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Notes & Tips
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Continuous fluorescence-based measurement of redox-driven sodium ion translocation
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Valentin Muras a, Björn Claussen a, Manikandan Karuppasamy b,c, Christiane Schaffitzel b,c, Julia Steuber a,⇑
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a
Department of Microbiology, University of Hohenheim (Stuttgart), Garbenstrasse 30, 70599 Stuttgart, Germany European Molecular Biology Laboratory (EMBL), Grenoble Outstation, 6 rue Jules Horowitz, 38042 Grenoble, France c Unit for Virus Host-Cell Interactions, University Grenoble Alpes-EMBL-CNRS, 6 rue Jules Horowitz, 38042 Grenoble, France b
a r t i c l e
i n f o
Article history: Received 26 February 2014 Received in revised form 8 May 2014 Accepted 15 May 2014 Available online xxxx Keywords: Sodium transport Sodium green Proteoliposomes Reconstitution Sodium ion-translocating NADH:quinone oxidoreductase (Na+-NQR) Fluorescence
a b s t r a c t Investigation of the mechanism of sodium ion pumping enzymes requires methods to follow the translocation of sodium ions by the purified and reconstituted proteins in vitro. Here, we describe a protocol that allows following the accumulation of Na+ in proteoliposomes by the Na+-translocating NADH: quinone oxidoreductase (Na+-NQR) from Vibrio cholerae using the sodium-sensitive fluorophor sodium green. In the presence of a regenerative system for its substrate NADH, the Na+-NQR accumulates Na+ in the proteoliposomes which is visible as a change in fluorescence. Ó 2014 Elsevier Inc. All rights reserved.
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The sodium translocating Na+-NQR1 from Vibrio cholerae is a membrane-embedded sodium ion transporter made up of six subunits and belongs to the class of primary sodium ion pumps [1]. The energy that enables transport of Na+ against a chemical gradient is derived from the oxidation of NADH with the final electron acceptor ubiquinone. The exact mechanism of electron transfer-driven sodium ion translocation by the Na+-NQR has not yet been elucidated, though critical subunits and cofactors have been identified [2,3]. Two methods for the determination of in vitro Na+ transport by NADH:quinone oxidoreductases have been described so far. They differ with respect to the Na+ isotope used (23Na or 22Na) and the corresponding detection method (atomic absorption spectroscopy or scintillation counting) [4], but they both represent discontinuous assays where the internal Na+ in proteoliposomes is quantified after separation of external Na+ by a chromatographic step. Here we describe a technique for continuous measurement of Na+ transport by reconstituted Na+-NQR. The method uses the sodium-sensitive fluorophor sodium green that has previously been used for studying Na+ transport by the Na+-translocating
⇑ Corresponding author. Fax: +49 711 45922238. E-mail address: [email protected] (J. Steuber). Abbreviations used: Na + -NQR, sodium ion-translocating NADH:quinone oxidoreductase; Q1, ubiquinone-1; DTT, dithiothreitol. 1
F1FO ATPase [5]. In the case of Na+-NQR, the method critically depends on the continuous regeneration of NADH with the help of lactate dehydrogenase and lactate. Na+-NQR was reconstituted into proteoliposomes using a dilution method as described in [6]. One milligram of Na+-NQR and 0.6 lg sodium green (S-6900, Molecular Probes) were added to a lipid film consisting of 40 mg of L-a-phosphatidylcholine (from soybean, Type II-S, 14–23% as choline) that were dried with a rotary evaporator in a round bottom flask. Subsequently the liposome/micelle mixture was treated with a tip sonicator (3 20 s). Reconstitution of Na+-NQR into liposomes was achieved by diluting the liposome/micelle mixture with a 50-fold volume of reconstitution buffer. The proteoliposomes were collected by ultracentrifugation (150,000g, 45 min, 4 °C) and resuspended in 2 ml reconstitution buffer. Excess sodium green was then removed from the external buffer by gel filtration on a NAP-10 column (Amersham Biosciences). Rates of NADH oxidation and ubiquinone-1 (Q1) reduction of Na+-NQR were determined as described [6], with the following modifications: NADH and Q1 (each 100 lM) were mixed in 200 mM KCl, 5% glycerol, 10 mM Tris/HCl, pH 8.0, containing 20 mM lactic acid and 5.5 U L-lactate dehydrogenase (from rabbit muscle, Roche). The reaction was started by the addition of 10 lg Na+-NQR.
http://dx.doi.org/10.1016/j.ab.2014.05.012 0003-2697/Ó 2014 Elsevier Inc. All rights reserved.
Please cite this article in press as: V. Muras et al., Continuous fluorescence-based measurement of redox-driven sodium ion translocation, Anal. Biochem. (2014), http://dx.doi.org/10.1016/j.ab.2014.05.012
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Notes & Tips / Anal. Biochem. xxx (2014) xxx–xxx
Proteoliposomes with incorporated sodium green (200 ll) were mixed with 1 ml reconstitution buffer containing 1 mM Q1, 20 mM L-lactic acid, and 0.1 mg ml 1 L-lactate dehydrogenase (550 U mg 1). Na+ uptake was initialized by the addition of 0.5 mM NADH. Changes in sodium green fluorescence were followed in a Horiba (Fluorolog) fluorospectrophotometer with an excitation wavelength of 488 nm and an emission wavelength of 540 nm. For normalization of the data, the final fluorescence emission intensity at the end of the reaction without added NaCl was set as 100%. The dissociation constant (KD) of sodium green (20 nM) toward sodium was determined in reconstitution buffer from the fluorescence increase at 535 nm at increasing Na+ concentrations. Data were fitted to a one-site binding model equation Y = (Fmax X)/(Kd + X) using the curve fit routine of the software Origin, where X = concentration of the free ligand (Na+), Fmax = maximum fluorescence emission, and Kd = equilibrium dissociation constant [7]. To follow sodium ion transport in the course of NADH oxidation by the Na+-NQR, the membrane-impermeable form of sodium green [8] was used to ensure that an increase of fluorescence exclusively resulted from Na+ that was accumulated in the proteoliposomes. The proteoliposomes had an average size of approximately 50 nm in diameter and maintained a membrane potential generated by the Na+-NQR, confirming their integrity (supplementary material, Figs. S1 and S2). The tightness of the liposomes was confirmed using the sodium ionophor gramicidin D. Addition of 10 mM NaCl to intact proteoliposomes did not affect sodium green fluorescence. Only on addition of 5 lM gramicidin, the fluorescence increased by approximately 10% which resulted from binding of Na+ to sodium green entrapped in the liposomes. The increase by 10% is in accordance with the observed fluorescence increase of sodium green followed in a titration from 0 to 100 mM NaCl (supplementary material, Fig. S3). First trials to monitor translocation of sodium ions into Na+-NQR liposomes with incorporated sodium green were not successful. There was no increase in fluorescence, and we reasoned that the amount of accumulated sodium ions during steady state in the Na+-NQR liposomes was too low, considering a KD of sodium green toward Na+ of 17 mM (supplementary material, Fig. S3). Both leakage of Na+ from the proteoliposomes and deceleration of Na+ transport due to substrate limitation may result in low internal Na+ concentrations. The transport of sodium ions by the Na+-NQR is inevitably linked to the availability of NADH, the electron donor, and ubiquinone, the electron acceptor. The stoichiometry of the reaction catalyzed by Na+-NQR is proposed to be one transported Na+ per electron. For each oxidized NADH, one ubiquinone is reduced to ubiquinol and two sodium ions are translocated [9]. Stoichiometric reduction of quinone with NADH is difficult to
achieve in vitro, probably due to leakage of electrons from reduced cofactors to dioxygen [10]. We reasoned that by improving the coupling ratio between NADH oxidation and quinone reduction, the Na+/electron ratio would also increase, promoting the accumulation of Na+ in the Na+-NQR liposomes. We simultaneously followed the oxidation of NADH and reduction of quinone by detergent-solubilized Na+-NQR in the buffer used for translocation experiments. After initiating NADH oxidation by the addition of Na+-NQR, we observed a rapid decrease in the concentration of NADH as it was oxidized to NAD+. There was a rapid but substoichiometric reduction of quinone, indicating that most of the quinone present in the assay either was not reduced or was rapidly reoxidized by O2 present in the assay buffer (Fig. 1A). In the presence of lactic acid and lactate dehydrogenase, NAD+ formed by the Na+-NQR was reduced to NADH, leading to the formation of an equilibrium state after 100 s in which pyruvate, lactate, NADH, and NAD+ were present (Fig. 1B). Interestingly, the presence of the regenerative system resulted in an extended quinone reduction phase up to 90 s, leading to the almost complete and stable conversion of quinone to quinol in the assay. After 100 s, the concentration of NADH increased again, since complete conversion of quinone to quinol prevented further oxidation of NADH by the Na+-NQR. The regeneration of NADH during turnover resulted in improved (net) quinone reduction rates by the Na+-NQR. In the presence of the regenerative system, NADH-driven uptake of Na+ could be followed with Na+-NQR liposomes loaded with sodium green. On initial addition of NADH an increase in fluorescence was observed which was stimulated by externally added Na+ (Fig. 2A). This suggests that the overall Na+/electron ratio was increased if NADH was continuously provided. Importantly, the rate and the final fluorescence signal intensity were higher in the presence of Na+, indicating binding of Na+ to sodium green in the inner lumen of the proteoliposomes. To demonstrate that the increase in fluorescence intensity resulted from uptake of sodium ions which were transported into the liposomes, a control was performed with an inactive variant of Na+-NQR, NqrE-E95A. The NqrE-E95A variant of Na+-NQR oxidizes NADH and reduces ubiquinone, albeit at lower rates than the wild-type enzyme, but fails to build up a membrane potential when reconstituted in liposomes [2], suggesting that the NQR-NqrE-E95A variant also lacks Na+ transport activity. With liposomes containing this variant, NADH addition promoted an increase in fluorescence which was very similar with or without NaCl added to the external lumen. After 150 s the same final fluorescence intensity was reached, irrespective of the Na+ concentration, indicating that no (net) uptake of Na+ had occurred. However, the increase in sodium green fluorescence observed even with the NqrE-E95A variant required an explanation. We
Fig.1. Spectrophotometric assay for Na+-NQR with continuous regeneration of NADH by lactate dehydrogenase. Rates of NADH oxidation (black trace) and quinone reduction (gray trace) by 10 lg Na+-NQR in the presence of 10 mM NaCl and 20 mM lactic acid were followed spectrophotometrically at 340 and 282 nm, respectively. (A) Without lactate dehydrogenase; (B) with 5.5 U lactate dehydrogenase.
Please cite this article in press as: V. Muras et al., Continuous fluorescence-based measurement of redox-driven sodium ion translocation, Anal. Biochem. (2014), http://dx.doi.org/10.1016/j.ab.2014.05.012
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Fig.2. The fluorescence-based sodium ion translocation assay allows discrimination between active and inactive Na+-NQR. Uptake of Na+ into proteoliposomes was followed continuously using the fluorophor sodium green in the presence of a system for regeneration of NADH. After equilibration, the reaction was started by adding NADH, indicated by the strong perturbation of the fluorescence signal. Black trace, with 50 mM NaCl; gray trace, without added NaCl. Proteoliposomes were prepared with wild-type Na+-NQR (A) or its variant carrying the E95A mutation on subunit NqrE (B). 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221
reasoned that it was caused by substrate turnover (NADH oxidation or Q1 reduction) and analyzed the effect of these substrates on fluorescence emission of liposomes loaded with sodium green. While addition of NADH did not affect sodium green fluorescence, we found that Q1 resulted in a strong quenching of the signal (supplementary material, Fig. S4). Partial dequenching was observed on reduction of ubiquinone to ubiquinol by dithiothreitol (DTT), which explains the fluorescence increase observed with the Na+-NQR in the absence of NaCl or with the NqrE-E95A variant with or without added NaCl. Despite this contribution of quinol formation to the overall increase in sodium green fluorescence, the assay allows discrimination between active and inactive Na+-NQR from a comparison of the kinetic behavior. With wildtype Na+-NQR, two distinct phases were observed, namely a very fast, initial increase in fluorescence, followed by a second, slower phase. In contrast, the fast, initial phase was not observed with the inactive NqrE-E95A variant. This reflected the slower, residual quinol formation activity of the NqrE-E95A variant which was not coupled to Na+ transport. We developed an assay to continuously monitor sodium ion translocation by Na+-NQR reconstituted into liposomes with incorporated sodium green. The following points should be considered when applying this method: First, due to the low affinity of sodium green toward Na+ (KD = 17 mM), the sodium translocation efficiency must be improved in order to accumulate sufficient amounts of sodium ions in the liposomes to detect changes in fluorescence. For the redox-driven Na+-NQR, this was achieved using a regenerative system to continuously re-reduce NAD+ to NADH. Second, ubiquinol formed as a substrate during turnover by the Na+-NQR increased the fluorescence emission intensity of sodium green which could be misinterpreted as Na+ uptake activity of the enzyme. Therefore, a control reaction without added Na+ must always be performed in parallel using the same batch of Na+-NQR liposomes under the same assay conditions. The residual Na+ concentration within the proteoliposomes should be kept as low as possible and must be determined by AAS prior to measurements since the sensitivity depends on the internal sodium concentration in the liposomes at the start of the reaction. In our preparations, the Na+ concentration before addition of NaCl was around 100 lM which was sufficiently low to monitor Na+ binding to the fluorophor. Half-maximum fluorescence emission intensity of sodium green was observed around 20 mM Na+; therefore the assay gives valid results in a concentration range from 0.1–10 mM Na+ in the lumen
of the proteoliposomes. If these points are considered, the assay described here is a valuable tool to rapidly test variants of the Na+-NQR for their Na+ transport activity, and to study the influence of other important parameters such as pH, osmolality, or an applied transmembrane potential on Na+ transport by the Na+-NQR. Clearly, the method can be applied to study other Na+-translocating membrane proteins as well.
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Acknowledgments
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This work was supported by contract research of the BadenWürttemberg Stiftung, Forschungsprogramm P-LS-Meth/4 (to J. S. Q1 and C. S.), and by the Deutsche Forschungsgemeinschaft (DFG) Q2 Grant FR 1321/3-1 (to J.S.).
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Appendix A. Supplementary data
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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ab.2014.05.012.
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References
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[1] M.S. Casutt et al., Localization and function of the membrane-bound riboflavin in the Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) from Vibrio cholerae, J. Biol. Chem. 285 (35) (2010) 27088–27099. [2] O. Juarez et al., Acid residues in the transmembrane helices of the Na+pumping NADH:quinone oxidoreductase from Vibrio cholerae involved in sodium translocation, Biochemistry 48 (40) (2009) 9516–9524. [3] R. Nedielkov et al., NMR reveals double occupancy of quinone-type ligands in the catalytic quinone binding site of the Na+-translocating NADH:quinone oxidoreductase from Vibrio cholerae, J. Biol. Chem. 288 (42) (2013) 30597– 30606. [4] A.C. Gemperli, P. Dimroth, J. Steuber, The respiratory complex I (NDH I) from Klebsiella pneumoniae, a sodium pump, J. Biol. Chem. 277 (37) (2002) 33811– 33817. [5] C. von Ballmoos, P. Dimroth, A continuous fluorescent method for measuring Na+ transport, Anal. Biochem. 335 (2) (2004) 334–337. [6] M. Tao et al., Oxidant-induced formation of a neutral flavosemiquinone in the Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) from Vibrio cholerae, Biochim. Biophys. Acta 1777 (7–8) (2008) 696–702. [7] E.C. Hulme, M.A. Trevethick, Ligand binding assays at equilibrium: validation and interpretation, Br. J. Pharmacol. 161 (6) (2010) 1219–1237. [8] Molecular Probes, product information, sodium green indicator (2001). [9] A.V. Bogachev, R.A. Murtazina, V.P. Skulachev, The Na+/e-stoichiometry of the Na+-motive NADH:quinone oxidoreductase in Vibrio alginolyticus, FEBS Lett. 409 (3) (1997) 475–477. [10] X.D. Pfenninger-Li et al., NADH:ubiquinone oxidoreductase of Vibrio alginolyticus: purification, properties, and reconstitution of the Na+ pump, Biochemistry 35 (20) (1996) 6233–6242.
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No. of Pages 3, Model 5G
5 June 2014 Analytical Biochemistry xxx (2014) xxx–xxx 1
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
2
Notes & Tips
6 4 7 5
Continuous fluorescence-based measurement of redox-driven sodium ion translocation
8
Valentin Muras a, Björn Claussen a, Manikandan Karuppasamy b,c, Christiane Schaffitzel b,c, Julia Steuber a,⇑
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a
Department of Microbiology, University of Hohenheim (Stuttgart), Garbenstrasse 30, 70599 Stuttgart, Germany European Molecular Biology Laboratory (EMBL), Grenoble Outstation, 6 rue Jules Horowitz, 38042 Grenoble, France c Unit for Virus Host-Cell Interactions, University Grenoble Alpes-EMBL-CNRS, 6 rue Jules Horowitz, 38042 Grenoble, France b
a r t i c l e
i n f o
Article history: Received 26 February 2014 Received in revised form 8 May 2014 Accepted 15 May 2014 Available online xxxx Keywords: Sodium transport Sodium green Proteoliposomes Reconstitution Sodium ion-translocating NADH:quinone oxidoreductase (Na+-NQR) Fluorescence
a b s t r a c t Investigation of the mechanism of sodium ion pumping enzymes requires methods to follow the translocation of sodium ions by the purified and reconstituted proteins in vitro. Here, we describe a protocol that allows following the accumulation of Na+ in proteoliposomes by the Na+-translocating NADH: quinone oxidoreductase (Na+-NQR) from Vibrio cholerae using the sodium-sensitive fluorophor sodium green. In the presence of a regenerative system for its substrate NADH, the Na+-NQR accumulates Na+ in the proteoliposomes which is visible as a change in fluorescence. Ó 2014 Elsevier Inc. All rights reserved.
30 31 32 33 34 35 36 37
38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58
The sodium translocating Na+-NQR1 from Vibrio cholerae is a membrane-embedded sodium ion transporter made up of six subunits and belongs to the class of primary sodium ion pumps [1]. The energy that enables transport of Na+ against a chemical gradient is derived from the oxidation of NADH with the final electron acceptor ubiquinone. The exact mechanism of electron transfer-driven sodium ion translocation by the Na+-NQR has not yet been elucidated, though critical subunits and cofactors have been identified [2,3]. Two methods for the determination of in vitro Na+ transport by NADH:quinone oxidoreductases have been described so far. They differ with respect to the Na+ isotope used (23Na or 22Na) and the corresponding detection method (atomic absorption spectroscopy or scintillation counting) [4], but they both represent discontinuous assays where the internal Na+ in proteoliposomes is quantified after separation of external Na+ by a chromatographic step. Here we describe a technique for continuous measurement of Na+ transport by reconstituted Na+-NQR. The method uses the sodium-sensitive fluorophor sodium green that has previously been used for studying Na+ transport by the Na+-translocating
⇑ Corresponding author. Fax: +49 711 45922238. E-mail address: [email protected] (J. Steuber). Abbreviations used: Na + -NQR, sodium ion-translocating NADH:quinone oxidoreductase; Q1, ubiquinone-1; DTT, dithiothreitol. 1
F1FO ATPase [5]. In the case of Na+-NQR, the method critically depends on the continuous regeneration of NADH with the help of lactate dehydrogenase and lactate. Na+-NQR was reconstituted into proteoliposomes using a dilution method as described in [6]. One milligram of Na+-NQR and 0.6 lg sodium green (S-6900, Molecular Probes) were added to a lipid film consisting of 40 mg of L-a-phosphatidylcholine (from soybean, Type II-S, 14–23% as choline) that were dried with a rotary evaporator in a round bottom flask. Subsequently the liposome/micelle mixture was treated with a tip sonicator (3 20 s). Reconstitution of Na+-NQR into liposomes was achieved by diluting the liposome/micelle mixture with a 50-fold volume of reconstitution buffer. The proteoliposomes were collected by ultracentrifugation (150,000g, 45 min, 4 °C) and resuspended in 2 ml reconstitution buffer. Excess sodium green was then removed from the external buffer by gel filtration on a NAP-10 column (Amersham Biosciences). Rates of NADH oxidation and ubiquinone-1 (Q1) reduction of Na+-NQR were determined as described [6], with the following modifications: NADH and Q1 (each 100 lM) were mixed in 200 mM KCl, 5% glycerol, 10 mM Tris/HCl, pH 8.0, containing 20 mM lactic acid and 5.5 U L-lactate dehydrogenase (from rabbit muscle, Roche). The reaction was started by the addition of 10 lg Na+-NQR.
http://dx.doi.org/10.1016/j.ab.2014.05.012 0003-2697/Ó 2014 Elsevier Inc. All rights reserved.
Please cite this article in press as: V. Muras et al., Continuous fluorescence-based measurement of redox-driven sodium ion translocation, Anal. Biochem. (2014), http://dx.doi.org/10.1016/j.ab.2014.05.012
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Notes & Tips / Anal. Biochem. xxx (2014) xxx–xxx
Proteoliposomes with incorporated sodium green (200 ll) were mixed with 1 ml reconstitution buffer containing 1 mM Q1, 20 mM L-lactic acid, and 0.1 mg ml 1 L-lactate dehydrogenase (550 U mg 1). Na+ uptake was initialized by the addition of 0.5 mM NADH. Changes in sodium green fluorescence were followed in a Horiba (Fluorolog) fluorospectrophotometer with an excitation wavelength of 488 nm and an emission wavelength of 540 nm. For normalization of the data, the final fluorescence emission intensity at the end of the reaction without added NaCl was set as 100%. The dissociation constant (KD) of sodium green (20 nM) toward sodium was determined in reconstitution buffer from the fluorescence increase at 535 nm at increasing Na+ concentrations. Data were fitted to a one-site binding model equation Y = (Fmax X)/(Kd + X) using the curve fit routine of the software Origin, where X = concentration of the free ligand (Na+), Fmax = maximum fluorescence emission, and Kd = equilibrium dissociation constant [7]. To follow sodium ion transport in the course of NADH oxidation by the Na+-NQR, the membrane-impermeable form of sodium green [8] was used to ensure that an increase of fluorescence exclusively resulted from Na+ that was accumulated in the proteoliposomes. The proteoliposomes had an average size of approximately 50 nm in diameter and maintained a membrane potential generated by the Na+-NQR, confirming their integrity (supplementary material, Figs. S1 and S2). The tightness of the liposomes was confirmed using the sodium ionophor gramicidin D. Addition of 10 mM NaCl to intact proteoliposomes did not affect sodium green fluorescence. Only on addition of 5 lM gramicidin, the fluorescence increased by approximately 10% which resulted from binding of Na+ to sodium green entrapped in the liposomes. The increase by 10% is in accordance with the observed fluorescence increase of sodium green followed in a titration from 0 to 100 mM NaCl (supplementary material, Fig. S3). First trials to monitor translocation of sodium ions into Na+-NQR liposomes with incorporated sodium green were not successful. There was no increase in fluorescence, and we reasoned that the amount of accumulated sodium ions during steady state in the Na+-NQR liposomes was too low, considering a KD of sodium green toward Na+ of 17 mM (supplementary material, Fig. S3). Both leakage of Na+ from the proteoliposomes and deceleration of Na+ transport due to substrate limitation may result in low internal Na+ concentrations. The transport of sodium ions by the Na+-NQR is inevitably linked to the availability of NADH, the electron donor, and ubiquinone, the electron acceptor. The stoichiometry of the reaction catalyzed by Na+-NQR is proposed to be one transported Na+ per electron. For each oxidized NADH, one ubiquinone is reduced to ubiquinol and two sodium ions are translocated [9]. Stoichiometric reduction of quinone with NADH is difficult to
achieve in vitro, probably due to leakage of electrons from reduced cofactors to dioxygen [10]. We reasoned that by improving the coupling ratio between NADH oxidation and quinone reduction, the Na+/electron ratio would also increase, promoting the accumulation of Na+ in the Na+-NQR liposomes. We simultaneously followed the oxidation of NADH and reduction of quinone by detergent-solubilized Na+-NQR in the buffer used for translocation experiments. After initiating NADH oxidation by the addition of Na+-NQR, we observed a rapid decrease in the concentration of NADH as it was oxidized to NAD+. There was a rapid but substoichiometric reduction of quinone, indicating that most of the quinone present in the assay either was not reduced or was rapidly reoxidized by O2 present in the assay buffer (Fig. 1A). In the presence of lactic acid and lactate dehydrogenase, NAD+ formed by the Na+-NQR was reduced to NADH, leading to the formation of an equilibrium state after 100 s in which pyruvate, lactate, NADH, and NAD+ were present (Fig. 1B). Interestingly, the presence of the regenerative system resulted in an extended quinone reduction phase up to 90 s, leading to the almost complete and stable conversion of quinone to quinol in the assay. After 100 s, the concentration of NADH increased again, since complete conversion of quinone to quinol prevented further oxidation of NADH by the Na+-NQR. The regeneration of NADH during turnover resulted in improved (net) quinone reduction rates by the Na+-NQR. In the presence of the regenerative system, NADH-driven uptake of Na+ could be followed with Na+-NQR liposomes loaded with sodium green. On initial addition of NADH an increase in fluorescence was observed which was stimulated by externally added Na+ (Fig. 2A). This suggests that the overall Na+/electron ratio was increased if NADH was continuously provided. Importantly, the rate and the final fluorescence signal intensity were higher in the presence of Na+, indicating binding of Na+ to sodium green in the inner lumen of the proteoliposomes. To demonstrate that the increase in fluorescence intensity resulted from uptake of sodium ions which were transported into the liposomes, a control was performed with an inactive variant of Na+-NQR, NqrE-E95A. The NqrE-E95A variant of Na+-NQR oxidizes NADH and reduces ubiquinone, albeit at lower rates than the wild-type enzyme, but fails to build up a membrane potential when reconstituted in liposomes [2], suggesting that the NQR-NqrE-E95A variant also lacks Na+ transport activity. With liposomes containing this variant, NADH addition promoted an increase in fluorescence which was very similar with or without NaCl added to the external lumen. After 150 s the same final fluorescence intensity was reached, irrespective of the Na+ concentration, indicating that no (net) uptake of Na+ had occurred. However, the increase in sodium green fluorescence observed even with the NqrE-E95A variant required an explanation. We
Fig.1. Spectrophotometric assay for Na+-NQR with continuous regeneration of NADH by lactate dehydrogenase. Rates of NADH oxidation (black trace) and quinone reduction (gray trace) by 10 lg Na+-NQR in the presence of 10 mM NaCl and 20 mM lactic acid were followed spectrophotometrically at 340 and 282 nm, respectively. (A) Without lactate dehydrogenase; (B) with 5.5 U lactate dehydrogenase.
Please cite this article in press as: V. Muras et al., Continuous fluorescence-based measurement of redox-driven sodium ion translocation, Anal. Biochem. (2014), http://dx.doi.org/10.1016/j.ab.2014.05.012
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Fig.2. The fluorescence-based sodium ion translocation assay allows discrimination between active and inactive Na+-NQR. Uptake of Na+ into proteoliposomes was followed continuously using the fluorophor sodium green in the presence of a system for regeneration of NADH. After equilibration, the reaction was started by adding NADH, indicated by the strong perturbation of the fluorescence signal. Black trace, with 50 mM NaCl; gray trace, without added NaCl. Proteoliposomes were prepared with wild-type Na+-NQR (A) or its variant carrying the E95A mutation on subunit NqrE (B). 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221
reasoned that it was caused by substrate turnover (NADH oxidation or Q1 reduction) and analyzed the effect of these substrates on fluorescence emission of liposomes loaded with sodium green. While addition of NADH did not affect sodium green fluorescence, we found that Q1 resulted in a strong quenching of the signal (supplementary material, Fig. S4). Partial dequenching was observed on reduction of ubiquinone to ubiquinol by dithiothreitol (DTT), which explains the fluorescence increase observed with the Na+-NQR in the absence of NaCl or with the NqrE-E95A variant with or without added NaCl. Despite this contribution of quinol formation to the overall increase in sodium green fluorescence, the assay allows discrimination between active and inactive Na+-NQR from a comparison of the kinetic behavior. With wildtype Na+-NQR, two distinct phases were observed, namely a very fast, initial increase in fluorescence, followed by a second, slower phase. In contrast, the fast, initial phase was not observed with the inactive NqrE-E95A variant. This reflected the slower, residual quinol formation activity of the NqrE-E95A variant which was not coupled to Na+ transport. We developed an assay to continuously monitor sodium ion translocation by Na+-NQR reconstituted into liposomes with incorporated sodium green. The following points should be considered when applying this method: First, due to the low affinity of sodium green toward Na+ (KD = 17 mM), the sodium translocation efficiency must be improved in order to accumulate sufficient amounts of sodium ions in the liposomes to detect changes in fluorescence. For the redox-driven Na+-NQR, this was achieved using a regenerative system to continuously re-reduce NAD+ to NADH. Second, ubiquinol formed as a substrate during turnover by the Na+-NQR increased the fluorescence emission intensity of sodium green which could be misinterpreted as Na+ uptake activity of the enzyme. Therefore, a control reaction without added Na+ must always be performed in parallel using the same batch of Na+-NQR liposomes under the same assay conditions. The residual Na+ concentration within the proteoliposomes should be kept as low as possible and must be determined by AAS prior to measurements since the sensitivity depends on the internal sodium concentration in the liposomes at the start of the reaction. In our preparations, the Na+ concentration before addition of NaCl was around 100 lM which was sufficiently low to monitor Na+ binding to the fluorophor. Half-maximum fluorescence emission intensity of sodium green was observed around 20 mM Na+; therefore the assay gives valid results in a concentration range from 0.1–10 mM Na+ in the lumen
of the proteoliposomes. If these points are considered, the assay described here is a valuable tool to rapidly test variants of the Na+-NQR for their Na+ transport activity, and to study the influence of other important parameters such as pH, osmolality, or an applied transmembrane potential on Na+ transport by the Na+-NQR. Clearly, the method can be applied to study other Na+-translocating membrane proteins as well.
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Acknowledgments
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This work was supported by contract research of the BadenWürttemberg Stiftung, Forschungsprogramm P-LS-Meth/4 (to J. S. Q1 and C. S.), and by the Deutsche Forschungsgemeinschaft (DFG) Q2 Grant FR 1321/3-1 (to J.S.).
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Appendix A. Supplementary data
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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ab.2014.05.012.
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References
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[1] M.S. Casutt et al., Localization and function of the membrane-bound riboflavin in the Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) from Vibrio cholerae, J. Biol. Chem. 285 (35) (2010) 27088–27099. [2] O. Juarez et al., Acid residues in the transmembrane helices of the Na+pumping NADH:quinone oxidoreductase from Vibrio cholerae involved in sodium translocation, Biochemistry 48 (40) (2009) 9516–9524. [3] R. Nedielkov et al., NMR reveals double occupancy of quinone-type ligands in the catalytic quinone binding site of the Na+-translocating NADH:quinone oxidoreductase from Vibrio cholerae, J. Biol. Chem. 288 (42) (2013) 30597– 30606. [4] A.C. Gemperli, P. Dimroth, J. Steuber, The respiratory complex I (NDH I) from Klebsiella pneumoniae, a sodium pump, J. Biol. Chem. 277 (37) (2002) 33811– 33817. [5] C. von Ballmoos, P. Dimroth, A continuous fluorescent method for measuring Na+ transport, Anal. Biochem. 335 (2) (2004) 334–337. [6] M. Tao et al., Oxidant-induced formation of a neutral flavosemiquinone in the Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) from Vibrio cholerae, Biochim. Biophys. Acta 1777 (7–8) (2008) 696–702. [7] E.C. Hulme, M.A. Trevethick, Ligand binding assays at equilibrium: validation and interpretation, Br. J. Pharmacol. 161 (6) (2010) 1219–1237. [8] Molecular Probes, product information, sodium green indicator (2001). [9] A.V. Bogachev, R.A. Murtazina, V.P. Skulachev, The Na+/e-stoichiometry of the Na+-motive NADH:quinone oxidoreductase in Vibrio alginolyticus, FEBS Lett. 409 (3) (1997) 475–477. [10] X.D. Pfenninger-Li et al., NADH:ubiquinone oxidoreductase of Vibrio alginolyticus: purification, properties, and reconstitution of the Na+ pump, Biochemistry 35 (20) (1996) 6233–6242.
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Please cite this article in press as: V. Muras et al., Continuous fluorescence-based measurement of redox-driven sodium ion translocation, Anal. Biochem. (2014), http://dx.doi.org/10.1016/j.ab.2014.05.012
