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N Carrasco

Publications and source records attributed to N Carrasco.

At least 55 records · Page 3Linked to original sources
PubMed1987

lac permease of Escherichia coli: arginine-302 as a component of the postulated proton relay.

The lac permease of Escherichia coli was modified by site-directed mutagenesis such that Arg-302 in putative helix IX was replaced with Leu. In addition, Ser-300 (helix IX) was replaced with Ala, and Lys-319 in putative helix X was replaced with Leu. Permease with Leu at position 302 manifests properties that are similar to those of permease with Arg in place of His-322 [Püttner, I. B., Sarkar, H. K., Poonian, M. S., & Kaback, H. R. (1986) Biochemistry 25, 4483]. Thus, permease with Leu-302 is markedly defective in active lactose transport, efflux, exchange, and counterflow but catalyzes downhill influx of lactose at high substrate concentrations without H+ translocation. In contrast, permease molecules with Ala at position 300 or Leu at position 319 catalyze lactose/H+ symport in a manner indistinguishable from that of wild-type permease. By molecular modeling, Arg-302 may be positioned in helix IX so that it faces the postulated His-322/Glu-325 ion pair in helix X. In this manner, the guanidino group in Arg-302 may interact with the imidazole of His-322 and thereby play a role in the H+ relay suggested to be involved in lactose/H+ symport [Carrasco, N., Antes, L. M., Poonian, M. S., & Kaback, H. R. (1986) Biochemistry 25, 4486].

Amino Acid Sequence↗
PubMed1986

lac permease of Escherichia coli: histidine-322 and glutamic acid-325 may be components of a charge-relay system.

When Glu-325 in the lac permease of Escherichia coli is replaced with Ala, lactose/H+ symport is abolished. Thus, the altered permease catalyzes neither uphill lactose accumulation nor efflux. Remarkably, however, permease with Ala-325 catalyzes exchange and counterflow at completely normal rates. Taken together with the results presented in the accompanying paper [Püttner, I. B., Sarkar, H. K., Poonian, M. S., & Kaback, H. R. (1986) Biochemistry (preceding paper in this issue)], the findings suggest that the His-322 and Glu-325 may be components of a charge-relay system that plays an important role in the coupled translocation of lactose and H+.

Binding Sites↗
PubMed1985

Functional and immunochemical characterization of a mutant of Escherichia coli energy uncoupled for lactose transport.

Right-side-out cytoplasmic membrane vesicles from Escherichia coli ML 308-22, a mutant "uncoupled" for beta-galactoside/H+ symport [Wong, P. T. S., Kashket, E. R., & Wilson, T. H. (1970) Proc. Natl. Acad. Sci. U.S.A. 65, 63], are specifically defective in the ability to catalyze accumulation of methyl 1-thio-beta-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 (ML 308-225). Alternatively, ML 308-22 vesicles bind p-nitrophenyl alpha-D-galactopyranoside and monoclonal antibody 4B1 to the same extent as ML 308-225 vesicles and catalyze facilitated diffusion and equilibrium exchange as well as ML 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, 2531]. That is, the mutation has no effect on the rate or extent of counterflow when external substrate is saturating but stimulates the efficiency of counterflow when external substrate is below the apparent Km. Moreover, although replacement of protium with deuterium stimulates counterflow in ML 308-225 vesicles when external substrate is subsaturating, the isotope has no effect on the mutant vesicles under the same conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1985

Direct measurement of the electrogenic activity of o-type cytochrome oxidase from Escherichia coli reconstituted into planar lipid bilayers.

Turnover of o-type cytochrome oxidase purified from Escherichia coli and reconstituted into proteoliposomes leads to the generation of a transmembrane electrical potential (interior negative) by means of vectorial electron flow. In the experiments reported here, purified oxidase is reconstituted in planar lipid bilayers formed at the tip of patch pipets, and open-circuit membrane potentials generated by electron transfer are measured directly. Potentials of up to 4 mV (substrate side positive) are generated in the presence of reduced phenazine methosulfate or ubiquinol-1, and with both substrates, electrogenic activity is inhibited by cyanide. Furthermore, the membrane potential generated during oxidase turnover is inhibited progressively with applied voltages (substrate side positive), decreasing almost to zero at an applied voltage of 150 mV.

Electrochemistry↗
PubMed1984

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.

Cell Membrane↗
PubMed1984

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)

Antibodies, Monoclonal↗
PubMed1984

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.

Antibodies, Monoclonal↗
PubMed1984

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.

Amino Acid Sequence↗
PubMed1982

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↗