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R Seckler

Publications and source records attributed to R Seckler.

42 records · Page 3Linked to original sources
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

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↗
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↗
PubMed1982

Purification and properties of the pyruvate dehydrogenase complex from Salmonella typhimurium and formation of hybrids with the enzyme complex from Escherichia coli.

The pyruvate dehydrogenase (Pyruvate:lipoamide oxidoreductase (decarboxylating and acceptor acetylating), EC 1.2.4.1) complex from Salmonella typhimurium was purified, characterized and compared to the enzyme complex from Escherichia coli. No difference could be found in the molecular weights of the native enzyme complexes or in the single polypeptide chains of the enzymes of the two organisms. Values of 100 000, 87 000 and 56 000 were obtained for the polypeptide chains of the pyruvate dehydrogenase, the dihydrolipoamide transacetylase (acetyl-CoA:dihydrolipoamide S-acetyltransferase, EC 2.3.1.12) and the dihydrolipoamide dehydrogenase (NADH:lipoamide oxidoreductase, EC 1.6.4.3) components, respectively. Complete cross-reactivity was found with antibodies directed against the pyruvate dehydrogenase complex from E. coli and electron micrographs of both enzyme complexes reveal identical structures. A high Michaelis constant for pyruvate with a Km = 6 . 10(-4) M and a somewhat weaker cooperativity as compared to the enzyme from E. coli reflect some minor differences, while the binding of the cofactor thiamine diphosphate (Km = 1 . 10(-6) M) is identical for both enzyme complexes. Reassociation to a fully active complex molecule works with equal facility between the pyruvate dehydrogenase component and a dihydrolipoamide transacetylase: dihydrolipoamide dehydrogenase subcomplex from either organism in all possible combinations.

Acetyltransferases↗
PubMedDate not supplied

Interaction of Salmonella phage P22 with its O-antigen receptor studied by X-ray crystallography.

The O-antigenic repeating units of the Salmonella cell surface lipopolysaccharides (serotypes A, B and D1) serve as receptors for phage P22. This initial binding step is mediated by the tailspike protein (TSP), which is present in six copies on the base plate of the phage. In addition to the binding activity, TSP also displays a low endoglycolytic activity, cleaving the alpha(1,3)-O-glycosidic bond between rhamnose and galactose of the O-antigenic repeats. The crystal structure of TSP in complex with receptor fragments allowed to identify the receptor binding site for the octasaccharide product of the enzymatic action of TSP on delipidated LPS and the active site consisting of Asp392, Asp395 and Glu359. The structure comprises a large right-handed parallel beta-helix of 13 turns. These fold independently in the trimer, whereas the N-terminus forms a cap-like structure and the C-terminal parts of the three polypeptide strands merge to a single common domain. In addition, TSP has served as model system for the folding of large, multisubunit proteins. Its folding pathway is influenced by a large number of point mutations, classified as lethal, temperature sensitive or general suppressor mutations, which influence the partitioning between aggregation and the productive folding pathway.

Antibody Specificity↗