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Biomedical subjects

H R Kaback

Publications and source records attributed to H R Kaback.

At least 199 records · Page 11Linked to original sources
PubMed1985

Site-specific mutagenesis of histidine residues in the lac permease of Escherichia coli.

The lacY gene of Escherichia coli, which encodes the lac permease, has been modified by oligonucleotide-directed, site-specific mutagenesis such that each of the four histidine residues in the molecule is replaced with an arginine residue. Replacement of histidine-35 and histidine-39 with arginine has no apparent effect on permease activity. In contrast, replacement of either histidine-205 or histidine-322 by arginine causes a dramatic loss of transport activity, although the cells contain a normal complement of permease molecules, as determined by immunoadsorption assays. Interestingly, although substitution of histidine-205 or histidine-322 by arginine results in the loss of ability to catalyze active lactose transport, permease molecules with arginine at residue 322 appear to facilitate downhill lactose movements at high concentrations of the disaccharide. The results provide strong support for the contention that histidine residues in the lac permease play an important role in the coupling between lactose and proton translocation.

Arginine↗
PubMed1985

Direct measurement of the electrogenic activity of o-type cytochrome oxidase from Escherichia coli reconstituted into planar lipid bilayers.

Turnover of o-type cytochrome oxidase purified from Escherichia coli and reconstituted into proteoliposomes leads to the generation of a transmembrane electrical potential (interior negative) by means of vectorial electron flow. In the experiments reported here, purified oxidase is reconstituted in planar lipid bilayers formed at the tip of patch pipets, and open-circuit membrane potentials generated by electron transfer are measured directly. Potentials of up to 4 mV (substrate side positive) are generated in the presence of reduced phenazine methosulfate or ubiquinol-1, and with both substrates, electrogenic activity is inhibited by cyanide. Furthermore, the membrane potential generated during oxidase turnover is inhibited progressively with applied voltages (substrate side positive), decreasing almost to zero at an applied voltage of 150 mV.

Electrochemistry↗
PubMed1984

Morphology of proteoliposomes reconstituted with purified lac carrier protein from Escherichia coli.

Proteoliposomes reconstituted with purified lac carrier protein from Escherichia coli were ultra-rapidly frozen and examined by freeze-fracture-etch electron microscopy. The proteoliposomes are greater than 95% unilamellar, and the majority are 30-150 nm in diameter. Fracture faces of proteoliposomes (at a protein:lipid molecular ratio of about 1:2500) display 7.0-nm diameter globular intramembrane particles uniformly distributed on convex and concave surfaces. Calculations of particle composition suggest that each intramembrane particle probably contains one or two molecules of the 46.5-kDa transmembranous lac carrier protein, depending on the correction factor for the thickness of the metal deposited to form the platinum/carbon replicas. Etched surfaces of the proteoliposomes are smooth. Incubation of the proteoliposomes with monoclonal antibody 4B1, which binds to an epitope in the lac carrier on the exterior of the proteoliposomes, dramatically alters the intramembrane particle distribution. After incubation with antibody, the convex (inner monolayer) fracture faces are nearly devoid of intramembrane particles, and an overall 4-fold reduction in the total number of intramembrane particles is observed.

Cell Membrane↗
PubMed1984

Cytochrome o type oxidase from Escherichia coli. Characterization of the enzyme and mechanism of electrochemical proton gradient generation.

Cytochrome o type oxidase purified from the membrane of Escherichia coli consists of four polypeptides (Mr 66000, 35000, 22000, and 17000), and the monomeric form predominates in octyl beta-D-glucopyranoside. The oxidase complex contains two b-type cytochromes (b-558 and b-563) and 2 mol of heme/mol of enzyme. Cytochrome o utilizes ubiquinol-1 and a number of other artificial electron donors as substrates but does not oxidize reduced cytochrome c or ferrocyanide. Activity is highly dependent upon exogenous phospholipids and/or Tween 20, and the quinone analogues 2-heptyl-4-hydroxyquinoline N-oxide and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole are potent inhibitors. Proteoliposomes were formed by detergent dilution or dialysis in the presence of the oxidase and phospholipids, followed by freeze-thaw/sonication. Vesicles formed by this means are unilamellar and contain a random distribution of 85-90-A intramembranous particles on the convex and concave fracture surfaces. During oxidase turnover, the reconstituted system generates a proton electrochemical gradient (interior negative and alkaline) of -115 to -140 mV; however, respiratory control is minimal (i.e., respiratory control ratios of about 1.5 are observed). By using a glass electrode to measure changes in external pH and the fluorescence of entrapped 8-hydroxy-1,3,6-pyrenetrisulfonate to measure changes in internal pH, it is apparent that during ubiquinol oxidation, protons are released on the external surface of the membrane and consumed on the internal surface. In contrast, with N,N,N',-N'-tetramethyl-p-phenylenediamine, an electron donor that carries few protons at neutral pH, little change in external pH is observed until the protonophore carbonyl cyanide m-chlorophenylhydrazone is added, at which point the medium becomes alkaline. The results taken as a whole are consistent with the concept that oxidase turnover generates an electrical potential (interior negative) due to vectorial electron flow from the outer to the inner surface of the membrane. The pH gradient (interior alkaline), on the other hand, appears to result from scalar (i.e., nonvectorial) reactions that consume and release protons at the inner and/or outer surfaces of the membrane, respectively. In other words, cytochrome o oxidase from Escherichia coli does not appear to catalyze vectorial proton translocation.

Cell Membrane↗
PubMed1984

Monoclonal antibodies against the lac carrier protein from Escherichia coli. 1. Functional studies.

The effects of various monoclonal antibodies against purified lac carrier protein on carrier-mediated lactose transport were studied in right-side-out membrane vesicles and in proteoliposomes reconstituted with purified lac carrier protein. Out of more than 60 monoclonal antibodies tested, only one antibody, designated 4B1, inhibits transport. Furthermore, the nature of the inhibition is highly specific in that the antibody inhibits only those transport reactions that involve net proton translocation (i.e., active transport, carrier-mediated influx and efflux under nonenergized conditions, and lactone-induced proton influx). In contrast, the antibody has little effect on equilibrium exchange and no effect on generation of the proton electrochemical gradient or on the ability of the carrier to bind a high-affinity ligand. Clearly, therefore, the antibody alters the relationship between lactose and proton translocation at the level of the lac carrier protein. When entrance counterflow is studied with external [1-14C]lactose at saturating and subsaturating concentrations, it is apparent that antibody 4B1 mimics the effects of deuterium oxide [Viitanen, P., Garcia, M.L., Foster, D.L., Kaczorowski, G. J., & Kaback, H.R. (1983) Biochemistry 22, 2531]. That is, the antibody has no effect on the rate or extent of counterflow when external lactose is saturating but stimulates the efficiency of counterflow when external lactose is below the apparent Km. It seems likely, therefore, that the antibody either inhibits the rate of deprotonation or alters the equilibrium between protonated and deprotonated forms of the carrier. Monovalent Fab fragments prepared from antibody 4B1 inhibit transport in a manner that is similar qualitatively to that of the intact antibody.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1984

Monoclonal antibodies against the lac carrier protein from Escherichia coli. 2. Binding studies with membrane vesicles and proteoliposomes reconstituted with purified lac carrier protein.

Monoclonal antibodies 4B1 and 5F7 bind to distinct, nonoverlapping epitopes in the lac carrier protein. By use of immunofluorescence microscopy and radiolabeled monoclonal antibodies and Fab fragments, it is shown that both 4B1 and 5F7 bind to spheroplasts and to right-side-out vesicles, but only to a small extent to inside-out vesicles. Clearly, therefore, the lac carrier protein has an asymmetric orientation within the cytoplasmic membrane of Escherichia coli, and both epitopes are located on the periplasmic surface. In right-side-out vesicles, radiolabeled 4B1 binds with a stoichiometry of 1 mol of antibody per 2 mol of lac carrier protein, while radiolabeled 4B1 Fab fragments bind 1:1. Importantly, the intact antibody and its Fab fragments bind to proteoliposomes reconstituted with purified lac carrier protein with a stoichiometry very similar to that observed in right-side-out membrane vesicles. Thus, it seems highly likely that the orientation of the lac carrier protein in the reconstituted system is similar to that in the bacterial cytoplasmic membrane, at least with respect to 4B1 epitope.

Antibodies, Monoclonal↗
PubMed1984

Site-directed mutagenesis of cys148 in the lac carrier protein of Escherichia coli.

The lac y gene of Escherichia coli which encodes the lac carrier protein has been modified by oligonucleotide-directed, site-specific mutagenesis such that cys148 is converted to a glycine residue. Cells bearing the mutated lac y gene exhibit initial rates of lactose transport that are about 4-fold lower than cells bearing the wild type gene on a recombinant plasmid. Furthermore, transport activity is less sensitive to inactivation by N-ethylmaleimide, and strikingly, galactosyl 1-thio-beta-D-galactopyranoside affords no protection against inactivation. The findings suggest that although cys148 is essential for substrate protection against sulfhydryl inactivation, it is not obligatory for lactose: proton symport and that another sulfhydryl group elsewhere within the lac carrier protein may be required for full activity.

DNA, Single-Stranded↗
PubMed1984

Intramolecular dislocation of the COOH terminus of the lac carrier protein in reconstituted proteoliposomes.

A dodecapeptide corresponding to the carboxyl terminus of the lac carrier of Escherichia coli was synthesized, coupled to thyroglobulin, and the conjugate was used to generate site-directed polyclonal antibodies. The antibodies react with the carboxyl-terminal peptide and with the lac carrier protein, while monoclonal antibody 4B1 reacts with intact lac carrier protein, but not with the carboxyl-terminal peptide. Antibody 4B1 binds preferentially to right-side-out membrane vesicles relative to inside-out vesicles, confirming the presence of the 4B1 epitope on the periplasmic surface of the membrane. Alternatively, anti-carboxyl-terminal antibody binds preferentially to inside-out vesicles, demonstrating that the carboxyl terminus of the lac carrier protein is on the cytoplasmic surface. Surprisingly, both antibodies bind to proteoliposomes reconstituted with purified lac carrier protein, and quantitative binding assays indicate that the epitopes are equally accessible. When proteoliposomes containing purified lac carrier protein are digested with carboxypeptidases A and B, binding of anti-carboxyl-terminal antibodies decreases by greater than 80%, while binding of antibody 4B1 and various transport activities remain essentially unchanged. It is suggested that during reconstitution, the lac carrier protein undergoes intramolecular dislocation of the carboxyl terminus with no significant effect on its catalytic activity.

Amino Acid Sequence↗
PubMed1984

Functional molecular weight of the lac carrier protein from Escherichia coli as studied by radiation inactivation analysis.

Cytoplasmic membrane vesicles prepared from Escherichia coli containing multiple copies of the lac y gene were frozen in liquid nitrogen before or after generation of a proton electrochemical gradient (interior negative and alkaline) and irradiated with a high-energy electron beam at -135 degrees C. Subsequently, the lac carrier protein was extracted into octyl beta-D-glucopyranoside, reconstituted into proteoliposomes, and assayed for transport activity. Under all conditions tested, activity decreased as a single exponential function of radiation dosage, allowing straightforward application of target theory for determination of functional molecular mass. When lac carrier activity solubilized from nonenergized vesicles was assayed, the results obtained were consistent with a functional molecular size of 45-50 kDa, a value similar to the size of the protein as determined by other means. Similar values were obtained when the octyl beta-D-glucopyranoside extract was irradiated, and the target size observed for D-lactate dehydrogenase was in good agreement with the molecular size of this enzyme. Strikingly, when the same procedures were carried out with vesicles that were energized with appropriate electron donors prior to freezing and irradiation, a functional molecular size of 85-100 kDa was obtained for the lac carrier with no change in the target size of D-lactate dehydrogenase. In contrast, when the vesicles were energized under conditions in which the proton electrochemical gradient was collapsed, the target mass of the lac carrier returned to 45-50 kDa. The results indicate that the functional mass of the lac carrier protein is no greater than a dimer and suggest that the proton electrochemical gradient may cause an alteration in subunit interactions.

Escherichia coli↗
PubMed1984

Purified reconstituted lac carrier protein from Escherichia coli is fully functional.

Proteoliposomes reconstituted with lac carrier protein purified from the plasma membrane of Escherichia coli catalyze each of the translocation reactions typical of the beta-galactoside transport system (i.e., active transport, counterflow, facilitated influx and efflux) with turnover numbers and apparent Km values comparable to those observed in right-side-out membrane vesicles. Furthermore, detailed kinetic studies show that the reconstituted system exhibits properties analogous to those observed in membrane vesicles. Imposition of a membrane potential (delta psi, interior negative) causes a marked decrease in apparent Km (by a factor of 7 to 10) with a smaller increase in Vmax (approximately equal to 3-fold). At submaximal values of delta psi, the reconstituted carrier exhibits biphasic kinetics, with one component manifesting the kinetic parameters of active transport and the other exhibiting the characteristics of facilitated diffusion. Finally, at low lactose concentrations, the initial velocity of influx varies linearly with the square of the proton electro-chemical gradient. The results provide quantitative support for the contention that a single polypeptide species, the product of the lac y gene, is responsible for each of the transport reactions typical of the beta-galactoside transport system.

Diffusion↗
PubMed1984

Quantitative association between electrical potential across the cytoplasmic membrane and early gentamicin uptake and killing in Staphylococcus aureus.

The relationship between the magnitude of the transmembrane electrical potential and the uptake of [14C]gentamicin was examined in wild-type Staphylococcus aureus in the logarithmic phase of growth. The electrical potential (delta psi) and the pH gradient across the cell membrane were determined by measuring the equilibrium distribution of [3H]tetraphenyl-phosphonium and [14C]acetylsalicylic acid, respectively. Incubation in the presence of the H+-ATPase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD) led to an increase in delta psi with no measurable effect on the pH gradient at external pHs ranging from 5.0 to 6.5, and the effect on delta psi was DCCD concentration dependent. In separate experiments, gentamicin uptake and killing were studied in the same cells under identical conditions. At pH 5.0 (delta psi = -140 mV), no gentamicin uptake occurred. In the presence of 40 and 100 microM DCCD, delta psi was increased to -162 and -184 mV, respectively, and gentamicin uptake was observed in a manner that was also dependent on the DCCD concentration. At pH 6.0 (delta psi = -164 mV), gentamicin uptake occurred in the absence of the carbodiimide but was enhanced in a concentration-dependent fashion by 40 and 100 microM DCCD (delta psi = -174 and -216 mV, respectively). In all cases increased gentamicin uptake was associated with an enhanced bactericidal effect. The results indicate that initiation of gentamicin uptake requires a threshold level of delta psi (-155 mV) and that above this level drug uptake is directly dependent on the magnitude of delta psi.

Biological Transport↗
PubMed1984

Factors influencing the accumulation of tetraphenylphosphonium cation in HeLa cells.

Exposure of HeLa cells to tetraphenylphosphonium cation (TPP+) results in a rapid accumulation intracellularly, and a steady-state level is reached within 10 min. Accumulation of [3H]TPP+ in HeLa cells is reduced under the following conditions: (i) after preincubation of cells in buffered saline or in medium containing two- to fourfold higher concentrations of amino acids, (ii) exposure to the alkylating agent L-1-tosylamido-2-phenyl-ethylchloromethyl ketone, (iii) ouabain-mediated inhibition of the Na+, K+ ATPase, and (iv) high external K+ concentrations. In contrast, addition of serum increases the uptake of TPP+. In synchronized cells, intracellular levels of TPP+ differ at various stages of cell cycle and are lowest in mitosis.

Biological Transport, Active↗