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J K Wright

Publications and source records attributed to J K Wright.

At least 91 records · Page 5Linked to original sources
PubMed1985

Properties of a mutant lactose carrier of Escherichia coli with a Cys148----Ser148 substitution.

The cysteine residue at position 148 in the lactose carrier protein of Escherichia coli has been replaced by serine using oligonucleotide-directed, site-specific mutagenesis of the lac Y gene. The mutant carrier is incorporated into the cytoplasmic membrane to the same extent as the wild-type carrier, confers a lactose-positive phenotype on cells, and actively transports lactose and other galactosides. However, the maximum rate of transport for several substrates is reduced by a factor of 6-10 while the apparent affinity is reduced by a factor of 2-4. Carrier activity in the mutant is much less sensitive to sulfhydryl reagents (HgCl2, p-(chloromercuri)benzenesulfonate and N-ethylmaleimide) than in the wild type, and beta-D-galactosyl 1-thio-beta-D-galactoside does not protect the mutant carrier against slow inactivation by N-ethylmaleimide. It is concluded that the Cys148 residue is not essential for carrier-catalyzed galactoside: proton symport and that its alkylation presumbly prohibits access of the substrate to the binding site by steric hindrance. A serine residue at position 148 in the amino acid sequence appears to alter the protein structure in such a way that one or more sulfhydryl groups elsewhere in the protein become accessible to alkylating agents thereby inhibiting transport. Recently, Trumble et al. [(1984) Biochem. Biophys. Res. Commun. 119, 860-867] arrived at similar conclusions by investigating a mutant carrier with a Cys148----Gly148 replacement.

Amino Acid Sequence↗
PubMed1985

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.

Biological Transport, Active↗
PubMed1984

Localization of the galactoside binding site in the lactose carrier of Escherichia coli.

The location of flurophores specifically bound to the lactose/H+ carrier of Escherichia coli was ascertained by the use of various collisional quenchers. The reporter groups were (1) the pyrenyl residue of N-(1-pyrenyl)maleimide attached to the essential cysteine residue 148, which is presumably at or near the galactoside binding site, and (2) the dansyl moieties of a series of fluorescent substrate molecules. The accessibility of these fluorophores from the lipid phase was assessed by nitroxyl-labelled fatty acids and phospholipids. By using a series of nitroxyl-labelled fatty acids carrying the quencher at different positions in the acyl chain, the position of a quenchable fluorophore with respect to the membrane normal can be determined. The accessibility of fluophores from the aqueous phase was assessed by using a water-soluble quencher, the N-methylpicolinium ion. The results of quenching studies suggest that the galactoside binding site is located within the carrier and that this binding site communicates with the aqueous phase through a pore.

Affinity Labels↗
PubMed1984

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)

Antibodies↗
PubMed1984

Purification of the lactose:H+ carrier of Escherichia coli and characterization of galactoside binding and transport.

The lactose carrier, a galactoside:H+ symporter in Escherichia coli, has been purified from cytoplasmic membranes by pre-extraction of the membranes with 5-sulfosalicylate, solubilization in dodecyl-O-beta-D-maltoside, Ecteola-column chromatography, and removal of residual impurities by anti-impurity antibodies. Subsequently, the purified carrier was reincorporated into E. coli phospholipid vesicles. Purification was monitored by tracer N-[3H]ethylmaleimide-labeled carrier and by binding of the substrate p-nitrophenyl-alpha-D-galactopyranoside. All purified carrier molecules were active in substrate binding and the purified protein was at least 95% pure by several criteria. Substrate binding to the purified carrier in detergent micelles and in reconstituted proteoliposomes yielded a stoichiometry close to one molecule substrate bound per polypeptide chain. Large unilamellar proteoliposomes (1-5-micron diameter) were prepared from initially small reconstituted vesicles by freeze-thaw cycles and low-speed centrifugation. These proteoliposomes catalyzed facilitated diffusion and active transport in response to artificially imposed electrochemical proton gradients (delta mu H+) or one of its components (delta psi or delta pH). Comparison of the steady-state level of galactoside accumulation and the nominal value of the driving gradients yielded cotransport stoichiometries up to 0.7 proton/galactoside, suggesting that the carrier protein is the only component required for active galactoside transport. The half-saturation constants for active uptake of lactose (KT = 200 microM) or beta-D-galactosyl-1-thio-beta-D-galactoside (KT = 50-80 microM) by the purified carrier were found to be similar to be similar to those measured in cells or cytoplasmic membrane vesicles. The maximum rate for active transport expressed as a turnover number was similar in proteoliposomes and cytoplasmic membrane vesicles (kcat = 3-4 s-1 for lactose) but considerably smaller than in cells (kcat = 40-60 s-1). Possible reasons for this discrepancy are discussed.

Bacterial Proteins↗
PubMed1983

Chemical modification of the lactose carrier of Escherichia coli by plumbagin, phenylarsinoxide or diethylpyrocarbonate affects the binding of galactoside.

The effects of chemical modification of the lactose carrier of Escherichia coli on galactoside binding (in overproducing strains) and on transport was examined. Both the modifying reagents diethylpyrocarbonate and rose bengal and the thiol reagents phenylarsinoxide and plumbagin can completely inhibit the binding of the substrate p-nitrophenyl alpha-D-galactopyranoside to the carrier. If care is taken to inhibit galactoside binding only partially, the loss of transport is found to parallel the loss of binding sites. The modified carrier molecules are completely inactive, while the remaining active carrier molecules evince normal transport and binding parameters. The binding of galactoside protects the carrier partially against these forms of chemical modification. In view of these observations, the results of previous chemical modification studies [Padan, E., Patel, L. and Kaback, H.R. (1979) Proc. Natl Acad. Sci. USA, 76, 6221-6225; Konings, W.N. and Robillard, G.T. (1982) Proc. Natl Acad. Sci. USA, 79, 5480-5484] must be re-interpreted. Our results stress the utility of studying substrate binding, the first step in the transport cycle.

Arsenicals↗
PubMed1983

Does the lactose carrier of Escherichia coli function as a monomer?

The purified lactose carrier of Escherichia coli (product of the lac Y gene) is shown to be a monomer in detergent micelles of dodecyl-O-beta-D-maltoside. The negative-dominant phenotype of mutant carriers (lacY-d mutants) could not be verified by measurements of the rate of galactoside transport in lacY+/Y-d diploid strains. It is proposed that the membrane-embedded carrier functions as a monomer in galactoside-H+ symport.

Biological Transport↗
PubMed1983

Peptide-specific antibody locates the COOH terminus of the lactose carrier of Escherichia coli on the cytoplasmic side of the plasma membrane.

The carboxyl-terminal decapeptide NH2-Leu-Leu-Arg-Arg-Gln-Val-Asn-Glu-Val-Ala-OH of the lactose carrier protein, the product of the lac Y gene of Escherichia coli, was synthesized, and specific anti-peptide antibodies were raised in rabbits. These antibodies bind to membrane-bound lactose carrier showing that the carboxyl terminus is accessible from the aqueous phase. The antibodies bind only to the surface of inverted cytoplasmic membrane vesicles (but not to closed, right-side-out membrane vesicles), demonstrating that the carboxyl terminus of the carrier protein is directed towards the cytoplasmic side of the plasma membrane in cells. The carboxyl terminus is a potent immunogenic epitope on the purified, detergent-solubilized carrier. Binding of peptide-specific antibodies to the carrier protein inhibits neither substrate binding nor translocation.

Amino Acid Sequence↗
PubMed1983

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 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 of the phosphotransferase system interacted directly with the lactose carrier. The binding of III to lactose carrier required the presence of the non-phosphorylated form of III and substrates of the carrier and exhibited a stoichiometry of 1.2+/- 0.2 mol III/mol lactose carrier. The K(d) of lactose carrier for III was 10 +/- 5 microM. III is apparently unable to interact with a mutant lactose carrier which still binds but does not transport galactosides. The binding of III 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 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.

Journal Article↗
PubMed1983

Self-mutilation.

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Abdomen↗
PubMed1983

Immune complexes in systemic sclerosis; detection by C1q binding, K-cell inhibition and Raji cell radioimmunoassays.

Thirty-four patients with systemic sclerosis (SS) were investigated for the presence of circulating immune complexes by means of a fluid phase C1q binding assay, K-cell inhibition assay and a Raji cell radioimmunoassay and the results compared with those obtained in 21 patients with systemic lupus erythematosus (SLE) and 52 normal healthy controls. Patients with SS showed an incidence of circulating immune complexes comparable to that found in SLE, with 20 patients (58.5%), giving a positive result with at least one of the assays. The presence of circulating immune complexes in patients with SS was found to be associated with both elevation of serum IgG and IgA levels and extensive visceral involvement by the disease. These findings raise the possibility that circulating immune complexes could be involved in the pathogenesis of SS.

Adult↗
PubMed1983

Serum-induced enhancement of peripheral blood mononuclear cell-mediated cytotoxicity towards human target cells in systemic sclerosis.

Sera from 7 of 37 patients with systemic sclerosis (SS) were found to markedly enhance cytotoxicity in several established human target cell lines when co-cultured with normal human peripheral blood mononuclear cells (PBM). Fractionation studies indicated that the cytotoxicity-inducing activity resided in the IgG-containing fractions of serum and that the effector cells were Fc-receptor positive. By contrast, sera from 27 normal controls produced little or no cytotoxicity when co-cultured with the same target cell lines and normal PBM. Any slight enhancement of cytotoxicity that occurred with a single target cell line was, on fractionation, either labile or associated with albumin containing fractions. These findings raise the possibility that antibody-dependent cellular cytotoxicity (ADCC) could provide a pathogenetic mechanism in some patients with SS.

Adult↗
PubMed1983

Serum-induced enhancement of peripheral blood mononuclear cell mediated cytotoxicity towards human target cells in systemic lupus erythematosus.

Sera from 6 out of 18 patients with systemic lupus erythematosus (SLE) were found to be capable of producing marked increases of cytotoxicity in several established human target cell lines when co-cultured with normal human peripheral blood mononuclear cells (PBM). Fractionation studies indicated that the cytotoxicity-inducing activity resided in IgG containing fractions and that the effector cells were Fc-receptor positive. By contrast, sera from 27 normal controls produced little or no cytotoxicity when similarly co-cultured with the same target cell lines and normal PBM. Any slight reactivity that occasionally occurred against a single cell line was, on serum fractionation, either labile or associated with albumen containing fractions. These findings raise the possibility that antibody-dependent cellular cytotoxicity could provide an additional pathogenetic mechanism in patients with SLE.

Adult↗
PubMed1982

A preliminary study of the treatment of trochanteric fractures of the femur with the Kenwright nail.

We present a preliminary study of the results of treatment of 106 fractures of the trochanteric region of the femur, using a new expanding compression nail, the Kenwright nail. The device obtains a secure hold in the femoral head and compresses the fracture line, to give very firm fixation, enabling patients to bear weight immediately with much more confidence than with conventional methods of fixation. Earlier discharge from hospital has been possible, with the release of valuable beds. In no case has the nail cut out. A similar study of its use in intracapsular fractures will be reported.

Aged↗
PubMed1982

Lactose carrier protein of Escherichia coli. Reconstitution of galactoside binding and countertransport.

A procedure for the reconstitution of the lactose carrier protein, a galactoside:proton symporter in Escherichia coli, is described. Starting from cytoplasmic membranes derived from carrier-overproducing strains, essentially all proteins including 89% of the carrier are solubilized by a mixture of dodecyl/tetradecyl polyoxyethylene (n = 9.5) ether and dodecyl O-beta-D-maltoside. In the micellar state the carrier binds substrates with reduced affinity. Addition of E. coli phospholipids and removal of detergents by a hydrophobic column yields small vesicles (50-60-nm diameter). In these vesicles, about 70% of the carrier is recovered and reconstituted carrier is identical to native carrier in terms of substrate binding. After fusion of the small vesicles into larger vesicles (1-5 micrometers), rapid countertransport of galactosides is demonstrated. Attempts to show active galactoside transport by the imposition of artificial electrical potential or pH gradients were unsuccessful, most likely because the reconstituted vesicles are in fact highly permeable to protons.

Bacterial Proteins↗
PubMed1982

Spontaneous lymphocyte-mediated (NK cell) cytotoxicity in systemic sclerosis: a comparison with antibody-dependent lymphocyte (K cell) cytotoxicity.

Spontaneous (NK cell) and antibody-dependent (K cell) cytotoxicity were investigated in 39 patients with systemic sclerosis (SS) and compared with that found in 52 normal controls. Cr-labelled Chang liver cells were used as targets in assays utilising both whole blood (WB) and peripheral blood mononuclear cells (PBM) as effectors. Patients with SS, who were severely affected by extensive visceral disease, were found to have significant impairment of both NK (p less than 0.005; p less than 0.05) and K (p less than 0.001; p less than 0.05) cell cytotoxicity by both effector systems, when compared with normal controls. These findings, which seem to be part of a wider defect in cell-mediated immunity, may provide a possible explanation for the described association of malignancy with systemic sclerosis.

Adult↗