Lac Carrier Research Articles

Substrate and inhibitor specificity of the lactate carrier of human neutrophils.

The substrate and inhibitor specificity of the lactic acid (Lac) transport system of human neutrophils was investigated. The ability of a variety of compounds to inhibit the influx of [14C]lactate, presumably reflecting competition by substrate analogues for binding at the external translocation site, was taken as an index of affinity for the Lac carrier. pH-state techniques were utilized to assess transportability. Results indicate a relatively low order of selectivity, the neutrophil H+(+)lactate- cotransport system demonstrating a broad acceptance of short-chain unsubstituted and substituted alkyl monocarboxylates as well as aromatic monocarboxylates. There was a slight preference for oxo, Cl, and OH substituents over other groups at the two-position of short chain alkyl fatty acids: all were readily transported across the plasma membrane at rates approaching that of L-lactate itself. Aromatic acids were not transported inward by the carrier although these compounds did permeate via simple nonionic diffusion. The neutrophil Lac carrier can be blocked by a number of cyanocinnamate derivatives, the classical inhibitors of monocarboxylate transport in mitochondria, and by dithiol compounds and sulfhydryl-reactive agents. This constellation of biochemical properties is similar to the features that characterize other well described H+(+)lactate- cotransport systems in red blood cells, Ehrlich ascites tumor cells, hepatocytes, and cardiac sarcolemmal vesicles, although significant differences exist when comparisons are made to the Na(+)-dependent lactate transporter of the kidney proximal tubule.

Lactic acid secretion by human neutrophils. Evidence for an H+ + lactate- cotransport system.

The pathway by which L-lactate (Lac) crosses the plasma membrane of isolated human neutrophils was investigated. The influx of [14C]Lac from a 2 mM Lac, 145 mM Cl-, 5.6 mM glucose medium was approximately 1.5 meq/liter of cell water.min and was sensitive to the organomercurial agent mersalyl (apparent Ki approximately 20 microM), to alpha-cyano-4-hydroxycinnamate (CHC), the classical inhibitor of monocarboxylate transport in mitochondria, and to UK-5099 (apparent Ki approximately 40 microM), a more potent analogue of CHC. Transport was also strongly blocked (greater than 80%) by 1 mM of either 3,5-diiodosalicylic acid, MK-473 (an indanyloxyacetate derivative), or diphenyl-amine-2-carboxylate, and by 0.4 mM pentachlorophenol, but not by 1 mM ethacrynic acid, furosemide, or the disulfonic stilbenes SITS or H2DIDS. One-way [14C]Lac efflux from steady-state cells amounted to approximately 6 meq/liter.min and was likewise affected by the agents listed above. Influx, which was membrane potential insensitive and Na+ independent, displayed a strong pH dependence: extracellular acidification enhanced uptake while alkalinization inhibited the process (pK' approximately 5.7 at 2 mM external Lac). The rate of [14C]Lac influx was a saturable function of external Lac, the Km being approximately 7 mM. Steady-state cells exhibited an intracellular Lac content of approximately 5 mM and secreted lactic acid into the bathing medium a a rate of approximately 4 meq/liter.min. Secretion was completely suppressed by 1 mM mersalyl which inactivates the carrier, leading to an internal accumulation of Lac. That the Lac carrier truly mediates an H+ + Lac- cotransport (or formally equivalent Lac-/OH- exchange) was documented by pH-stat techniques wherein an alkalinization of poorly buffered medium could be detected upon the addition of Lac; these pH changes were sensitive to mersalyl. Thus, the Lac carrier of neutrophils possesses several features in common with other monocarboxylate transport systems in erythrocytes and epithelia.

30] Methods for the study of the melibiose carrier of Escherichia coli

This chapter describes methods used for measuring the transport of both the sugar and cation substrates. Some of these methods such as the preparation of radioactive melibiose and the construction of the Li+-selective electrode were devised specifically for studies of the melibiose transport system. The melibiose carrier of Escherichia coli is a representative of the class of carriers that require a cation for cotransport. An unusual feature of this transport system is that it is able to utilize Na + , Li + , or H + as the coupling cation depending on the conditions. Another interesting property of the carrier is that different sugar substrates show different specificities for the cation requirement. A variety of strains of E. coli has been isolated for use in experiments on the melibiose carrier. It is essential to start with a strain from which the lactose carrier (lac Y) has been deleted, as the lac carrier also is capable of transporting melibiose. The growth of normal E. coli cells on melibiose minimal medium is strongly inhibited by 10 mM Li + and it is therefore possible to isolate Li + -resistant mutants. This organism is dependent on Na + or Li + for growth on melibiose as it has lost the ability to transport the disaccharide with protons.

32] Purification, reconstitution, and characterization of the lac permease of Escherichia coli

Publisher Summary Transport of β-galactosides in Escherichia coli is catalyzed by the product of the lac Y gene, the lac permease or lac carrier protein, which translocates substrate with H + in a symport (cotransport) reaction. The lac permease has been identified as a membrane protein chemically and functionally in 1970. The lacY gene has been cloned into a recombinant plasmid, its product amplified and synthesized in vitro, and the sequence of the protein is deduced from the DNA sequence. This chapter describes a detailed purification scheme that takes the lac permease from membrane to molecule in a fully functional state. Membranes from a strain of E. coli containing multiple copies of the lac Y gene are first sequentially extracted with high concentrations of urea and cholate to affect about a 3-fold purification of the permease in situ. Reconstitution of purified lac permease into proteoliposomes and a variety of functional assays are described in the chapter.

33] Preparation of monoclonal antibodies and site-directed polyclonal antibodies against the lac permease of Escherichia coli

Publisher Summary This chapter discusses the prepration of monoclonal antibodies and site-detected polyclonal antibodies against the lac Permease of E.coli. The lac permease (that is, lac carrier protein) of Escherichia coli is an intrinsic membrane protein, the product of the lac Y gene that catalyzes the translocation of β -galactosides with hydrogen ion in a symport or cotransport reaction. The permease can be solubilized from the membrane, purified to homogeneity, reconstituted into phospholipid vesicles, and shown to catalyze all of the transport reactions typical of the native membrane with comparable activities and kinetic properties. To test the predictions, to determine the orientation of the permease in native and reconstituted membranes, and to obtain information regarding structure or function relationships, monocionai antibodies (Mabs) against purified lac permease and polyclonal antibodies directed against synthetic peptides corresponding to portions of the permease have been utilized. The preparation of Mabs against purified immunogens such as lac permease is relatively straightforward. Advantageous aspect of Mabs is their potential for inhibiting catalytic activity. However, the most significant advantages of site-directed polyclonal antibodies are that they are directed against a predefined epitope and that their generation takes less time and less effort than Mabs.

Coordinate expression of a small polypeptide with the lactose carrier of Escherichia coli.

Induction of the lac operon in wild type Escherichia coli strains results in synthesis of a 16-kDa inner membrane protein in addition to the known products of the lacZ, lacY, and lacA genes. Cells carrying the lacY gene on a multicopy plasmid overproduce this 16-kDa polypeptide as well as the Lac carrier, the membrane protein product of the lacY gene. However, [35S]methionine labeling of minicells carrying the lacY plasmid shows that the 16-kDa protein is not synthesized from the plasmid DNA. We have purified and partially characterized the 16-kDa protein. It is an acidic membrane protein of apparent Mr = 15,800 whose amino-terminal sequence (NH2-Met-Arg-Asn-Phe-Asp-Leu-) does not match any known lac operon DNA sequence. Using antibody prepared to the purified 16-kDa protein, we have quantitatively analyzed conditions under which this protein is made and have shown that the amount of 16-kDa protein which appears in the membrane is proportional to lac operon expression. Hybridization of a synthetic oligodeoxyribonucleotide probe complementary to the 5' end of 16-kDa protein mRNA shows that its synthesis is regulated at the level of transcription.

Morphology of proteoliposomes reconstituted with purified lac carrier protein from Escherichia coli.

Proteoliposomes reconstituted with purified lac carrier protein from Escherichia coli were ultra-rapidly frozen and examined by freeze-fracture-etch electron microscopy. The proteoliposomes are greater than 95% unilamellar, and the majority are 30-150 nm in diameter. Fracture faces of proteoliposomes (at a protein:lipid molecular ratio of about 1:2500) display 7.0-nm diameter globular intramembrane particles uniformly distributed on convex and concave surfaces. Calculations of particle composition suggest that each intramembrane particle probably contains one or two molecules of the 46.5-kDa transmembranous lac carrier protein, depending on the correction factor for the thickness of the metal deposited to form the platinum/carbon replicas. Etched surfaces of the proteoliposomes are smooth. Incubation of the proteoliposomes with monoclonal antibody 4B1, which binds to an epitope in the lac carrier on the exterior of the proteoliposomes, dramatically alters the intramembrane particle distribution. After incubation with antibody, the convex (inner monolayer) fracture faces are nearly devoid of intramembrane particles, and an overall 4-fold reduction in the total number of intramembrane particles is observed.

Intramolecular dislocation of the COOH terminus of the lac carrier protein in reconstituted proteoliposomes.

A dodecapeptide corresponding to the carboxyl terminus of the lac carrier of Escherichia coli was synthesized, coupled to thyroglobulin, and the conjugate was used to generate site-directed polyclonal antibodies. The antibodies react with the carboxyl-terminal peptide and with the lac carrier protein, while monoclonal antibody 4B1 reacts with intact lac carrier protein, but not with the carboxyl-terminal peptide. Antibody 4B1 binds preferentially to right-side-out membrane vesicles relative to inside-out vesicles, confirming the presence of the 4B1 epitope on the periplasmic surface of the membrane. Alternatively, anti-carboxyl-terminal antibody binds preferentially to inside-out vesicles, demonstrating that the carboxyl terminus of the lac carrier protein is on the cytoplasmic surface. Surprisingly, both antibodies bind to proteoliposomes reconstituted with purified lac carrier protein, and quantitative binding assays indicate that the epitopes are equally accessible. When proteoliposomes containing purified lac carrier protein are digested with carboxypeptidases A and B, binding of anti-carboxyl-terminal antibodies decreases by greater than 80%, while binding of antibody 4B1 and various transport activities remain essentially unchanged. It is suggested that during reconstitution, the lac carrier protein undergoes intramolecular dislocation of the carboxyl terminus with no significant effect on its catalytic activity.

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)

Monoclonal antibodies against the lac carrier protein from Escherichia coli. 2. Binding studies with membrane vesicles and proteoliposomes reconstituted with purified lac carrier protein.

Monoclonal antibodies 4B1 and 5F7 bind to distinct, nonoverlapping epitopes in the lac carrier protein. By use of immunofluorescence microscopy and radiolabeled monoclonal antibodies and Fab fragments, it is shown that both 4B1 and 5F7 bind to spheroplasts and to right-side-out vesicles, but only to a small extent to inside-out vesicles. Clearly, therefore, the lac carrier protein has an asymmetric orientation within the cytoplasmic membrane of Escherichia coli, and both epitopes are located on the periplasmic surface. In right-side-out vesicles, radiolabeled 4B1 binds with a stoichiometry of 1 mol of antibody per 2 mol of lac carrier protein, while radiolabeled 4B1 Fab fragments bind 1:1. Importantly, the intact antibody and its Fab fragments bind to proteoliposomes reconstituted with purified lac carrier protein with a stoichiometry very similar to that observed in right-side-out membrane vesicles. Thus, it seems highly likely that the orientation of the lac carrier protein in the reconstituted system is similar to that in the bacterial cytoplasmic membrane, at least with respect to 4B1 epitope.

Site-directed mutagenesis of cys148 in the lac carrier protein of Escherichia coli

The lac y gene of Escherichia coli which encodes the lac carrier protein has been modified by oligonucleotide-directed, site-specific mutagenesis such that cys148 is converted to a glycine residue. Cells bearing the mutated lac y gene exhibit initial rates of lactose transport that are about 4-fold lower than cells bearing the wild type gene on a recombinant plasmid. Furthermore, transport activity is less sensitive to inactivation by N-ethylmaleimide, and strikingly, galactosyl 1-thio-β-D-galactopyranoside affords no protection against inactivation. The findings suggest that although cys148 is essential for substrate protection against sulfhydryl inactivation, it is not obligatory for lactose:proton symport and that another sulfhydryl group elsewhere within the lac carrier protein may be required for full activity.

Purified reconstituted lac carrier protein from Escherichia coli is fully functional.

Proteoliposomes reconstituted with lac carrier protein purified from the plasma membrane of Escherichia coli catalyze each of the translocation reactions typical of the beta-galactoside transport system (i.e., active transport, counterflow, facilitated influx and efflux) with turnover numbers and apparent Km values comparable to those observed in right-side-out membrane vesicles. Furthermore, detailed kinetic studies show that the reconstituted system exhibits properties analogous to those observed in membrane vesicles. Imposition of a membrane potential (delta psi, interior negative) causes a marked decrease in apparent Km (by a factor of 7 to 10) with a smaller increase in Vmax (approximately equal to 3-fold). At submaximal values of delta psi, the reconstituted carrier exhibits biphasic kinetics, with one component manifesting the kinetic parameters of active transport and the other exhibiting the characteristics of facilitated diffusion. Finally, at low lactose concentrations, the initial velocity of influx varies linearly with the square of the proton electro-chemical gradient. The results provide quantitative support for the contention that a single polypeptide species, the product of the lac y gene, is responsible for each of the transport reactions typical of the beta-galactoside transport system.

Functional molecular weight of the lac carrier protein from Escherichia coli as studied by radiation inactivation analysis.

Cytoplasmic membrane vesicles prepared from Escherichia coli containing multiple copies of the lac y gene were frozen in liquid nitrogen before or after generation of a proton electrochemical gradient (interior negative and alkaline) and irradiated with a high-energy electron beam at -135 degrees C. Subsequently, the lac carrier protein was extracted into octyl beta-D-glucopyranoside, reconstituted into proteoliposomes, and assayed for transport activity. Under all conditions tested, activity decreased as a single exponential function of radiation dosage, allowing straightforward application of target theory for determination of functional molecular mass. When lac carrier activity solubilized from nonenergized vesicles was assayed, the results obtained were consistent with a functional molecular size of 45-50 kDa, a value similar to the size of the protein as determined by other means. Similar values were obtained when the octyl beta-D-glucopyranoside extract was irradiated, and the target size observed for D-lactate dehydrogenase was in good agreement with the molecular size of this enzyme. Strikingly, when the same procedures were carried out with vesicles that were energized with appropriate electron donors prior to freezing and irradiation, a functional molecular size of 85-100 kDa was obtained for the lac carrier with no change in the target size of D-lactate dehydrogenase. In contrast, when the vesicles were energized under conditions in which the proton electrochemical gradient was collapsed, the target mass of the lac carrier returned to 45-50 kDa. The results indicate that the functional mass of the lac carrier protein is no greater than a dimer and suggest that the proton electrochemical gradient may cause an alteration in subunit interactions.

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.

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.

Mechanism of lactose translocation in proteoliposomes reconstituted with lac carrier protein purified from Escherichia coli. 2. Deuterium solvent isotope effects.

Mechanism of lactose translocation in proteoliposomes reconstituted with lac carrier protein purified from Escherichia coli. 2. Deuterium solvent isotope effects.

Mechanism of lactose translocation in proteoliposomes reconstituted with lac carrier protein purified from Escherichia coli. 1. Effect of pH and imposed membrane potential on efflux, exchange, and counterflow.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTMechanism of lactose translocation in proteoliposomes reconstituted with lac carrier protein purified from Escherichia coli. I. Effect of pH and imposed membrane potential on efflux, exchange, and counterflowMaria Luisa Garcia, Paul Viitanen, David L. Foster, and H. Ronald KabackCite this: Biochemistry 1983, 22, 10, 2524–2531Publication Date (Print):May 10, 1983Publication History Published online1 May 2002Published inissue 10 May 1983https://doi.org/10.1021/bi00279a033RIGHTS & PERMISSIONSArticle Views204Altmetric-Citations65LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (2 MB) Get e-Alerts Get e-Alerts

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.

Mechanism of lactose transport in Escherichia coli membrane vesicles: evidence for the involvement of a histidine residue(s) in the response of the lac carrier to the proton electrochemical gradient

Mechanism of lactose transport in Escherichia coli membrane vesicles: evidence for the involvement of a histidine residue(s) in the response of the lac carrier to the proton electrochemical gradient

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.