Mechanism of lactose translocation in proteoliposomes reconstituted with lac carrier protein purified from Escherichia coli. 2. Deuterium solvent isotope effects.
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Deuterium↗
Biomedical subjects
H R Kaback
Publications and source records attributed to H R Kaback.
Structure of the lac carrier protein of Escherichia coli.
Circular dichroic measurements on the lac carrier protein purified from the cytoplasmic membrane of Escherichia coli indicate that 85 +/- 5% of the amino acid residues comprising this integral membrane protein are arranged in helical secondary structures. Analysis of the sequential hydropathic character of this protein by the method of Kyte and Doolittle (J. Mol. Biol. (1982) 157, 105-132) indicates that the protein is composed of at least 12 hydrophobic segments with a mean length of 24 +/- 4 residues/segment. Approximately 70% of the 417 amino acids in the lac carrier are found in these domains. The hydropathic profile, together with the circular dichroic measurements, suggest that the 12 hydrophobic segments are largely in a helical conformation. If the segments are assumed to be alpha-helical, the mean length of each domain approximates the thickness of the most hydrophobic portion of the lipid bilayer. Based on these considerations, it is proposed that the lac carrier protein consists of at least 12 alpha-helical segments that traverse the membrane in a perpendicular sense, i.e. in a fashion similar to bacteriorhodopsin.
Circular Dichroism↗
The lac carrier protein in Escherichia coli.
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Antibodies, Monoclonal↗
Topology of the lac carrier protein in the membrane of Escherichia coli.
Proteolysis of topologically sealed right-side-out and inside-out membrane vesicles from Escherichia coli with chymotrypsin, trypsin, or papain inactivates lac carrier function in a symmetrical manner. Concomitantly, the electrophoretic mobility of lac carrier protein photoaffinity labeled in situ with p-nitro[2-3H]phenyl-alpha-D-galactopyranoside is altered from a relative Mr of 33,000 to 20,000, and the time course of proteolysis is almost identical in vesicles of opposite polarities. In contrast, solubilization of the vesicles in NaDodSO4 followed by proteolysis causes fragmentation of the Mr 33,000 band into material that electrophoreses at the solvent front. Notably, proteolysis has no effect whatsoever on the ability of the lac carrier protein to bind substrate, as judged by photoaffinity-labeling experiments. Furthermore, the electrophoretic patterns of samples proteolyzed prior to photoaffinity labeling are the same as those observed when the procedures are reversed. These results show that the lac carrier protein spans the membrane and indicate that the binding site resides within a segment that is embedded in the bilayer.
Affinity Labels↗
Reconstitution of active transport in proteoliposomes containing cytochrome o oxidase and lac carrier protein purified from Escherichia coli.
Most active transport across the bacterial cell membrane is driven by a proton electrochemical gradient (delta-muH+, interior negative and alkaline) generated via electron transfer through a membrane-bound respiratory chain. This phenomenon is now reproduced in vitro with proteoliposomes containing only two proteins purified from the membrane of Escherichia coli. An o-type cytochrome oxidase was extracted from membranes of a cytochrome d terminal oxidase mutant with octyl beta-D-glucopyranoside after sequential treatment with urea and cholate and was purified to homogeneity by ion-exchange chromatography. The purified oxidase contains four polypeptides (MrS 66,000, 35,000, 22,000, and 17,000), two b-type cytochromes (b558 and b563), and 16-17 nmol of heme b per mg of protein, and it catalyzes the oxidation of ubiquinol and other electron donors with specific activities 20- to 30-fold higher than crude membranes. The lac carrier protein was purified as described. Proteoliposomes were formed in the presence of the oxidase and lac carrier protein by detergent dilution, followed by freeze-thaw/sonication. The system generates a delta-muH+ (interior negative and alkaline) with ubiquinol as electron donor and the magnitude of delta-muH+ is dependent on the concentration of cytochrome o in the proteoliposomes. Furthermore, the proteoliposomes transport lactose against a concentration gradient to an extent that is commensurate with the magnitude of delta-muH+ generated. The results provide powerful additional support for the "chemiosmotic hypothesis" and demonstrate that purified lac carrier protein retains the ability to function in a physiological manner.
Biological Transport, Active↗
Macrophage membrane potential changes associated with gamma 2b/gamma 1 Fc receptor-ligand binding.
We have studied the effects of specific ligands of the receptor for the IgG Fc fragment (FcR) on the membrane potential (delta psi) of the macrophage cell line J774 by the [3H]tetraphenylphosphonium ion equilibration technique. We observe a membrane depolarization with binding of FcR ligands that is dependent on the degree of receptor crosslinking. Binding of the FcR by monovalent ligands is not sufficient to induce a significant drop in delta psi, but a sustained depolarization lasting approximately equal to 20 min occurs with insoluble multivalent ligands. This FcR-mediated depolarization can be inhibited by substitution of Na+ from the cell incubation medium with monovalent choline cation, indicating that depolarization is due to Na+ influx into the cell. The extracellular Ca2+ does not play a significant role in membrane depolarization. The depolarization response is not triggered by monoclonal antibodies directed against three other major macrophage surface antigens. The cell depolarization mediated by FcR ligands is followed by a prolonged hyperpolarization that can be partially blocked by ouabain and quinine, indicating that the hyperpolarization response is a result of a combination of a Na+,K+-ATPase activity and a Ca2+-activated K+ conductance. These data support our hypothesis that the mouse macrophage IgG FcR is a ligand-dependent ion channel.
Animals↗
Mouse macrophage Fc receptor for IgG gamma 2b/gamma 1 in artificial and plasma membrane vesicles functions as a ligand-dependent ionophore.
We tested the effect of specific ligands to the mouse macrophage IgG gamma 2b/gamma 1 Fc fragment receptor (FcR) on ion permeability of plasma membrane vesicles prepared from J774 macrophages by nitrogen cavitation. The monoclonal antibody directed against IgG gamma 2b/gamma 1 FcR (gamma 2b/gamma 1 FcR), 2.4G2 IgG, and soluble and immobilized immunocomplexes induces a dramatic cation flow through plasma membrane vesicles, as measured by [3H]tetraphenylphosphonium+ accumulation. Challenge with the monovalent 2.4G2 Fab also produces an ion flow but the effect is smaller by a factor of 2, and three other monoclonal antibodies directed against major surface antigens of mouse macrophages produce no net ion flow. Membrane vesicles incubated with FcR ligands do not discriminate between Na+ and K+ and show low permeability to Ca2+. gamma 2b/gamma 1 FcR was purified by using monoclonal 2.4G2 and reconstituted into proteoliposomes. Under these circumstances, the purified receptor increased the cation permeability of the proteoliposomes in the presence of specific ligands. The data indicate that the gamma 2b/gamma 1 FcR of J774 macrophages functions as a ligand-dependent ionophore. The ion influx into macrophages mediated by the FcR may play an important role as a signal for internalization of membranes and stimulus-secretion coupling.
Animals↗
Membrane potential in anaerobically growing Staphylococcus aureus and its relationship to gentamicin uptake.
The electrical potential (delta psi) across the cytoplasmic membranes of Staphylococcus aureus cells growing under aerobic and anaerobic conditions was determined by measuring the equilibrium distribution of [3H]tetraphenyl phosphonium. In conjunction, gentamicin uptake and killing were studied in the same cells under identical conditions. Under aerobic conditions, delta psi was -169 mV, gentamicin uptake was readily demonstrable, and the number of viable cells decreased by almost four orders of magnitude in the presence of antibiotic. In contrast, delta psi was -142 mV anaerobically, gentamicin uptake was essentially nonexistent, and the aminoglycoside had no effect on viability. Remarkably, when the ionophore nigericin was added under anaerobic conditions, delta psi increased to the level observed aerobically, gentamicin uptake tripled to about 18% of the aerobic level, and viability decreased by one order of magnitude. The results are consistent with other observations (Mates et al., Proc. Natl. Acad. Sci. U.S.A. 79:6693-6697, 1982), indicating that the relationship between delta psi and gentamicin uptake is gated, and suggest that diminution of delta psi may be an important factor in aminoglycoside resistance under anaerobic conditions.
Anaerobiosis↗
Mechanism of lactose transport in Escherichia coli membrane vesicles: evidence for the involvement of histidine residue(s) in the response of the lac carrier to the proton electrochemical gradient.
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Biological Transport↗
Direct measurement of lactose/proton symport in Escherichia coli membrane vesicles: further evidence for the involvement of histidine residue(s).
Addition of lactose to Escherichia coli ML 308-225 membrane vesicles under nonenergized conditions induces transient alkalinization of the medium, and the initial rate of proton influx is stimulated by valinomycin and abolished by nigericin or carbonyl cyanide m-chlorophenylhydrazone. A functional lac y gene product is absolutely required as the effect is not observed in ML 308-225 vesicles treated with N-ethylmaleimide nor with vesicles from uninduced Escherichia coli ML 30. Furthermore, the magnitude of the phenomenon is enhanced about 3-fold in vesicles from Escherichia coli T206, which contain amplified levels of the lac carrier protein. Kinetic parameters for lactose-induced proton influx are the same as those determined for lactose-facilitated diffusion, and quantitative comparison of the initial rates of the two fluxes indicates that the stoichiometry between protons and lactose is 1:1. Treatment of ML 308-225 vesicles with diethyl pyrocarbonate causes inactivation of lactose-induced proton influx. Remarkably, however, treatment with the histidine reagent enhances the rate of lactose-facilitated diffusion in a manner suggesting that the altered lac carrier catalyzes lactose influx without the symport of protons. The results are consistent with the hypothesis that acylation of a histidyl residue(s) in the lac carrier protein dissociates lactose influx from proton influx and indicate that this residue(s) play(s) an important role in the pathway of proton translocation.
Biological Transport↗
Lactose-proton symport by purified lac carrier protein.
The lac carrier protein of Escherichia coli was purified by an improved procedure and its activity assayed by a rapid filter method. Following reconstitution of the carrier by octyl glucoside dilution, proteoliposomes were concentrated by filtration on a microporous filter. Lactose accumulation by adsorbed or entrapped proteoliposomes is driven by an artificially imposed pH gradient (interior alkaline), by a membrane potential (interior negative), or by a combination of both forces. Activity is almost completely abolished by the protonophore carbonyl cyanide m-chlorophenylhydrazone or by the competitive inhibitor thiodigalactoside. Addition of lactose to proteoliposomes under appropriate conditions results in alkalinization of the external medium. This effect is not observed with liposomes devoid of lac carrier or in the presence of proton conducting agents. The results provide a strong indication that the lac gamma gene product is the only protein in the cytoplasmic membrane of Escherichia coli required for lactose-proton symport.
Biological Transport↗
Simultaneous reconstitution of Escherichia coli membrane vesicles with D-lactate and D-amino acid dehydrogenases.
Purified preparations of D-amino acid dehydrogenase [Olsiewski, P.J., Kaczorowski, G. J., & Walsh, C. T. (1980) J. Biol. Chem. 225, 4487] and D-lactate dehydrogenase [Kohn, L.D., & Kaback, H.R. (1973) J. Biol. Chem. 248, 7012] bind independently to right-side-out and inverted Escherichia coli vesicles and to phosphatidylcholine liposomes without detectable competition. The reconstituted vesicles catalyze D-lactate- and D-alanine-dependent respiration (O2 uptake), proton translocation, and proton/lactose symport. The enzymes do not share common sites of association on either face of the E. coli membrane, and binding of both enzymes to the bilayer appears to be due to nonspecific affinity for the surface rather than specific binding to proteinaceous receptors. Each enzyme, however, appears to reduce a common proton translocating step in the membrane-bound respiratory chain, and substrate-derived electrons are transferred through a common rate-determining redox component that precedes the site of proton translocation. The results suggest that although binding is nonspecific, there is a common site for proton translocation in the membrane between the flavin-linked dehydrogenases and the cytochromes and that this site is accessible by distinct routes of electron transfer from primary dehydrogenases on either surface of the membrane.
Biological Transport↗
A method for rapid, continuous monitoring of solute uptake and binding.
A method has been developed for dynamically monitoring the free concentration of diffusible, tritiated solutes. The technique utilizes particles of a solid scintillator microencapsulated in gel beads that are permeable to diffusible label. Since tritium beta radioactivity has an effective range in water of only a few micrometers, only label that is free to diffuse through the gel can excite the scintillator, while sequestered label is effectively excluded. Thus, the scintillation light output monitors the freely diffusible concentration of label exclusively. A simple, preliminary encapsulation technique is described and tested, and the theory behind the method is discussed with regard to the time resolution attainable for a given label concentration and type of encapsulation. The feasibility of the method is demonstrated by measuring the uptake of [3H]tetraphenylphosphonium by Escherichia coli membrane vesicles in response to the generation of a membrane potential (interior negative).
Glucose↗
In vitro synthesis of the membrane-bound D-lactate dehydrogenase of Escherichia coli.
Synthesis of the membrane-bound, flavin-linked D-lactate dehydrogenase of Escherichia coli has been studied by using a recombinant plasmid containing the dld gene [Young, I. G., Jaworowski, A., & Poulis, M. (1982) Biochemistry (following paper in this issue)]. Expression of the cloned dld gene was achieved either in vivo with transformed minicells or in vitro with a fractionated transcription/translation system. In both instances, a product is observed that is specifically immunoprecipitated by gamma-globulin prepared against the purified enzyme and comigrates with authentic D-lactate dehydrogenase on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Furthermore, the product is catalytically active and binds to membrane vesicles during or after synthesis. Thus, it seems likely that the protein is synthesized in mature form and binds to the membrane without a leader peptide sequence. Interestingly, addition of flavin adenine dinucleotide to the in vitro reaction mixtures causes a 2-fold increase in the synthesis of the enzyme, suggesting that the cofactor plays a regulatory role in the synthesis of the apoprotein. Finally, L factor, a protein involved in regulation of protein elongation, has an inhibitory effect on the expression of the dld gene and a stimulatory effect on the expression of the ndh gene (encoding NADH dehydrogenase).
DNA, Bacterial↗
Membrane potential and gentamicin uptake in Staphylococcus aureus.
At pH 5.0, the electrical potential (delta psi, interior negative) across the plasma membrane of Staphylococcus aureus exhibits a minimum of -85 to -90 mV; the pH gradient (delta pH, interior alkaline) across the membrane approximates a maximum of about -100 mV. Under these conditions, uptake of the aminoglycoside gentamicin is negligible, and viability of the organism is not impaired by the antibiotic. In contrast, at pH 7.5, at which delta psi is about -130 mV and delta pH is 0, gentamicin uptake is observed and the drug markedly decreases viability. Dramatically, when the ionophore nigericin is added at pH 5.0, gentamicin uptake is induced, there is a striking decrease in viability, and the effect is associated with an increase in delta psi at the expense of delta pH. Consistently, valinomycin, which dissipates delta psi in the presence of potassium, abolishes gentamicin uptake and killing. In addition, from pH 5.0 to pH 7.5, there is a direct relationship between the magnitude of delta psi and both gentamicin uptake and its bactericidal effect. However, a threshold delta psi of -75 to -90 mV is apparently necessary to initiate uptake and killing. These observations provide a strong indication that delta psi plays a critical role in the uptake and antibacterial action of gentamicin and suggest that nigericin-like ionophores may be clinically useful in synergy with aminoglycosides.
Biological Transport, Active↗
Preparation, characterization, and properties of monoclonal antibodies against the lac carrier protein from Escherichia coli.
Monoclonal antibodies directed against the lac carrier protein purified from the membrane of Escherichia coli were prepared by somatic cell fusion of mouse myeloma cells with splenocytes from an immunized mouse. Several clones produce antibodies that react with the purified protein as demonstrated by solid-phase radioimmunoassay and by immunoblotting experiments; culture supernatants from the clones inhibit active transport of lactose in isolated membrane vesicles. Five stable clones were selected for expansion, formal cloning, and production of ascites fluid, and the antibodies secreted in vivo by each clone also were found to inhibit lactose transport. Antibody from hybridoma 4B1, an IgG2a immunoglobulin, inhibits active transport of lactose in proteoliposomes reconstituted with purified lac carrier and in right-side-out membrane vesicles. In contrast, the antibody has no effect on the generation of the proton electrochemical gradient by membrane vesicles nor does it alter the ability of vesicles containing the lac carrier to bind p-nitrophenyl-alpha-D-galactopyranoside. In order to achieve 50% inhibition of transport activity, a 2- to 3-fold molar excess of antibody to lac carrier is required, regardless of the amount of lac carrier in the membrane. Thus, the concentration of antibody required for a given degree of inhibition is proportional to the amount of lac carrier in the membrane. Finally, antibody-induced inhibition occurs within seconds, an observation suggesting that the epitope is accessible on the surface of the membrane.
Animals↗
Purification and reconstitution of functional lactose carrier from Escherichia coli.
The lactose carrier protein of Escherichia coli was purified by a simple procedure employing differential solubilization and ion-exchange chromatography and reconstituted into liposomes by octylglucoside dilution. The proteoliposomes exhibited both membrane potential-driven lactose transport and lactose counterflow. Furthermore, the purified protein was identified as the product of the lac y gene. These and other results demonstrate that the lactose carrier is the only polypeptide species essential for energy-coupled lactose transport and counterflow.
Amino Acids↗
Reconstitution of Escherichia coli membrane vesicles with D-amino acid dehydrogenase.
When purified D-amino acid dehydrogenase [Olsiewski, P. J., Kaczorowski, G. J., & Walsh, C. T. (1980) J. Biol. Chem. 255, 4487] is incubated with right-side-out membrane vesicles from Escherichia coli, the enzyme binds to the membrane in a time- and concentration-dependent manner. As a result, the vesicles acquire the ability to oxidize D-alanine and catalyze D-alanine-dependent active transport. Similarly, incubation of D-amino acid dehydrogenase with inside-out vesicles results in binding of enzyme and D-alanine oxidase activity. Antibody inhibition studies indicate that the enzyme is bound exclusively to the inner cytoplasmic surface of the membrane in native vesicles (i.e., membrane vesicles prepared from cells induced for D-amino acid dehydrogenase). In contrast, similar studies with reconstituted vesicles demonstrate that enzyme binds to the surface exposed to the medium regardless of the orientation of the membrane. Thus, enzyme bound to right-side-out vesicles is located on the opposite side of the membrane from where it is normally found. Remarkably, in the presence of D-alanine, reconstituted right-side-out and inside-out vesicles generate electrochemical proton gradients of similar magnitude but opposite polarity, indicating that enzyme bound to either surface of the membrane is physiologically functional. The results suggest that vectorial proton translocation via the respiratory chain occurs at a point distal to the site where electrons enter the respiratory chain from the primary dehydrogenase, a conclusion that is inconsistent with the notion that the dehydrogenase forms part of a proton-translocating loop.
Alanine↗