Sidedness of native membrane vesicles of Escherichia coli and orientation of the reconstituted lactose: H+ carrier

The orientation of the lactose:H+ carrier of Escherichia coli in various preparations of native and reconstituted vesicles is determined with two impermeant, macromolecular probes: antibodies directed against the C-terminal decapeptide of the carrier and carboxypeptidase A (EC 3.4.17.1). Two methods are employed. Method I is based upon the digestion of all accessible and, therefore, presumably external, C termini of the carrier with carboxypeptidase A and detection of the remaining, internal C termini with 125I-labelled anti-(C-terminus) antibody after electrophoresis of the carrier in the presence of sodium dodecyl sulfate and transfer to nitrocellulose filters. Method II is based upon the binding of 125I-labelled anti-(C-terminus) antibody to the external C termini of the carrier in vesicles and the subsequent isolation of bound antibody by centrifugation. The labelled antibodies are calibrated using a preparation of inside-out vesicles prepared by high-pressure lysis of strain T206. The carrier content is determined by substrate binding. Because the C terminus of the carrier is known to reside on the cytoplasmic side of the membrane, these methods can also be used to determine the sidedness of various preparations of membrane vesicles. Spheroplasts are confirmed to contain carrier molecules of a single orientation, corresponding to that in right-side-out vesicles. In contrast, in purified cytoplasmic membrane vesicles and in crude membrane preparations obtained by sonication or by high-pressure lysis, 96% of the C termini are accessible to carboxypeptidase A, even after repeated sonication. This implies that nearly all carrier molecules in these preparations possess an orientation opposite to that in the cell or in right-side-out vesicles. In proteoliposomes containing carrier reconstituted or purified and reconstituted by two different methods, only 48% of the carrier molecules are oriented in the same way as in the cell. Subjecting such proteoliposomes to cycles of freezing and thawing or to sonication results in a reshuffling of carrier molecules between the inside-out and right-side-out populations while maintaining 41% in the right-side-out orientation. Digestion of the C terminus of the carrier with carboxypeptidase A does not alter either galactoside binding or countertransport. Thus carrier molecules of the inside-out orientation cannot be selectively inactivated. Additionally, an antiserum directed against the purified carrier is demonstrated to contain nearly exclusively anti-(C-terminus) antibodies, which can, in principle, be used in Method I.(ABSTRACT TRUNCATED AT 400 WORDS)     

The kinetic mechanism of galactoside/H+ cotransport in Escherichia coli

To determine the kinetic mechanism of galactoside active transport by the lactose/H+ cotransporter of Escherichia coli, galactoside binding and transport are studied in the absence and presence of delta mu H+. For several reasons, the substrate beta-D-galactosyl-1-thi-beta-D-galactoside (GalSGal) is preferred over lactose. In the absence of delta mu H+, the cotransporter retains high affinity for GalSGal, and the affinity is the same on both sides of the membrane. At physiological pH, the cotransporter is protonated and the dissociation constant for H+ may be 50 pM. The cosubstrates bind in a random fashion. An isomerization of the cotransporter corresponding to reorientation of the binding sites is rate-determining. When delta mu H+ is imposed, two reorientations become faster, and one becomes slower. The affinity of the cotransporter for GalSGal on both sides of the membrane is unchanged. The inability of the cotransporter to bring the accumulation of galactoside into equilibrium with delta mu H+ at high galactoside concentrations can be explained without postulating uncoupled fluxes of galactoside or H+ across the membrane (leaks). The formation of the ternary carrier-H+-galactoside complex on the cytoplasmic side of the membrane with increasing internal levels of sugar and the rapidity of galactoside exchange inhibit net influx of galactoside and favor exchange. Net transport is slow at high galactoside levels. Thus, the cotransporter can self-regulate transport without uncoupling H+ and galactoside fluxes. Because the values of delta mu H+ during binding and transport studies were measured, these results can be subjected to a quantitative analysis.     

Substrate recognition at the cytoplasmic and extracellular binding site of the lactose transport protein of Streptococcus thermophilus

The lactose transport protein (LacS) of Streptococcus thermophilus catalyzes the uptake of lactose in an exchange reaction with intracellularly formed galactose. The interactions between the substrate and the cytoplasmic and extracellular binding site of LacS have been characterized by assaying binding and transport of a range of sugars in proteoliposomes, in which the purified protein was reconstituted with a unidirectional orientation. Specificity for galactoside binding is given by the spatial configuration of the C-2, C-3, C-4, and C-6 hydroxyl groups of the galactose moiety. Except for a C-4 methoxy substitution, replacement of the hydroxyl groups for bulkier groups is not tolerated at these positions. Large hydrophobic or hydrophilic substitutions on the galactose C-1 alpha or beta position did not impair transport. In fact, the hydrophobic groups increased the binding affinity but decreased transport rates compared with galactose. Binding and transport characteristics of deoxygalactosides from either side of the membrane showed that the cytoplasmic and extracellular binding site interact differently with galactose. Compared with galactose, the IC(50) values for 2-deoxy- and 6-deoxygalactose at the cytoplasmic binding site were increased 150- and 20-fold, respectively, whereas they were the same at the extracellular binding site. From these and other experiments, we conclude that the binding sites and translocation pathway of LacS are spacious along the C-1 to C-4 axis of the galactose moiety and are restricted along the C-2 to C-6 axis. The differences in affinity at the cytoplasmic and extracellular binding site ensure that the transport via LacS is highly asymmetrical for the two opposing directions of translocation.     

Lactose transport system of Streptococcus thermophilus. Functional reconstitution of the protein and characterization of the kinetic mechanism of transport.

The kinetic mechanism of the lactose transport system of Streptococcus thermophilus was studied in membrane vesicles fused with cytochrome c oxidase containing liposomes and in proteoliposomes in which cytochrome c oxidase was coreconstituted with the lactose transport protein. Selective manipulation of the components of the proton (and sodium) motive force indicated that both a membrane potential and a pH gradient could drive transport. The galactoside/proton stoichiometry was close to unity. Experiments which discriminate between the effects of internal pH and delta pH as driving force on galactoside/proton symport showed that the carrier is highly activated at alkaline internal pH values, which biases the transport system kinetically toward the pH component of the proton motive force. Galactoside efflux increased with increasing pH with a pKa of about 8, whereas galactoside exchange (and counterflow) exhibited a pH optimum around 7 with pKa values of 6 and 8, respectively. Imposition of delta pH (interior alkaline) retarded the rate of efflux at any pH value tested, whereas the rate of exchange was stimulated by an imposed delta pH at pH 5.8, not affected at pH 7.0, and inhibited at pH 8.0 and 9.0. The results have been evaluated in terms of random and ordered association/dissociation of galactoside and proton on the inner surface of the membrane. Imposition of delta psi (interior negative) decreased the rate of efflux but had no effect on the rate of exchange, indicating that the unloaded transport protein carries a net negative charge and that during exchange and counterflow the carrier recycles in the protonated form.     

The mechanism of inducer exclusion. Direct interaction between purified III of the phosphoenolpyruvate:sugar phosphotransferase system and the lactose carrier of Escherichia coli

A hypothesis for the regulation of some sugar transport systems by the bacterial phosphoenolpyruvate:sugar transport system postulates an interaction between III^Glc of this system and the carrier whose activity is regulated. We have studied this interaction in more detail, employing one of these transport systems, the lactose carrier of Escherichia coli. Purified III^Glc of the phosphotransferase system interacted directly with the lactose carrier. The binding of III^Glc to lactose carrier required the presence of the non-phosphorylated form of III^Glc and substrates of the carrier and exhibited a stoichiometry of 1.2± 0.2 mol III^Glc/mol lactose carrier. The K_d of lactose carrier for III^Glc was 10 ± 5 µM. III^Glc is apparently unable to interact with a mutant lactose carrier which still binds but does not transport galactosides. The binding of III^Glc to the lactose carrier results in a 3.5-fold increase in the apparent affinity of galactosides for the carrier. Significantly, the binding of III^Glc to the lactose carrier results in an inhibition of galactoside translocation both in membrane vesicles and liposomes reconstituted with the purified lactose carrier. This inhibition may thus be the basis for the well-documented phenomenon of inducer exclusion.     

Kinetics of ATP synthesis catalyzed by the H(+)-ATPase from chloroplasts (CF0F1) reconstituted into liposomes and coreconstituted with bacteriorhodopsin.

The H(+)-ATPase from chloroplasts (CF0F1) was isolated, purified and reconstituted into liposomes from phosphatidylcholine/phosphatidic acid. A transmembrane pH difference, delta pH, and a transmembrane electric potential difference, delta psi, were generated by an acid/base transition. The rate of ATP synthesis was measured at constant delta pH and constant delta psi as a function of temperature between 5 degrees C and 45 degrees C. The activation energy was 55 kJ mol-1. CF0F1 was coreconstituted with bacteriorhodopsin at a molar ratio of approximately 1:170 in the same type of liposomes. Illumination of the proteoliposomes leads to proton transport into the vesicles generating a constant delta pH = 1.8. The dependence of the rate of ATP synthesis on ADP concentration was measured with CF0F1 in the oxidized state, E(ox), and in the reduced state, E(red). The results can be described by Michaelis-Menten kinetics with the following parameters: Vmax = 0.5 s-1, Km = 8 microM for E(ox) and Vmax = 2.0 s-1, Km = 8 microM for E(red).     

Characterization of purified, reconstituted site-directed cysteine mutants of the lactose permease of Escherichia coli

lac permease mutated at each of the 8 cysteinyl residues in the molecule was solubilized from the membrane, purified, and reconstituted into proteoliposomes. The transport activity of proteoliposomes reconstituted with each mutant permease relative to the wild-type is virtually identical with that reported for intact cells and/or right-side-out membrane vesicles. Moreover, a double mutant containing Ser in place of both Cys148 and Cys154 exhibits significant ability to catalyze active lactose transport. The results provide strong confirmation for the contention that cysteinyl residues in lac permease do not play an important role in the transport mechanism. The effect of sulfhydryl oxidant 5-hydroxy-2-methyl-1,4-naphthoquinone on lactose transport in proteoliposomes reconstituted with wild-type or mutant permeases was also investigated, and the results indicate that inactivation is probably due to formation of a covalent adduct with Cys148 and/or Cys154 rather than disulfide formation. Thus, it seems unlikely that sulfhydryl-disulfide interconversion functions to regulate permease activity.     

Transport by reconstituted lactose carrier from parental and mutant strains ofEscherichia coli

Summary The lactose transport carrier from parental (X71/F'W3747) and mutant cells (54/F'5441) was reconstituted into proteoliposomes. Transport by the counterflow assay showed slightly greater activity in proteoliposomes prepared from extracts of the mutant membranes compared with that for the parental cell. The mutant carrier showed a threefold lowerK _m but similarV _max compared to the parent. On the other hand proteoliposomes from the mutant showed a defect in protonmotive force-driven accumulation, compared with the parent. With a pH gradient (inside alkaline) plus a membrane potential (inside negative) the parental proteoliposomes accumulated lactose 25-fold over the medium concentration while the mutant proteoliposomes accumulated sixfold. In a series of experiments proteoliposomes were exposed to proteolytic enzymes. Chrymotrypsin treatment resulted in 30% inhibition of counterflow activity for the reconstituted carrier from both parent and mutant. Papain produced 84% inhibition of transport by the reconstituted parental carrier but only 41% of that of the mutant. Trypsin and carboxypeptidase Y treatment had no effect on counterflow activity of either parent or mutant. Exposure of purified lactose carrier in proteoliposomes to carboxypeptidase Y resulted in the release of alanine and valine, the two C-terminal amino acids predicted from the DNA sequence.     

Characterization of the unidirectional transport of carnitine catalyzed by the reconstituted carnitine carrier from rat liver mitochondria

The carnitine carrier from rat liver mitochondria was purified by chromatography on hydroxyapatite and celite and reconstituted in egg yolk phospholipid vesicles by adsorbing the detergent on polystyrene beads. In the reconstituted system, in addition to the carnitine/carnitine exchange, the purified protein catalyzed a uni-directional transport (uniport) of carnitine measured as uptake into unloaded proteoliposomes as well as efflux from prelabelled proteoliposomes. In both cases the reaction followed a first-order kinetics with a rate constant of 0.023-0.026 min-1. Besides carnitine, also acylcarnitines were transported in the uniport mode. N-Ethylmaleimide inhibited the uni-directional transport of carnitine completely. The uniport of carnitine is not influenced by the delta pH and the electric gradient across the membrane. The activation energy for uniport was 115 kJ/mol and the half-saturation constant on the external side of the proteoliposomes was 0.53 mM. The maximal rate of the uniport at 25 degrees C was 0.2 mumol/min per mg protein, i.e. about 10 times lower than that of the reconstituted carnitine transport in exchange mode.     

The phospholipid requirement for activity of the lactose carrier of Escherichia coli

The transport activity of the lactose carrier of Escherichia coli has been reconstituted in proteoliposomes composed of different phospholipids. The maximal activity was observed with the natural E. coli lipid as well as mixtures containing phosphatidylethanolamine or phosphatidylserine. Phosphatidylcholine or mixtures of phosphatidylcholine with phosphatidylglycerol, phosphatidic acid, or cardiolipin showed low activity. The lactose carrier reconstituted with amino phospholipids of increasing degrees of methylation (dioleoylphosphatidylethanolamine, dioleoylmonomethylphosphatidylethanolamine, dioleoyldimethylphosphatidylethanolamine, and dioleoylphosphatidylcholine) revealed a progressive decrease in both counterflow and proton motive force-driven lactose uptake activities. Trinitrophenylation of phosphatidylethanolamine in the E. coli proteoliposomes resulted in a marked reduction in lactose carrier activity. Partial restitution of transport activity was obtained by detergent extraction of the carrier from these inactive proteoliposomes and reconstitution of the carrier into proteoliposomes containing normal E. coli lipid. These results suggest that the amino group of the amino phospholipids (e.g. phosphatidylethanolamine and phosphatidylserine) is required for the full function of the lactose carrier from E. coli.     

Kinetic analysis of lactose exchange in proteoliposomes reconstituted with purified lac permease

Lactose exchange catalyzed by purified lac permease reconstituted into proteoliposomes was analyzed with unequal concentrations of lactose on either side of the membrane and at low pH so as to prevent equilibration of the two pools. Exchange with external concentrations below 1.0 mM is a single-exponential process, and the apparent affinity constants for external and internal substrate are close to the apparent KMs reported for active transport and efflux, respectively [Viitanen, P.V., Garcia, M. L., & Kaback, H. R. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1629]. At external lactose concentrations above 1.0 mM, a second kinetic pathway becomes evident with an apparent affinity constant of about 6 mM which is similar to the apparent KM for facilitated influx. A second pathway is not observed with respect to internal lactose even when the concentration is increased up to 80 mM. Furthermore, high internal or external lactose concentrations do not inhibit the exchange reaction. Biphasic kinetics with respect to external lactose are retained in a mutant permease that catalyzes exchange but is defective in H(+)-coupled lactose transport. It is suggested that lac permease has more than one binding site and that this may be the underlying reason for the biphasic kinetics observed for both exchange and H(+)-coupled lactose transport.     

Monoclonal antibodies against the lac carrier protein from Escherichia coli. 1. Functional studies

The effects of various monoclonal antibodies against purified lac carrier protein on carrier-mediated lactose transport were studied in right-side-out membrane vesicles and in proteoliposomes reconstituted with purified lac carrier protein. Out of more than 60 monoclonal antibodies tested, only one antibody, designated 4B1, inhibits transport. Furthermore, the nature of the inhibition is highly specific in that the antibody inhibits only those transport reactions that involve net proton translocation (i.e., active transport, carrier-mediated influx and efflux under nonenergized conditions, and lactone-induced proton influx). In contrast, the antibody has little effect on equilibrium exchange and no effect on generation of the proton electrochemical gradient or on the ability of the carrier to bind a high-affinity ligand. Clearly, therefore, the antibody alters the relationship between lactose and proton translocation at the level of the lac carrier protein. When entrance counterflow is studied with external [1-14C]lactose at saturating and subsaturating concentrations, it is apparent that antibody 4B1 mimics the effects of deuterium oxide [Viitanen, P., Garcia, M.L., Foster, D.L., Kaczorowski, G. J., & Kaback, H.R. (1983) Biochemistry 22, 2531]. That is, the antibody has no effect on the rate or extent of counterflow when external lactose is saturating but stimulates the efficiency of counterflow when external lactose is below the apparent Km. It seems likely, therefore, that the antibody either inhibits the rate of deprotonation or alters the equilibrium between protonated and deprotonated forms of the carrier. Monovalent Fab fragments prepared from antibody 4B1 inhibit transport in a manner that is similar qualitatively to that of the intact antibody.(ABSTRACT TRUNCATED AT 250 WORDS)     

The mitochondrial tricarboxylate carrier of silver eel: chemical modification by sulfhydryl reagents

The tricarboxylate (or citrate) carrier was purified from eel liver mitochondria and functionally reconstituted into liposomes. Incubation of the proteoliposomes with various sulfhydryl reagents led to inhibition of the reconstituted citrate transport activity. Preincubation of the proteoliposomes with reversible SH reagents, such as mercurials and methanethiosulfonates, protected the eel liver tricarboxylate carrier against inactivation by the irreversible reagent N-(1-pyrenyl)maleimide (PM). Citrate and L-malate, two substrates of the tricarboxylate carrier, protected the protein against inactivation by sulfhydryl reagents and decreased the fluorescent PM bound to the purified protein. These results suggest that the eel liver tricarboxylate carrier requires a single population of free cysteine(s) in order to manifest catalytic activity. The reactive cysteine(s) is most probably located at or near the substrate binding site of the carrier protein.     

The nitrite transport protein NirC from Salmonella typhimurium is a nitrite/proton antiporter

In anaerobically grown bacteria, transport of nitrite is catalyzed by an integral membrane protein of the form ate-nitrite transporter family, NirC, which in Salmonella typhimurium plays a critical role in intracellular virulence. We present a functional characterization of the S. typhimurium nitrite transporter StmNirC in native membrane vesicles as well as purified and reconstituted into proteoliposomes. Using an electrophysiological technique based on solid supported membranes, we show nitrite induced translocation of negative charges into proteoliposomes reconstituted with purified StmNirC. These data demonstrate the electrogenicity of StmNirC and its substrate specificity for nitrite. Monitoring changes in ΔpH on everted membrane vesicles containing overexpressed StmNirC using acridine orange as a pH indicator we demonstrate that StmNirC acts as a secondary active transporter. It promotes low affinity transport of nitrite coupled to H(+) antiport with a pH independent profile in the pH range from 6 to 8. In addition to nitrite also nitrate is transported by StmNirC, but with reduced flux and complete absence of proton antiport activity. Taken together, these data suggest a bispecific anion selectivity of StmNirC with an ion specific transport mode. This may play a role in regulating nitrite transport under physiological conditions.     

The reconstituted isolated uncoupling protein is a membrane potential driven H+ translocator.

The isolated uncoupling protein (UCP) from brown fat adipose tissue mitochondria has been reconstituted into artificial phospholipid vesicles. Because of the high lability of H+ transport, several new steps have been introduced in the reconstitution; the detergent octyl-POE, the addition of phospholipids to mitochondria prior to solubilization and purification, the vesicle formation by rapid removal of detergent with polystyrene beads and of external salts by a mixed ion exchange. In the K+-loaded proteoliposomes, H+ influx can be induced by a diffusion potential on addition of valinomycin. H+ influx is inhibited to more than 90% by GTP addition, in the assay for UCP activity. By reversing delta psi with external K+, H+ efflux is measured, however, at a four times lower rate. In vesicles loaded with internal GTP, H+ influx is fully inhibited but can be activated by Dowex-OH treatment to an even higher rate than that found in the GTP-free vesicles. Binding studies with GTP show that most of the active UCP are oriented with the binding site outside as in mitochondria, and that in GTP-loaded vesicles GTP is also bound at the outside. The rate of H+ transport is linearly dependent on the membrane potential. Despite the ordered orientation, there is no 'valve' mechanism, since there is H+ efflux with a reversed potential. pH dependency is only small between pH 6.5 and 7.5, indicating that the H+-translocating site differs from the highly pH-dependent nucleotide-binding site. The turnover number of reconstituted UCP is commensurate with mitochondrial function and indicates a carrier instead of a channel-type H+ transport.(ABSTRACT TRUNCATED AT 250 WORDS)     

Partial purification of alanine carrier from rabbit small intestine brush border membrane and its functional reconstitution into proteoliposomes

An alanine transport carrier was partially purified from brush border membranes of rabbit small intestine. The alanine carrier activity was not solubilized with 0.4% deoxycholate but recovered in the detergent-insoluble fraction. The detergent-insoluble proteins were reconstituted into proteoliposomes with soybean phospholipids. The reconstituted proteoliposomes were capable of uptake of alanine driven by an electrochemical potential of Na+. The initial rate of alanine uptake into the proteoliposomes was 90 pmoles/mg protein/sec, which was 15-fold higher than that observed with the native membrane vesicles. The uptake of alanine was effectively suppressed by various neutral amino acids but not by either cationic or anionic amino acids.     

The effects of pH on proton sugar symport activity of the lactose permease purified from Escherichia coli

The lactose permease, which catalyzes galactoside-proton symport into Escherichia coli, has been purified and reconstituted in active form into artificial lipid vesicles. The roles of many detergents and phospholipids in solubilization and stabilization of the activity of the permease have been examined with a view to its eventual crystallization. Initial rates of uptake into reconstituted proteoliposomes determined by rapid mixing techniques proved that the activity of the permease can be comparable to that observed in the intact cell, while the best values for uptake rates obtained with conventional techniques were comparable to those reported for vesicles. The activity of the purified protein has been monitored over time periods of hours to weeks. It is shown that, under the best current conditions, the permease retains full activity for 1 to 2 weeks. Although this is still marginal for its crystallization, future improvements can now be assayed by rather stringent criteria. The mechanism of galactoside transport into reconstituted proteoliposome has been investigated by examining the effects of pH on influx into the vesicles. It is shown that the observed effects are entirely consistent with the predictions of a simple model of proton symport. The apparent increase in rate of uptake that is observed in the presence of a pH gradient is not so much due to an acceleration by a component of the protonmotive force as to the relaxation of inhibition by a product (internal protons) of the symport reaction.     

A conformational change in the lactose permease of Escherichia coli is induced by ligand binding or membrane potential.

Lactose transport in membrane vesicles containing lactose permease with a single Cys residue in place of Val 315 is inactivated by N-ethylmaleimide in a manner that is stimulated by substrate or by a H+ electrochemical gradient (delta microH+; Sahin-Tóth M, Kaback HR, 1993, Protein Sci 2:1024-1033). The findings are confirmed and extended in this communication. Purified, reconstituted Val 315-->Cys permease reacts with N-ethylmaleimide or hydrophobic fluorescent maleimides but not with a membrane impermeant thiol reagent, and beta-galactosides specifically stimulate the rate of labeling. Furthermore, the reactivity of purified Val 315-->Cys permease is enhanced by imposition of a membrane potential (delta psi, interior negative). The results indicate that either ligand binding or delta psi induces a conformational change in the permease that brings the N-terminus of helix X into an environment that is more accessible from the lipid phase.     

The lactose/H+ carrier of Escherichia coli: lac YUN mutation decreases the rate of active transport and mimics an energy-uncoupled phenotype.

The Escherichia coli K12 strain X71-54 carries the lac YUN allele, coding for a lactose/H+ carrier defective in the accumulation of a number of galactosides [Wilson, Kusch & Kashket (1970) Biochem. Biophys. Res. Commun. 40, 1409-1414]. Previous studies proposed that the lower accumulation in the mutant be due to a faulty coupling of H+ and galactoside fluxes via the carrier. Immunochemical characterization of the carriers in membranes from mutant and parent strains with an antibody directed against the C-terminal decapeptide of the wild-type carrier leads to the conclusion that the mutant carrier is similar to the wild-type in terms of apparent Mr, C-terminal sequence, and level of incorporation into the membrane. The pH-dependence of galactoside transport was compared in the mutant and the parent. At pH 8.0-9.0, mutant and parent behave similarly with respect to the accumulation of beta-D-galactosyl 1-thio-beta-D-galactoside and to the ability to grow on the carrier substrate melibiose. At pH 6.0, both the maximal velocity for active transport and the level of accumulation of beta-D-galactosyl-1-thio-beta-D-galactoside are lower in the mutant. The mutant also is unable to grow on melibiose at pH 5.5. However, at pH 6.0 and low galactoside concentrations, the symport stoichiometry is 0.90 H+ per galactoside in the mutant as compared with 1.07 in the parent. These observations suggest that symport is normal in the mutant and that the lower rate of transport in the mutant is responsible for the phenotype. At higher galactoside concentrations, accumulation is determined not only thermodynamically but also kinetically, contrary to a simple interpretation of the chemiosmotic theory. Therefore lower rates of active transport can mimic the effect of uncoupling H+ and galactoside symport. Examination of countertransport in poisoned cells at pH 6.0 reveals that the rate constants for the reorientation of the loaded and unloaded carrier are altered in the mutant. The reorientation of the unloaded carrier is slower in the mutant. However, the reorientation of the galactoside-H+-carrier complex is slower for substrates like melibiose, but faster for substrates like lactose. These findings suggest that lactose-like and melibiose-like substrates interact with the carrier in slightly different ways.     

Quenching of red cell tryptophan fluorescence by mercurial compounds

Intrinsic tryptophan fluorescence in red cell ghost membranes labeled with N-ethylmaleimide (N-EM) is quenched in a dose-dependent manner by the organic mercurial p-chloromercuribenzene sulfonate (p-CMBS). Fluorescence lifetime analysis shows that quenching occurs by a static mechanism. Binding of p-CMBS occurs by a rapid (less than 5 s) biomolecular association (dissociation constant K1 = 1.8 mM) followed by a slower unimolecular transition with forward rate constant k2 = 0.015 s-1 and reverse rate constant k-2 = 0.0054 s-1. Analysis of the temperature dependence of k2 gives delta H = 6.5 kcal/mol and delta S = -21 eu. The mercurial compounds p-chloromercuribenzoic acid, p-aminophenylmercuric acetate, and mercuric chloride quench red cell tryptophan fluorescence by the same mechanism as p-CMBS does; the measured k2 value was the same for each compound, whereas K1 varied. p-CMBS also quenches the tryptophan fluorescence in vesicles reconstituted with purified band 3, the red cell anion exchange protein, in a manner similar to that in ghost membranes. These experiments define a mercurial binding site on band 3 in ghosts treated with N-EM and establish the binding mechanism to this site. The characteristics of this p-CMBS binding site on band 3 differ significantly from those of the p-CMBS binding site involved in red cell water and urea transport inhibition.