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Keywords:

  • A1AO ATP synthase;
  • Archaea;
  • hyperthermophile;
  • Na+;
  • Pyrococcus

Abstract

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References

The rotor subunit c of the A1AO ATP synthase of the hyperthermophilic archaeon Pyrococcus furiosus contains a conserved Na+-binding motif, indicating that Na+ is a coupling ion. To experimentally address the nature of the coupling ion, we isolated the enzyme by detergent solubilization from native membranes followed by chromatographic separation techniques. The entire membrane-embedded motor domain was present in the preparation. The rotor subunit c was found to form an SDS-resistant oligomer. Under the conditions tested, the enzyme had maximal activity at 100 °C, had a rather broad pH optimum between pH 5.5 and 8.0, and was inhibited by diethystilbestrol and derivatives thereof. ATP hydrolysis was strictly dependent on Na+, with a Km of 0.6 mm. Li+, but not K+, could substitute for Na+. The Na+ dependence was less pronounced at higher proton concentrations, indicating competition between Na+ and H+ for a common binding site. Moreover, inhibition of the ATPase by N′,N′-dicyclohexylcarbodiimide could be relieved by Na+. Taken together, these data demonstrate the use of Na+ as coupling ion for the A1AO ATP synthase of Pyrococcus furiosus, the first Na+ A1AO ATP synthase described.

Abbreviation
DES

diethylstilbestrol

Membrane-bound, multisubunit, ion-translocating ATP synthases/ATPases are present in every domain of life. They arose from a common ancestor, but evolved into three distinct classes of ATP synthases/ATPases: the F1FO ATP synthase present in bacteria, mitochondria and chloroplasts, the A1AO ATP synthase present in archae, and the V1VO ATPase associated with cytoplasmic organelles such as vacuoles in eukaryotes [1–4]. A common feature of ATP synthases/ATPases is their organization into two domains, a hydrophilic and a membrane-bound domain, that are connected by (at least) two stalks, one central and one to two peripheral [5–12]. The hydrophilic domain catalyzes ATP hydrolysis [13–16], and the membrane-bound domain translocates ions from one side of the membrane to the other against their electrochemical gradient [17–20].

ATP synthases/ATPases are rotary machines that work as a pair of coupled motors: a chemically driven motor (F1/A1/V1) that is attached to the membrane, and an ion gradient-driven membrane-embedded motor (FO/AO/VO) [21–27]. The membrane-embedded motor is composed of a stator and a rotor. The rotor is composed of multiple copies of subunit c that form an oligomeric ring of noncovalently linked subunits, and rotation of the c ring is obligatorily coupled to ion flow across the membrane [28–31]. Most F1FO ATP synthases and V1VO ATPases use the proton as a coupling ion, but some use sodium ions instead. The ion-binding site is located in subunit c. The bacterial 8 kDa c subunit monomer folds in the membrane like a hairpin, with two transmembrane helices connected by a cytoplasmic loop, and a single carboxylic acid (aspartate or glutamate [D61 in Escherichia coli; E65 in Ilyobacter tartaricus]) has been identified as the protonatable group in the rotor subunit [32–37]. The recently determined high-resolution structure of the rotor from I. tartaricus revealed that the coordination sphere for Na+ is formed by side-chain oxygens of Gln32 and Glu65 of one subunit, and the hydroxyl group of Ser66 and the backbone carbonyl oxygen of Val63 of the neighboring subunit [38]. The motif is conserved in Na+ F1FO ATP synthases [39,40]. V1VO ATPases have a ≈ 16 kDa subunit c that arose by gene duplication and fusion, giving rise to a monomer with four transmembrane helices. The active carboxylate is conserved in helix four but not in helix two. The Na+ coordination sphere in the c ring of Enterococcus hirae is composed of helix two and four of one monomer. The side chains of T64, Q65, Q110 and E139, and in addition the backbone carbonyl of L61, form the Na+-binding pocket [41].

Na+-translocating ATPases have so far been found only in anaerobic prokaryotes such as Propionigenium modestum, I. tartaricus and Acetobacterium woodii (F1FO ATP synthase) and Caloramator fervidus, Clostridium paradoxum and E. hirae (annotated or described as ‘bacterial’ V1VO ATPases, but there is debate about the classification in the literature) [42–47]. They have been found to be advantageous in analyzing the mechanism of ion transport and the structure of the c ring. In contrast to F1FO ATP synthases and V1VO ATPases, Na+-dependent A1AO ATP synthases have not been described. Experimental confirmation of the use of Na+ as a coupling ion in A1AO ATP synthases has so far not been obtained, due to the lack of purified and coupled A1AO ATP synthases. All preparations but one, from Methanocaldococcus jannaschii, lacked the membrane-embedded motor [13]. To experimentally address the nature of the coupling ion in the A1AO ATP synthase, we have enriched and studied the A1AO ATP synthase from the hyperthermophilic archaeon Pyrococcus furiosus. This preparation contains the membrane-embedded motor, and we will present evidence that it is an Na+-dependent enzyme. This is the first description of an Na+ A1AO ATP synthase.

Results

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References

Genetic organization of the A1AO ATP synthase of P. furiosus

The genome of P. furiosus encodes only one ATP synthase, the A1AO ATP synthase. The ATP synthase is encoded by the genes atpHIKECFABD, which are organized in one cluster [48]. The genes encode the nine subunits H (atpH), a (atpI), c (atpK), E (atpE), C (atpC), F (atpF), A (atpA), B (atpB) and D (atpD). A hypothetical model of the subunit topology of A1AO ATP synthases is presented in Fig. 1. Subunits a and c form the membrane-embedded motor, with a being the stator, and multiple copies of subunit c forming the rotor. Unlike c subunits of F1FO ATP synthases but like those of V1VO ATPases, subunit c of P. furiosus has four transmembrane helices and a molecular mass of 15.8 kDa, as deduced from the DNA sequence. The active carboxylate is conserved in helix four but not in helix two. Most interestingly, sequence comparisons revealed that the residues involved in Na+ binding in E. hirae are fully conserved in P. furiosus (Fig. 2). Moreover, the other pyrococci whose genomes have been sequenced (Pyrococcus abyssi and Pyrococcus horikoshii) all contain the predicted Na+-binding site.

image

Figure 1.  Subunit composition and topology of A1AO ATP synthases. This hypothetical model is based on biochemical data summarized in Müller et al.[64,65]. Please note that the stoichiometry of the c subunits is unknown but is likely to be different in A1AO ATP synthases from different organisms [68].

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image

Figure 2.  Alignment of sequences from c subunits of Na+ ATPases. Depicted are the sequences of c subunits from the V1VO ATPase of E. hirae (E. hi), and the A1AO ATP synthases of P. furiosus (P. fu), P. horikoshii (P. ho), and P. abyssi (P. ab). Amino acids forming the Na+-binding site in E. hirae are boxed. These residues are conserved in pyrococci.

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Solubilization of the A1AO ATP synthase from membranes of P. furiosus

Washed membranes of cells of P. furiosus grown at 98 °C on complex medium had an ATPase activity of around 0.2 U·mg−1 protein. Different detergents were tested under various conditions for their ability to solubilize the A1AO ATP synthase from the washed membranes. Triton X-100, dodecylmaltoside, sodium cholate or Chaps were used at a concentration of 1 g·g−1 membrane protein, and the mixture was incubated for 2 h at 40 °C. With Chaps, 47% of the total activity was solubilized, and with dodecylmaltoside or Na+-cholate, 82% of the activity was solubilized (Table 1). Triton X-100 led to solubilization of 85% of the activity. A mixture of Triton X-100 and dodecylmaltoside (3 : 1) solubilized 76% of the activity. Higher amounts of detergent or different temperatures did not enhance the yield. Therefore, Triton X-100 was used for further solubilizations.

Table 1.   Solubilization of the P. furiosus A1AO ATP synthase with different detergents. Detergents were used at a concentration of 1 g·g−1 membrane protein; the total protein concentration was 12 mg·mL−1, and the volume was 1 mL.
DetergentATPase activity
Membrane (mU)SupernatantPelletTotal activity (mU)Recovery (%)a
mU%mU%
  1. a  One hundred per cent refers to membrane-bound activity. b  The detergents were used in a mixture comprising 1 part dodecylmaltoside/3 parts Triton X-100.

Dodecylmaltoside28023182471727899
Chaps280131471234425491
Triton X-100293249855519304104
Dodecylmaltoside/  Triton X-100 (1 : 3b)28021276562026896
Na+-cholate280230827828307110

Purification of the A1AO ATP synthase from membranes of P. furiosus

After MgCl2 had been added to the solubilisate to a final concentration of 50 mm, the ATP synthase was further purified by differential poly(ethylene glycol) precipitation (4.1%, 12.9%). This resulted in 80% loss of activity and four-fold enrichment. Subsequently, the precipitate was dissolved in buffer containing Triton X-100 and subjected to gel filtration (Superose 6 column). The ATPase eluted as a single peak with a specific activity of 5800 mU·mg−1 and an overall yield of 18% (Table 2).

Table 2.   Purification of the A1AO ATP synthase of P. furiosus.
Purification stepProtein (mg)ATPase activity (U)Specific activity (mU·mg−1)Enrichment (fold)Yield (%)
Membranes464871871100
TX-100 supernatant7570933580.5
Poly(ethylene glycol) precipitation2518720420.5
Gel filtration2.715.658003118

Subunit composition

A representative SDS/PAGE result is shown in Fig. 3. The preparation contained the A1AO ATP synthase subunits a (75 kDa, identified by western blot analysis with an antibody against subunit a of M. jannaschii), A [69 kDa, identified by MALDI-TOF MS (score 94)], B (52 kDa, identified by western blot analysis with an antibody against subunit B of M. jannaschii), C (42 kDa; the mass of the protein corresponds to the calculated mass), D [25 kDa, identified by MALDI-TOF MS (score 90)], E [23 kDa, identified by MALDI-TOF MS (score 111)], and c (16 kDa, identified by western blot analysis with an antibody against subunit c of M. jannaschii). It should be noted that the gene atpA is 3042 bp long and encodes a 113 kDa protein. However, it contains an intein sequence that is not present in the mature protein. Interestingly, the c oligomer was partly SDS-resistant and could not be resolved from subunit a using 10% SDS/PAGE. It had an apparent mass of 75 kDa, and was identified in a western blot with a antibody against subunit c from M. jannaschii. Moreover, MALDI-TOF MS analysis of the polypeptides around 75 kDa clearly identified subunits a and c.

image

Figure 3.  Subunit composition of the A1AO ATP synthase preparation. The preparation was subjected to SDS/PAGE on 10% gels. Proteins were stained with Coomassie Brilliant Blue. Subunits are indicated on the left, and a molecular mass marker on the right.

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In addition to the ATP synthase subunits, the preparations contained variable amounts of contaminating proteins. The polypeptide with the apparent mass of 130 kDa could not be identified by MALDI-TOF MS; the polypeptide disappeared after heating of the sample in SDS and mercaptoethanol to temperatures of 90 °C or 100 °C for 10 min or 120 °C for 3 min, indicating that it might be an aggregation product. The 97 kDa protein was identified by MALDI-TOF MS as an amylopullulanase. The 60 kDa protein represents a single subunit of the thermosome, an archaeal chaperonin. The 50 kDa protein was identified as a hypothetical oligopeptide transport system permease protein, and the 44 kDa protein as a ‘hypothetical protein’. The proteins with masses of 30 and 28 kDa could not be identified by MALDI-TOF MS.

Catalytic properties of the purified A1AO ATP synthase

ATPase activity was detected over a wide pH range, with optimal activity between pH 5.5 and 8.0 (Fig. 4A). pH values lower than 5.5 decreased the activity very strongly (23% activity at pH 5); the maximum was reached at pH 6.0, but the decrease was less pronounced (92% activity at pH 7.0). The enzyme was practically inactive at temperatures below 60 °C, and higher temperatures led to increasing activities. The highest activity was obtained at 100 °C, the highest temperature tested (Fig. 4B). Apart from ATP, the enzyme hydrolyzed ITP (99% activity) and even GTP (64% activity) with rather high rates, but UTP (31% activity) and CTP (14% activity) with low rates. Measurements were started after 3 min of incubation at 100 °C by the addition of 2.5 mm Na2-ATP, Na2-ITP, Na2-CTP, Na3-UTP or Li2-GTP. Neither ADP nor pyrophosphate (Na4P2O7·10 H2O) were hydrolyzed. Divalent cations were required for activity. Optimal activity was obtained with MgCl2; MnCl2 (72%) was not superior over MgCl2, as has been described for other A1AO ATPases [49–52]. Zn2+ could replace Mg2+ to some extent (73%), but Ca2+, Ni2+ and Cu2+ were less effective (47%, 36% or 12%). Divalent cations were used at a concentration of 5 mm. The Km value for Mg-ATP was determined to be 0.63 mm, and the maximal velocity was 1.7 U·mg−1(Fig. 5).

image

Figure 4.  pH (A) and temperature (B) dependence of ATPase activity. After 3 min at 100 °C, the reaction was started by addition of Na2-ATP to a final concentration of 2.5 mm. The buffer contained 100 mm Mes, 100 mm Tris, 5 mm MgCl2, 200 mm KCl and 40 mm NaHSO3. One hundred per cent activity corresponds to 1 U·mg−1 in (A) and 1.7 U·mg−1 in (B). The pH in (B) was 6.0.

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image

Figure 5.  Kinetics of ATP hydrolysis. (A) After 3 min at 100 °C, the reaction was started by addition of Na2-ATP to final concentrations of 0–7 mm. The buffer contained 100 mm Mes, 100 mm Tris, 5 mm MgCl2, 200 mm KCl and 40 mm NaHSO3, and the pH was adjusted to pH 6. The initial rates of activity are plotted against the ATP concentration. (B) A double reciprocal plot of the data presented in (A).

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Inhibitor sensitivity of the A1AO ATP synthase

Previously, it has been shown that ATP hydrolysis as catalyzed by the A1AO ATP synthase from the archaeon Methanosarcina mazei was inhibited by diethylstilbestrol (DES) and its derivatives (hexestrol, dienestrol), but not by DES-dipropionate and trans-stilbene [53]. DES inhibited ATP hydrolysis with an IC50 of 0.36 mm. Of the DES derivatives tested, dienestrol and hexestrol were also potent inhibitors of the A1AO ATP synthase from P. furiosus, with IC50 values of 0.52 and 0.59 mm, respectively. Trans-stilbene did not inhibit ATP hydrolysis, and DES-dipropionate only inhibited at concentrations above 1 mm(Fig. 6). Inhibitors such as vanadate, azide or nitrate had no effect on ATP hydrolysis. Amiloride and derivatives (hexamethylene amiloride, ethylisopropyl amiloride, phenamil and benzamil), inhibitors of Na+/H+-antiporters [54], Na+ F1FO ATP synthases [46,55] and Na+-driven flagellar motors [56] or Na+ channels [57], could not be tested for their inhibitory effect, because they were not stable at the high temperatures required.

image

Figure 6.  Inhibition of ATP hydrolysis by DES and derivatives. After preincubation with the inhibitor for 30 min at room temperature, and incubation for 3 min at 100 °C, the reaction was started by addition of Na2-ATP to a final concentration of 2.5 mm. The buffer contained 100 mm Tris base, 100 mm maleic acid and 5 mm MgCl2. The pH was adjusted to 8.0 with KOH or HCl. DES and derivatives were added as an ethanolic solution; controls received the solvent only. Δ, trans-stilbene; ▪, DES; •, dienestrol; ◆, DES-dipropionate; bsl00072, hexestrol.

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Na+ dependence of ATP hydrolysis

To test the dependence of catalytic activity on Na+, ATP hydrolysis was determined in a buffer to which no Na+ had been added and that was prepared under special conditions to decrease the Na+ level as much as possible. The Na+ concentration in the buffer, as determined by an Na+-specific electrode, was 0.2 mm. Under these conditions, ATP hydrolysis was very low (0.091 U·mg−1). However, ATPase activity was restored upon addition of NaCl (Fig. 7). Maximal activity was obtained at 5 mm Na+. The stimulation of ATP hydrolysis by Na+ followed Michaelis–Menten kinetics. From a double reciprocal plot, the Km for Na+ was determined to be 0.6 mm. Li+, but not K+, could substitute for Na+. These data provide evidence that the A1AO ATP synthase of P. furiosus uses Na+ as coupling ion. The stimulating effect of Na+ was much weaker at more acidic pH values (Fig. 7). This has also been seen before with Na+ F1FO ATP synthases [45,55,58], and indicates that the A1AO ATP synthase from P. furiosus can transport both Na+ and H+.

image

Figure 7.  Na+ dependence of ATP hydrolysis. The buffer contained 100 mm Tris base, 100 mm maleic acid and 5 mm MgCl2 at pH 5.5 (bsl00072) or pH 8 (▪). The NaCl concentration was as indicated. The reaction was started by the addition of 2.5 mm K2-ATP.

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Protection from N ′,N ′-dicyclohexylcarbodiimide inhibition by Na+

Like F1FO ATP synthases and V1VO ATPases, A1AO ATP synthases are inhibited by N′,N′-dicyclohexylcarbodiimide. A special feature of Na+-translocating ATPases/ATP synthases is protection from N ′,N ′-dicyclohexylcarbodiimide inhibition by Na+, which is due to competition between Na+ and N ′,N ′-dicyclohexylcarbodiimide for a common binding site, i.e. the active carboxylate of subunit c[55,58]. When tested at 0.2 mm Na+, the A1AO ATPase of P. furiosus was inhibited by N ′,N ′-dicyclohexylcarbodiimide, indicating that the A1 and AO domains in this preparation are functionally coupled. Half-maximal inhibition was observed at 100 µm(Fig. 8A). However, the presence of 5 mm Na+ in the buffer during the preincubation period with N ′,N ′-dicyclohexylcarbodiimide completely protected the enzyme from N ′,N ′-dicyclohexylcarbodiimide inhibition (Fig. 8B).

image

Figure 8.   (A) Inhibition of ATP hydrolysis by N′,N′-dicyclohexylcarbodiimide. After preincubation with increasing concentrations of N′,N′-dicyclohexylcarbodiimide (0–1 mm) in the absence of Na+ for 30 min at room temperature, and incubation for 3 min at 100 °C, the reaction was started by addition of Na2-ATP to a final concentration of 2.5 mm. N ′,N ′-Dicyclohexylcarbodiimide was added as an ethanolic solution; controls received the solvent only. One hundred per cent corresponds to 0.083 U·mg−1. (B) Na+ protects against N ′,N ′-dicyclohexylcarbodiimide inhibition. The enzyme was preincubated in buffer without additional Na+ (bsl00066) or with 5 mm Na+(•) and N ′,N ′-dicyclohexylcarbodiimide as indicated. After preincubation for 30 min at room temperature, and incubation for 3 min at 100 °C, the reaction was started by addition of Na2-ATP to a final concentration of 2.5 mm. N ′,N ′-Dicyclohexylcarbodiimide was added as an ethanolic solution; controls received the solvent only. One hundred per cent corresponds to 0.88 U·mg−1.

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Discussion

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References

In anaerobic environments, microbes often grow on substrates that allow for the synthesis of only 1–4 mol of ATP [59], and in addition, the concentration of membrane-permeable organic acids such as acetate or succinate will counteract proton-based energetics, due to uncoupling effects [60,61]. It has long been known that anaerobic members of the third domain of life, the Archaea, such as the methanogens, couple metabolic activities to the generation of primary, transmembrane electrochemical Na+ gradients [13,62,63], but Na+-driven ATP synthesis could not be demonstrated unequivocally using whole cells [64]. However, sequence comparisons indicated that the Na+-binding motif in subunit c of F1FO ATP synthases and V1VO ATPases is present in subunit c of some A1AO ATP synthases, i.e. those from the methanogens, pyrococci, Thermoplasmatales, Archaeoglobales and halobacteria [65]. Unfortunately, a dependence on Na+ of ATP synthesis or ATP hydrolysis using purified enzymes could not be demonstrated, due to the lack of purified, intact A1AO ATP synthases [48].

Here, we have established a protocol to enrich the A1AO ATP synthase of P. furiosus. The enzyme contained the membrane-embedded motor, and the A1 and AO domains were functionally coupled, as is evident from the inhibition of ATP hydrolysis by N′,N′-dicyclohexylcarbodiimide and the Na+ dependence of ATP hydrolysis. The enzyme exhibited the highest ATPase activities at 100 °C, an outstanding feature of a membrane-embedded macromolecular transport machinery. The A1AO ATP synthase from P. furiosus is one of the most interesting members of the class of A1AO ATP synthases/ATPases. It has a V1VO-type c subunit that could contribute to the thermostability of the rotor. Furthermore, the c subunit has only one ion-binding site in four transmembrane helices, and this would argue for a function of the enyzme as an ATPase, not an ATP synthase. However, ATP synthesis was demonstrated in vesicle preparations [66], and as the A1AO ATP synthase genes are the only ATP synthase genes on the chromosome [67], this would argue for the A1AO ATP synthase catalyzing ATP synthesis in vivo, despite the V1VO-type c subunit. The structural basis for ATP synthesis in the P. furiosus enzyme is unknown, but should reside in the membrane-embedded motor [68]. Unfortunately, the preparation described here contained thermosomes that interfered with structural analyses. Attempts to remove the thermosomes by pH changes, washing the membranes with NaCl, LiCl or CaCO3, anion exchange chromatography and several gel filtrations (Superose 6, S300, S400 and S1000) were unsuccessful.

Most important, we have established that the A1AO ATP synthase of P. furiosus uses Na+ as a coupling ion. This is based on the Na+ dependence of ATP hydrolysis, the protection from N′,N′-dicyclohexylcarbodiimide inhibition by Na+, and the presence of an Na+-binding motif in subunit c. The Na+-binding motif is identical to the motif established experimentally for the 16 kDa c subunit of E. hirae[41]. Na+ is bound by only one subunit c. In contrast, the Na+-binding site in the F1FO ATP synthase from I. tartaricus is formed by two subunits, and this bridging of subunit c by Na+ is thought to be the structural basis for SDS resistance of the c ring of Na+ F1FO ATP synthases [69]. Interestingly, we observed partial SDS resistance of the c ring of P. furiosus. As the analogy with the E. hirae c ring would argue against subunit bridging by Na+, a more general mechanism may enable SDS resistance in the P. furiosus c ring.

The presence of an Na+-dependent A1AO ATP synthase gives the first hint of Na+-based bioenergetics in P. furiosus. This hyperthermophile is known to grow by fermentation of sugars. During fermentation, the electrochemical ion gradient across the membrane is established by the A1AO ATP synthase. In addition, P. furiosus can also couple hydrogen oxidation to the synthesis of ATP by a chemiosmotic mechanism [66]. A potential role of Na+ in the bioenergetics of P. furiosus was not addressed in previous studies, but this is not excluded. A hydrogenase of the Ech type has been discussed as a primary Na+ pump in some methanogens [70]. A membrane-bound hydrogenase is present in P. furiosus, and could couple hydrogen oxidation to the generation of transmembrane primary Na+ potential. As outlined above, Na+-based bioenergetics are of advantage in alkaline [71], anaerobic [61] and hot environments [72], and may contribute to successful life at high temperatures.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References

Materials

All chemicals were reagent grade and were purchased from Merck AG (Darmstadt, Germany). N′,N′-Dicyclohexylcarbodiimide and Triton X-100 were from Sigma Chemical Co. (Deisenhofen, Germany).

Organism

P. furiosus (DSM 3638) was obtained from the Deutsche Sammlung für Mikroorganismen und Zellkulturen, Braunschweig, Germany. For purification of the ATPase, P. furiosus was grown in a 300 L fermenter at 98 °C in the medium described [73]. The fermenter was pressurized to 2 bar with N2/CO2 (80 : 20). The gas flowthrough was adjusted to 1–7 L·min−1, depending on the growth phase. Growth was monitored by cell counts. Cells were harvested in the late exponential growth phase by centrifugation (10 000 g; 20 min; 4 °C) in a Sorvall Superspeed RC2-B. The pellets were stored at − 80 °C.

Preparation of membranes

Cells, 10–20 g, were resuspended in buffer containing 25 mm Tris (pH 7.5), 5 mm MgCl2, 0.1 mm phenylmethanesulfonyl fluoride and 0.1 mg DNase·mL−1. After homogenization, the cells were disrupted by three passages through a French pressure cell at 1200 psiG. Cell debris was removed by centrifugation (11 000 g; 30 min). Membranes were recovered from the extract by centrifugation (120 000 g; 2 h; Beckman L100K, 50.2 Ti rotor) and were washed in 100 mm Hepes (pH 7.0), 5 mm MgCl2, 10% glycerol (v/v), 100 mm NaCl and 0.1 mm phenylmethanesulfonyl fluoride. The supernatant after this wash step contained negligible ATP hydrolysis activity. The washed membrane pellet was resuspended in 15–20 mL of membrane buffer containing 100 mm Hepes (pH 7.0), 5 mm MgCl2, 10% glycerol (v/v) and 0.1 mm phenylmethanesulfonyl fluoride. The protein concentration was determined as previously described [74,75].

Solubilization and purification of the A1AO ATPase

Membrane proteins (29 mg·mL−1, 16 mL) were solubilized with Triton X-100 at a concentration of 3% (v/v) (1 g of detergent per 1 g of membrane protein). After 2 h of occasional mixing at 40 °C, the membranes were solubilized overnight at room temperature on a swayer. After centrifugation (120 000 g; 100 min; Beckman L100K, 50.2 Ti rotor), 0.5 mL of 1 m MgCl2 was added to 10 mL of solubilisate. The ATP synthase was further purified by a two-step precipitation. First, contaminating proteins were precipitated with polyethylene glycol 6000 (4.1%, w/w). The precipitate was removed by centrifugation (120 000 g; 1 h; Beckman L100K, 50.2 Ti rotor). The ATP synthase was then precipitated with polyethylene glycol 6000 at a concentration of 12.9% (w/w). The precipitate was collected by centrifugation (120 000 g; 1 h; Beckman L100K, 50.2 Ti rotor) and dissolved in 2–3 mL of buffer [50 mm Tris/HCl (pH 7.5), 5 mm MgCl2 and 0.05% Triton X-100 (v/v)]. Insoluble material was removed by centrifugation (7700 g; 10 min; Hettich centrifuge Mikro22). The supernatant was applied to a Superose 6 column, equilibrated with 50 mm Tris/HCl (pH 7.5), 5 mm MgCl2, 10% glycerol (v/v), and 0.1% Triton X-100. All steps were performed at 4 °C.

ATPase activity

ATPase activity was measured in an assay mixture containing 100 mm Mes, 100 mm Tris (pH 6.0), 40 mm NaHSO3, 5 mm MgCl2, 200 mm KCl and enzyme solution. After incubation for 3 min at 100 °C, the reaction was started by addition of Na2ATP to a final concentration of 2.5 mm. The ATPase activity was determined by a discontinuous assay following the ATP-dependent formation of inorganic phosphate as previously described [76]. For inhibitor studies, the reaction mixture was preincubated for at least 30 min at room temperature before the reaction was started by addition of ATP. Stock solutions of N′,N′-dicyclohexylcarbodiimide, DES and derivatives were prepared fresh for each experiment in ethanol. Amiloride, benzamil, ethylisopropyl amiloride or hexamethylene amiloride were dissolved in dimethylsulfoxide; controls received the solvent only. For determination of ATPase activity in the absence of sodium ions, the activity was assayed in 100 mm Tris base, 100 mm maleic acid and 5 mm MgCl2. The pH was adjusted with KOH or HCl.

Immunologic investigations

Western blotting with SDS/PAGE gels was performed as previously described [77]. The nitrocellulose sheets were applied to different antisera and treated with alkaline phosphatase-conjugated goat anti-(mouse IgGs) in a reaction mixture made up of 0.0075% (w/v) Nitro Blue tetrazolium chloride and 0.03% (w/v) 5-bromo-4-chloro-3-indolyl phosphate in 100 mm Tris/HCl, 100 mm NaCl and 5 mm MgCl2 (pH 8.8).

Tryptic digest and MALDI-TOF MS analysis

The products of the A1AO ATPase were excised from a gel and subjected to in-gel digestion protocols as previously described [78,79]. After 12 h, the supernatant was removed, and the remaining peptides were extracted three times with 50% acetonitrile/5% formic acid (v/v). All fractions were dried in a speed vacuum concentrator prior to MALDI-TOF MS analysis. Delayed extraction MALDI-TOF mass spectra were recorded on a voyager delayed extraction STR instrument (Applied Biosystems, Darmstadt, Germany). Spectra were externally calibrated with a Sequazyme peptide mass standard kit (ABI). Proteins were identified using mascot (http://www.matrixscience.com). Protein scores greater than 77 are significant (P < 0.05).

Determination of the Na+ concentration

Na+ concentrations were determined with a sodium electrode Orion 84-11ROSS (Thermo Electron Corporation, Witchford, UK). All standards for calibration (ionic strength adjustor, electrode rinse solution; sodium electrode storage solution) were prepared freshly for each measurement in distilled water, and plastic laboratory-ware was used. The calibration curve was measured at room temperature from 0 to 200 µm NaCl.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References

This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB472).

References

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References
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