-purified mitochondria were obtained from crude extracts prepared as described by Casolo et al. (2002), except for the absence of bovine serum albumin in all the preparation steps. Crude mitochondria were loaded on a Percoll discontinuous gradient (40 –21 –13% w/v) and centrifuged in a swinging-bucket rotor at 20,000 £ g for 40 min. The purified mitochondria were carefully collected at the 40 –21% interface. To remove Percoll, the purified mitochondria were washed in 250 ml of 20 mM 3-[N-morpholino]propanesulfonic acid – KOH (pH 7.2), 0.3 M mannitol, 1 mM EDTA and centrifuged three times at 21,000 £ g for 10 min. The final pellet was resuspended in 10 mM BTP – HCl (pH 7.0), 0.25 M sucrose. Matrix proteins, in the supernatant, were obtained from purified mitochondria after three ultrasonic irradiations for 30 s (100 W) and separation of the membrane fraction by ultracentrifugation (100,000 £ g for 40 min). 2.2. Ion exchange chromatography Matrix proteins (ca. 6 mg protein) were loaded on a Mono Q column equilibrated with 50 mM BTP – HCl (pH 7.0). Adsorbed proteins were eluted by a continuous gradient (0 – 300 mM KCl). Fractions showing PPiase activity were pooled and dialyzed overnight against 2 mM BTP – HCl (pH 6.5) and then lyophilized. 2.3. Non-denaturing preparative gel electrophoresis (ELFE) Electro-endosmotic preparative electrophoresis was performed with an ELFE apparatus (Zancani et al., 1995), using a non-denaturing polyacrylamide gel (10% w/v). Lyophilized fractions from ion exchange chromatography (600 – 800 mg protein)
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were resuspended in 7.5% (w/v) glycerol and 0.0025% (w/v) bromophenol blue (1 ml final volume) and loaded on the gel at 4 8C. The current was set at 10 mA and, after the elution of the bromophenol blue peak, the fractions were collected every 20 min (corresponding to ca. 3 ml). Fractions showing PPiase activity were pooled and used either to perform further enzymatic activities or dialyzed against 2 mM BTP – HCl (pH 6.5) and then lyophilized.
inorganic phosphate as a standard. In the experiments on the effect of the antibody raised against the a/b-subunit of ATP synthase on PPiase activity, the fractions pooled from ELFE (20 ml) were preincubated with the antibody (1 ml) for 30 min at 30 8C. The pyrophosphatase activity was detected in a final volume of 400 ml as described above.
2.4. SDS-PAGE and Western blotting
Characterization of polypeptides from polyacrylamide gel by MALDI-TOF mass spectrometry was performed on trypsin digested peptides. In short, proteins bands were excised from SDS-PAGE, cysteines were reduced and alkylated with iodoacetammide (Shevchenko and Shevchenko, 2001). The samples were then digested with porcine trypsin (Promega) in 40 mM ammonium bicarbonate at 37 8C for 6 – 8 h. The reaction was stopped by freezing. Tryptic peptides were extracted by ZipTip C18 (Millipore) reverse phase material, directly eluted and crystallized in a 50% (v/v) acetonitrile/ water saturated solution of a-cyano-4-hydroxycinnamic acid. MALDI mass spectra were recorded in the positive ion mode with delayed extraction on a Reflex IV timeof-flight instrument equipped with a MTP multiprobe inlet and a 337 nm nitrogen laser. Mass spectra were obtained by averaging 50– 200 individual laser shots. Calibration of the spectra was internally performed by a two-point linear fit using the autolysis products of trypsin at m=z 842.50 and 2211.10. A database search with the peptide masses was performed against the NCBInr database using the peptide search algorithm MASCOT (Matrix Science). Fragments generated by post-source decay (PSD) experiments were analyzed using the database search algorithm MS-Tag (http://prospector.ucsf.edu/ ucsfhtml3.2/mstag.htm).
Protein fractions from purified mitochondria (10 mg), matrix proteins (10 mg), ion exchange chromatography peak (ca. 5 mg) and the lyophilized peak eluted from ELFE (ca. 1 mg) were resuspended in 50 mM Tris –HCl (pH 6.8), 7.5% (w/v) glycerol, 2% (w/v) SDS and 0.0025% (w/v) bromophenol blue in a final volume of 50 ml. After incubation at 95 8C for 3 min, the samples were loaded on 12% (w/v) SDS-PAGE. The gels were initially stained with Coomassie Brilliant Blue R-250 and then with silver nitrate. Western blotting was performed by electroeluting the protein from the gel to a nitrocellulose membrane. Immunodetection was performed using rabbit polyclonal antibodies raised against mitochondrial (Vianello et al., 1997), vacuolar (Maeshima and Yoshida, 1989), soluble (du Jardin et al., 1995) PPiases or the a/b-subunit of the ATP synthase from mammalian mitochondria (Tomasetig et al., 2002). Immunochemical reactions were detected by the staining obtained using alkaline phosphatase-conjugate secondary antibody. 2.5. Enzymatic assays Pyrophosphatase activity was performed at 25 8C in a final volume of 400 ml of 20 mM Hepes –Tris (pH 7.5), 0.25 M sucrose, 50 mM KCl, 1.5 mM MgSO4 and 100 mM EGTA. The reactions were started by the addition of 200 mM PPi and were stopped after either 30 min (fractions pooled after ion exchange chromatography) or 1 h (fractions pooled from ELFE) by the addition of Fiske-Subarrow reagent (Cross et al., 1978). The absorbance values at 740 nm were converted to PPi hydrolyzed as half Pi released, calculated by a calibration curve using
2.6. MALDI-TOF mass spectrometry analysis
2.7. Protein determination Protein determinations were performed by the method of Bradford (1976). Absorbance values obtained at 595 nm were referred to a calibration curve obtained with bovine serum albumin as a standard.
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2.8. Data presentation Profiles of PPiase activity from proteins purified by chromatography and ELFE are reported as typical curves. Values in tables are means of at least three independent experiments ^ standard deviations.
3. Results 3.1. Purification of mitochondrial PPiase Fig. 1 shows the elution profile of PPiase activity after purification of mitochondrial matrix proteins by ion exchange chromatography (Mono Q). This PPiase activity was evident as a single peak ranging from 40 –42 to 58– 60 fractions. The 42– 43 to 52 – 53 fractions were then pooled. Their proteins were resolved by SDS-PAGE and coloured by silver stain (inset). The patterns reveal that the proteins still present in this pool were strongly diminished (Chr lane), when compared with total mitochondrial proteins (MtP) or mitochondrial matrix proteins (MP). The same sample of proteins (fractions from 42 –43 to 52– 53) was, therefore, further purified by non-denaturing ELFE (Fig. 2). Even in this case, PPiase activity eluted as a single sharp peak. The analysis of the proteins (from 38 to 40 fractions) by SDS-PAGE (always coloured by silver stain) showed a single protein band with a molecular mass
Fig. 1. Elution profile of PPiase activity after ion exchange (Mono Q) chromatography of mitochondrial matrix proteins. Inset shows SDS-PAGE of mitochondrial proteins (MtP), matrix proteins (MP) and chromatography-purified proteins (Chr). On the left, standard molecular mass markers are shown.
of ca. 55 kDa (inset, lane a). These pooled fractions were, hence, utilized for the biochemical and immunochemical characterization. The purification steps of PPiase can also be followed as increase of specific activity (Table 1). The crude extract (matrix proteins) exhibited a PPiase activity which increased 2.8-fold after ion exchange chromatography and 27-fold after nondenaturing ELFE. 3.2. Biochemical characterization of PPiase activity The only substrate utilized by the purified protein was PPi, because a nucleotide diphosphate (ADP) or triphosphates (ATP, CTP, GTP and UTP) induced only a negligible phosphohydrolytic activity (Table 2). Considering that soluble F1 from beef heart ATP synthase, depleted of adenine nucleotides, can hydrolyze PPi to values of ca. 0.2 nmol min21 mg21 protein (Tuena de Go´mez-Puyou et al., 1993), the PPiase activity of the purified protein was very high. This
Fig. 2. Elution profile of PPiase activity of chromatography-purified proteins (fractions pooled from chromatography from 42–43 to 52– 53) by ELFE. Inset shows: (a) SDS-PAGE of purified protein corresponding to the peak of activity; (b) Western blotting of the same protein that cross-reacted with polyclonal antibody raised against the a/b-subunit of ATP synthase from beef heart mitochondria. On the left, standard molecular mass markers are shown. Arrow indicates the purified protein at 55 kDa.
M. Zancani et al. / Mitochondrion 3 (2003) 111–118 Table 1 Purification steps of pea stem mitochondrial PPiase Step
Specific activity [nmol PPi (min £ mg protein)21]
Crude extract (matrix proteins) Chromatography (Mono Q)a ELFEa
189 ^ 30 529 ^ 45 5191 ^ 284
a
The specific activity increased 2.8- and 27-fold after chromatography (Mono Q) or ELFE, respectively.
PPiase had an absolute requirement for Mg2þ, while it was completely insensitive to monovalent cations (Table 3). Similarly, some divalent cations unaffected this activity, except for Mn2þ that caused a 40% inhibition. The activity showed a pH dependence with a maximum in the range between 8.0 and 9.5 (Fig. 3). The enzyme was inhibited by imidodiphosphate (ca. 70%) and aminomethylenediphosphate (ca. 45%), while other diphosphonates had minor effects (Table 4). In addition, PPiase activity was inhibited by both F2 and Ca2þ, which caused, at 100 mM concentration, ca. 70 –80% inhibition (Fig. 4). 3.3. Immunochemical characterization of PPiase The purified protein, exhibiting PPiase activity, did not cross-react with polyclonal antibodies raised against vacuolar, mitochondrial or soluble PPiases, respectively. Conversely, this protein cross-reacted with an antibody for the a/b-subunit of ATP synthase from beef heart mitochondria (Fig. 2, inset, lane b). In agreement, this antibody inhibited of ca. 40% PPiase activity (5.23 and 3.14 mmol PPi min21 mg21 protein in control and antibody-treated
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Table 3 Effect of monovalent and divalent cations on PPiase activity from purified fractions Cation
mmol PPi (min £ mg protein)21
None (control) Kþ Rbþ Csþ Liþ Naþ
5.19 ^ 0.28 5.25 ^ 0.29 5.13 ^ 0.28 5.12 ^ 0.28 4.39 ^ 0.24 4.82 ^ 0.26
Co2þ Cu2þ Mn2þ Zn2þ
5.02 ^ 0.27 5.41 ^ 0.30 2.98 ^ 0.16 4.79 ^ 0.26
The purified protein utilized in these assays was obtained by pooling the fractions from 38 to 40 after elution by ELFE. Final concentration of monovalent or divalent cations was 20 mM and 100 mM, respectively. No PPiase activity was detected in the absence of Mg2þ; none of the above divalent cations were able to substitute for Mg2þ.
samples, respectively). Nevertheless, the purified protein was subjected to mass spectrometry to further confirm this result. 3.4. Mass spectrometry characterization of mitochondrial PPiase The monoisotopic mass list recorded from the tryptic peptides of the 55 kDa band from the Coomassie stained SDS-PAGE fitted a gene
Table 2 Phosphohydrolytic activity of pea stem mitochondrial purified protein Substrate
mmol hydrolyzed (min £ mg protein)21
200 mM PPi 1 mM ATP 1 mM ADP 1 mM CTP 1 mM GTP 1 mM UTP
5.20 ^ 0.28 0.07 ^ 0.02 0.26 ^ 0.03 0.18 ^ 0.02 0.07 ^ 0.01 0.22 ^ 0.03
The purified protein utilized in these assays was obtained by pooling the fractions from 38 to 40 after elution by ELFE.
Fig. 3. PPiase activity as function of pH of the incubation medium. Mean values and standard deviations are calculated from three independent experiments.
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Table 4 Effect of diphosphonates on PPiase activity from purified fractions Diphosphonate
mmol PPi (min £ mg protein)21
Control Aminomethylenediphosphate Aminobuthylene-1-hydroxy-1,1-diphosphate Imidodiphosphate Aminohexane-1-hydroxy-1,1-diphosphate Dichloromethylenediphosphate Ethane-1-hydroxy-1,1-diphosphate
5.18 ^ 0.28 2.87 ^ 0.16 5.01 ^ 0.27 1.26 ^ 0.69 4.31 ^ 0.24 4.85 ^ 0.27 4.49 ^ 0.25
The purified protein utilized in these assays was obtained by pooling the fractions from 38 to 40 after elution by ELFE. The final concentration of inhibitors was 1 mM, except for aminomethylenediphosphate (10 mM).
product encoding a probable b-subunit protein (gil7436097) of Hþ-transporting ATP synthase (EC 3.6.1.34) from P. sativum mitochondria. The mass finger print data had a confidence value greater than 95% ðP , 0:05Þ in identifying this specific polypeptide. This identification was further confirmed by PSD experiments on two relevant peptide species on the MALDI-TOF spectra. The ion species at 1173.70 amu (MHþ) was selected and returned a fragmentation pattern fitting the sequence VVDLLAPYQR. A second PSD experiment was performed on the ionic peptide species at 1149.66 amu (MHþ) giving a fragmentation profile fitting the sequence TEHFLPIHR: The overall mass spectrometry data cover about 30% of the assigned sequence (Table 5). An initial signal sequence peptide was predicted to localize the protein in the mitochondria (software prediction Mitoprot II). The theoretical molecular weight, 60.4 kDa, calculated from the database sequence, fits quite well with the value of 55 kDa estimated from the SDS-PAGE once the initial Nterminus 38 aminoacids of the signal peptide (Table 5) are removed from the calculation.
1999), sustains some PPi-requiring reactions related to tonoplast energization (Maeshima, 2000; Rea et al., 1992), sucrose degradation (Stitt, 1990; Kruger, 1997) and glycolysis (Stitt, 1990; Davies, 1997), acting at the same time as regulator of the pyrophosphate fructose-6-phosphate phosphotransferase (Sung et al., 1988). PPi is also compartmentalized in the mitochondrial matrix, which contains a comparable level of PPi (0.2 mM), and where it can accomplish both roles as energetic substrate or as regulator of metabolic pathways (Casolo et al., 2002). Indeed, PPi could be synthesized/hydrolyzed by a mitochondrial Hþ-PPiase, which seems to act near to the thermodynamic equilibrium (Zancani et al., 1998). This allows PPi to be synthesized by dissipating the electrochemical proton gradient, established across the inner membrane by substrate oxidation (Casolo et al., 2002). Conversely, PPi may be consumed to maintain this gradient, particularly under condition of oxygen shortage (Vianello and Macrı`, 1999), as already suggested for the tonoplast
4. Discussion In higher plant cells, PPi plays a crucial role either as energetic substrate or as regulator of metabolic pathways (Stitt, 1998; Plaxton, 1996). Its cytoplasmic concentration (0.2 – 0.3 mM) is regulated by some soluble PPiases (Rojas-Beltran et al.,
Fig. 4. Effect of increasing concentration of F2 (upper panel) and total Ca2þ (lower panel) on PPase activity.
M. Zancani et al. / Mitochondrion 3 (2003) 111–118 Table 5 Sequence coverage by trypsin digestion peptide mass fingerprint of P. sativum b-subunit of the mitochondrial ATP synthase 1 51 101 151 201 251 301 351 401 451 501 551
MASRRLVSSL IRSSLRRSSS KPSISASTSR LTSQSRASPC GYLLNRVAHY ATSAAAAALP PSPPPAKKEG PGGGKITDEF TGKGAIGHVC QVIGAVVDVR FEEGLPPILT SLGLLGSHET RFWTLEVAQF GRGRVRTIAM DATEGVVRGW RVLNTGSPIS VPVGRANPWA YHEVIGEPID EKGELKTEHF LPIHREGPSF VEQATEQEIL VTGIKVVDLL APYQRGGKIG LFGGAGVGKT VLIMELINNV AKAHGGFSVF AGVGERTREG NDLYREMIES GVIKLGDKQS ESKCALVYGQ MNGPPGARAR VGLTGLTVAE HFRDAEGQDV LLFVDNIFRF TQANSEVSRL LGRIPSAVGY QPTLATDLGG LQERITTPKK GSITPGQAIY VPADDLTDLA PATTFAHLDA TTVLSRTISE LGIYPVVDPL DSTSRMLSPL ILGEAHYETA RGVQKVLQNY KNLQGIIAIL GMDELSEDDK LTVARARKIQ RFLSQPFHVA EVFTGGPGKY VDLKENITSF QGVLDGKYDD LSEQSFYMVG GIEEVIAKAE KIAKESAASS S
The protein regions covered in the mass fingerprint are shown in bold. Peptides sequences confirmed by fragmentation analysis and by post-source decay (PSD) are underlined. The predicted mitochondrial leader sequence is shown in italics.
Hþ-PPiase (Macrı` et al., 1995; Carystinos et al., 1995). In addition, free PPi acts as a powerful competitive inhibitor of succinate dehydrogenase, whose inhibition is released by chelation of PPi with Mg2þ (Casolo et al., 1998). For these reasons, it is possible to speculate that also the matrix concentration of PPi may be maintained under control by specific soluble PPiases, distinct from the abovementioned Hþ-PPiases (Casolo et al., 2002). However, no other PPiase activities have been hitherto described in plant mitochondria. We now show that a matrix protein, with a molecular mass of 55 kDa, exhibits the ability to hydrolyze PPi. This PPiase activity is Mg2þ requiring, is not stimulated by monovalent cations and is strongly inhibited by F2, Ca2þ, aminomethylenediphosphate and imidodiphosphate, all typical PPiase inhibitors. Apart from the molecular mass, the other features are typical of soluble PPiases (Cooperman et al., 1992). The protein does not cross-react with antibodies raised
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against vacuolar Hþ-PPiase, soluble PPiase and mitochondrial Hþ-PPiase, respectively. The molecular mass of this PPiase is identical to that identified for a thylakoid PPiase (55 kDa), whose activity is also Mg 2þ requiring and is not stimulated by Kþ (Jiang et al., 1997). The primary structure of the soluble pea stem PPiase has a high degree of homology, as stated, with the pea mitochondrial b-subunit of ATP synthase. On the other hand, the antibody raised against the a/bsubunit of the ATP synthase inhibits PPiase activity. Hitherto, a PPiase activity has only been described for soluble F1 from beef heart (Tuena de Go´mez-Puyou et al., 1993). This complex is able to carry both the synthesis and hydrolysis of PPi. In particular, F1 possesses three PPi-binding sites, which comprise one high affinity binding site and two lower affinity sites (Issartel et al., 1987). It seems that PPi mimics ADP and, therefore, that they share the same binding site in F1. However, the PPiase activity exhibited by the pea stem b-subunit is higher than that recovered for F1 from beef heart (Tuena de Go´mez-Puyou et al., 1993). These results can be rationalized in two ways: first, the 55 kDa protein of pea mitochondria can be considered as a soluble PPiase, distinct from the cytoplasmic one; second, it may be simply considered as a b-subunit of the main ATP synthase, which performs a dual function as soluble PPiase or as subunit of the more complex ATP synthase. On the other hand, it has already been suggested that mitochondrial (and chloroplast?) PPiase could be soluble parts of Hþ pumps (Baltscheffsky et al., 1999).
Acknowledgements This research was supported by the University of Udine. Thanks are due to: Dr M. Maeshima (Nagoya University, Japan) for antibody against mung bean tonoplast PPiase; Dr P. du Jardin (University of Gembloux, Belgium) for antibody against soluble PPiase from potato; Prof. G. Lippe (University of Udine, Italy) and Prof. F. Zanotti (University of Bari, Italy) for the antibodies against the a/b-subunit of ATP synthase.
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The b-subunit of pea stem mitochondrial ATP synthase exhibits PPiase activity Marco Zancania, Valentino Casoloa, Carlo Peressona, Giorgio Federicib, Andrea Urbanib, Francesco Macrı`a, Angelo Vianelloa,* a
Sezione di Biologia Vegetale, Dipartimento di Biologia ed Economia Agro-Ind., Universita` di Udine, via Cotonificio 108, Udine 33100, Italy Laboratorio di Biochimica Clinica, Ospedale Pediatrico del Bambino Gesu`—IRCCS, Piazza S. Onofrio 4, Rome 00166, Vatican City State
b
Received 12 February 2003; received in revised form 8 July 2003; accepted 29 July 2003
Abstract A soluble protein with a molecular mass of 55 kDa has been purified from etiolated pea stem mitochondria. The protein exhibits a Mg2þ-requiring PPiase activity, with an optimum at pH 9.0, which is not stimulated by monovalent cations, but inhibited by F2, Ca2þ, aminomethylenediphosphate and imidodiphosphate. The protein does not cross-react with polyclonal antibodies raised against vacuolar, mitochondrial or soluble PPiases, respectively. Conversely, it cross-reacts with an antibody for the a/b-subunit of the ATP synthase from beef heart mitochondria. The purified protein has been analyzed by MALDI-TOF mass spectrometry and the results, covering the 30% of assigned sequence, indicate that it corresponds to the b-subunit of the ATP synthase of pea mitochondria. It is suggested that this enzymatic protein may perform a dual function as soluble PPiase or as subunit of the more complex ATP synthase. q 2003 Elsevier B.V. and Mitochondria Research Society. All rights reserved. Keywords: ATP synthase; b-Subunit; MALDI-TOF; Mitochondria; Pisum sativum; PPiase
1. Introduction
Abbreviations: BTP, 1,3-bis[tris(hydroxymethyl)methyl amino]propane; EDTA, ethylene-diamminetetraacetic acid; EGTA, ethylene glycol-bis(2-aminoethylether)-N,N,N0 ,N0 -tetra acetic acid; ELFE, electro-endosmotic preparative electrophoresis; MALDI, matrix-assisted laser desorption-ionization; PAGE, polyacrylamide gel electrophoresis; PSD, post-source decay; SDS, sodium dodecyl sulphate; TOF, time-of-flight. * Corresponding author. Tel.: þ39-432-558-781; fax: þ 39-432558-784. E-mail address: [email protected] (A. Vianello).
Inorganic pyrophosphatases (PPiases) (EC 3.6.1.1) are ubiquitous enzymes that catalyze the hydrolysis of pyrophosphate (diphosphate, PPi) to orthophosphate (Pi). The known PPiases can be subdivided into three main families: (1) tightly membrane-bound Hþpumping PPiases; (2) earlier known family of soluble PPiases; (3) recently found family of soluble PPiases (Baltscheffsky et al., 1999). Their presence in both prokaryotic and eukaryotic organisms raises the question whether PPi could be a direct ancestor of
1567-7249/$20.00 q 2003 Elsevier B.V. and Mitochondria Research Society. All rights reserved. doi:10.1016/S1567-7249(03)00105-3
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ATP, as an energy source, in the origin and evolution of life. In mammalian cells, PPi is considered a simple byproduct of a number of biosynthetic reactions (e.g. DNA, RNA synthesis, amino acid activation, activation of fatty acids, etc.), which has to be continuously removed by cytoplasmic soluble PPiases to drive forward these reactions (Kornberg, 1957). In higher plant cells, PPi is always produced by the same type of reactions, but the cytosol of these cells does not contain soluble PPiases, thus permitting an accumulation of a significant amount of PPi (0.2 – 0.3 mM) (Weiner et al., 1987). This cytosolic PPi can then act as an energy donor for at least three reactions, namely PPi-dependent proton pumping at the level of tonoplast (Maeshima, 2000; Rea et al., 1992), sucrose degradation via sucrose synthase, UDP-glucose phosphorylase and phosphofructokinase (Stitt, 1990; Kruger, 1997), and entry into glycolysis via the pyrophosphate fructose-6phosphate phosphotransferase (Stitt, 1990; Davies, 1997). These PPi-consuming reactions are ubiquitous in higher plants and operate in parallel with the corresponding ATP-dependent enzymes. Therefore, PPi is now considered an energetic alternative to ATP in plant cells (Stitt, 1998). This situation could be related to the sessile nature of plants and, consequently, to the need of having a more flexible metabolism. Higher plant mitochondria have a matrix concentration of PPi (0.2 mM) which appears to be in equilibrium with that of the cytoplasm by an exchange mediated by the adenine nucleotide translocase (Casolo et al., 2002). The mitochondrial PPi can be produced/utilized by a Hþ-PPiase, which seems to be loosely bound to the inner surface of the inner membrane (Vianello and Macrı`, 1999). This enzyme has a Hþ/PPi stoichiometry of two (Zancani et al., 1998) and a catalytic subunit of 35 kDa, which is probably part of a more complex multimeric enzyme (Zancani et al., 1995). On the basis of radiation inactivation analysis, the functional masses of enzymatic and proton translocation of mitochondrial Hþ-PPiase are 170 and 156 kDa, respectively (Jiang et al., 2000). In the light of the above considerations, it appears clear that higher plant mitochondria have an appreciable PPi metabolism. In this framework, we have now
identified a soluble (matrix) PPiase, which presents a high degree of homology with the b-subunit of the ATP synthase.
2. Materials and methods 2.1. Isolation of purified mitochondria and matrix proteins Pea stem Percoll-purified mitochondria were obtained from crude extracts prepared as described by Casolo et al. (2002), except for the absence of bovine serum albumin in all the preparation steps. Crude mitochondria were loaded on a Percoll discontinuous gradient (40 –21 –13% w/v) and centrifuged in a swinging-bucket rotor at 20,000 £ g for 40 min. The purified mitochondria were carefully collected at the 40 –21% interface. To remove Percoll, the purified mitochondria were washed in 250 ml of 20 mM 3-[N-morpholino]propanesulfonic acid – KOH (pH 7.2), 0.3 M mannitol, 1 mM EDTA and centrifuged three times at 21,000 £ g for 10 min. The final pellet was resuspended in 10 mM BTP – HCl (pH 7.0), 0.25 M sucrose. Matrix proteins, in the supernatant, were obtained from purified mitochondria after three ultrasonic irradiations for 30 s (100 W) and separation of the membrane fraction by ultracentrifugation (100,000 £ g for 40 min). 2.2. Ion exchange chromatography Matrix proteins (ca. 6 mg protein) were loaded on a Mono Q column equilibrated with 50 mM BTP – HCl (pH 7.0). Adsorbed proteins were eluted by a continuous gradient (0 – 300 mM KCl). Fractions showing PPiase activity were pooled and dialyzed overnight against 2 mM BTP – HCl (pH 6.5) and then lyophilized. 2.3. Non-denaturing preparative gel electrophoresis (ELFE) Electro-endosmotic preparative electrophoresis was performed with an ELFE apparatus (Zancani et al., 1995), using a non-denaturing polyacrylamide gel (10% w/v). Lyophilized fractions from ion exchange chromatography (600 – 800 mg protein)
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were resuspended in 7.5% (w/v) glycerol and 0.0025% (w/v) bromophenol blue (1 ml final volume) and loaded on the gel at 4 8C. The current was set at 10 mA and, after the elution of the bromophenol blue peak, the fractions were collected every 20 min (corresponding to ca. 3 ml). Fractions showing PPiase activity were pooled and used either to perform further enzymatic activities or dialyzed against 2 mM BTP – HCl (pH 6.5) and then lyophilized.
inorganic phosphate as a standard. In the experiments on the effect of the antibody raised against the a/b-subunit of ATP synthase on PPiase activity, the fractions pooled from ELFE (20 ml) were preincubated with the antibody (1 ml) for 30 min at 30 8C. The pyrophosphatase activity was detected in a final volume of 400 ml as described above.
2.4. SDS-PAGE and Western blotting
Characterization of polypeptides from polyacrylamide gel by MALDI-TOF mass spectrometry was performed on trypsin digested peptides. In short, proteins bands were excised from SDS-PAGE, cysteines were reduced and alkylated with iodoacetammide (Shevchenko and Shevchenko, 2001). The samples were then digested with porcine trypsin (Promega) in 40 mM ammonium bicarbonate at 37 8C for 6 – 8 h. The reaction was stopped by freezing. Tryptic peptides were extracted by ZipTip C18 (Millipore) reverse phase material, directly eluted and crystallized in a 50% (v/v) acetonitrile/ water saturated solution of a-cyano-4-hydroxycinnamic acid. MALDI mass spectra were recorded in the positive ion mode with delayed extraction on a Reflex IV timeof-flight instrument equipped with a MTP multiprobe inlet and a 337 nm nitrogen laser. Mass spectra were obtained by averaging 50– 200 individual laser shots. Calibration of the spectra was internally performed by a two-point linear fit using the autolysis products of trypsin at m=z 842.50 and 2211.10. A database search with the peptide masses was performed against the NCBInr database using the peptide search algorithm MASCOT (Matrix Science). Fragments generated by post-source decay (PSD) experiments were analyzed using the database search algorithm MS-Tag (http://prospector.ucsf.edu/ ucsfhtml3.2/mstag.htm).
Protein fractions from purified mitochondria (10 mg), matrix proteins (10 mg), ion exchange chromatography peak (ca. 5 mg) and the lyophilized peak eluted from ELFE (ca. 1 mg) were resuspended in 50 mM Tris –HCl (pH 6.8), 7.5% (w/v) glycerol, 2% (w/v) SDS and 0.0025% (w/v) bromophenol blue in a final volume of 50 ml. After incubation at 95 8C for 3 min, the samples were loaded on 12% (w/v) SDS-PAGE. The gels were initially stained with Coomassie Brilliant Blue R-250 and then with silver nitrate. Western blotting was performed by electroeluting the protein from the gel to a nitrocellulose membrane. Immunodetection was performed using rabbit polyclonal antibodies raised against mitochondrial (Vianello et al., 1997), vacuolar (Maeshima and Yoshida, 1989), soluble (du Jardin et al., 1995) PPiases or the a/b-subunit of the ATP synthase from mammalian mitochondria (Tomasetig et al., 2002). Immunochemical reactions were detected by the staining obtained using alkaline phosphatase-conjugate secondary antibody. 2.5. Enzymatic assays Pyrophosphatase activity was performed at 25 8C in a final volume of 400 ml of 20 mM Hepes –Tris (pH 7.5), 0.25 M sucrose, 50 mM KCl, 1.5 mM MgSO4 and 100 mM EGTA. The reactions were started by the addition of 200 mM PPi and were stopped after either 30 min (fractions pooled after ion exchange chromatography) or 1 h (fractions pooled from ELFE) by the addition of Fiske-Subarrow reagent (Cross et al., 1978). The absorbance values at 740 nm were converted to PPi hydrolyzed as half Pi released, calculated by a calibration curve using
2.6. MALDI-TOF mass spectrometry analysis
2.7. Protein determination Protein determinations were performed by the method of Bradford (1976). Absorbance values obtained at 595 nm were referred to a calibration curve obtained with bovine serum albumin as a standard.
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2.8. Data presentation Profiles of PPiase activity from proteins purified by chromatography and ELFE are reported as typical curves. Values in tables are means of at least three independent experiments ^ standard deviations.
3. Results 3.1. Purification of mitochondrial PPiase Fig. 1 shows the elution profile of PPiase activity after purification of mitochondrial matrix proteins by ion exchange chromatography (Mono Q). This PPiase activity was evident as a single peak ranging from 40 –42 to 58– 60 fractions. The 42– 43 to 52 – 53 fractions were then pooled. Their proteins were resolved by SDS-PAGE and coloured by silver stain (inset). The patterns reveal that the proteins still present in this pool were strongly diminished (Chr lane), when compared with total mitochondrial proteins (MtP) or mitochondrial matrix proteins (MP). The same sample of proteins (fractions from 42 –43 to 52– 53) was, therefore, further purified by non-denaturing ELFE (Fig. 2). Even in this case, PPiase activity eluted as a single sharp peak. The analysis of the proteins (from 38 to 40 fractions) by SDS-PAGE (always coloured by silver stain) showed a single protein band with a molecular mass
Fig. 1. Elution profile of PPiase activity after ion exchange (Mono Q) chromatography of mitochondrial matrix proteins. Inset shows SDS-PAGE of mitochondrial proteins (MtP), matrix proteins (MP) and chromatography-purified proteins (Chr). On the left, standard molecular mass markers are shown.
of ca. 55 kDa (inset, lane a). These pooled fractions were, hence, utilized for the biochemical and immunochemical characterization. The purification steps of PPiase can also be followed as increase of specific activity (Table 1). The crude extract (matrix proteins) exhibited a PPiase activity which increased 2.8-fold after ion exchange chromatography and 27-fold after nondenaturing ELFE. 3.2. Biochemical characterization of PPiase activity The only substrate utilized by the purified protein was PPi, because a nucleotide diphosphate (ADP) or triphosphates (ATP, CTP, GTP and UTP) induced only a negligible phosphohydrolytic activity (Table 2). Considering that soluble F1 from beef heart ATP synthase, depleted of adenine nucleotides, can hydrolyze PPi to values of ca. 0.2 nmol min21 mg21 protein (Tuena de Go´mez-Puyou et al., 1993), the PPiase activity of the purified protein was very high. This
Fig. 2. Elution profile of PPiase activity of chromatography-purified proteins (fractions pooled from chromatography from 42–43 to 52– 53) by ELFE. Inset shows: (a) SDS-PAGE of purified protein corresponding to the peak of activity; (b) Western blotting of the same protein that cross-reacted with polyclonal antibody raised against the a/b-subunit of ATP synthase from beef heart mitochondria. On the left, standard molecular mass markers are shown. Arrow indicates the purified protein at 55 kDa.
M. Zancani et al. / Mitochondrion 3 (2003) 111–118 Table 1 Purification steps of pea stem mitochondrial PPiase Step
Specific activity [nmol PPi (min £ mg protein)21]
Crude extract (matrix proteins) Chromatography (Mono Q)a ELFEa
189 ^ 30 529 ^ 45 5191 ^ 284
a
The specific activity increased 2.8- and 27-fold after chromatography (Mono Q) or ELFE, respectively.
PPiase had an absolute requirement for Mg2þ, while it was completely insensitive to monovalent cations (Table 3). Similarly, some divalent cations unaffected this activity, except for Mn2þ that caused a 40% inhibition. The activity showed a pH dependence with a maximum in the range between 8.0 and 9.5 (Fig. 3). The enzyme was inhibited by imidodiphosphate (ca. 70%) and aminomethylenediphosphate (ca. 45%), while other diphosphonates had minor effects (Table 4). In addition, PPiase activity was inhibited by both F2 and Ca2þ, which caused, at 100 mM concentration, ca. 70 –80% inhibition (Fig. 4). 3.3. Immunochemical characterization of PPiase The purified protein, exhibiting PPiase activity, did not cross-react with polyclonal antibodies raised against vacuolar, mitochondrial or soluble PPiases, respectively. Conversely, this protein cross-reacted with an antibody for the a/b-subunit of ATP synthase from beef heart mitochondria (Fig. 2, inset, lane b). In agreement, this antibody inhibited of ca. 40% PPiase activity (5.23 and 3.14 mmol PPi min21 mg21 protein in control and antibody-treated
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Table 3 Effect of monovalent and divalent cations on PPiase activity from purified fractions Cation
mmol PPi (min £ mg protein)21
None (control) Kþ Rbþ Csþ Liþ Naþ
5.19 ^ 0.28 5.25 ^ 0.29 5.13 ^ 0.28 5.12 ^ 0.28 4.39 ^ 0.24 4.82 ^ 0.26
Co2þ Cu2þ Mn2þ Zn2þ
5.02 ^ 0.27 5.41 ^ 0.30 2.98 ^ 0.16 4.79 ^ 0.26
The purified protein utilized in these assays was obtained by pooling the fractions from 38 to 40 after elution by ELFE. Final concentration of monovalent or divalent cations was 20 mM and 100 mM, respectively. No PPiase activity was detected in the absence of Mg2þ; none of the above divalent cations were able to substitute for Mg2þ.
samples, respectively). Nevertheless, the purified protein was subjected to mass spectrometry to further confirm this result. 3.4. Mass spectrometry characterization of mitochondrial PPiase The monoisotopic mass list recorded from the tryptic peptides of the 55 kDa band from the Coomassie stained SDS-PAGE fitted a gene
Table 2 Phosphohydrolytic activity of pea stem mitochondrial purified protein Substrate
mmol hydrolyzed (min £ mg protein)21
200 mM PPi 1 mM ATP 1 mM ADP 1 mM CTP 1 mM GTP 1 mM UTP
5.20 ^ 0.28 0.07 ^ 0.02 0.26 ^ 0.03 0.18 ^ 0.02 0.07 ^ 0.01 0.22 ^ 0.03
The purified protein utilized in these assays was obtained by pooling the fractions from 38 to 40 after elution by ELFE.
Fig. 3. PPiase activity as function of pH of the incubation medium. Mean values and standard deviations are calculated from three independent experiments.
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Table 4 Effect of diphosphonates on PPiase activity from purified fractions Diphosphonate
mmol PPi (min £ mg protein)21
Control Aminomethylenediphosphate Aminobuthylene-1-hydroxy-1,1-diphosphate Imidodiphosphate Aminohexane-1-hydroxy-1,1-diphosphate Dichloromethylenediphosphate Ethane-1-hydroxy-1,1-diphosphate
5.18 ^ 0.28 2.87 ^ 0.16 5.01 ^ 0.27 1.26 ^ 0.69 4.31 ^ 0.24 4.85 ^ 0.27 4.49 ^ 0.25
The purified protein utilized in these assays was obtained by pooling the fractions from 38 to 40 after elution by ELFE. The final concentration of inhibitors was 1 mM, except for aminomethylenediphosphate (10 mM).
product encoding a probable b-subunit protein (gil7436097) of Hþ-transporting ATP synthase (EC 3.6.1.34) from P. sativum mitochondria. The mass finger print data had a confidence value greater than 95% ðP , 0:05Þ in identifying this specific polypeptide. This identification was further confirmed by PSD experiments on two relevant peptide species on the MALDI-TOF spectra. The ion species at 1173.70 amu (MHþ) was selected and returned a fragmentation pattern fitting the sequence VVDLLAPYQR. A second PSD experiment was performed on the ionic peptide species at 1149.66 amu (MHþ) giving a fragmentation profile fitting the sequence TEHFLPIHR: The overall mass spectrometry data cover about 30% of the assigned sequence (Table 5). An initial signal sequence peptide was predicted to localize the protein in the mitochondria (software prediction Mitoprot II). The theoretical molecular weight, 60.4 kDa, calculated from the database sequence, fits quite well with the value of 55 kDa estimated from the SDS-PAGE once the initial Nterminus 38 aminoacids of the signal peptide (Table 5) are removed from the calculation.
1999), sustains some PPi-requiring reactions related to tonoplast energization (Maeshima, 2000; Rea et al., 1992), sucrose degradation (Stitt, 1990; Kruger, 1997) and glycolysis (Stitt, 1990; Davies, 1997), acting at the same time as regulator of the pyrophosphate fructose-6-phosphate phosphotransferase (Sung et al., 1988). PPi is also compartmentalized in the mitochondrial matrix, which contains a comparable level of PPi (0.2 mM), and where it can accomplish both roles as energetic substrate or as regulator of metabolic pathways (Casolo et al., 2002). Indeed, PPi could be synthesized/hydrolyzed by a mitochondrial Hþ-PPiase, which seems to act near to the thermodynamic equilibrium (Zancani et al., 1998). This allows PPi to be synthesized by dissipating the electrochemical proton gradient, established across the inner membrane by substrate oxidation (Casolo et al., 2002). Conversely, PPi may be consumed to maintain this gradient, particularly under condition of oxygen shortage (Vianello and Macrı`, 1999), as already suggested for the tonoplast
4. Discussion In higher plant cells, PPi plays a crucial role either as energetic substrate or as regulator of metabolic pathways (Stitt, 1998; Plaxton, 1996). Its cytoplasmic concentration (0.2 – 0.3 mM) is regulated by some soluble PPiases (Rojas-Beltran et al.,
Fig. 4. Effect of increasing concentration of F2 (upper panel) and total Ca2þ (lower panel) on PPase activity.
M. Zancani et al. / Mitochondrion 3 (2003) 111–118 Table 5 Sequence coverage by trypsin digestion peptide mass fingerprint of P. sativum b-subunit of the mitochondrial ATP synthase 1 51 101 151 201 251 301 351 401 451 501 551
MASRRLVSSL IRSSLRRSSS KPSISASTSR LTSQSRASPC GYLLNRVAHY ATSAAAAALP PSPPPAKKEG PGGGKITDEF TGKGAIGHVC QVIGAVVDVR FEEGLPPILT SLGLLGSHET RFWTLEVAQF GRGRVRTIAM DATEGVVRGW RVLNTGSPIS VPVGRANPWA YHEVIGEPID EKGELKTEHF LPIHREGPSF VEQATEQEIL VTGIKVVDLL APYQRGGKIG LFGGAGVGKT VLIMELINNV AKAHGGFSVF AGVGERTREG NDLYREMIES GVIKLGDKQS ESKCALVYGQ MNGPPGARAR VGLTGLTVAE HFRDAEGQDV LLFVDNIFRF TQANSEVSRL LGRIPSAVGY QPTLATDLGG LQERITTPKK GSITPGQAIY VPADDLTDLA PATTFAHLDA TTVLSRTISE LGIYPVVDPL DSTSRMLSPL ILGEAHYETA RGVQKVLQNY KNLQGIIAIL GMDELSEDDK LTVARARKIQ RFLSQPFHVA EVFTGGPGKY VDLKENITSF QGVLDGKYDD LSEQSFYMVG GIEEVIAKAE KIAKESAASS S
The protein regions covered in the mass fingerprint are shown in bold. Peptides sequences confirmed by fragmentation analysis and by post-source decay (PSD) are underlined. The predicted mitochondrial leader sequence is shown in italics.
Hþ-PPiase (Macrı` et al., 1995; Carystinos et al., 1995). In addition, free PPi acts as a powerful competitive inhibitor of succinate dehydrogenase, whose inhibition is released by chelation of PPi with Mg2þ (Casolo et al., 1998). For these reasons, it is possible to speculate that also the matrix concentration of PPi may be maintained under control by specific soluble PPiases, distinct from the abovementioned Hþ-PPiases (Casolo et al., 2002). However, no other PPiase activities have been hitherto described in plant mitochondria. We now show that a matrix protein, with a molecular mass of 55 kDa, exhibits the ability to hydrolyze PPi. This PPiase activity is Mg2þ requiring, is not stimulated by monovalent cations and is strongly inhibited by F2, Ca2þ, aminomethylenediphosphate and imidodiphosphate, all typical PPiase inhibitors. Apart from the molecular mass, the other features are typical of soluble PPiases (Cooperman et al., 1992). The protein does not cross-react with antibodies raised
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against vacuolar Hþ-PPiase, soluble PPiase and mitochondrial Hþ-PPiase, respectively. The molecular mass of this PPiase is identical to that identified for a thylakoid PPiase (55 kDa), whose activity is also Mg 2þ requiring and is not stimulated by Kþ (Jiang et al., 1997). The primary structure of the soluble pea stem PPiase has a high degree of homology, as stated, with the pea mitochondrial b-subunit of ATP synthase. On the other hand, the antibody raised against the a/bsubunit of the ATP synthase inhibits PPiase activity. Hitherto, a PPiase activity has only been described for soluble F1 from beef heart (Tuena de Go´mez-Puyou et al., 1993). This complex is able to carry both the synthesis and hydrolysis of PPi. In particular, F1 possesses three PPi-binding sites, which comprise one high affinity binding site and two lower affinity sites (Issartel et al., 1987). It seems that PPi mimics ADP and, therefore, that they share the same binding site in F1. However, the PPiase activity exhibited by the pea stem b-subunit is higher than that recovered for F1 from beef heart (Tuena de Go´mez-Puyou et al., 1993). These results can be rationalized in two ways: first, the 55 kDa protein of pea mitochondria can be considered as a soluble PPiase, distinct from the cytoplasmic one; second, it may be simply considered as a b-subunit of the main ATP synthase, which performs a dual function as soluble PPiase or as subunit of the more complex ATP synthase. On the other hand, it has already been suggested that mitochondrial (and chloroplast?) PPiase could be soluble parts of Hþ pumps (Baltscheffsky et al., 1999).
Acknowledgements This research was supported by the University of Udine. Thanks are due to: Dr M. Maeshima (Nagoya University, Japan) for antibody against mung bean tonoplast PPiase; Dr P. du Jardin (University of Gembloux, Belgium) for antibody against soluble PPiase from potato; Prof. G. Lippe (University of Udine, Italy) and Prof. F. Zanotti (University of Bari, Italy) for the antibodies against the a/b-subunit of ATP synthase.
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