- Email: [email protected]
Modifications by Na+ and K+, and the site of Ag+ inhibition in the Na+-translocating NADH-quinone reductase from a marine Vibrio alginolyticus
Biochimica et Biophysica Acta, 1183 (1993) 201-205 © 1993 Elsevier Science Publishers B.V. All rights reserved 0005-2728/93/$06.00
201
BBABIO 43919
Modifications by Na + and K +, and the site of Ag + inhibition in the Na÷-translocating NADH-quinone reductase from a marine Vibrio alginolyticus Tsutomu Unemoto *, Tatsuya Ogura and Maki Hayashi Laboratory of Membrane Biochemistry, Faculty of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263 (Japan) (Received 22 April 1993)
Key words: Monovalent cation; Modification; Sodium ion pump; NADH-quinone reductase; Argentous inhibition; Silver; (Marine bacterium); (V. alginolyticus) Modifications by monovalent cations and the site of Ag + inhibition in the Na÷-translocating NADH-quinone reductase from a marine Fibrio alginolyticus were examined by kinetic methods using a purified/3-subunit and a whole a/3y-complex. The activity of FAD-containing /3-subunit for 2-methyl-l,4-naphthoquinone (menadione) and ubiquinone-1 (Q-l) was stimulated by either Na ÷ or K ÷ due to the decrease in Kin, which was saturated at about 200 mM. The /3-subunit in the complex, however, was already in a state of high-affinity for these quinones, even in the absence of salts, and the activity was further stimulated by either Na + or K ÷, due to the increase in maximum velocity, which was saturated at about 200 mM. The formation of ubiquinol-1 from Q-1 catalyzed by the complex was not fully activated by K + alone and specifically required Na ÷ for maximum activation. Since the requirement for Na + was greatly decreased in the presence of 200 mM K +, the Na+-dependent reaction catalyzed by FMN-containing a-subunit was saturated by low concentrations of Na + when the /3-subunit was in a activated state. Ag ÷, a specific inhibitor of this enzyme, was found to act as a competitive inhibitor for quinones at the active site of the/3-subunit. Since the K i for Ag + (2 nM) was extraordinarily lower than the K m for menadione (15/zM), Ag ÷ was considered to be useful as a specific reagent for the analysis of the active site of the/3-subunit.
Introduction The respiratory chain of a marine bacterium, 14brio alginolyticus, contains an N a + - d e p e n d e n t N A D H quinone reductase that functions as an electrogenic Na + p u m p (for reviews, see Refs. 1,2). The Na+-de pendent activation of the respiratory-chain-linked N A D H - q u i n o n e reductase was first demonstrated in V. alginolyticus [3]. This enzyme was purified and was found to be composed of three subunits, a, /3 and y, with the apparent molecular masses of 52, 46 and 32 kDa, respectively [4-6]. The FAD-containing /3-subunit reacted with N A D H and reduced 2-methyl-l,4naphthoquinone (menadione) and ubiquinone-1 ( Q - l ) by a one-electron transfer pathway, which was stimulated by either Na ÷ or K + [4]. On the other hand, the whole complex of N A D H - q u i n o n e reductase (a/3ycomplex) reduced Q-1 to ubiquinol-1, which was
strongly activated by Na + but not by K + [4]. Menadione, however, was reduced to semiquinone radicals by the complex and the activity was stimulated by either Na ÷ or K ÷. Apparently, menadione directly interacted with the/3-subunit within the complex. Thus, the reaction catalyzed by the FMN-containing a-subunit specifically required Na ÷ for the activity and was assigned to be the coupling site of Na ÷ translocation [6]. Although the activities catalyzed by the /3-subunit and by the complex were differently modified by monovalent cations, the mechanisms of these modifications have not been studied in detail. Furthermore, the complex was specifically inhibited by nanomolar concentrations of Ag ÷ [7], but the m o d e of inhibitory action of Ag ÷ remained unsolved. Therefore, the modifications by monovalent cations and the site of Ag ÷ inhibition in the Na ÷-translocating N A D H - q u i n o n e reductase were analyzed by kinetic methods using the purified /3-subunit and the a/3y-complex. Materials and Methods
* Corresponding author. Abbreviations: Menadione, 2-methyl-1,4-naphthoquinone; Q-l, ubiquinone-1.
Chemicals. Liponox DCH, an alkyl polyoxyethylene ether detergent [4], was kindly supplied by Lion
202 (Kanagawa, Japan). Ubiquinone-1 (Q-l) was kindly supplied by Eizai (Tokyo, Japan). Other reagents used were of the highest commercial grade available. Enzyme assays. Menadione reductase activity was assayed at 30°C from the decrease in absorbance at 340 nm with menadione as an electron acceptor. The standard assay mixture contained 0.2 mM NADH, 0.1 mM menadione, 200 mM NaCI, 0.1% (w/v) Liponox DCH, 20 mM Tris-HCl (pH 8.0) and enzyme in a total volume of 1.0 ml. The reaction was started by the addition of enzyme and the initial velocity was calculated based on the absorption coefficient of 6.22 mM-1 cm -1. Quinone reductase activity was assayed at 30°C by following the formation of ubiquinol-1 from Q-1 as previously described [2]. The standard assay mixture contained 0.1 mM NADH, 15/zM Q-l, 200 mM NaCl, 0.015% Liponox DCH, 20 mM Tris-HCI (pH 8.0) and enzyme in a total volume of 2.0 ml. Changes in absorbance difference at the wavelength pair, 242-270.5 nm, were recorded with a Hitachi 557 two wavelength spectrophotometer, and the rate of ubiquinol-1 formation was calculated based on the absorption coefficient of 9.6 mM -1 cm-1. When the /~-subunit was used as the enzyme, Q-1 reductase activity was measured from the decrease in absorbance at 340 nm. To examine the effects of Na ÷ and K +, chloride salts were used throughout the experiments. For kinetic analyses, the concentrations of NADH and Liponox DCH in the reaction mixture were adjusted to 0.1 mM and 0.015%, respectively, in all experiments. For the determination of inhibitory effect of Ag ÷, the enzyme was preincubated with Ag ÷ for 3 min, and then the reaction was started by the addition of electron acceptor and NADH. 1 U of activity was defined as the amount of enzyme catalyzing the oxidation of 1 izmol NADH or the reduction of 1 ~mol Q-1 in 1 rain. Purification of enzymes. The growth of V. alginolyticus 138-2 and the preparation of the membrane fraction were performed as previously described [7]. Purifications of the Na÷-dependent NADH-quinone reductase (afly-complex) and the fl-subunit were performed as previously described [4-6] with slight modifications. For the extraction of quinone reductase from the membrane, pretreatment of the membrane with 2% (w/w) sodium cholate [4] was omitted to minimize enzyme inactivation, and the enzyme was extracted from the membrane with 1% (w/v) Liponox DCH containing 10% (w/v) glycerol, 20 mM Tris-HCl (pH 7.0), 0.1 mM EDTA, 10 mM NaC1 at the concentration of 5 mg membrane protein/ml. The Liponox extracts were applied to DEAE-Sephacel column and the active fraction was eluted with 0.3 M NaC1 (DEAE-Sephacel fraction) as described [5]. The DEAE-Sephacel fraction was further purified with TSK-gel DEAE-5PW
column (2.15 x 15 cm, Toyo Soda, Tokyo), where the quinone reductase complex and the fl-subunit were separated as described [6]. Each fraction was further purified by a HiLoad Superdex 200 column (1.6 × 60 cm, Pharmacia) with a buffer system containing 10 mM Tris-HC1 (pH 7.0), 0.1% (w/v) Liponox DCH, 5% (w/v) glycerol. Other methods. SDS-PAGE was performed by using discontinuous buffer system of Laemmli [8]. For kinetic analyses, the value of each experimental point was obtained as the mean of at least three determinations. The line of best fit for each set of double-reciprocal plots, and kinetic constants and their standard errors were calculated using the program Enzyme Kinetics (Trinity Software, Campton, NH, USA) with a Macintosh computer. Protein was determined by the method of Bradford [9] with bovine serum albumin as standard. Results
Purification of enzymes The Na+-translocating NADH-quinone reductase complex and the fl-subunit were purified by a simple modified method as described in Materials and Methods. The purified quinone reductase complex had a specific activity of about 65 U / m g protein as measured with the quinone reductase assay, and the two preparations of the /~-subunit had specific activities of 92 and 220 U / m g protein, respectively, as measured with the menadione reductase assay. On SDS-PAGE, the quinone reductase complex contained a-, fl- and ysubunits as major proteins, and the fl-subunit was not contaminated with a- and y-subunits. Although the specific activities of the present enzymes were lower than those reported in our previous paper [6], the present enzymes were suited for investigating enzymatic properties and were used in the following experiments.
Modifications of the fl-subunit by monovalent cations The fl-subunit catalyzed the reduction of menadione by a one-electron transfer pathway to produce semiquinone radicals [4]. Under the standard assay conditions, the activity in the absence of salts corresponding to 30.6 U / m g protein was stimulated about 3-fold in the presence of 200 mM NaCI or KC1. The stimulating effect of NaCl was slightly better than that of KCI especially at concentrations above 200 mM, but no significant difference was observed between NaC1 and KCI up to 200 raM, Thus, the/~-subunit showed no specific requirement for monovalent cations for the stimulation of menadione reductase activity. Fig. 1 shows the effect of NaCI on the activity of the fl-subunit for menadione. The increase in NaCl decreased K m values for menadione without affecting
203
0.12 ~ 0.09
oo,
_0.U I/,°,
(0)~0/25) / /
"'c"°-
//
~. 002I ~ ~ ~ I
0.02
"
NaCI lmM)
I
I
I
0.04 0.06 0.08 llMenadlone [,uM"I]
I
t
0.05
0.10
Fig. 1. Double-reciprocal plots of initial velocity vs. menadione concentration at fixed levels of NaCl. The reaction mixture contained 0.1 mM NADH, 0.015% Liponox DCH, 20 mM Tris-HCI (pH 8.0) and the fl-subunit in the presence of varied concentrations of menadione (/zM) and NaCl (mM) indicated in the figure. Initial velocity (v) was expressed in U / m g protein. The /3-subunit with a specific activity of 92 U / m g protein for menadione under the standard assay conditions was used.
" ~ ' ~ 1200) I
I
0.10
I
0.15
0.20
t/Q-1 [pM"1] Fig. 2. Double-reciprocal plots of initial velocity vs. Q-1 concentration at fixed levels of NaCL The reaction was carried out under the conditions described in Fig. 1, except that Q-1 (p.M) was used as the electron acceptor. The /3-subunit with a specific activity of 220 U / m g protein for menadione was used. In the absence of NaCl, the other four data points lie outside the frame.
Modifications of the NADH-quinone reductase complex by monovalent cations the maximum velocity (V). The apparent K m for menadione in the absence of salts was calculated to be 196 _ 18/zM, which decreased to 15.3 + 1.4/~M in the presence of 200 mM NaCl, whereas the apparent V was maintained at about 75 U / m g protein. Therefore, the stimulating effect of NaC1 was due to the decrease in K m for menadione. Essentially the same results were obtained with KCI (data not shown). The /3-subunit also reduced Q-1 by a one-electron transfer pathway without producing detectable amounts of ubiquinol-1 [4]. When the activity for Q-1 was determined from the decrease in NADH under the standard quinone reductase assay, the activity in the absence of salts (4.0 U / m g protein) was stimulated about 10-fold in the presence of either 200 mM NaCl or KC1. In this case, the salt-dependent activation curve did not conform to Michaelis-Menten kinetics and the stimulating effects of salts were insufficient, especially at concentrations below 50 mM, exhibiting a rather sigmoid shape. Fig. 2 shows the effect of NaCl on the activity of the /3-subunit for Q-1. In the absence of salts, the K m value for Q-1 corresponded to 93/~M and the V value approached 1 /3 of that obtained in the presence of 200 mM NaCl (150 U / m g protein). At 75 mM NaC1 and above, K m values for Q-1 decreased with the increase in NaCl without significantly affecting V. In the presence of 200 mM NaCl, the K m for Q-1 was estimated to be 24.7 5-4.8 /~M. Thus, the stimulation of the /3-subunit for Q-1 at concentrations below 75 mM was due to both the decrease in K m and the increase in V. However, the mode of activation at higher concentrations was mainly due to the decrease in K m for Q-1 (Fig. 2), which was very similar to that for menadione (Fig. 1). Essentially the same results were obtained with KC1 (data not shown).
Under the standard assay conditions, the activity of NADH-quinone reductase complex for menadione in the absence of salts (26.6 U / m g protein) was stimulated about 5-fold in the presence of 200 mM NaC1 or KCI. Fig. 3 shows the effects of NaCl on the activity of the complex for menadione. In contrast to the case of the/3-subunit, NaCl stimulated the activity of the complex by increasing V without affecting the K m for menadione. The V value approached to 182 U / m g protein at 200 mM NaCl or KCI. The Km value in the absence of salts was estimated to be 15.7 5- 0.9 /~M, which was identical with that of the 3-subunit in the presence of 200 mM NaC1 or KC1. Therefore, the /3-subunit was maintained in a state of high-affinity for menadione within the complex even in the absence of salts. Further stimulation of the/3-subunit in the complex was due to the salt-dependent increase in V. In contrast to the case of menadione, the complex showed no activity for Q-1 in the absence of salts and specifically required Na + for maximum activity (Fig. 4).
i00.t ..c, ,m., , 0 y _0041 /
/ (200)
oo8-oo3
o o3
l/Menadlone
[p,M"1]
Fig. 3. Double-reciprocal plots of initial velocity vs. menadione concentration at fixed levels of NaCI. The reaction was carried out as described in Fig. 1, except that the complex with a specific activity of 65 U / m g protein for Q-1 was used as the enzyme. The initial velocity was determined from the decrease in NADH.
204 8O 200mM KCI
I¢
Q.
+ NaCl
Ag+(
NaCI
60
n
~
-"" 0 . 0 6
O~
Io
•~- 0 . 0 4
M
.-_ 4 0
C
-~ 20 I
~ 0.02"
KCl
i
o
,
,
,
J
50 100 150 Salt concentration (raM)
o
200
Fig. 4. Effects of monovalent cations on the activity of the complex for Q-1. The activity was assayed under the conditions of standard quinone reductase assay, except that the chloride salts of monvalent cations were varied as described in the figure.
The concentrations of NaC1 and KC1 to give a halfmaximum velocity (M1/2) were estimated to be about 54 and 78 mM, respectively. In the presence of 200 mM KCI, the MI/2 for NaCI was reduced to 4.3 mM. These results were essentially similar to those observed with the membrane-bound N A D H oxidase from I4 alginolyticus [10]. Thus, the Na÷-dependent NADHquinone reductase segment in the respiratory chain of I4 alginolyticus apparently acted as a limiting step in the salt-dependent activation. Fig. 5 shows the effects of NaC1 on the activity of the complex for Q-1. The primary plots all intersected at a point below the baseline and the activation by NaCI was due to the increase in both the K m for Q-1 and I/". The K m and V values at 200 mM NaCI were calculated to be 12.9 + 1.8 /zM and 110 + 9 U / m g protein. Such type of activation was not observed with KCI. In the presence of 200 mM KC1, however, the K m for Q-1 was estimated to be 3.0 + 0.3 /zM, but the V value was only 12% of that in the presence of 200 mM NaC1. Under that conditions, the addition of small amounts of NaCI greatly increased V value, which was accompanied with the increase in K m. Thus, in the
0.12
..c,,m.l
I
I
I
Fig. 6. Effects of Ag ÷ on the activity of the complex for menadione.
The reaction was carried out as described in Fig. 3, except that the reaction mixture contained 200 mM NaCI and Ag +(nM) as indicated in the figure. presence of 200 mM KCI, the specific requirement for Na ÷ of the reaction catalyzing the formation of ubiquinol-1 was saturated with low concentrations of Na ÷ as was expected from Fig. 4.
The site of Ag ÷ inhibition Since the complex is strongly inhibited by nanomolar concentrations of Ag ÷ [7], the effects of Ag ÷ on the activity of the complex for menadione were examined. As shown in Fig. 6, Ag + acted as a competitive inhibitor for menadione. The secondary plots of slope vs. Ag ÷ concentration gave a straight line in this concentration range and the K i value was estimated to be 1.8 nM. Although this experiments were carried out in the presence of 200 mM NaC1, an identical K i value was obtained in the presence of 200 mM KC1. Therefore, the inhibitory effect of Ag ÷ was not influenced by the species of monovalent cations. Furthermore, such a strong Ag ÷ inhibition was observed with the complex even in the absence of salts. When the /3-subunit was used as the enzyme, the same type of competitive inhibition was observed in the presence of 200 mM NaC1 or KCI, and the K i value for Ag ÷ was estimated to be 2.1 nM. In the absence of salts, however, higher concentrations of Ag + were required to competitively inhibit the activity. The K i Na+/K +
0.08
I
0.02 0.04 0.06 0.08 0.10 llMenadione [laM-1]
~ A~
K÷
~ 0.04'
I
-0.20
I
0.20 0.40 11Q-1 [pM"1]
Fig. 5. Double-reciprocal plots of initial velocity vs. Q-1 concentration at fixed levels of NaCI. T h e reaction was carried out as described in Fig. 3, except that Q-1 was used as the electron acceptor. T h e initial velocity was determined from the formation of ubiquinol-1.
AJ~: High-affinity type A ~ V : High-affinity and
high-velocity type
Fig. 7. Modifications by monovalent cations of the/3-subunit and the N A D H - q u i n o n e reductase complex.
205 value for Ag + in the absence of salts was estimated to be 320 nM, which was more than 100-fold higher than that in the presence of 200 mM NaCI or KC1. The inhibitory effects of Ag + for Q-1 reduction were essentially similar to those for menadione reduction, and the strong Ag + inhibition was observed only in the presence of salts. These results indicated that Ag ÷ interacted at the quinone-binding site of the/3-subunit and that the inhibitory effects of Ag ÷ were directly related to the affinity of the/3-subunit for quinone substrates.
Discussion From the kinetic results of salt modifications of the /3-subunit and the a/3y-complex, the stimulating effects of monovalent cations may be formulated as shown in Fig. 7. The purified /3-subunit with a low affinity for menadione and Q-1 in the absence of salts increases its affinity by the increase in Na ÷ or K ÷, which is saturated at about 200 mM. On the other hand, the fl-subunit in the complex is already in a state of high-affinity for these substrates (designated as A/3 in the figure) even in the absence of salts. The activity of the/3-subunit in the complex is further stimulated by the addition of Na + or K ÷ due to the increase in V. Thus, in the presence of saturated concentrations of Na + or K + (about 200 mM), the/3-subunit in the complex is maintained in a state of high-affinity and high-velocity type (A/3V). The formation of ubiquinol-1 catalyzed by the complex, however, is not fully activated by K ÷ alone and specifically requires Na ÷ for maximum activation. Since the requirement for Na ÷ greatly decreases in the presence of 200 mM K +, the Na+-dependent reaction catalyzed by a-subunit is saturated by low concentrations of Na + when the /3-subunit is in an activated state. Thus, the necessity of high concentrations of Na ÷ for the activation of the complex may well be explained by the requirement of about 200 mM Na ÷ for full activation of the/3-subunit in the complex. It is interesting to note that the purified /3-subunit with a low affinity for quinones is converted into a high-affinity type as a constituent part of the complex even in the absence of salts. Thus, the mode of salt modification of the fl-subunit was altered depending on the conditions of its existence. We previously reported that the affinity of the /3-subunit for Q-1 is modified by the y-subunit [6]. Therefore, the y-subunit seems to play an important role for the activation of the/3-subunit in the complex. The Na+-translocating NADH-quinone reductase is very sensitive to Ag + [7]. In this paper, the site of Ag + inhibition was located at the quinone-binding site of the /3-subunit. The strength of Ag + inhibition was correlated with the affinity of the /3-subunit for quinones, and the K i value for Ag + (2 nM) was extraordinarily lower than the K m value for mena-
dione (15/zM). Thus, the affinity of Ag + for the active site was 7500-fold higher than that of menadione. Ag + does not have any structural resemblance to quinones and its inhibitory effects are prevented by thiol compounds. Therefore, Ag + is very likely to interact with a specified thiol group within the active site that directly participates in the catalytic activity. Ag + may be useful as a specific reagent for the analysis of active site of the/3-subunit. Semeykina and Skulachev [11] reported that very low concentrations of Ag + inhibit the uphill Na ÷ transport coupled to the Na +-pumping NADH-quinone reductase and terminal oxidase of Bacillus F l U . Ag + was also shown to induce an increse in Na ÷ permeability of the membrane vesicles. When 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO), a specific inhibitor of Na + pump, was added before Ag +, such an Ag+-in duced increase in Na + permeability was prevented. From these, it was suggested that Ag + uncoupled the electron and Na + transport so that the Ag+-modified NADH-quinone reductase operated as an Na + channel rather than an Na + pump. In the Na+-translocat ing NADH-quinone reductase from V. alginlolyticus, the Na+-dependent reaction catalyzed by the FMNcontaining a-subunit was assigned to be the coupling site of Na + transport, which was specifically inhibited by HQNO [1,2,6]. Since Ag + and HQNO interact at a quite different site within the complex, the formation of Na + channel by the Ag+-modified enzyme is unlikely with this enzyme. Further studies are required to solve these problems.
Acknowledgement A part of this work was supported by a grant-in-aid from the Japanese Ministry of Educations, Science and Culture.
References 1 Unemoto, T., Tokuda, H. and Hayashi, M. (1990) in The Bacteria, Vol. XII, Bacterial Energetics (Kruiwich, T.A., ed.), pp. 33-54, Academic Press, San Diego. 2 Unemoto, T. and Hayashi, M. (1989) J. Bioenerg. Biomembr. 21, 649-662. 3 Unemoto, T. and Hayashi, M. (1979) J. Biochem. 85, 1461-1467. 4 Hayashi, M. and Unemoto, T. (1984) Biochim. Biophys. Acta 767, 470-478. 5 Hayashi, M. and Unemoto, T. (1986) FEBS I_#.tt. 202, 327-330. 6 Hayashi, M. and Unemoto, T. (1987) Biochim. Biophys. Acta 890, 47-54. 7 Hayashi, M., Miyoshi, T., Sato, M. and Unemoto, T. (1992) Biochim. Biophys. Acta 1099, 145-151. 8 Laemmli, U.K. (1970) Nature 227, 680-685. 9 Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. 10 Unemoto, T., Hayashi, M. and Hayashi, M. (1977) J. Biochem. 82, 1389-1395. 11 Semeykina, A.L. and Skulachev, V.P. (1990) FEBS Lett. 269, 69-72.
201
BBABIO 43919
Modifications by Na + and K +, and the site of Ag + inhibition in the Na÷-translocating NADH-quinone reductase from a marine Vibrio alginolyticus Tsutomu Unemoto *, Tatsuya Ogura and Maki Hayashi Laboratory of Membrane Biochemistry, Faculty of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263 (Japan) (Received 22 April 1993)
Key words: Monovalent cation; Modification; Sodium ion pump; NADH-quinone reductase; Argentous inhibition; Silver; (Marine bacterium); (V. alginolyticus) Modifications by monovalent cations and the site of Ag + inhibition in the Na÷-translocating NADH-quinone reductase from a marine Fibrio alginolyticus were examined by kinetic methods using a purified/3-subunit and a whole a/3y-complex. The activity of FAD-containing /3-subunit for 2-methyl-l,4-naphthoquinone (menadione) and ubiquinone-1 (Q-l) was stimulated by either Na ÷ or K ÷ due to the decrease in Kin, which was saturated at about 200 mM. The /3-subunit in the complex, however, was already in a state of high-affinity for these quinones, even in the absence of salts, and the activity was further stimulated by either Na + or K ÷, due to the increase in maximum velocity, which was saturated at about 200 mM. The formation of ubiquinol-1 from Q-1 catalyzed by the complex was not fully activated by K + alone and specifically required Na ÷ for maximum activation. Since the requirement for Na + was greatly decreased in the presence of 200 mM K +, the Na+-dependent reaction catalyzed by FMN-containing a-subunit was saturated by low concentrations of Na + when the /3-subunit was in a activated state. Ag ÷, a specific inhibitor of this enzyme, was found to act as a competitive inhibitor for quinones at the active site of the/3-subunit. Since the K i for Ag + (2 nM) was extraordinarily lower than the K m for menadione (15/zM), Ag ÷ was considered to be useful as a specific reagent for the analysis of the active site of the/3-subunit.
Introduction The respiratory chain of a marine bacterium, 14brio alginolyticus, contains an N a + - d e p e n d e n t N A D H quinone reductase that functions as an electrogenic Na + p u m p (for reviews, see Refs. 1,2). The Na+-de pendent activation of the respiratory-chain-linked N A D H - q u i n o n e reductase was first demonstrated in V. alginolyticus [3]. This enzyme was purified and was found to be composed of three subunits, a, /3 and y, with the apparent molecular masses of 52, 46 and 32 kDa, respectively [4-6]. The FAD-containing /3-subunit reacted with N A D H and reduced 2-methyl-l,4naphthoquinone (menadione) and ubiquinone-1 ( Q - l ) by a one-electron transfer pathway, which was stimulated by either Na ÷ or K + [4]. On the other hand, the whole complex of N A D H - q u i n o n e reductase (a/3ycomplex) reduced Q-1 to ubiquinol-1, which was
strongly activated by Na + but not by K + [4]. Menadione, however, was reduced to semiquinone radicals by the complex and the activity was stimulated by either Na ÷ or K ÷. Apparently, menadione directly interacted with the/3-subunit within the complex. Thus, the reaction catalyzed by the FMN-containing a-subunit specifically required Na ÷ for the activity and was assigned to be the coupling site of Na ÷ translocation [6]. Although the activities catalyzed by the /3-subunit and by the complex were differently modified by monovalent cations, the mechanisms of these modifications have not been studied in detail. Furthermore, the complex was specifically inhibited by nanomolar concentrations of Ag ÷ [7], but the m o d e of inhibitory action of Ag ÷ remained unsolved. Therefore, the modifications by monovalent cations and the site of Ag ÷ inhibition in the Na ÷-translocating N A D H - q u i n o n e reductase were analyzed by kinetic methods using the purified /3-subunit and the a/3y-complex. Materials and Methods
* Corresponding author. Abbreviations: Menadione, 2-methyl-1,4-naphthoquinone; Q-l, ubiquinone-1.
Chemicals. Liponox DCH, an alkyl polyoxyethylene ether detergent [4], was kindly supplied by Lion
202 (Kanagawa, Japan). Ubiquinone-1 (Q-l) was kindly supplied by Eizai (Tokyo, Japan). Other reagents used were of the highest commercial grade available. Enzyme assays. Menadione reductase activity was assayed at 30°C from the decrease in absorbance at 340 nm with menadione as an electron acceptor. The standard assay mixture contained 0.2 mM NADH, 0.1 mM menadione, 200 mM NaCI, 0.1% (w/v) Liponox DCH, 20 mM Tris-HCl (pH 8.0) and enzyme in a total volume of 1.0 ml. The reaction was started by the addition of enzyme and the initial velocity was calculated based on the absorption coefficient of 6.22 mM-1 cm -1. Quinone reductase activity was assayed at 30°C by following the formation of ubiquinol-1 from Q-1 as previously described [2]. The standard assay mixture contained 0.1 mM NADH, 15/zM Q-l, 200 mM NaCl, 0.015% Liponox DCH, 20 mM Tris-HCI (pH 8.0) and enzyme in a total volume of 2.0 ml. Changes in absorbance difference at the wavelength pair, 242-270.5 nm, were recorded with a Hitachi 557 two wavelength spectrophotometer, and the rate of ubiquinol-1 formation was calculated based on the absorption coefficient of 9.6 mM -1 cm-1. When the /~-subunit was used as the enzyme, Q-1 reductase activity was measured from the decrease in absorbance at 340 nm. To examine the effects of Na ÷ and K +, chloride salts were used throughout the experiments. For kinetic analyses, the concentrations of NADH and Liponox DCH in the reaction mixture were adjusted to 0.1 mM and 0.015%, respectively, in all experiments. For the determination of inhibitory effect of Ag ÷, the enzyme was preincubated with Ag ÷ for 3 min, and then the reaction was started by the addition of electron acceptor and NADH. 1 U of activity was defined as the amount of enzyme catalyzing the oxidation of 1 izmol NADH or the reduction of 1 ~mol Q-1 in 1 rain. Purification of enzymes. The growth of V. alginolyticus 138-2 and the preparation of the membrane fraction were performed as previously described [7]. Purifications of the Na÷-dependent NADH-quinone reductase (afly-complex) and the fl-subunit were performed as previously described [4-6] with slight modifications. For the extraction of quinone reductase from the membrane, pretreatment of the membrane with 2% (w/w) sodium cholate [4] was omitted to minimize enzyme inactivation, and the enzyme was extracted from the membrane with 1% (w/v) Liponox DCH containing 10% (w/v) glycerol, 20 mM Tris-HCl (pH 7.0), 0.1 mM EDTA, 10 mM NaC1 at the concentration of 5 mg membrane protein/ml. The Liponox extracts were applied to DEAE-Sephacel column and the active fraction was eluted with 0.3 M NaC1 (DEAE-Sephacel fraction) as described [5]. The DEAE-Sephacel fraction was further purified with TSK-gel DEAE-5PW
column (2.15 x 15 cm, Toyo Soda, Tokyo), where the quinone reductase complex and the fl-subunit were separated as described [6]. Each fraction was further purified by a HiLoad Superdex 200 column (1.6 × 60 cm, Pharmacia) with a buffer system containing 10 mM Tris-HC1 (pH 7.0), 0.1% (w/v) Liponox DCH, 5% (w/v) glycerol. Other methods. SDS-PAGE was performed by using discontinuous buffer system of Laemmli [8]. For kinetic analyses, the value of each experimental point was obtained as the mean of at least three determinations. The line of best fit for each set of double-reciprocal plots, and kinetic constants and their standard errors were calculated using the program Enzyme Kinetics (Trinity Software, Campton, NH, USA) with a Macintosh computer. Protein was determined by the method of Bradford [9] with bovine serum albumin as standard. Results
Purification of enzymes The Na+-translocating NADH-quinone reductase complex and the fl-subunit were purified by a simple modified method as described in Materials and Methods. The purified quinone reductase complex had a specific activity of about 65 U / m g protein as measured with the quinone reductase assay, and the two preparations of the /~-subunit had specific activities of 92 and 220 U / m g protein, respectively, as measured with the menadione reductase assay. On SDS-PAGE, the quinone reductase complex contained a-, fl- and ysubunits as major proteins, and the fl-subunit was not contaminated with a- and y-subunits. Although the specific activities of the present enzymes were lower than those reported in our previous paper [6], the present enzymes were suited for investigating enzymatic properties and were used in the following experiments.
Modifications of the fl-subunit by monovalent cations The fl-subunit catalyzed the reduction of menadione by a one-electron transfer pathway to produce semiquinone radicals [4]. Under the standard assay conditions, the activity in the absence of salts corresponding to 30.6 U / m g protein was stimulated about 3-fold in the presence of 200 mM NaCI or KC1. The stimulating effect of NaCl was slightly better than that of KCI especially at concentrations above 200 mM, but no significant difference was observed between NaC1 and KCI up to 200 raM, Thus, the/~-subunit showed no specific requirement for monovalent cations for the stimulation of menadione reductase activity. Fig. 1 shows the effect of NaCI on the activity of the fl-subunit for menadione. The increase in NaCl decreased K m values for menadione without affecting
203
0.12 ~ 0.09
oo,
_0.U I/,°,
(0)~0/25) / /
"'c"°-
//
~. 002I ~ ~ ~ I
0.02
"
NaCI lmM)
I
I
I
0.04 0.06 0.08 llMenadlone [,uM"I]
I
t
0.05
0.10
Fig. 1. Double-reciprocal plots of initial velocity vs. menadione concentration at fixed levels of NaCl. The reaction mixture contained 0.1 mM NADH, 0.015% Liponox DCH, 20 mM Tris-HCI (pH 8.0) and the fl-subunit in the presence of varied concentrations of menadione (/zM) and NaCl (mM) indicated in the figure. Initial velocity (v) was expressed in U / m g protein. The /3-subunit with a specific activity of 92 U / m g protein for menadione under the standard assay conditions was used.
" ~ ' ~ 1200) I
I
0.10
I
0.15
0.20
t/Q-1 [pM"1] Fig. 2. Double-reciprocal plots of initial velocity vs. Q-1 concentration at fixed levels of NaCL The reaction was carried out under the conditions described in Fig. 1, except that Q-1 (p.M) was used as the electron acceptor. The /3-subunit with a specific activity of 220 U / m g protein for menadione was used. In the absence of NaCl, the other four data points lie outside the frame.
Modifications of the NADH-quinone reductase complex by monovalent cations the maximum velocity (V). The apparent K m for menadione in the absence of salts was calculated to be 196 _ 18/zM, which decreased to 15.3 + 1.4/~M in the presence of 200 mM NaCl, whereas the apparent V was maintained at about 75 U / m g protein. Therefore, the stimulating effect of NaC1 was due to the decrease in K m for menadione. Essentially the same results were obtained with KCI (data not shown). The /3-subunit also reduced Q-1 by a one-electron transfer pathway without producing detectable amounts of ubiquinol-1 [4]. When the activity for Q-1 was determined from the decrease in NADH under the standard quinone reductase assay, the activity in the absence of salts (4.0 U / m g protein) was stimulated about 10-fold in the presence of either 200 mM NaCl or KC1. In this case, the salt-dependent activation curve did not conform to Michaelis-Menten kinetics and the stimulating effects of salts were insufficient, especially at concentrations below 50 mM, exhibiting a rather sigmoid shape. Fig. 2 shows the effect of NaCl on the activity of the /3-subunit for Q-1. In the absence of salts, the K m value for Q-1 corresponded to 93/~M and the V value approached 1 /3 of that obtained in the presence of 200 mM NaCl (150 U / m g protein). At 75 mM NaC1 and above, K m values for Q-1 decreased with the increase in NaCl without significantly affecting V. In the presence of 200 mM NaCl, the K m for Q-1 was estimated to be 24.7 5-4.8 /~M. Thus, the stimulation of the /3-subunit for Q-1 at concentrations below 75 mM was due to both the decrease in K m and the increase in V. However, the mode of activation at higher concentrations was mainly due to the decrease in K m for Q-1 (Fig. 2), which was very similar to that for menadione (Fig. 1). Essentially the same results were obtained with KC1 (data not shown).
Under the standard assay conditions, the activity of NADH-quinone reductase complex for menadione in the absence of salts (26.6 U / m g protein) was stimulated about 5-fold in the presence of 200 mM NaC1 or KCI. Fig. 3 shows the effects of NaCl on the activity of the complex for menadione. In contrast to the case of the/3-subunit, NaCl stimulated the activity of the complex by increasing V without affecting the K m for menadione. The V value approached to 182 U / m g protein at 200 mM NaCl or KCI. The Km value in the absence of salts was estimated to be 15.7 5- 0.9 /~M, which was identical with that of the 3-subunit in the presence of 200 mM NaC1 or KC1. Therefore, the /3-subunit was maintained in a state of high-affinity for menadione within the complex even in the absence of salts. Further stimulation of the/3-subunit in the complex was due to the salt-dependent increase in V. In contrast to the case of menadione, the complex showed no activity for Q-1 in the absence of salts and specifically required Na + for maximum activity (Fig. 4).
i00.t ..c, ,m., , 0 y _0041 /
/ (200)
oo8-oo3
o o3
l/Menadlone
[p,M"1]
Fig. 3. Double-reciprocal plots of initial velocity vs. menadione concentration at fixed levels of NaCI. The reaction was carried out as described in Fig. 1, except that the complex with a specific activity of 65 U / m g protein for Q-1 was used as the enzyme. The initial velocity was determined from the decrease in NADH.
204 8O 200mM KCI
I¢
Q.
+ NaCl
Ag+(
NaCI
60
n
~
-"" 0 . 0 6
O~
Io
•~- 0 . 0 4
M
.-_ 4 0
C
-~ 20 I
~ 0.02"
KCl
i
o
,
,
,
J
50 100 150 Salt concentration (raM)
o
200
Fig. 4. Effects of monovalent cations on the activity of the complex for Q-1. The activity was assayed under the conditions of standard quinone reductase assay, except that the chloride salts of monvalent cations were varied as described in the figure.
The concentrations of NaC1 and KC1 to give a halfmaximum velocity (M1/2) were estimated to be about 54 and 78 mM, respectively. In the presence of 200 mM KCI, the MI/2 for NaCI was reduced to 4.3 mM. These results were essentially similar to those observed with the membrane-bound N A D H oxidase from I4 alginolyticus [10]. Thus, the Na÷-dependent NADHquinone reductase segment in the respiratory chain of I4 alginolyticus apparently acted as a limiting step in the salt-dependent activation. Fig. 5 shows the effects of NaC1 on the activity of the complex for Q-1. The primary plots all intersected at a point below the baseline and the activation by NaCI was due to the increase in both the K m for Q-1 and I/". The K m and V values at 200 mM NaCI were calculated to be 12.9 + 1.8 /zM and 110 + 9 U / m g protein. Such type of activation was not observed with KCI. In the presence of 200 mM KC1, however, the K m for Q-1 was estimated to be 3.0 + 0.3 /zM, but the V value was only 12% of that in the presence of 200 mM NaC1. Under that conditions, the addition of small amounts of NaCI greatly increased V value, which was accompanied with the increase in K m. Thus, in the
0.12
..c,,m.l
I
I
I
Fig. 6. Effects of Ag ÷ on the activity of the complex for menadione.
The reaction was carried out as described in Fig. 3, except that the reaction mixture contained 200 mM NaCI and Ag +(nM) as indicated in the figure. presence of 200 mM KCI, the specific requirement for Na ÷ of the reaction catalyzing the formation of ubiquinol-1 was saturated with low concentrations of Na ÷ as was expected from Fig. 4.
The site of Ag ÷ inhibition Since the complex is strongly inhibited by nanomolar concentrations of Ag ÷ [7], the effects of Ag ÷ on the activity of the complex for menadione were examined. As shown in Fig. 6, Ag + acted as a competitive inhibitor for menadione. The secondary plots of slope vs. Ag ÷ concentration gave a straight line in this concentration range and the K i value was estimated to be 1.8 nM. Although this experiments were carried out in the presence of 200 mM NaC1, an identical K i value was obtained in the presence of 200 mM KC1. Therefore, the inhibitory effect of Ag ÷ was not influenced by the species of monovalent cations. Furthermore, such a strong Ag ÷ inhibition was observed with the complex even in the absence of salts. When the /3-subunit was used as the enzyme, the same type of competitive inhibition was observed in the presence of 200 mM NaC1 or KCI, and the K i value for Ag ÷ was estimated to be 2.1 nM. In the absence of salts, however, higher concentrations of Ag + were required to competitively inhibit the activity. The K i Na+/K +
0.08
I
0.02 0.04 0.06 0.08 0.10 llMenadione [laM-1]
~ A~
K÷
~ 0.04'
I
-0.20
I
0.20 0.40 11Q-1 [pM"1]
Fig. 5. Double-reciprocal plots of initial velocity vs. Q-1 concentration at fixed levels of NaCI. T h e reaction was carried out as described in Fig. 3, except that Q-1 was used as the electron acceptor. T h e initial velocity was determined from the formation of ubiquinol-1.
AJ~: High-affinity type A ~ V : High-affinity and
high-velocity type
Fig. 7. Modifications by monovalent cations of the/3-subunit and the N A D H - q u i n o n e reductase complex.
205 value for Ag + in the absence of salts was estimated to be 320 nM, which was more than 100-fold higher than that in the presence of 200 mM NaCI or KC1. The inhibitory effects of Ag + for Q-1 reduction were essentially similar to those for menadione reduction, and the strong Ag + inhibition was observed only in the presence of salts. These results indicated that Ag ÷ interacted at the quinone-binding site of the/3-subunit and that the inhibitory effects of Ag ÷ were directly related to the affinity of the/3-subunit for quinone substrates.
Discussion From the kinetic results of salt modifications of the /3-subunit and the a/3y-complex, the stimulating effects of monovalent cations may be formulated as shown in Fig. 7. The purified /3-subunit with a low affinity for menadione and Q-1 in the absence of salts increases its affinity by the increase in Na ÷ or K ÷, which is saturated at about 200 mM. On the other hand, the fl-subunit in the complex is already in a state of high-affinity for these substrates (designated as A/3 in the figure) even in the absence of salts. The activity of the/3-subunit in the complex is further stimulated by the addition of Na + or K ÷ due to the increase in V. Thus, in the presence of saturated concentrations of Na + or K + (about 200 mM), the/3-subunit in the complex is maintained in a state of high-affinity and high-velocity type (A/3V). The formation of ubiquinol-1 catalyzed by the complex, however, is not fully activated by K ÷ alone and specifically requires Na ÷ for maximum activation. Since the requirement for Na ÷ greatly decreases in the presence of 200 mM K +, the Na+-dependent reaction catalyzed by a-subunit is saturated by low concentrations of Na + when the /3-subunit is in an activated state. Thus, the necessity of high concentrations of Na ÷ for the activation of the complex may well be explained by the requirement of about 200 mM Na ÷ for full activation of the/3-subunit in the complex. It is interesting to note that the purified /3-subunit with a low affinity for quinones is converted into a high-affinity type as a constituent part of the complex even in the absence of salts. Thus, the mode of salt modification of the fl-subunit was altered depending on the conditions of its existence. We previously reported that the affinity of the /3-subunit for Q-1 is modified by the y-subunit [6]. Therefore, the y-subunit seems to play an important role for the activation of the/3-subunit in the complex. The Na+-translocating NADH-quinone reductase is very sensitive to Ag + [7]. In this paper, the site of Ag + inhibition was located at the quinone-binding site of the /3-subunit. The strength of Ag + inhibition was correlated with the affinity of the /3-subunit for quinones, and the K i value for Ag + (2 nM) was extraordinarily lower than the K m value for mena-
dione (15/zM). Thus, the affinity of Ag + for the active site was 7500-fold higher than that of menadione. Ag + does not have any structural resemblance to quinones and its inhibitory effects are prevented by thiol compounds. Therefore, Ag + is very likely to interact with a specified thiol group within the active site that directly participates in the catalytic activity. Ag + may be useful as a specific reagent for the analysis of active site of the/3-subunit. Semeykina and Skulachev [11] reported that very low concentrations of Ag + inhibit the uphill Na ÷ transport coupled to the Na +-pumping NADH-quinone reductase and terminal oxidase of Bacillus F l U . Ag + was also shown to induce an increse in Na ÷ permeability of the membrane vesicles. When 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO), a specific inhibitor of Na + pump, was added before Ag +, such an Ag+-in duced increase in Na + permeability was prevented. From these, it was suggested that Ag + uncoupled the electron and Na + transport so that the Ag+-modified NADH-quinone reductase operated as an Na + channel rather than an Na + pump. In the Na+-translocat ing NADH-quinone reductase from V. alginlolyticus, the Na+-dependent reaction catalyzed by the FMNcontaining a-subunit was assigned to be the coupling site of Na + transport, which was specifically inhibited by HQNO [1,2,6]. Since Ag + and HQNO interact at a quite different site within the complex, the formation of Na + channel by the Ag+-modified enzyme is unlikely with this enzyme. Further studies are required to solve these problems.
Acknowledgement A part of this work was supported by a grant-in-aid from the Japanese Ministry of Educations, Science and Culture.
References 1 Unemoto, T., Tokuda, H. and Hayashi, M. (1990) in The Bacteria, Vol. XII, Bacterial Energetics (Kruiwich, T.A., ed.), pp. 33-54, Academic Press, San Diego. 2 Unemoto, T. and Hayashi, M. (1989) J. Bioenerg. Biomembr. 21, 649-662. 3 Unemoto, T. and Hayashi, M. (1979) J. Biochem. 85, 1461-1467. 4 Hayashi, M. and Unemoto, T. (1984) Biochim. Biophys. Acta 767, 470-478. 5 Hayashi, M. and Unemoto, T. (1986) FEBS I_#.tt. 202, 327-330. 6 Hayashi, M. and Unemoto, T. (1987) Biochim. Biophys. Acta 890, 47-54. 7 Hayashi, M., Miyoshi, T., Sato, M. and Unemoto, T. (1992) Biochim. Biophys. Acta 1099, 145-151. 8 Laemmli, U.K. (1970) Nature 227, 680-685. 9 Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. 10 Unemoto, T., Hayashi, M. and Hayashi, M. (1977) J. Biochem. 82, 1389-1395. 11 Semeykina, A.L. and Skulachev, V.P. (1990) FEBS Lett. 269, 69-72.