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H R Kaback

Publications and source records attributed to H R Kaback.

At least 271 records · Page 15Linked to original sources
PubMed1977

Energetics and molecular biology of active transport in bacterial membrane vesicles.

Bacterial membrane vesicles retain the same sidedness as the membrane in the intact cell and catalyze active transport of many solutes by a respiration-dependent mechanism that does not involve the generation of utilization of ATP or other high-energy phosphate compounds. In E. coli vesicles, most of these transport systems are coupled to an electrochemical gradient of protons (deltamuH+, interior negative and alkaline) generated primarily by the oxidation of D-lactate or reduced phenazine methosulfate via a membrane-bound respiratory chain. Oxygen or, under appropriate conditions, fumarate or nitrate can function as terminal electron acceptors, and the site at which deltamuH+ is generated is located before cytochrome b1 in the respiratory chain. Certain (N-dansyl)aminoalkyl-beta-D-galactopyranosides (Dns-gal) and N(2-nitro-4-azidophenyl)aminoalkyl 1-thio-beta-D-galactopyranosides (APG) are competitive inhibitors of lactose transport but are not transported themselves. Various fluorescence techniques, direct binding assays, and photoinactivation studies demonstrate that the great bulk of the lac carrier protein (ca. 95%) does not bind ligand in the absence of energy-coupling. Upon generation of a deltamuH+ (interior negative and alkaline), binding of Dns-gal and APG-dependent photoinactivation are observed. The data indicate that energy is coupled to the initial step in the transport process, and suggest that the lac carrier protein may be negatively charged.

Bacteria↗
PubMed1977

Microenvironment of the binding site in the lac carrier protein.

Studies with a homologous series of (N-dansyl)aminoalkyl-1-thio-beta-D-galactopyranosides containing two to six methylene carbons bridging the galactosyl and dansyl ends of the molecules are described. The compounds were utilized in radioactive and nonradioactive form, and binding of each homologue to membrane vesicles isolated from Escherichia coli ML 308-225 was measured directly by flow dialysis in the presence of D-lactate. The results are compared with the D-lactate-induced fluorescence enhancement observed with each dansylgalactoside and with the ability of N-methylpicolinium perchlorate to quench the fluorescence of the bound homologues. The binding affinity of the lac carrier protein for the probes varies directly with the length of the alkyl linkage, and the same number of binding sites is observed with each homologue. In contrast, however, the increase in fluorescence observed upon binding varies dramatically as the alkyl chain is increased in length, with the fluorescence exhibiting maximal values at two and six methylene carbons and a minimum at four methylene carbons. Furthermore, quenching by N-methylpicolinium perchlorate exhibits an inverse relationship and maximum quenching is observed with the 4 carbon homologue. Possible reasons for this behavior are discussed.

Bacterial Proteins↗
PubMed1977

Effects of thyrotropin on the thyroid cell membrane: hyperpolarization induced by hormone-receptor interaction.

Cultured thyroid cells accumulate the lipophilic cation triphenylmethylphosphonium, indicating that there is an electrical potential (interior negative) across the plasma membrane. Thyrotropin stimulates the uptake of the lipophilic cation 3-fold, and the proton conductor carbonylcyanide-m-chlorophenylhydrazone causes efflux of triphenylmethylphosphonium accumulated in the presence or absence of thyrotropin. The stimulatory effect of thyrotropin on triphenylmethylphosphonium accumulation is not mimicked by human chorionic gonadotropin, a glycoprotein hormone with a similar structure whose target organ is not the thyroid, and the effect is abolished if the thyrotropin-receptor activity of the cells is destroyed by treatment with trypsin. Analogous effects are observed with thyroid plasma membrane vesicles which are essentially devoid of mitochondrial and soluble enzyme activities. Triphenylmethylphosphonium uptake and stimulation by thyrotropin occurs when NaCl, KCl, or Tris.HCl concentration gradients are artifically imposed across the vesicle membrane ([salt](out) > [salt](in)). It seems likely, therefore, that triphenylmethylphosphonium uptake is driven by a chloride diffusion potential (interior negative) and that thyrotropin either increases the permeability of the membrane to anions or decreases its permeability to cations. Thyrotropin-stimulated triphenylmethylphosphonium uptake in the vesicle preparations reaches a quasi steady-state within 3 min; in contrast, thyrotropin-stimulated adenylate cyclase activity is negligible during this period of time, becomes measurable after about 4 min, and is optimal after 12-15 min. Thus, a primary mode of action of thyrotropin on the thyroid cell may be an alteration in the electrical potential across the plasma membrane. The relevance of this observation to the mechanism of action of other glycoprotein hormones, certain bacterial toxins, and interferon is discussed.

Animals↗
PubMed1976

Equilibrium between two forms of the lac carrier protein in energized and nonenergized membrane vesicles from Escherichia coli.

p-Nitrophenyl alpha-D-galactopyranoside is a competitive inhibitor of lactose transport in membrane vesicles prepared from Escherichia coli ML 308-225 (Ki congruent to 6.6 muM) but is not accumulated by the vesicles. Binding of p-nitrophenyl alpha-D-[6-3H]galactopyranoside to membrane vesicles has been measured by flow dialysis. In the presence of D-lactate, ligand binds to the vesicles with a KD of about 6 muM, and a total of 2.3 nmol per mg of membrane protein is bound at saturation. In the absence of D-lactate, a small amount of binding can be detected (approximately 0.2 nmol per mg of membrane protein) with a similar affinity constant (KD congruent to 9 muM). Binding inthe presence or absence of D-lactate is dependent upon a functional lac y gene product and upon the structural integrity of the vesicle membrane and is reversed by p-hydroxymercuribenzenesulfonate. Agents such as 2,4-dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone, and valinomycin, alone or in combination, abolish D-lactate-dependent binding but do not affect binding in the absence of electron donors. The results confirm previous observations that the bulk of the lac carrier protein is unable to bind ligand unless the membrane is energized. and they also corroborate observations that a small amount of binding occurs in the absence of energy coupling. The findings are discussed in terms of a model in which the lac carrier protein exists in a state of dynamic equilibrium between two forms: (i) a low affinity, cryptic form which predominates in the absence of energy coupling; and (ii) a high affinity form, accessible from the external surface of the membrane, which predominates in the presence of an electrochemical gradient of protons (interior negative and alkaline).

Bacterial Proteins↗
PubMed1976

Effect of lipids on the reconstitution of D-lactate oxidase in Escherichia coli membrane vesicles.

An unsaturated fatty acid auxotroph lacking D-lactate dehydrogenase activity has been isolated from Escherichia coli ML 308-225 dld-3. While NADH oxidase activity in membrane vesicles prepared from the mutant cells grown in a variety of unsaturated fatty acids is comparable to that of previously isolated fatty acid auxotrophs, D-lactate oxidase activity is absent. However, D-lactate oxidase ativity can be restored when vesicles are incubated with a purified preparation of D-lactate dehydrogenase obtained from wild type cells. The effect of altering the fatty acid composition of the membrane on the reconstitution of D-lactate oxidase activity was examined. Binding of purified D-lactate dehydrogenase was not affected by either the lipid composition of the membrane vesicles or the temperature during reconstitution. However, the reconstitution of D-lactate oxidase activity was strongly influenced by the fatty acid composition of the membrane lipids. The temperature dependence of the reconstituted activity was analyzed. Temperature transitions were not observed with membrane vesicles supplemented with oleic or linolenic acid but palmitelaidic acid-enriched vesicles exhibited a transition temperature at 30 degrees. Attempts to reconstitute the elaidic-supplemented vesicles at temperatures below 30 degrees failed to yield active D-lactate oxidase and the vesicles aggregated. At 42 degrees, aggregation did not occur and D-lactate oxidase activity was obtained to a level of 10% of that found with membranes from the parent strain (ML 308-225). These results suggest that although binding of D-lactate dehydrogenase is independent of the physical state of the membrane, reconstitution of the D-lactate oxidase activity in membrane vesicles is dependent upon the fatty acid composition of the phospholipid, hence the physical state of the membrane.

Cell Membrane↗
PubMed1976

The role of the carbodiimide-reactive component of the adenosine-5'-triphosphatase complex in the proton permeability of Escherichia coli membrane vesicles.

Membrane vesicles isolated from wild-type and dicyclohexylcarbodiimide-resistant strains of Escherichia coli exhibit identical respiration-dependent transport activities, and in both cases, this activity is abolished by extraction of the vesicles with 1.0 M guanidine-HCl. Transport activity of extracted wild-type vesicles is completely restored by exposing the vesicles to lipophilic or water-soluble carbodiimides, while transport activity of the mutant vesicles is not restored by exposure to lipophilic carbodiimides. Strikingly, however, complete reactivation of transport in mutant vesicles is observed with water-soluble carbodiimides. Similarly, the Ca2+, Mg2+-stimulated ATPase activity of wild-type vesicles is inhibited by both classes of carbodiimides, while the ATPase activity of mutant vesicles is inhibited by water-soluble carbodiimides, but resistant to inhibition by lipophilic carbodiimides. The carbodiimide-reactive component of the membraneous Ca2+, Mg2+-stimulated ATPase complex in wildtype vesicles is readily labeled with N,N'-dicyclohexyl[14C]-carbodiimide, while the analogous component in mutant vesicles is not reactive. Alternatively, when vesicles are treated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide [14C]methiodide, a water-soluble carbodiimide, the carbodiimide-reactive component is labeled to a similar degree in both preparations. The results suggest that the altered carbodiimide-reactive proteolipid in the dicyclohexylcarbodiimide-resistant mutant is specifically defective in its ability to react with lipophilic carbodiimides. In addition, these and other findings indicate that the increase in proton permeability observed on extraction of isolated membrane vesicles with chaotropic agents is due exclusively to an effect on the carbodiimide-reactive component of the Ca2+, Mg2+-stimulated ATPase complex.

Adenosine Triphosphatases↗
PubMed1976

Energy-dependent binding of dansylgalactoside to the lac carrier protein: direct binding measurements.

High specific activity 6'-N-[3H]dansyl)aminohexyl 1-thio-beta-D-galactopyranoside (Dns6-Gal) has been synthesized, and its binding to Escherichia coli membrane vesicles measured directly by flow dialysis. With ML 308-225 vesicles containing the lac carrier protein, specific binding is not detected in the absence of D-lactate or reduced phenazine methosulfate. In the presence of these electron donors, binding is observed, and the binding constant and number of binding sites are approximately 4 muM and 1.5 nmol/mg of membrane protein, respectively. These values are in excellent agreement with those obtained by fluorescence titration. p-Chloromercuribenzenesulfonate, which directly inactivates the lac carrier protein, and carbonylcyanide m-chlorophenylhydrazone, which collapses the membrane potential, cause release of bound Dns6-Gal. Moreover, significant binding is not observed with membrane vesicles that are devoid of the lac carrier protein. The results provide qualitative and quantitative confirmation of previous studies which indicate that changes in dansylgalactoside fluorescence observed on "energization" of membrane vesicles reflect binding of the probe to the lac carrier protein.

4-Chloromercuribenzenesulfonate↗
PubMed1976

The electrochemical gradient of protons and its relationship to active transport in Escherichia coli membrane vesicles.

Membrane vesicles isolated from E. coli generate a trans-membrane proton gradient of 2 pH units under appropriate conditions when assayed by flow dialysis. Using the distribution of weak acids to measure the proton gradient (deltapH) and the distribution of the lipophilic cation triphenyl-methylphosphonium to measure the electrical potential across the membrane (delta psi), the vesicles are shown to generate an electrochemical proton gradient (deltamuH+) of approximately-180 mV at pH 5.5 in the presence of ascorbate and phenazine methosulfate, the major component of which is a deltapH of about -110mV. As external pH is increased, deltapH decreases, reaching 0 at pH 7.5 and above, while delta psi remains at about-75 mV and internal pH remains at pH 7.5. Moreover, the ability of various electron donors to drive transport is correlated with their ability to generate deltamuH+. In addition, deltapH and delta psi can be varied reciprocally in the presence of valinomycin and nigericin. These data and others (manuscript in preparation) provide convincing support for the role of chemiosmotic phenomena in active transport.

Acetates↗
PubMed1975

Lifetime and rotational relaxation time of dansylgalactoside bound to the lac carrier protein.

The results presented in this paper demonstrate that the excited state lifetime, anisotropy, and rotational relaxation time of 2'-(N-dansyl)aninoethyl 1-thio-beta-D-galactopyranoside (DG2) increase when the probe is bound specifically to the lac carrier protein in "energized" Escherichia coli membrane vesicles. Although the probe also binds nonspecifically to the vesicle membrane, such binding is independent of the lac carrier protein and is unaffected by "energization" of the vesicles. The experiments provide further evidence that the dansylgalactosides are useful probes for the beta-galactoside transport system and support the hypothesis that the changes in dansylgalactoside fluorescence observed on "energization" of membrane vesicles reflect changes in the binding of the probe.

Bacterial Proteins↗
PubMed1975

Photoinactivation of the beta-galactoside transport system in Escherichia coli membrane vesicles with an impermeant azidophenylgalactoside.

2'-N-(2-Nitro-4-azidophenyl) aminoethyl-1-thio-beta-D-galactopyranoside (APG2) is a competitive inhibitor of lactose transport in membrane vesicles isolated from Escherichia coli ML 308-225, exhibiting an apparent Ki of 30 to 40 muM, but is not transported. When irradiated with visible light in the presence of D-lactate, APG2 irreversibly inactivates the lac transport system. Imposition of a membrane potential (positive outside) by valinomycin-induced potassium efflux also causes APG2 photoinactivation. Strikingly, photoinactivation is not observed in the absence of D-lactate or a potassium diffusion gradient. Kinetic studies of the inactivation process yield a KD of 35 muM. Since lactose protects against the inactivation, it is apparent that these effects are specific for the lac transport system. The results show that APG2 inactivates from the outer surface of the vesicle membrane and support the previous hypothesis that the lac carrier protein is unable to bind external substrate in the absence of energy coupling.

Amino Acids↗
PubMed1975

Reversible inactivation of vectorial phosphorylation by hydroxybutynoate in Escherichia coli membrane vesicles.

The acetylenic hydroxy acid 2-hydroxy-3-butynoate causes irreversible inactivation of the Escherichia coli membrane-bound flavoenzyme D-lactic dehyrogenase, and thus blocks D-lactate dependent active transport in isolated membrane vesicles [Walsh, C. T., Abeles, R. H., and Kaback H. R. (1972), J. Biol. Chem. 247, 7858]. The inactivator is a suicide substrate for the dehydrogenase, undergoing a small number of turnovers before partitioning between oxidation and inactiviation. It is now demonstrated that reactive product molecules of 2-keto-3-butynoate can diffuse in the membranes to a component of the phosphotransferase system and cause time-dependent and covalent inactivation of phosphoenolpyruvate-dependent hexose uptake. Membrane vesicles from double mutants with low levels of both D- and L-lactic dehydrogenase lose only 30 percent of their hexose uptake capacity on exposure to hydroxybutynoate under conditions sufficient to fully inactivate hexose transport in wild type vesicles. Transport of 1-[14C]hydroxybutynoate into vesicles is followed by rapid covalent labeling of membrane proteins by the reactive, enzymatically generating keto acid oxidation product. Incubation of hydroxybutynoate-inactivated vesicles (5% residual activity) for 20 min in buffer with 10 mM dithiothreitol results in reactivation of 63% of the hexose transport activity, a 12-fold increase in activity. No reactivation occurs if the vesicular phosphotransferase system is inactivated by keto acid derived from membrane oxidation of the olefinic congener 2-hydroxy-3-butenoate. In contrast to thiol reactivation of acetylenic-blocked glucose transport, blockage of D-lactate-stimulated proline uptake is not alleviated, stressing different modes of inactivation of the phosphotransferase system compared to the membranous lactate dehydrogenases.

Biological Transport, Active↗
PubMed1975

Transport in an Escherichia coli fatty acid auxotroph. A novel case of catabolite repression.

The transport of alpha-methylglucoside and a variety of amino acids including histidine, leucine, lysine, proline, and threonine are reduced 5- to 50-fold in Escherichia coli K1061, an unsaturated fatty acid auxotroph, when cells are grown in medium containing glucose as a carbon source and supplemented with either cis-vaccenic acid or cis-9, 10-methylenehexadecanoic acid. Cells grown in glucose medium containing oleate, palmitoleate, or cis-7-hexadecenoate do not exhibit this effect and transport alpha-methylglucoside and amino acids at rates comparable to those of a wild type strain. For cis-vaccenate- and cis-9, 10-methylenehexadecanoate-grown cells the capacity to transport amino acids and alpha-methylglucoside is normal when glycerol or succinate is the energy supply or when cyclic adenosine 3':5'-monophosphate is added to cultures growing on glucose. These results suggest that in this strain several transport systems are affected by catabolite repression, but the phenomenon is unique since repression requires not only glucose but specific unsaturated fatty acids, cis-vaccenic acid, or cis-9, 20-methylenehexadecanoic acid.

Biological Transport, Active↗
PubMed1975

Localization of D-lactate dehydrogenase in native and reconstituted Escherichia coli membrane vesicles.

In the preceding paper the preparation and characterization of antiserum to purified D-lactate are described. In this paper the effects of the antibody on D-lactate dehydrogenase activity and D-lactate-dependent active transport in native Escherichia coli ML 308-225 membrane vesicles and ML 308-225dld-3 vesicles reconstituted with D-lactate dehydrogenase are described. The results demonstrate that D-lactate dehydrogenase is inaccessible to antibody in native ML 308-225 vesicles, but readily accessible to antibody in reconstituted dld-3 vesicles. The findings indicate that D-lactate dehydrogenase is located on the inner surface of native ML 308-225 vesicles and on the outer surface of reconstituted dld-3 vesicles. The results with the native vesicle preparations also provide further evidence that virtually none of the vesicles is inverted or sufficiently damaged to allow access of antibody to D-lactate dehydrogenase. In addition, experiments are presented which demonstrate that an impermeable electron carrier, reduced 5-N-methylphenazonium-3-sulfonate, drives active transport in native ML 308-225 vesicles as well as its permeable analogue reduced phenazine methosulfate. Thus, reduction of the respiratory chain from either side of the vesicle membrane is able to drive active transport. Ca2+, Mg2+-stimulated ATPase is also inaccessible to antibody in ML 308-225 vesicles unless the preparation is subjected to ultrasonic sound, incubated in Tris buffer at pH 9.0, or homogenized vigorously. Moreover, as opposed to D-lactate dehydrogenase and cytochrome b1, ATPase is readily lost from the membrane during the preparation of vesicles.

Animals↗
PubMed1975

Immunochemical properties of the membrane-bound D-lactate dehydrogenase from Escherichia coli.

The preparation, characterization, and purification of antibody against the membrane-bound D-lactate dehydrogenase solubilized and purified from Escherichia coli ML 308-225 are described. The antibody is highly specific for the flavin-linked D-lactate dehydrogenase, and incubation of the enzyme with antiserum results in marked inhibition of enzymatic activity. By means of a radioimmune assay, it is demonstrated that membrane vesicles prepared from E. coli ML 308-225dld-3 contain catalytically inactive material which cross-reacts with native D-lactate dehydrogenase. In the following paper, the effects of this antiserum on D-lactate dehydrogenase activity and D-lactate-dependent active transport in native and reconstituted membrane vesicles are examined.

Animals↗