1136
BIOCHEMICAL SOCIETY TRANSACTIONS
Beechey, R. B., Hubbard, S.A., Linnett; P. E., Mitchell, A. D. & Munn, E. A. (1975)Biochem.J. 148,533-537 Esch, F. S. &Allison, W. S.(1978)J. Biol. Chem. 253,6100-6106 Linnett, P. E., Mitchell, A. D., Osselton, M. D., Mulheirn,L. J. &Beechey, R. B. (1978)Biochem. J. 170,503-510 Lowe, P. N., Baum, H. & Beechey, R. B. (1979) Biochem. Soc. Trans. 7,237-239 Monsan, P., Puzo, B. & Mazarguil, H. (1975) Biochimie 57,1281-1292
Hydrogen-Dependent Proton Translocation by Membrane Vesicles from Escherichia coli ROBERT W. JONES Department of Biochemistry, Medical Sciences Institute, University of Dundee, Dundee DD1 4HN, Scotland, U.K.
Proton translocation coupled to bacterial fumarate reduction has been demonstrated with a number of electron donors (Gutowski & Rosenberg, 1977; Kroger, 1978; Miki & Wilson, 1978). Escherichia coli can obtain energy for anaerobic growth from hydrogendependent reduction of fumerate (Bernhard & Gottschalk, 1978; Macy et al., 1976; Yamamoto & Ishimoto, 1978). This reaction might therefore be expected to translocate protons (Jones et al., 1978). Quenching of the fluorescence of the acridine dye atebrin has been used to demonstrate the presence of a pH difference, ApH, across the membrane of chloroplasts (Schuldiner et al., 1972), submitochondrial particles (Azzone & Massari, 1973) and inverted membrane vesicles from E. coli(Haddock & Downie, 1974; Haddock & Kendall-Tobias, 1975). The quenching of atebrin fluorescence was used in the experiments described here to show that hydrogen-dependent fumarate reduction by inverted membrane vesicles from E. coli translocates protons. E. coli strain A1002 was grown at 37°C in a 5-litre bottle without stirring so as to maintain anaerobic conditions. The growth medium used was that of Cohen & Rickenberg (1956) supplemented with: glucose, peptone and fumarate (each OS%, w/v); isoleucine, valine and methionine (each 20figlml) ; 1mM-MgC1, ; 1f i ~ - ( N H ~ ) ~ M o , o ~ ~
I
Fumarate
Fumarate Ateirin N T H
,i~
-
Y'
5min
Fig. 1. Hydrogen-plus-fumarate-dependentatebrin-fluorescence quenching by inverted membrane vesiclesfrom E. coli Inverted membrane vesicles (3.4mg) were suspended in 2.5ml of 300rn~-KCl/lSmMacid], pH7.5, MgCl,/lOmM-Hepes ~4-(2-hydroxyethyl)-l-piperazine-ethanesulphonic and bubbled with (a) hydrogen or (6) nitrogen for 10min. Additions, where indicated, were: 4,u~-atebrin; 0.4m~-sodium fumarate, 1mM-NADH; 2-n-heptyl-4-hydroxyquinoline N-oxide (HOQNO, 2,uglml); 0 . 5 m ~ - A T p ;2p~-tetrachlorosalicylanilide
(TCS). 1979
583rd MEETING, CAMBRIDGE
1137
and 1,uM-K2SeO3.Preparation of inverted membrane vesicles and fluorescence measurements were as described previously (Haddock & Downie, 1974). Fig. l(a) shows that the addition of fumarate to a hydrogen-saturated suspension of inverted membrane vesicles of E. coli causes the quenching of atebrin fluorescence. The return of fluorescence after a few minutes is probably due to exhaustion of hydrogen, since addition of NADH restores the quenching. Addition of the respiratory inhibitor 2-n-heptyl-4-hydroxyquinolineN-oxide causes the fluorescence to return. Quenching is restored by the addition of ATP as the ATPase pumps protons into the intravesicular space while hydrolysing ATP (West & Mitchell, 1974). Addition of the uncoupler tetrachlorosalicylanilide (Hamilton, 1968) allows protons to equilibrate across the membrane and fluorescence is again observed. Addition of fumarate to a N,-saturated suspension of vesicles does not give rise to fluorescence quenching (Fig. Ib), showing that the quenching in Fig. l(a) is hydrogen-dependent. Hydrogen-dependent quenching was also observed with oxygen and nitrate as electron acceptors, and fumarate-dependent quenching was observed with NADH and formate as electron donors (results not shown). Hydrogen-plus-fumarate-dependentquenching was abolished by 2-n-hept yl-4-hydroxyquinoline N-oxide, tetrachlorosalicylanilide and CO, an inhibitor of several bacterial hydrogenases (Mortensen & Chen, 1974). There is evidence that acridine-fluorescence quenching is a qualitative probe for ApH (Schuldiner et al., 1972;Elema et al., 1978), but its value in the quantitative determination of ApH has been questioned (Elema et al., 1978). It can be concluded that hydrogen-dependent fumarate reduction by membrane vesicles from E. coli is coupled to an inward translocation of protons. This would be an outward translocation in intact cells. This work was supported by the Science Research Council. Auone, G. F. & Massari, S. (1973)Biochim. Biophys. Acta 301,195-226 Bernhard, Th. & Gottschalk, G. (1978)Arch. Microbiol. 116,235-238 Cohen, G . N. & Rickenberg, H. W. (1956)Ann. Znst. Pasteur, Paris, 91,693-720 Elema, R. P.,Michels, P. A. M. & Konings, W. N. (1978)Eur. J. Biochem. 92,381-387 Gutowski, S . J. & Rosenberg, H. (1977)Biochem. J. 164,265-267 Haddock, B. A. & Downie, J. A. (1974)Biochem. J. 142,703-706 Haddock, B. A. & Kendall-Tobias,N. W. (1975)Biochem. J. 152,655-659 Hamilton, W. A. (1968)J.Gen. Microbiol. 50,441-458 Jones, R. W., Haddock, B. A. & Garland, P. B. (1978)in The Proton and CalciumPumps (Azzone, G. F., Avron, M., Metcalfe, J. C., Quagliariello, E. & Siliprandi,N., eds.), pp. 71-80,Elsevier/ North Holland, Amsterdam and New York Kroger, A. (1978)Biochim. Biophys. Acta 505, 129-145 Macy, J., Kulla, H. & Gottschalk, G. (1976)J.Bacteriol. 125,423-428 Miki, K. & Wilson, T. H. (1978)Biochem. Biophys. Res. Commun. 83,1570-1575 Mortenson, L. E. & Chen, J.-S. (1974)Microbial Iron Metabolism (Neilands, J. B., ed.), pp. 231-282, Academic Press, New York Schuldiner, S., Rottenberg, H. & Avron, M. (1972)Eur. J. Biochem. 25,64-70 West, I. & Mitchell, P. (1974)FEBSLett. 40,1-4 Yamamoto, Y. & Ishimoto, M. (1978)J.Biochem. (Tokyo) 84,673-679
Cell-Density Control of Amino Acid Transport in Simian Virus 40-Transformed 3T3 Cells GIUSEPPE PIEDIMONTE, MARIAROSARIA TRAMACERE and ANGEL0 F. BORGHETTI Instituto di Patologia Generale, Universith di Parma, Via Gramsci 14,43100 Parma, Italy
Changes in membrane transport of low-molecular-weight nutrients are among the cellsurface alterations that are involved in the control of normal cell growth (Pardee, 1975)and in viral transformation (Patterson, 1974). In 3T3 cells, amino acid transport
VOl. 7
BIOCHEMICAL SOCIETY TRANSACTIONS
Beechey, R. B., Hubbard, S.A., Linnett; P. E., Mitchell, A. D. & Munn, E. A. (1975)Biochem.J. 148,533-537 Esch, F. S. &Allison, W. S.(1978)J. Biol. Chem. 253,6100-6106 Linnett, P. E., Mitchell, A. D., Osselton, M. D., Mulheirn,L. J. &Beechey, R. B. (1978)Biochem. J. 170,503-510 Lowe, P. N., Baum, H. & Beechey, R. B. (1979) Biochem. Soc. Trans. 7,237-239 Monsan, P., Puzo, B. & Mazarguil, H. (1975) Biochimie 57,1281-1292
Hydrogen-Dependent Proton Translocation by Membrane Vesicles from Escherichia coli ROBERT W. JONES Department of Biochemistry, Medical Sciences Institute, University of Dundee, Dundee DD1 4HN, Scotland, U.K.
Proton translocation coupled to bacterial fumarate reduction has been demonstrated with a number of electron donors (Gutowski & Rosenberg, 1977; Kroger, 1978; Miki & Wilson, 1978). Escherichia coli can obtain energy for anaerobic growth from hydrogendependent reduction of fumerate (Bernhard & Gottschalk, 1978; Macy et al., 1976; Yamamoto & Ishimoto, 1978). This reaction might therefore be expected to translocate protons (Jones et al., 1978). Quenching of the fluorescence of the acridine dye atebrin has been used to demonstrate the presence of a pH difference, ApH, across the membrane of chloroplasts (Schuldiner et al., 1972), submitochondrial particles (Azzone & Massari, 1973) and inverted membrane vesicles from E. coli(Haddock & Downie, 1974; Haddock & Kendall-Tobias, 1975). The quenching of atebrin fluorescence was used in the experiments described here to show that hydrogen-dependent fumarate reduction by inverted membrane vesicles from E. coli translocates protons. E. coli strain A1002 was grown at 37°C in a 5-litre bottle without stirring so as to maintain anaerobic conditions. The growth medium used was that of Cohen & Rickenberg (1956) supplemented with: glucose, peptone and fumarate (each OS%, w/v); isoleucine, valine and methionine (each 20figlml) ; 1mM-MgC1, ; 1f i ~ - ( N H ~ ) ~ M o , o ~ ~
I
Fumarate
Fumarate Ateirin N T H
,i~
-
Y'
5min
Fig. 1. Hydrogen-plus-fumarate-dependentatebrin-fluorescence quenching by inverted membrane vesiclesfrom E. coli Inverted membrane vesicles (3.4mg) were suspended in 2.5ml of 300rn~-KCl/lSmMacid], pH7.5, MgCl,/lOmM-Hepes ~4-(2-hydroxyethyl)-l-piperazine-ethanesulphonic and bubbled with (a) hydrogen or (6) nitrogen for 10min. Additions, where indicated, were: 4,u~-atebrin; 0.4m~-sodium fumarate, 1mM-NADH; 2-n-heptyl-4-hydroxyquinoline N-oxide (HOQNO, 2,uglml); 0 . 5 m ~ - A T p ;2p~-tetrachlorosalicylanilide
(TCS). 1979
583rd MEETING, CAMBRIDGE
1137
and 1,uM-K2SeO3.Preparation of inverted membrane vesicles and fluorescence measurements were as described previously (Haddock & Downie, 1974). Fig. l(a) shows that the addition of fumarate to a hydrogen-saturated suspension of inverted membrane vesicles of E. coli causes the quenching of atebrin fluorescence. The return of fluorescence after a few minutes is probably due to exhaustion of hydrogen, since addition of NADH restores the quenching. Addition of the respiratory inhibitor 2-n-heptyl-4-hydroxyquinolineN-oxide causes the fluorescence to return. Quenching is restored by the addition of ATP as the ATPase pumps protons into the intravesicular space while hydrolysing ATP (West & Mitchell, 1974). Addition of the uncoupler tetrachlorosalicylanilide (Hamilton, 1968) allows protons to equilibrate across the membrane and fluorescence is again observed. Addition of fumarate to a N,-saturated suspension of vesicles does not give rise to fluorescence quenching (Fig. Ib), showing that the quenching in Fig. l(a) is hydrogen-dependent. Hydrogen-dependent quenching was also observed with oxygen and nitrate as electron acceptors, and fumarate-dependent quenching was observed with NADH and formate as electron donors (results not shown). Hydrogen-plus-fumarate-dependentquenching was abolished by 2-n-hept yl-4-hydroxyquinoline N-oxide, tetrachlorosalicylanilide and CO, an inhibitor of several bacterial hydrogenases (Mortensen & Chen, 1974). There is evidence that acridine-fluorescence quenching is a qualitative probe for ApH (Schuldiner et al., 1972;Elema et al., 1978), but its value in the quantitative determination of ApH has been questioned (Elema et al., 1978). It can be concluded that hydrogen-dependent fumarate reduction by membrane vesicles from E. coli is coupled to an inward translocation of protons. This would be an outward translocation in intact cells. This work was supported by the Science Research Council. Auone, G. F. & Massari, S. (1973)Biochim. Biophys. Acta 301,195-226 Bernhard, Th. & Gottschalk, G. (1978)Arch. Microbiol. 116,235-238 Cohen, G . N. & Rickenberg, H. W. (1956)Ann. Znst. Pasteur, Paris, 91,693-720 Elema, R. P.,Michels, P. A. M. & Konings, W. N. (1978)Eur. J. Biochem. 92,381-387 Gutowski, S . J. & Rosenberg, H. (1977)Biochem. J. 164,265-267 Haddock, B. A. & Downie, J. A. (1974)Biochem. J. 142,703-706 Haddock, B. A. & Kendall-Tobias,N. W. (1975)Biochem. J. 152,655-659 Hamilton, W. A. (1968)J.Gen. Microbiol. 50,441-458 Jones, R. W., Haddock, B. A. & Garland, P. B. (1978)in The Proton and CalciumPumps (Azzone, G. F., Avron, M., Metcalfe, J. C., Quagliariello, E. & Siliprandi,N., eds.), pp. 71-80,Elsevier/ North Holland, Amsterdam and New York Kroger, A. (1978)Biochim. Biophys. Acta 505, 129-145 Macy, J., Kulla, H. & Gottschalk, G. (1976)J.Bacteriol. 125,423-428 Miki, K. & Wilson, T. H. (1978)Biochem. Biophys. Res. Commun. 83,1570-1575 Mortenson, L. E. & Chen, J.-S. (1974)Microbial Iron Metabolism (Neilands, J. B., ed.), pp. 231-282, Academic Press, New York Schuldiner, S., Rottenberg, H. & Avron, M. (1972)Eur. J. Biochem. 25,64-70 West, I. & Mitchell, P. (1974)FEBSLett. 40,1-4 Yamamoto, Y. & Ishimoto, M. (1978)J.Biochem. (Tokyo) 84,673-679
Cell-Density Control of Amino Acid Transport in Simian Virus 40-Transformed 3T3 Cells GIUSEPPE PIEDIMONTE, MARIAROSARIA TRAMACERE and ANGEL0 F. BORGHETTI Instituto di Patologia Generale, Universith di Parma, Via Gramsci 14,43100 Parma, Italy
Changes in membrane transport of low-molecular-weight nutrients are among the cellsurface alterations that are involved in the control of normal cell growth (Pardee, 1975)and in viral transformation (Patterson, 1974). In 3T3 cells, amino acid transport
VOl. 7
