Alternative-substrate inhibition and the kinetic mechanism of the beta-galactoside/proton symport of Escherichia coli.
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The effects of competing alternative substrates on the rate of uptake by galactoside/proton symport were investigated. These experiments produced a decrease in apparent maximum velocity with increased alternative-substrate concentration that cannot be accounted for by a simple ordered mechanism. This, together with non-linearities in the variation of the apparent kinetic constants with alternative-substrate concentration, can be accounted for by a random mechanism for galactoside and proton binding. (+info)
Dependence on pH of substrate binding to lactose carrier in Escherichia coli cytoplasmic membranes.
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Lactose permease in Escherichia mediates proton-substrate cotransport. The molecular mechanism of this process is not understood. We examined the effect of proton concentration on the binding of a substance analogue to the carrier. The dissociation constant of p-nitrophenyl-alpha-galactoside from the carrier was dependent on pH, with an apparent pKa of 9.7. (+info)
Topology of the lac carrier protein in the membrane of Escherichia coli.
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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. (+info)
Involvement of the bacterial groM gene product in bacteriophage T7 reproduction. II. A reduced level of ion concentrations causes the blockage of T7 maturation in K-12-M cells.
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Cellular leakage observed in Escherichia coli K-12-M shortly after T7 infection might be the cause of arrested phage morphogenesis. We observed in this strain, but not in the normal host, a drastic reduction of the intracellular concentration of potassium (60%), magnesium (40%), putrescine (90%), and spermidine (40%), whereas ATP was not significantly reduced. Leakage started about 1 min after the addition of phage and was arrested 3 to 5 min postinfection. Larger molecules such as o-nitrophenyl-beta-D-galactopyranoside could not enter the cells, showing that the permeability of the membrane was not generally affected. To prevent their leakage, we increased the outside concentrations of several small molecules and ions. The yield of progeny phage was substantially increased by the addition of 100 mM MgSO4. (+info)
The transient kinetics of uptake of galactosides into Escherichia coli.
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The uptake of galactosides into Escherichia coli via the lactose permease was studied in the time range 0.01-10s by rapid mixing and quenched flow. An initial transient was observed under two conditions. Firstly, a lag in the approach to the steady state was observed at low galactoside concentrations (less than Km). Secondly, a burst of uptake was observed when anaerobic cell suspensions were mixed with aerobic substrate solutions. However, the cause of the burst of uptake appears to be a burst in the rate of respiration. The rate of galactoside uptake during this phase is 10-fold greater than during the steady state. (+info)
Enzyme-enhancement immunoassay: a homogeneous assay for polyvalent ligands and antibodies.
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A homogeneous enzyme immunoassay for proteins has been developed that avoids the need for a labeled antigen. The technique involves antibody labeled with beta-galactosidase (EC 3.2.1.23), succinylated antibody, and a macromolecular o-nitrophenyl-beta-galactoside substrate. The enzyme-labeled antibody and the succinylated antibody form an immune complex in the presence of sample antigen. An enzyme within this negatively charged microenvironment produces a product that forms a second light-scattering phase, whereas the product produced by free enzyme remains soluble. Thus the antigen modulates the rate of increase in light scattering. The technique has been applied to assays for human immunoglobulin G and C-reactive protein as well as for specific antibodies. (+info)
Sugar transport by the bacterial phosphotransferase system. Regulation of other transport systems (lactose and melibiose).
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The role of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) in the phenomenon of inducer exclusion was examined in whole cells of Salmonella typhimurium which carried the genes of the Escherichia coli lactose operon on an episome. In the presence of the PTS substrate methyl alpha-D-glucopyranoside, the extent of accumulation of the lactose analog methyl beta-D-thiogalactopyranoside was reduced. A strain carrying a mutation in the gene for Enzyme I was hypersensitive to the PTS effect, while a crr mutant strain was completely resistant. Influx, efflux, and exchange of galactosides via the lactose "permease" were inhibited by methyl alpha-glucoside. This inhibition occurred in the presence of metabolic energy poisons, and therefore does not involve either the generation of metabolic energy or energy-coupling to the lactose transport system. When the cellular content of the lactose permease was increased by induction with isopropyl beta-D-thiogalactopyranoside, cells gradually became less sensitive to inducer exclusion. The extent of inhibition of methyl beta-thiogalactoside accumulation by methyl alpha-glucoside was shown to be dependent on the relative cellular content of the PTS and lactose system. The data were consistent with an hypothesis involving partial inactivation of galactoside transport due to interaction between a component of the PTS and the lactose permease. By examination of the effects of the PTS and lactose uptake and melibiose permease-mediated uptake of methyl beta-thiogalactoside, it was further shown that the manner in which inducer exclusion is expressed is independent on the routes available to the non-PTS sugar for exit from the cell. (+info)
Properties of the lactose transport system in Klebsiella sp. strain CT-1.
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Highly purified [D-glucose-1-14C]lactose has been used to study the transport of lactose by Klebsiella sp. strain CT-1. Strain CT-1 transports lactose by a lactose-inducible system that exhibited an apparent Km of 6 mM lactose and an apparent Vmax of 140 nmol/min per mg of cell protein. Lactose uptake was inhibited competitively by o-nitrophenyl-beta-D-galactoside with a Ki value of 8 mM, but was not inhibited by thio-beta-methyl-galactoside. D-Glucose, D-mannose, 2-deoxyglucose, and alpha-methyl-D-glucoside also inhibited lactose uptake. Phosphoenolpyruvate-dependent hydrolysis of o-nitrophenyl-beta-D-galactoside and lactose-dependent release of pyruvate from phosphoenolpyruvate by benzene-treated CT-1 cells showed that CT-1 transports lactose by a phosphoenolpyruvate:sugar phosphotransferase system. Correlations between the growth rate of CT-1 on lactose and properties of the transport system indicated that transport is the rate limiting step in utilization of lactose. (+info)