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H R Kaback

Publications and source records attributed to H R Kaback.

At least 289 records · Page 16Linked to original sources
PubMed1975

Differentiation between binding and transport of dansylgalactosides in Escherichia coli.

The results presented in this paper confirm and extend previous observations which indicate that fluorescent dansylgalactsodes bind to the beta-galactoside carrier protein but do not penetrate the cytoplasmic membrane. The conclusion is supported by the following observations. (a) Although 2'-(N-dansyl)aminoethyl-beta-D-thiogalactopyranoside and 2'-(N-dansyl)aminoethyl-beta-D-galactopyranoside are competitive inhibitors of lactose transport in intact cells of Escherichia coli and induce the in vitro synthesis of beta-galactosidase, they do not induce beta-galactosidase in vivo. (b) p-Chloromercuribenzenesulfonate does not cause efflux of lactose from the intravesicular pool, but causes rapid reversal of D-lactate-induced dansylgalactoside fluorescence. (c) Dansylgalactosides inhibit dilution-induced, carrier-mediated lactose efflux.

4-Chloromercuribenzenesulfonate↗
PubMed1975

Energy-dependent binding of dansylgalactosides to the beta-galactoside carrier protein.

Fluorescent beta-galactosides (1-(N-dansyl)amino-beta-D-galactopyranoside (DG0), 2'-(N-dansyl)aminoethyl-beta-D-thiogalactopyranoside (DG2), 2'-(N-dansyl)aminoethyl-beta-D-galactopyranoside (oxy-DG2), and 6'-(N-dansyl)aminohexyl-beta-D-thiogalactopyranoside (DG6)) competitively inhibit lactose transport by membrane vesicles from Escherichia coli ML 309-225, but are not actively transported. An increase in the fluorescence of these dansylgalactosides is observed upon addition of D-lactate, imposition of a membrane diffusion potential (positive outside), or dilution-induced, carrier-mediated lactose efflux. The increase is not observed with 2'-(N-dansyl)aminoethyl-beta-D-thioglucopyranoside nor with membrane vesicles lacking the beta-galactoside transport system. Moreover, the D-lactate-induced fluorescence increase is blocked or rapidly reversed by addition of beta-galactosides, sulfhydryl reagents, inhibitors of D-lactate oxidation, or uncoupling agents. The fluorescence increase exhibits an emission maximum at 500 nm and excitation maxima at 345 nm and at 292 nm. The latter excitation maximum is absent unless D-lactate is added, indicating that the bound dansylgalactoside molecules are excited by energy transfer from the membrane proteins. Titration of vesicles with dansylgalactosides in the presence of D-lactate demonstrates that the lac carrier protein constitutes 3 to 4% oof the total membrane protein, and that the affinity of the carrier for substrate is directly related to the length of the alkyl chain between the galactosidic and the dansyl moieties of the dansylgalactosides. In addition, there is excellent agreement between the affinity constants of the various dansylgalactosides as determined by fluorimetric titration and their apparent Kis for lactose transport (KDs and/or apparent Kis are approximately 550, 3o, 40, and 5 muM FOR DG0, DG2, oxy-DG2, and DG6, respectively). Polarization of fluorescence measurements with DG2 and DG6 demonstrate a dramatic increase in polarization on addition of D-lactate which is reversed by addition of lactose or anaerobiosis. These findings provide strong evidence for the contention that the fluorescence changes observed on "energization" of the membrane are due to binding of the dansylgalactosides per se, rather than binding followed by transfer into the hydrophobic interior of the membrane

Bacterial Proteins↗
PubMed1975

Photoinactivation of the beta-galactoside transport system in Escherichia coli membrane vesicles with 2-nitro-4-azidophenyl-1-thio-beta-D-galactopyranoside.

2-Nitro-4-azidophenyl-1-thio-beta-D-galactopyranoside (azidophenylgalactoside) is a competitive inhibitor of lactose transport in membrane vesicles isolated from Escherichia coli ML 308-225, exhibiting an apparent Ki of 75 muM. The initial rate and steady state level of [3H]azidophenylgalactoside accumulation are markedly stimulated by the addition of D-lactate to vesicles containing the lac transport system, and kinetic studies reveal an apparent Km of 75 muM. Membrane vesicles devoid of the lac transport system do not take up significant amounts of azidophenylgalactoside in the presence or absence of D-lactate. When exposed to visible light in the presence of D-lactate, azidophenylgalactoside irreversibly inactivates the lac transport system. Strikingly, photolytic inactivation is not observed in the absence of D-lactate. Kinetic studies of the inactivation process yield a KD of 77 muM. Since lactose protects against inactivation and azidophenylgalactoside does not inactivate amino acid transport, it is apparent that these effects are specific for the lac transport system. The results are consistent with the proposal that the lac carrier protein is inaccessible to substrate in the absence of energy coupling.

Amino Acids↗
PubMed1975

Ubiquinone-mediated coupling of NADH dehydrogenase to active transport in membrane vesicles from Escherichia coli.

Addition of ubiquinone-1 to E. coli ML 308-225 membrane vesicles dramatically increases coupling between NADH oxidation and active transport such that initial rates and steady-state levels of lactose and amino-acid accumulation are comparable to those observed during D-lactate oxidation. Similar but less dramatic effects are observed with the quinone and succinate or L-lactate. In the presence of NADH and ubiquinone-1, the vesicles also generate a membrane potential (interior negative) that is similar in magnitude to that observed in the presence of D-lactate. Stimulation of NADH-dependent transport by ubiquinone-1 cannot be accounted for by increased rates of oxidation of NADH, and the effect of the quinone on NADH-dependent lactose transport is not observed in vesicles depleted of NADH dehydrogenase activity. Thus, it is apparent that ubiquinone-1 shunts electrons from NADH dehydrogenase [NADH:(acceptor)oxidoreductase; EC 1.6.99.3] to the portion of the respiratory chain containing the energy-coupling site. The findings demonstrate unequivocally that inefficient coupling of NADH oxidation to active transport cannot be due to the presence of inverted vesicles. In addition, they provide further support for specific localization of the energy-coupling site.

Amino Acids↗
PubMed1975

Reversible effects of chaotropic agents on the proton permeability of Escherichia coli membrane vesicles.

Extraction of E. coli ML 308-225 membrane vesicles with chaotropic agents causes the vesicles to become specifically permeable to protons. As a result, the vesicles no longer generate a membrane potential, interior negative, and they do not catalyze respiration-dependent lactose or proline transport. Treatment of the extracted vesicles with various carbodiimides decreases the permeability of the vesicle membrane to protons, causing them to regain their ability to generate a membrane potential. By this means, active transport is completely reactivated. Exposure of the vesicles to carbodiimides prior to extraction with chaotropic agents makes transport activity impervious to the effects of the chaotropes.

Biological Transport, Active↗
PubMed1975

Vinylglycolate resistance in Escherichia coli.

Escherichia coli K-12 vinylglycolate-resistant mutants have been isolated and characterized. Two of the mutants, JSH 150 and JSH 151, have been determined to be double mutants, lacking both membrane-bound L-and D-lactate dehydrogenases. The lactate transport system is intact in all strains; both radioactive lactate and vinylglycolate are actively taken up. Likewise, the phosphoenolypyruvate-dependent phosphotransferase system for hexose uptake is active. Vinylglycolate, previously shown to inhibit the phosphoenolpyruvate-dependent phosphotransferase system, has very little effect in the double mutants. The extent of vinylglycolate inhibition in other mutants seems directly related to the activity of the lactate dehydrogenases. This indicates that vinylglycolate is oxidized to 2-keto-3-butenoate before inactivating the phosphoenolpyruvate-dependent phosphotransferase system. These results were found in whole cells and confirmed in isolated membrane vesicles.

Biological Transport, Active↗
PubMed1975

Physiological suppression of a transport defect in Escherichia coli mutants deficient in Ca2+, Mg2+-stimulated adenosine triphosphatase.

Transport properties of membrane vesicles isolated from two adenosine triphosphatase-deficient mutants of Escherichia coli, NR70 and DL54, were compared with those of vesicles prepared from the corresponding parental strains. As reported previously (Rosen, 1973; Altendorf et al., 1974), vesicles prepared from these mutants grown under aerobic conditions exhibited defective amino acid transport, and activity was restored after treatment with dicyclohexylcarbodiimide. In sharp contrast, however, vesicles isolated from the same mutants grown anaerobically in the presence of nitrate exhibited completely normal transport activity when assayed under either anaerobic or aerobic conditions. Suppression of the transport defect was not due to the manner by which the vesicles were prepared, and the adenosine triphosphatase deficiency was not ameliorated by anaerobic growth in the presence of nitrite. Finally, the transport activity of vesicles prepared from the mutants grown under aerobic conditions was relatively resistant to the effect of 1.0 M guanidine hydrochloride extraction, whereas the activity of vesicles prepared from mutants grown anaerobically was totally refractory to the effect of the chaotrope.

Adenosine Triphosphatases↗
PubMed1974

Transport studies in bacterial membrane vesicles.

The use of bacterial membrane vesicles as an experimental system for the study of active transport has been discussed. Vesicles are prepared from osmotically sensitized bacteria, and consist of osmotically intact, membranebound sacs without internal structure. They retain litle or no cytoplasm. Under appropriate conditions, these vesicles catalyze the transport of a variety of solutes at rates which are comparable, in many cases, to those of intact cells. Two general types of transport systems have been elucidated in the vesicle system: (i) group translocation systems which catalyze vectorial covalent reactions; and (ii) respirationlinked transport systems that catalyze the active transport of a whole range of metabolites against an electrochemical or osmotic gradient. In E. coli membrane vesicles, the respiration-linked transport systems are coupled primarily to the oxidation of (D)-lactate to pyruvate, catalyzed by a flavin-linked, membrane-bound (D)-lactate dehydrogenase which has been purified to homogeneity. Electrons derived from (D)-lactate or certain artificial electron donors are transferred to oxygen by means of a membrane-bound respiratory chain, and respiration is coupled to active transport within a segment of the respiratory chain between the primary dehydrogenase and cytochrome. b(l). The great majority of the individual membrane vesicles in the population catalyze active transport, and the generation or hydtolysis of ATP is not involved. Under anaerobic conditions, fumarate or nitrate can be utilized in place of oxygen as terminal electron acceptors. With the exception that (D)-lactate is not always the most effective electron donor for active transport, vesicles prepared from a number of other organisms catalyze transport in a similar manner. Fluorescent dansylgalactosides are useful molecular probes of active transport in the vesicle system. These compounds are competitive inhibitors of beta-galactoside transport, but are not transported themselves. Fluorescence studies indicate that the lac carrier protein constitutes approximately 3 to 6 percent of the total membrane protein, and that it is not accessible to the external medium unless the membrane is "energized." Thus, energy is coupled to one of the initial steps in the transport process. Studies with a photoaffinity-labeled galactoside provide independent support for this conclusion. When membrane vesicles prepared from a (D)-lactate dehydrogenase mutant of E. coli are treated with (D)-lactate dehydrogenase, the enzyme binds to the vesicles and they regain the capacity to catalyze (D)-lactate oxidation and (D)-lactate-dependent active transport. The maximal specific transport activity obtained in the reconstituted system is similar in magnitude to that of wildtype vesicles. Titration studies with dansylgalactoside demonstrate that there is at least a seven- to eightfold excess of lac carrier protein relative to (D)-lactate dehydrogenase. Evidence is presented indicating that the enzyme is bound to the inner surface of native membrane vesicles and to the outer surface of reconstituted vesicles, and that the flavin coenzyme moiety is critically involved in binding. Possible mechanisms of respirationlinked active transport are discussed.

Anaerobiosis↗
PubMed1974

Determination of the absolute number of Escherichia coli membrane vesicles that catalyze active transport.

Transport of vinylglycolate (2-hydroxy-3-butenoic acid) via the lactate transport system is the limiting step for covalent labeling of membrane vesicles prepared from E. coli ML 308-225. Thus, the rate and extent of vinylglycolate labeling is stimulated about 10-fold by ascorbate-phenazine methosulfate, and stimulation is abolished by 2,4-dinitrophenol and by phospholipase treatment, neither of which affect the rate of vinylglycolate oxidation. [(3)H]Vinylglycolate of high specific activity has been prepared, and vesicles have been labeled with this compound in the presence of ascorbate-phenazine methosulfate. Examination of these preparations by high resolution radioautography in the electron microscope demonstrates that virtually all of the vesicles are labeled. The experiments provide a strong indication that most, if not all, of the membrane vesicles in these preparations catalyze active transport.

Ascorbic Acid↗
PubMed1974

D-lactate dehydrogenase binding in Escherichia coli dld- membrane vesicles reconstituted for active transport.

When membrane vesicles prepared from a D-lactate dehydrogenase mutant of E. coli ML 308-225 are treated with a homogeneous preparation of D-lactate dehydrogenase, the enzyme binds to the vesicles and they regain the capacity to catalyze D-lactate oxidation and D-lactate-dependent active transport. Although membranebound enzyme increases linearly with addition of increasing quantities of enzyme, reconstituted transport activity and D-lactate oxidation are saturable functions of the amount of enzyme bound. The maximal specific transport activity obtained in the reconstituted system is similar in magnitude to that of wild type vesicles. Titration studies with 2-(N-dansyl)-aminoethyl-beta-D-thiogalactoside demonstrate that there is at least a 7- to 8-fold excess of lac carrier protein relative to D-lactate dehydrogenase. Hydroxybutynoate-inactivated enzyme does not bind to the vesicles, indicating that the coenzyme moiety is critically involved in binding. Conformational changes are also apparently involved since 0.6 M guanidine.HCl is required for optimal binding and reconstitution. The relative unreactivity of reconstituted vesicles towards vinylglycolic acid suggests that D-lactate dehydrogenase is bound to the outer surface of the reconstituted vesicles.

Binding Sites↗