882
BIOCHEMICAL SOCIETY TRANSACTIONS
The Effect of Protein-Functional-Group Reagents on D-Gluconate Transport in Bacillus subtilis MAIRIN P. O’SULLIVAN and MICHAEL N. McKILLEN Department of Biochemistry, University of Dublin, Trinity College, Dublin 2, Ireland
It appears that shock-resistant transport systems from Escherichia coli are extremely sensitive to the action of thiol-group reagents such as N-ethylmaleimide and p-chloromercuribenzoate, whereas shock-releasable binding proteins are unaffected by thiolgroup reagents (Kaback & Barnes, 1971; Boos, 1974; Berger & Heppel, 1974). Since Gram-positive organisms such as Bacillus subtilis have not been shown to possess shocksensitivetransport systems, we undertook an investigation of the effect of thiol- and other protein-functional-group reagents on D-gluconate transport in B. subtilis to devise a procedure to label covalently the solute-binding site of the D-gluconate carrier (Fox &Kennedy, 1965;Fournier &Pardee, 1974).D-Gluconate is transported in this organism by an inducible, highly specific, energy-dependent transport system subject to control by catabolite repression (McKillen & Rountree, 1973; Baxter et al., 1974, 1975). The procedures for growth and harvesting of the cells, the induction and assay of D-gluconate transport and the treatment of cells with functional-group reagents were described previously (Fournier et al., 1972; Baxter et al., 1974; Fournier & Pardee, 1974). As shown in Table 1,D-gluconatetransport was sensitiveto thiol-, &-amino-and hydroxylgroup reagents. This sensitivity to N-ethylmaleimide and p-chloromercuribenzoate was particularly striking, and, taken with the kinetic evidence (Baxter et af., 1974), pointed to the presence of thiol groups in the carrier system. To exclude the possibility that the thiol-group reagents exerted their inhibiting action by preventing the generation of the energized membrane state (see Mitchell, 1976), we investigated the effect of N-ethylmaleimide on endogenous respiration in intact cells and on both NADH oxidation and ATPase (adenosine triphosphatase) activity of isolated membrane vesicles in oitro. N-Ethylmaleimide(1 m)inhibited endogenous respiration by 95 % in intact cells, but the oxidation of ~ O ~ M - N A D byHvesicles (Konings & Freese, 1972)was insensitive to 1mMN-ethylmaleimide, as was the (Ca2+/Mg2+)-stimulatedATPasecomplex activity in oitro (H. Graham, unpublished work). The inhibition of endogenous respiration could, however, be restored to 70% of normal by supplying 100mM-L-malate to suitably Table 1. Effect of protein-functional-group reagents on D-gluconate transport in B. subtilis SB-26 Cells were treated with each reagent for the desired time at the indicated pH and the reaction was stopped by the addition of excess of B-mercaptoethanol (for N-ethylmaleimide), cysteine (for iodoacetate), lysine (for ethyl acetimidate) or serine (for phenylmethane sulphonyl fluoride). The cells were then extensively washed and resuspended at pH7.2 for the standard gluconate-transport assay. Exposure Inhibition of Reagent Concn. time (min) pH transport (%) N-Ethylmaleimide 0.01 mM 1 6.8 40 0.1 mM 1 6.8 96 l.0mM 1 6.8 96 10.0mM 1 6.8 96 p-Chloromercuribenzoate 0.01 mM 1 6.8 25 0.1 mM 1 6.8 94 Iodoacetate 0.1 mM 1 6.5 65 Phenylmethane sulphonyl 0.1 mM 5 7.2 76 fluoride lOmM 5 7.2 98 50m~ 10 8.5* 42 Ethyl acetimidate * Glycine/KOH buffer (0.124~),pH8.5, replaced the 0.124~-potassiumphosphate buffer. 1976
564th MEETING, DUBLIN
883
Table 2. Effect of N-ethylmaleimide on the protonophore-resistant component of gluconate transport in B. subtilis SB-26 Cells were incubated, as appropriate, with 0.1 mM-N-ethylmaleimide for 1min at pH6.8 at 3 7 T , and the reaction stopped by the addition of 0.1 m@-mercaptoethanol. Cells were then harvested, washed and resuspended at pH7.2 and incubated in the presence of the respective protonophore for lOmin before the assay of gluconate transport by the standard procedure. Inhibition of Inhibition of *Inhibition of protonogluconate protonophore re- phore-resistant gluconate transport in sistant gluconate transport by 0.01 mMtransport by N-ethylmaleimide absence of N-ethylmale- 0.1 m-N-ethyl- in presence of 10mM-DProtonophore imide (%) maleimide (%) gluconate (%) Tetrachlorosalicylanilide 98 61 91 (2OPM) Carbonyl cyanide 99 52 94 p-triiluoromet hoxyphen y 1hydrazone ( 2 0 , ~ ~ ) Carbonyl cyanide m-chlorophenylhydrazone 97 71 72 (2OPM) * Cells were exposed to 10mM-D-gluconatefor 1 min before the addition of N-ethylmaleimide. induced cells (Fournier et al., 1972), but was not relieved by the addition of exogenous D-glucose, D-gluconate, citrate, 2-oxogluratate, fumarate, succinate or pyruvate. Similarly, gluconate transport was restored to 65 % of the control value by the presence of exogenous 100mM-L-malate. These results suggest that the primary effect of N-ethylmaleimide is to prevent the delivery of reducing equivalents to the respiratory redox chain. By treating the cells with protonophores (see Mitchell, 1976) we then investigated the action of Nethylmaleimide on the D-gluconate-facilitated diffusion system. As Table 2 shows, the protonophores tetrachlorosalicylanilide, carbonyl cyanide p-trifluoromethoxyphenylhydrazone and carbonyl cyanide m-chlorophenylhydrazone strongly inhibited D-gluconate transport in standard cell suspensions. The residual transport activity was presumably due to the transport system operating in an uncoupled mode (facilitated diffusion), and it is clear that N-ethylmaleimide treatment inhibits 50-70% of this residual activity. However, the inhibition of this residual activity could not be prevented or alleviated by the presence of l0mM-gluconate, and there is some suggestion that gluconate exposure rendered the system more susceptible to N-ethylmaleimide action. Abendano & Kepes (1973) reported that the D-glucuronate-transport system of E. coli was also made more susceptible to N-ethylmaleimide inactivation in the presence of the permeant. The present results imply that thiol groups are probably not involved in the gluconate-binding site, but d o not exclude the presence of thiol groups elsewhere in the carrier. Clearly, any investigations concerned with the action of protein-functionalgroup reagents on transport systems must differentiate between the effect of these agents on the energization of the process and the carrier itself. We thank the Department of Education, Ireland, for a Research Maintenance Award ta M. P. 0 ’ s .
Abendano, J. J. & Kepes, A. (1973) Biochem. Biophys. Res. Commun. 54, 1342-1346 Baxter, A. L., Torrie, S. & McKillen, M. N. (1974) Biochem. SOC.Trans. 2, 1370-1372 Baxter, A. L., McKillen, M. N. & Syms, M. (1975) Biochem. SOC.Trans. 3,1205-1207 Berger, E. A. & Heppel, L. A. (1974) J. Biol. Chem. 249, 7747-7755 BOOS,W. (1974) Annu. Rev. Biochem. 43, 123-146
Vol. 4
884
BIOCHEMICAL SOCIETY TRANSACTIONS
Foumier, R. E. & Pardee, A. B. (1974) J. Biol. Chem. 249, 5948-5954 Fournier, R. E., McKillen, M. N., Pardee, A. B. & Willecke, K. (1972) J. Biol. Chem. 247, 5587-5595 Fox, C. F. & Kennedy, E. P. (1965) Proc. Natl. Acad. Sci. U.S.A. 54, 891-899 Kaback, H. R. & Barnes, E. M. (1971) J . Biol. Chem. 246, 5523-5531 Konings, W. N. & Freese, E. (1972) J. Biol. Chem. 247, 2408-2418 McKillen, M. N. & Rountree, J. H. (1973) Biochem. Soc. Trans. 1, 442-445 Mitchell, P. (1976) Biochem. SOC.Trans. 4, 399-430
The Energization of D-Gluconate Transport in Bacillus suhtilis A. LESLEY BAXTER and MICHAEL N. McKILLEN Department of Biochemistry, Trinity College, Dublin 2, Ireland
D-GlUCOnate is transported in Bacillus subtilis SB-26 by an inducible, highly specific, energy-dependent transport system that is subject to control by catabolite repression (McKillen & Rountree, 1973; Baxter et al., 1974, 1975). It has become fashionable in the past few years to discuss the energetic aspects of permeant transport in terms of the Mitchell chemi-osmotic coupling hypothesis (see Mitchell, 1976). It could be predicted that D-gluconate, which exists as an anion at pH7.2 (PIC:, 3.86), should be transported by coupling in an electroneutral mode, and with a proton symport responding only to ApH. Thus any agents or treatments which dissipate or prevent the generation of Ap (or its integral components) should have a profound effect on ~-gluconatetransport. It has become accepted dogma (see Simoni & Postma, 1975) in micro-organisms that the proton-translocating (Ca2+/Mg2+)-stimulated ATPase (adenosine triphosphatase) complex (EC 3.6.1.3) normally functions in the direction of ATP synthesis in aerobic conditions (oxidative phosphorylation), and under anaerobic conditions (or in bacteria lacking a respiratory redox chain) the ATPase functions to link the hydrolysis of ATP to permeant transport (and other energydependent membrane events). Thus the proton-translocating respiratory redox chain of an obligate aerobe such as B. subtilis must presumably be the only source for Ap generation under normal physiological conditions. Recently the situation has become more complex, for it has been shown that, in Escherichia coli at least, there are a number of transport systems which are apparently not energized by Ap set up by electron flow or ATP hydrolysis but instead are energized directly by phosphate-bond energy, derived from either glycolysis or oxidative phosphorylation, independent of (Ca2+/MgZ+)-stimulatedATPase action (Berger & Heppel, 1974). We have tested the effect of respiratory-redox-chain and ATPase-complex inhibitors, protonophores and ionophores on D-gluconate transport in an attempt to elucidate the nature of the energy-coupling mechanism for this system. The experimental procedures for the growth and harvesting of the cells and the induction and assay of the transport system were described previously (Fournier et al., 1972; Baxter et al., 1974). Theconcentrations of inhibitors chosen were, in the absence of extensive data for B. subtilis, essentially similar to those used by other workers for E. coli (see Harold, 1972) or mitochondria (see Harold, 1972; Racker, 1975). Cell suspensions were preincubated in the presence of each inhibitor for 5min and transport was initiated by the addition of ~-[U-'~C]gluconate. The inhibitor data are summarized in Table 1. With certain exceptions, D-gluconate transport was sensitive to respiratory-redox-chain and ATPase-complex inhibitors as well as to protonophores and ionophores. The NADH dehydrogenase of B. subtilis is apparently either insensitive to rotenone and piericidin A or alternatively these two inhibitors have difficulty of access to the complex. Hampton & Freese (1974) have shown that NADH oxidase activity in membrane vesicles of B. subtilis is insensitive to rotenone. All the other redox-chain inhibitors produced marked inhibition of D-gluconate transport (47-99 % inhibition).
1976
BIOCHEMICAL SOCIETY TRANSACTIONS
The Effect of Protein-Functional-Group Reagents on D-Gluconate Transport in Bacillus subtilis MAIRIN P. O’SULLIVAN and MICHAEL N. McKILLEN Department of Biochemistry, University of Dublin, Trinity College, Dublin 2, Ireland
It appears that shock-resistant transport systems from Escherichia coli are extremely sensitive to the action of thiol-group reagents such as N-ethylmaleimide and p-chloromercuribenzoate, whereas shock-releasable binding proteins are unaffected by thiolgroup reagents (Kaback & Barnes, 1971; Boos, 1974; Berger & Heppel, 1974). Since Gram-positive organisms such as Bacillus subtilis have not been shown to possess shocksensitivetransport systems, we undertook an investigation of the effect of thiol- and other protein-functional-group reagents on D-gluconate transport in B. subtilis to devise a procedure to label covalently the solute-binding site of the D-gluconate carrier (Fox &Kennedy, 1965;Fournier &Pardee, 1974).D-Gluconate is transported in this organism by an inducible, highly specific, energy-dependent transport system subject to control by catabolite repression (McKillen & Rountree, 1973; Baxter et al., 1974, 1975). The procedures for growth and harvesting of the cells, the induction and assay of D-gluconate transport and the treatment of cells with functional-group reagents were described previously (Fournier et al., 1972; Baxter et al., 1974; Fournier & Pardee, 1974). As shown in Table 1,D-gluconatetransport was sensitiveto thiol-, &-amino-and hydroxylgroup reagents. This sensitivity to N-ethylmaleimide and p-chloromercuribenzoate was particularly striking, and, taken with the kinetic evidence (Baxter et af., 1974), pointed to the presence of thiol groups in the carrier system. To exclude the possibility that the thiol-group reagents exerted their inhibiting action by preventing the generation of the energized membrane state (see Mitchell, 1976), we investigated the effect of N-ethylmaleimide on endogenous respiration in intact cells and on both NADH oxidation and ATPase (adenosine triphosphatase) activity of isolated membrane vesicles in oitro. N-Ethylmaleimide(1 m)inhibited endogenous respiration by 95 % in intact cells, but the oxidation of ~ O ~ M - N A D byHvesicles (Konings & Freese, 1972)was insensitive to 1mMN-ethylmaleimide, as was the (Ca2+/Mg2+)-stimulatedATPasecomplex activity in oitro (H. Graham, unpublished work). The inhibition of endogenous respiration could, however, be restored to 70% of normal by supplying 100mM-L-malate to suitably Table 1. Effect of protein-functional-group reagents on D-gluconate transport in B. subtilis SB-26 Cells were treated with each reagent for the desired time at the indicated pH and the reaction was stopped by the addition of excess of B-mercaptoethanol (for N-ethylmaleimide), cysteine (for iodoacetate), lysine (for ethyl acetimidate) or serine (for phenylmethane sulphonyl fluoride). The cells were then extensively washed and resuspended at pH7.2 for the standard gluconate-transport assay. Exposure Inhibition of Reagent Concn. time (min) pH transport (%) N-Ethylmaleimide 0.01 mM 1 6.8 40 0.1 mM 1 6.8 96 l.0mM 1 6.8 96 10.0mM 1 6.8 96 p-Chloromercuribenzoate 0.01 mM 1 6.8 25 0.1 mM 1 6.8 94 Iodoacetate 0.1 mM 1 6.5 65 Phenylmethane sulphonyl 0.1 mM 5 7.2 76 fluoride lOmM 5 7.2 98 50m~ 10 8.5* 42 Ethyl acetimidate * Glycine/KOH buffer (0.124~),pH8.5, replaced the 0.124~-potassiumphosphate buffer. 1976
564th MEETING, DUBLIN
883
Table 2. Effect of N-ethylmaleimide on the protonophore-resistant component of gluconate transport in B. subtilis SB-26 Cells were incubated, as appropriate, with 0.1 mM-N-ethylmaleimide for 1min at pH6.8 at 3 7 T , and the reaction stopped by the addition of 0.1 m@-mercaptoethanol. Cells were then harvested, washed and resuspended at pH7.2 and incubated in the presence of the respective protonophore for lOmin before the assay of gluconate transport by the standard procedure. Inhibition of Inhibition of *Inhibition of protonogluconate protonophore re- phore-resistant gluconate transport in sistant gluconate transport by 0.01 mMtransport by N-ethylmaleimide absence of N-ethylmale- 0.1 m-N-ethyl- in presence of 10mM-DProtonophore imide (%) maleimide (%) gluconate (%) Tetrachlorosalicylanilide 98 61 91 (2OPM) Carbonyl cyanide 99 52 94 p-triiluoromet hoxyphen y 1hydrazone ( 2 0 , ~ ~ ) Carbonyl cyanide m-chlorophenylhydrazone 97 71 72 (2OPM) * Cells were exposed to 10mM-D-gluconatefor 1 min before the addition of N-ethylmaleimide. induced cells (Fournier et al., 1972), but was not relieved by the addition of exogenous D-glucose, D-gluconate, citrate, 2-oxogluratate, fumarate, succinate or pyruvate. Similarly, gluconate transport was restored to 65 % of the control value by the presence of exogenous 100mM-L-malate. These results suggest that the primary effect of N-ethylmaleimide is to prevent the delivery of reducing equivalents to the respiratory redox chain. By treating the cells with protonophores (see Mitchell, 1976) we then investigated the action of Nethylmaleimide on the D-gluconate-facilitated diffusion system. As Table 2 shows, the protonophores tetrachlorosalicylanilide, carbonyl cyanide p-trifluoromethoxyphenylhydrazone and carbonyl cyanide m-chlorophenylhydrazone strongly inhibited D-gluconate transport in standard cell suspensions. The residual transport activity was presumably due to the transport system operating in an uncoupled mode (facilitated diffusion), and it is clear that N-ethylmaleimide treatment inhibits 50-70% of this residual activity. However, the inhibition of this residual activity could not be prevented or alleviated by the presence of l0mM-gluconate, and there is some suggestion that gluconate exposure rendered the system more susceptible to N-ethylmaleimide action. Abendano & Kepes (1973) reported that the D-glucuronate-transport system of E. coli was also made more susceptible to N-ethylmaleimide inactivation in the presence of the permeant. The present results imply that thiol groups are probably not involved in the gluconate-binding site, but d o not exclude the presence of thiol groups elsewhere in the carrier. Clearly, any investigations concerned with the action of protein-functionalgroup reagents on transport systems must differentiate between the effect of these agents on the energization of the process and the carrier itself. We thank the Department of Education, Ireland, for a Research Maintenance Award ta M. P. 0 ’ s .
Abendano, J. J. & Kepes, A. (1973) Biochem. Biophys. Res. Commun. 54, 1342-1346 Baxter, A. L., Torrie, S. & McKillen, M. N. (1974) Biochem. SOC.Trans. 2, 1370-1372 Baxter, A. L., McKillen, M. N. & Syms, M. (1975) Biochem. SOC.Trans. 3,1205-1207 Berger, E. A. & Heppel, L. A. (1974) J. Biol. Chem. 249, 7747-7755 BOOS,W. (1974) Annu. Rev. Biochem. 43, 123-146
Vol. 4
884
BIOCHEMICAL SOCIETY TRANSACTIONS
Foumier, R. E. & Pardee, A. B. (1974) J. Biol. Chem. 249, 5948-5954 Fournier, R. E., McKillen, M. N., Pardee, A. B. & Willecke, K. (1972) J. Biol. Chem. 247, 5587-5595 Fox, C. F. & Kennedy, E. P. (1965) Proc. Natl. Acad. Sci. U.S.A. 54, 891-899 Kaback, H. R. & Barnes, E. M. (1971) J . Biol. Chem. 246, 5523-5531 Konings, W. N. & Freese, E. (1972) J. Biol. Chem. 247, 2408-2418 McKillen, M. N. & Rountree, J. H. (1973) Biochem. Soc. Trans. 1, 442-445 Mitchell, P. (1976) Biochem. SOC.Trans. 4, 399-430
The Energization of D-Gluconate Transport in Bacillus suhtilis A. LESLEY BAXTER and MICHAEL N. McKILLEN Department of Biochemistry, Trinity College, Dublin 2, Ireland
D-GlUCOnate is transported in Bacillus subtilis SB-26 by an inducible, highly specific, energy-dependent transport system that is subject to control by catabolite repression (McKillen & Rountree, 1973; Baxter et al., 1974, 1975). It has become fashionable in the past few years to discuss the energetic aspects of permeant transport in terms of the Mitchell chemi-osmotic coupling hypothesis (see Mitchell, 1976). It could be predicted that D-gluconate, which exists as an anion at pH7.2 (PIC:, 3.86), should be transported by coupling in an electroneutral mode, and with a proton symport responding only to ApH. Thus any agents or treatments which dissipate or prevent the generation of Ap (or its integral components) should have a profound effect on ~-gluconatetransport. It has become accepted dogma (see Simoni & Postma, 1975) in micro-organisms that the proton-translocating (Ca2+/Mg2+)-stimulated ATPase (adenosine triphosphatase) complex (EC 3.6.1.3) normally functions in the direction of ATP synthesis in aerobic conditions (oxidative phosphorylation), and under anaerobic conditions (or in bacteria lacking a respiratory redox chain) the ATPase functions to link the hydrolysis of ATP to permeant transport (and other energydependent membrane events). Thus the proton-translocating respiratory redox chain of an obligate aerobe such as B. subtilis must presumably be the only source for Ap generation under normal physiological conditions. Recently the situation has become more complex, for it has been shown that, in Escherichia coli at least, there are a number of transport systems which are apparently not energized by Ap set up by electron flow or ATP hydrolysis but instead are energized directly by phosphate-bond energy, derived from either glycolysis or oxidative phosphorylation, independent of (Ca2+/MgZ+)-stimulatedATPase action (Berger & Heppel, 1974). We have tested the effect of respiratory-redox-chain and ATPase-complex inhibitors, protonophores and ionophores on D-gluconate transport in an attempt to elucidate the nature of the energy-coupling mechanism for this system. The experimental procedures for the growth and harvesting of the cells and the induction and assay of the transport system were described previously (Fournier et al., 1972; Baxter et al., 1974). Theconcentrations of inhibitors chosen were, in the absence of extensive data for B. subtilis, essentially similar to those used by other workers for E. coli (see Harold, 1972) or mitochondria (see Harold, 1972; Racker, 1975). Cell suspensions were preincubated in the presence of each inhibitor for 5min and transport was initiated by the addition of ~-[U-'~C]gluconate. The inhibitor data are summarized in Table 1. With certain exceptions, D-gluconate transport was sensitive to respiratory-redox-chain and ATPase-complex inhibitors as well as to protonophores and ionophores. The NADH dehydrogenase of B. subtilis is apparently either insensitive to rotenone and piericidin A or alternatively these two inhibitors have difficulty of access to the complex. Hampton & Freese (1974) have shown that NADH oxidase activity in membrane vesicles of B. subtilis is insensitive to rotenone. All the other redox-chain inhibitors produced marked inhibition of D-gluconate transport (47-99 % inhibition).
1976
