Transport of succinate in Escherichia coli. II. Characteristics of uptake and energy coupling with transport in membrane preparations (original) (raw)
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Proceedings of the National Academy of Sciences, 1970
The conversion of D-lactate to pyruvate in isolated membrane preparations of E. coli ML 308-225 markedly stimulates the transport of proline, glutamic acid, aspartic acid, asparagine, tryptophan, lysine, serine, alanine, and glycine. The uptake of histidine, phenylalanine, tyrosine, leucine, isoleucine, and valine by the membranes is also markedly stimulated by this conversion, although these amino acids are taken up much less effectively than those mentioned previously. The uptake of arginine, methionine, cystine, and cysteine is enhanced only about twofold in the presence of D-(-)-lactate, and these amino acids are not concentrated well by the membranes. With the exception of glutamate, aspartate, asparagine, and methionine, which are converted to other metabolites to varying extents in the intramembranal pool, each of the other amino acids was recovered from the membranes as the unchanged amino acid. Succinate, L-(+)-lactate, D,L-a-hydroxybutyrate, and DPNH partially replace D-(-)-lactate but are less effective. Previous work from this laboratory" 2 demonstrated that isolated membrane preparations from E. coli W6, in the absence of soluble proteins and nucleic acids, catalyzed the concentrative uptake of proline, whereas membranes prepared from E. coli W157, a proline transport mutant,3 did not. Proline uptake by these preparations was stimulated by glucose and was inhibited by anaerobiosis and by a variety of compounds known to uncouple oxidative phosphorylation or inhibit electron transport. Studies recently made with membranes prepared from E. coli 1\IL 308-225 have defined the energetics of the proline uptake system in much greater detail.4 D-(-)-Lactate markedly stimulates proline uptake with a 20to 30-fold increase over baseline levels. Of all the metabolites and cofactors tested, only succinate, L-(+)-lactate, D,L-a-hydroxybutyrate, and DPNH replace D-(-)-lactate to any extent whatsoever. Succinate stimulates proline uptake 8to 10-fold, and L-(+)-lactate, D,L-a-hydroxybutyrate, and DPNH stimulate only 3to 4-10(8
Proton-linked l-fucose transport in Escherichia coli
Biochemical Journal, 1987
Addition of L-fucose to energy-depleted anaerobic suspensions of Escherichia coli elicited an uncouplersensitive alkaline pH change diagnostic of L-fucose/H' symport activity. 2. L-Galactose or D-arabinose were also substrates, but not inducers, for the L-fucose/H+ symporter. 3. L-Fucose transport into subcellular vesicles was dependent upon respiration, displayed a pH optimum of about 5.5, and was inhibited by protonophores and ionophores. 4. These results showed that L-fucose transport into E. coli was energized by the transmembrane electrochemical gradient of protons. 5. Neither steady state kinetic measurements nor assays of L-fucose binding to periplasmic proteins revealed the existence of a second L-fucose transport system.
MECHANISM OF LACTOSE TRANSLOCATION IN MEMBRANE VESICLES FROM ESCHERICHIA COLI
Annals of The New York Academy of Sciences, 1980
The chemiosmotic hypothesis proposed by Peter Mitchell 1-5 has stimulated widespread interest in the role of the "protonmotive force" in bioenergetic processes. According to this hypothesis, energy derived from respiration or photochemical reactions is transformed into a transmembrane electrochemical gradient of protons ( A~T~~+ ) that represents the immediate driving force for the synthesis of ATP, active transport, and certain other energy-dependent processes.G In 1963, Mitchell postulated explicitly that ATrI+ drives the accumulation of B-galactosides in E.wherichia coli and that the active transport of these substrates occurs via coupled movements with protons (i.e., symport) .i By this means, a 8-galactoside-specific membrane protein (the product of the lac y gene) translocates substrate with protons, the substrate moving against and the proton( s) with their respective electrochemical gradients.
Biochemistry, 1980
These studies document the effects of the proton electrochemical gradient (ApH+, interior negative and alkaline) on the kinetics of various transport systems in right-side-out membrane vesicles from Escherichia coli, with particular emphasis on the @-galactoside transport system. Under completely deenergized conditions (Le., facilitated diffusion), the @-galactoside transport system exhibits a high apparent K, for either lactose or @-D-galactopyranosyl I-thio-@-Dgalactopyranoside, and generation of ApH+ via the respiratory chain results in at least a 100-fold decrease in the apparent K,. Furthermore, a low apparent K, is observed when the membrane potential (A*) or the pH gradient (SpH) is dissipated selectively with an appropriate ionophore and when either A* or ApH is imposed artifically across the membrane. Thus, either component of ApH+ is able to elicit the low apparent K, characteristic of the energized system. A detailed series of kinetic experiments is presented in which initial rates of lactose transport were studied as a function of lactose concentration under conditions where ApH and/or A* were varied systematically at pH 5.5 and 7.5. Surprisingly, the results demonstrate that the apparent K, remains constant from about -180 to -30 mV, while the maximum velocity of transport varies to the second power with either component of SpH+ at both pHs, even though the maximum velocity is about IO-fold higher at pH 7.5 over a comparable range of ApH+ values. Since a high apparent K , is observed under completely deenergized conditions, the findings appear to be paradoxical; however, studies carried out over an extended range of lactose concentrations demonstrate that when ApH+ is dissipated partially the system exhibits biphasic kinetics. M e m b r a n e vesicles prepared from Escherichia coli are essentially devoid of cytoplasmic constituents and retain the same polarity and configuration as the membrane in the intact cell Owen & Kaback, 1978, 1979a. Furthermore, in addition to catalyzing the vectorial phosphorylation of certain sugars via the phosphoenolpyruvate-
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1975
Escherichia coli and two partial revertants of that mutant were examined for the ability to generate a high energy membrane state with Dlactate or ATP, as measured by the quenching of the fluorescent dye quinacrine, 2. All three strains showed reductions in the aerobically-driven quenching of fluorescence compared to the wild type, but the reduction could be reversed by the addition of either N,N'-dicyclohexylcarbodiimide or the crude soluble ATPase of the wild type. 3. The mutant exhibited a decreased ability to accumulate sugars and amino acids and showed an increased permeability to protons. 4. One partial revertant showed a slight increase in active transport and a slight decrease in proton permeability. 5. The other partial revertant showed a large increase in transport ability and a large decrease in proton permeability. 6. A model is proposed in which the conformation of the Mg2+-ATPase is important in the utilization of energy derived from the electron transport chain and this function is independent of the catalytic activity of the Mg 2 รท-ATPase.
European Journal of Biochemistry, 1976
Active transport of amino acids by membrane vesicles from Eschrricizicr cnli, grown anaerobically on glucose in the presence of nitrate, can be energized under anaerobic conditions by electron transfer in the nitrate respiration system with formate as electron donor and nitrate as acceptor. A high rate of amino acid transport is also obtained under anaerobic conditions by electron transfer from formate to the nitrate analogue chlorate or to the membrane-impermeable electron acceptor ferricyanide. Electron transfer from formate to nitrate results in the generation of an electrical potential as is indicated by the uptake of the lipophilic cation triphenylmethylphosphonium. Ferricyanide accepts electrons from at least two sites of the nitrate respiration system. One of these sites appears to be nitrate reductase, because cytochrome 6, reduced by formate, is completely reoxidized by ferricyanide and glutamate transport energized by formate plus ferricyanide and formate plus nitrate are affected by the same electron transfer inhibitors. A second site of electron transfer to ferricyanide appears to be located prior to nitrate reductase in the nitrate respiration system, since formate is oxidized at a higher rate in the presence of ferricyanide than with nitrate while formate/ferricyanide energizes transport of amino acids at a lower rate than formate/nitrate. Moreover, electron transfer inhibitors block electron transfer from formate to nitrate to a significantly higher extent than from formate to ferricyanide. The effects of irradiation of the membrane vesicles with near ultraviolet light suggest that quinones play an essential role in the electron transfer from formate to nitrate or ferricyanide. Irradiation blocks completely formate-dependent nitrate and ferricyanide reduction and active transport driven by formate/nitrate and formate/ferricyanide, but has hardly any effect on the activity of formate dehydrogenase and on ascorbate/phenazine methosulphatejoxygen-driven transport. Similar effects of ferricyanide have been observed in membrane vesicles from E. coli, grown anaerobically in the presence of fumarate. In these membrane vesicles a high rate of lactose and triphenylmethylphosphonium uptake under anaerobic conditions is obtained by electron transfer from glycerol 1-phosphate to fumarate and also to ferricyanide and evidence has been presented for the involvement of cytochromes in these electron transfers.
Sucrose transport through maltoporin mutants of Escherichia coli
Protein Engineering Design & Selection, 2001
Maltoporin (LamB) and sucrose porin (ScrY) reside in the bacterial outer membrane and facilitate the passive diffusion of maltodextrins and sucrose, respectively. To gain further insight into the determinants of solute specificity, LamB mutants were designed to allow translocation of sucrose, which hardly translocates through wild-type LamB. Three LamB mutants were studied. (a) Based on sequence and structure alignment of LamB with ScrY, two LamB triple mutants were generated (R109D, Y118D, D121F; R109N, Y118D, D121F) to mimic the ScrY constriction. The crystal structure of the first of these mutants was determined to be 3.2 A and showed an increased ScrY-like cross-section except for D109 that protrudes into the channel. (b) Based on this crystal structure a double mutant was generated by truncation of the two residues that obstruct the channel most in LamB (R109A, Y118A). Analysis of liposome swelling and in vivo sugar uptake demonstrated substantial sucrose permeation through all mutants with the double alanine mutant performing best. The triple mutants did not show a well-defined binding site as indicated by sugar-induced ion current noise analysis, which can be explained by remaining steric interference as deduced from the crystal structure. Binding, however, was observed for the double mutant that had the obstructing residues truncated to alanines.
Succinate transport in Rhizobium leguminosarum
Journal of bacteriology, 1981
The transport of succinate was studied in an effective streptomycin-resistant strain of Rhizobium leguminosarum. High levels of succinate transport occurred when cells were grown on succinate, fumarate, or malate, whereas low activity was found when cells were grown on glucose, sucrose, arabinose, or pyruvate as the sole carbon source. Because of the rapid metabolism of succinate after transport into the cells, a succinate dehydrogenase-deficient mutant was isolated in which intracellular succinate accumulated to over 400 times the external concentration. Succinate transport was completely abolished in the presence of metabolic uncouplers but was relatively insensitive to sodium arsenate. Succinate transport was a saturable function of the succinate concentration, and the apparent Km and Vmax values for transport were determined in both the parent and the succinate dehydrogenase mutant. Malate and fumarate competitively inhibited succinate transport, whereas citrate and malonate had...
Biochemistry, 1985
Right-side-out cytoplasmic membrane vesicles from Escherichia coli M L 308-22, a mutant "uncoupled" for @-galactoside/H+ symport [Wong, P. T. S., Kashket, E. R., & Wilson, T. H. (1970) Proc. Natl. Acad. Sci. U.S.A. 65, 631, are specifically defective in the ability to catalyze accumulation of methyl l-thio-(3-D-galactopyranoside (TMG) in the presence of an H + electrochemical gradient (interior negative and alkaline). Furthermore, the rate of carrier-mediated efflux under nonenergized conditions is slow and unaffected by ambient pH from pH 5.5 to 7.5, and TMG-induced H+ influx is only about 15% of that observed in vesicles containing wild-type lac permease ( M L 308-225). Alternatively, M L 308-22 vesicles bind pnitrophenyl a-D-galactopyranoside and monoclonal antibody 4B 1 to the same extent as ML 308-225 vesicles and catalyze facilitated diffusion and equilibrium exchange as well as M L 308-225 vesicles. When entrance counterflow is studied with external substrate at saturating and subsaturating concentrations, it is apparent that the mutation simulates the effects of deuterium oxide [Viitanen, P., Garcia, M. L., Foster, D. L., Kaczorowski, G. J., & Kaback, H. R. (1983) Biochemistry 22, 253 11.