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H R Kaback

Publications and source records attributed to H R Kaback.

At least 235 records · Page 13Linked to original sources
PubMed1981

Effect of the proton electrochemical gradient on maleimide inactivation of active transport in Escherichia coli membrane vesicles.

Many active transport systems present in Escherichia coli membrane vesicles are inhibited by maleimides and other sulfhydryl reagents. These reagents do not interfere with the oxidation of reduced phenazine methosulfate or with the electrochemical proton gradient (delta muH+). The rate of inactivation is increased in the presence of reduced phenazine methosulfate, and it is shown that the electrochemical proton gradient is responsible for the effect. Furthermore, similar effects observed with the proline and melibiose transport systems. Thus, it appears that either the reactivity or accessibility of a sulfhydryl group(s) in each of these carriers is altered by the presence of a transmembrane delta muH+. The findings are consistent with the notion that delta muH+, in addition to acting as the immediate driving force the active transport, may bring about structural or conformational changes in certain membrane proteins that catalyze active transport.

Biological Transport, Active↗
PubMed1981

Relationship between the Na+/H+ antiporter and Na+/substrate symport in Bacillus alcalophilus.

The Na+/H+ antiporter of the obligate alkalophile Bacillus alcalophilus facilitates growth at alkaline pH and precludes growth below pH 8.5. Thus, nonalkalophilic mutant strains do not exhibit Na+/H+ antiport activity and, interestingly, such strains concomitantly lose the ability to catalyze Na+-dependent accumulation of alpha-aminoisobutyrate [Krulwich, T. A., Mandel, D. G. Bornstein, R. F. & Guffanti, A. A. (1979) Biochem. Biophys. Res. Commun. 91, 58-62]. Several other Na+-dependent transport systems are now documented in vesicles from the wild-type strain, and it is demonstrated that these systems are defective in vesicles from the nonalkalophilic mutant KM23. Surprisingly, the defect seems to result not from the loss of Na+/H+ antiport activity per se but from a pleiotropic defect in the Na+/substrate symporters themselves. Monensin, an ionophore that catalyzes Na+/H+ exchange, does not restore respiration-driven Na+/substrate symport in KM23 vesicles. Moreover, with KM23 vesicles, efflux of alpha-aminoisobutyrate, L-malate, and L-aspartate down their respective concentration gradients is not stimulated by Na+, in contrast to the observations with wild-type vesicles. Because monensin should ameliorate a simple defect in Na+/H+ antiport activity and the antiporter should not be required for Na+/substrate symport down a concentration gradient, the results suggest that there may be a direct relationship between the antiporter and various Na+/substrate symporters. One possibility is that the systems share a Na+-translocating subunit.

Aminoisobutyric Acids↗
PubMed1981

ATP-driven active transport in right-side-out bacterial membrane vesicles.

Membrane vesicles from Salmonella typhimurium induced for phosphoglycerate transport, were loaded with pyruvate kinase and ADP by lysing spheroplasts under appropriate conditions. Vesicles so prepared catalyze active transport of proline and serine in the presence of phosphoenolpyruvate; this activity is abolished by the protonophore carbonyl cyanide-m-chlorophenylhydrazone and by the H+-ATPase inhibitor N,N' dicyclohexylcarbodiimide but not by anoxia or cyanide. In contrast, D-lactate-driven active transport is abolished by the hydrazone and by anoxia or cyanide but not by the carbodiimide. Moreover, phosphoenolpyruvate does not drive transport effectively in vesicles that lack the phosphoglycerate transport system. The results are consistent with an overall mechanism in which phosphoenolpyruvate gains access to the interior of the vesicles by means of the phosphoglycerate transporter and is then acted on by pyruvate kinase to phosphorylate ADP. ATP formed inside of the vesicles is then hydrolyzed by the H+-ATPase, leading to the generation of a proton electrochemical gradient that drives H+/solute symport. By using pBR322 as vector and Escherichia coli as host, a fragment of S. typhimurium DNA coding for the phosphoglycerate transport system has been cloned. E. coli membrane vesicles containing the phosphoglycerate transport system also catalyze transport in the presence of phosphoenolpyruvate when they are loaded with pyruvate kinase and ADP.

Adenosine Triphosphatases↗
PubMed1980

Mechanism of the melibiose porter in membrane vesicles of Escherichia coli.

The melibiose transport system of Escherichia coli catalyzes sodium--methyl 1-thio-beta-D-galactopyranoside (TMG) symport, and the cation is required not only for respiration-driven active transport but also for binding of substrate to the carrier in the absence of energy and for carrier-mediated TMG efflux. As opposed to the proton--beta-galactoside symport system [Kaczorowski, G. J., & Kaback, H. R. (1979) Biochemistry 18, 3691], efflux and exchange of TMG occur at the same rate, implying that the rates of the two processes are limited by a common step, most likely the translocation of substrate across the membrane. Furthermore, the rate of exchange, as well as efflux, is influenced by imposition of a membrane potential (delta psi; interior negative), suggesting that the ternary complex between sodium, TMG, and the porter may bear a net positive charge. Consistently, energization of the vesicles leads to a large increase in the Vmax for TMG influx, with little or no change in the apparent Km of the process. It is proposed that the sodium gradient (Na+out < Na+in) and the delta psi (interior negative) may affect different steps in the overall mechanism of active TMG accumulation in the following manner: the sodium gradient causes an increased affinity for TMG on the outer surface of the membrane relative to the inside and the delta psi facilitates a reaction involved with the translocation of the positively charged ternary complex to the inner surface of the membrane.

Biological Transport↗
PubMed1980

Quantitative measurements of membrane potential in Escherichia coli.

By use of giant cells of Escherichia coli induced by growth in the presence of 6-amidinopenicillanic acid, membrane potentials have been measured by two completely independent techniques: directly with intracellular microelectrodes and indirectly from the steady-state distribution of [3H]tetraphenylphosphonium. Under a variety of conditions, the two methods yield values that agree very closely. Thus, with both techniques, the membrane potential approximates -85 mV (interior negative) at pH 5.0 and -142 mV at pH 8.0, with an average slope of -22 mV/pH unit over the range pH 5.0-7.0. A parallel study of membrane vesicles prepared from giant cells was undertaken using tetraphenylphosphonium distribution alone as a measure of membrane potential. The vesicles were found to exhibit a much smaller slope of membrane potential vs. extracellular pH (about -6 mV/pH unit) than intact giant cells. The results indicate that distribution studies with these lipophilic cations provide an excellent measure of membrane potential and are discussed in relation to calculations of H+/substrate stoichiometry for protonsymport systems in E. coli.

Amdinocillin↗
PubMed1980

Glucose 6-phosphate transport in membrane vesicles isolated from Escherichia coli: effect of imposed electrical potential and pH gradient.

Imposition of a membrane potential (delta psi, interior negative) or a pH gradient (delta pH, interior alkaline) across the membrane of Escherichia coli DF2000 leads to a marked, transient increase in glucose 6-phosphate transport that varies systematically with pH. Outwardly directed potassium diffusion gradients in the presence of valinomycin (i.e., generation of delta psi, interior negative) drive glucose 6-phosphate transport at pH 7.5 but much less effectively at pH 5.5, although the magnitudes of the transient delta psi generated are comparable at both pH values. Similarly, imposition of delta psi (interior negative) retards the rate of passive, carrier-mediated glucose 6-phosphate efflux down a concentration gradient at pH 7.5 but not at pH 5.5. In contrast, imposition of delta pH (interior alkaline) by means of outwardly directed acetate diffusion gradients drives glucose 6-phosphate accumulation at pH 5.5 but is relatively ineffective at pH 7.5. The results are independent of the pK of glucose 6-phosphate and provide strong support for the argument that the glucose 6-phosphate porter catalyzes an electrically neutral reaction at acid pH and an electrogenic reaction at alkaline pH. In addition, they are entirely consistent with the hypothesis that the proton/glucose 6-phosphate stoichiometry increases at alkaline pH [Rottenberg, H. (1976) FEBS Lett. 66, 159; Ramos, S., & Kaback, H.R. (1977) Biochemistry 16, 854, 4271].

Biological Transport, Active↗
PubMed1980

Resolution and identification of iron-containing antigens in membrane vesicles from Escherichia coli.

Iron-containing antigens present in membrane vesicles prepared from Escherichia coli ML 308-225 were analyzed by crossed immunoelectrophoresis following growth of the organism in the presence of 59Fe. Seven discrete antigens (or antigen complexes) are detected by autoradiography, and six contain primarily nonheme iron. Three immunoprecipitates are positively identified as NADH dehydrogenase (EC 1.6.99.3), NADPH dehydrogenase (EC 1.6.99.1), and glutamate dehydrogenase (EC 1.4.1.4) based on activity stains for these enzymes. Two other immunogens containing nonheme iron correspond to antigens no. 22 and 37 in the crossed immunoelectrophoresis reference pattern of Owen & Kaback [Owen, P., & Kaback, H. R. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 3148]. In addition, the immunoprecipitate corresponding to Braun's lipoprotein contains tightly bound iron.

Antigens, Bacterial↗
PubMed1980

Electrochemical proton gradient in inverted membrane vesicles from Escherichia coli.

Inverted membrane vesicles prepared from Escherichia coli ML 308-225 generate a transmembrane electrochemical proton gradient (delta mu H+; interior positive and acid) during oxidation of D-lactate, succinate, reduced phenazine methosulfate, or NADH or hydrolysis of ATP. Using the distribution of the lipophilic anion thiocyanate to measure the membrane potential (delta psi) and the permeant weak base methylamine to measure the pH gradient (delta pH), maximal values for delta psi H+ of approximately +160 mV are obtained. Many of the properties of delta psi H+ in inverted vesicles are similar to those described previously in right-side-out vesicles [Ramos, S., & Kaback, H.R. (1977) Biochemistry 16, 848]: (1) the magnitude of the delta psi (interior positive) generated in the presence of D-lactate or reduced phenazine methosulfate is similar to that observed in right-side-out vesicles but of opposite polarity and independent of pH from 5.5 to 8.0; (2) plots of delta pH vs. internal pH in the right-side-out vesicles are similar with D-lactate as the electron donor; (3) as observed with right-side-out vesicles, dissipation of delta psi or delta pH leads to a concomitant increase in the other parameter without a change in the rate of respiration; (4) inverted vesicles catalyze Na+ accumulation, and it is apparent that the process can be driven by either delta psi (interior positive) or delta pH (interior acid).

Biological Transport↗
PubMed1980

Specific labeling of the lac carrier protein in membrane vesicles of Escherichia coli by a photoaffinity reagent.

4-Nitrophenyl-alpha-D-galactopyranoside (NPG) was used as a photoaffinity reagent to specifically inactivate the beta-galactoside transport system in Escherichia coli ML 308-225 membrane vesicles. Photolysis of NPG produced time-dependent, irreversible loss of transport activity with corresponding incorporation of [3H]NPG into the membrane. Both processes were blocked by beta-D-galactopyranosyl 1-thio-beta-D-galactopyranoside, a high-affinity substrate of the lac carrier protein and inactivation of lactose transport was specific because NPG photolysis did not affect proline uptake or the ability of the vesicles to generate an electrochemical proton gradient. Arylation of the lac carrier protein was stoichiometric and resulted in the formation of 0.25 nmol of NPG adduct per mg of membrane protein. All attempts to regenerate transport activity by reillumination in the presence of externally added nucleophiles failed, indicating that arylation is functionally irreversible. When vesicles labeled with [3H]NPG under defined experimental conditions were solubilized and analyzed by gel electrophoresis, only one radioactive peak with an apparent molecular weight of 30,000 was observed, confirming that the reaction is highly specific. The results demonstrate that NPG is an active-site-directed photoaffinity label for the lac carrier protein.

Affinity Labels↗
PubMed1980

Membrane potential changes during mitogenic stimulation of mouse spleen lymphocytes.

By monitoring differences in accumulation of the lipophilic cation [(3)H]tetraphenylphosphonium in media containing low or high potassium concentrations [Lichtshtein, D., Kaback, H. R. & Blume, A. J. (1979) Proc. Natl. Acad. Sci. USA 76, 650-654], the membrane potential of lymphocytes from various sources has been estimated. On the basis of this method, the potential of normal mouse spleen lymphocytes (T and B cells) is -65 +/- 2 mV (mean +/- SEM, interior negative). During the course of mitogenic stimulation by concanavalin A, lipopolysaccharide, or fetal calf serum, the membrane potential of murine spleen lymphocytes changes systematically according to the following pattern: (i) early depolarization lasting 2-3 hr, (ii) repolarization over the next 7 hr, and (iii) a final hyperpolarization phase during the last 24-48 hr. During repolarization and hyperpolarization, moreover, there is a direct correlation between the membrane potential and DNA synthesis, as judged by [(3)H]thymidine incorporation. By using isolated T and B cells, it is observed that concanavalin A depolarizes T cells only, whereas lipopolysaccharide depolarizes B cells only. Thus, both mitogens exhibit the same specificity for depolarization as for mitogenic stimulation. On the basis of these observations, it is suggested that the transition of lymphocytes from a resting state to mitotic activity is initiated by depolarization of the plasma membrane.

Animals↗
PubMed1980

Electrochemical proton gradient in Micrococcus lysodeikticus cells and membrane vesicles.

Using the distribution of weak acids to measure the pH gradient (delta pH; interior alkaline) and the distribution of the lipophilic cation [3H]tetraphenylphosphonium+ to monitor the membrane potential (delta psi; interior negative), we studied the electrochemical gradient or protons (delta mu- H+) across the membrane of Micrococcus lysodeikticus cells and plasma membrane vesicles. With reduced phenazine methosulfate as electron donor, intact cells exhibited a relatively constant delta mu- H+ (interior negative and alkaline) of -193 mV to -223 mV from pH 5.5 to pH 8.5. On the other hand, in membrane vesicles under the same conditions, delta mu- H+ decreased from a maximum value of -166 mV at pH 5.5 to -107 mV at pH 8.0 and above. This difference is related to a differential effect of external pH on the components of delta mu- H+. In intact cells, delta pH decreased from about -86 mV (i.e., 1.4 units) at pH 5.5 to zero at pH 7.8 and above, and the decreases in delta pH was accompanied by a reciprocal increase in delta psi from -110 mV at pH 5.5 to -211 mV at pH 8.0 and above. In membrane vesicles, the decrease in delta pH with increasing external pH was similar to that described for intact cells; however, delta psi increased from -82 mV at pH 5.5 to only -107 mV at pH 8.0 and above.

Benzoates↗