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S Schuldiner

Publications and source records attributed to S Schuldiner.

17 recordsLinked to original sources
PubMed1979

Solubilization and reconstitution of the catecholamine transporter from bovine chromaffin granules.

The catecholamine transporter from bovine chromaffin granules has been solubilized by using low concentrations of sodium cholate in the presence of phospholipids. The functional solubilized protein has been incorporated into liposomes after removal of the detergent either by gel filtration or by dialysis. Reserpine-sensitive accumulation against a concentration gradient is achieved by artifically imposing a pH gradient across the membrane. In the reconstituted system adenosine 5'-triphosphate (ATP) serves as an energy source only at higher detergent concentrations. The proton-translocating adenosine triphosphatase (ATPase) is solubilized in parallel with the increasing efficiency of ATP as an energy source. Several criteria are proposed to distinguish between carrier-mediated (reserpine sensitive) and unmediated transport in the reconstituted system. The reserpine-sensitive process shows affinity and ss presented in this communication provide further support for the contention that concentrative uptake in biogenic amine storage vesicles is driven by a transmembrane pH gradient, which, in the native system, is generated by a proton-translocating ATPase. Moreover, the assays described provide a tool for the isolation and purification of the transport protein.

Adenosine Triphosphatases↗
PubMed1978

Energy transduction in the photosynthetic membranes of the cyanobacterium (blue-green alga) P-lectonema boryanum.

Membrane preparations isolated from the photosynthetic lamellae of the cyanobacterium Plectonema boryanum generate upon illumination a transmembrane pH gradient of approximately 2 to 3 pH units (acid inside), as determined from the distribution of either fluorescent or radioactive amines (9 aminoacridine and [14C]methylamine, respectively). Using the distribution of permeant ions to measure the electrical potential across the membrane, it was found that the latter is practically nil under conditions in which the deltapH is formed and photophosphorylation takes place. In agreement with the above findings cyclic photophosphorylation in this membrane preparation is inhibited by agents shown to collapse the deltapH but not by agents which should collapse the electrical potential. It is deduced that the pattern of proton movement in the photosynthetic lamellae of intact Plectonema spheroplasts corresponds to that of the cell-free membrane system, as both preparations show similar light dependent accumulation of fluorescent amine. It is concluded that the pattern of energy transduction in Plectonema photosynthetic lamellae is similar to that of chloroplast thylakoid membranes and not to that of bacterial cytoplasmic membranes. The evolutionary implications of the findings are discussed and a model for the directionality of H+ movements in the whole cell is presented.

Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone↗
PubMed1978

Role of a transmembrane pH gradient in epinephrine transport by chromaffin granule membrane vesicles.

ATP-driven transport and accumulation of epinephrine in chromaffin granule membrane vesicles isolated from bovine adrenal medulla is inhibited by the proton ionophores carbonylcyanide p-trifluoromethoxyphenylhydrazone and nigericin, but not by valinomycin. Moreover, an artificially imposed pH gradient (interior acid) is able to drive this reserpine-sensitive transport system in the absence of ATP. Dicyclohexylcarbodiimide, an inactivator of the chromaffin granule membrane-bound ATPase, completely inhibits ATP-dependent epinephrine accumulation, but has much less effect when an imposed pH gradient is the driving force for epinephrine transport. The findings provide a strong indication that a pH gradient (interior acid) is the immediate driving force for epinephrine uptake in these storage granules and suggest that ATP-driven epinephrine transport is the result of two processes: (i) generation of a proton electrochemical gradient (interior acid and positive) by the membrane-bound, proton-translocating ATPase; and (ii) pH gradient-driven accumulation of the catecholamine.

Animals↗
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↗
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

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

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↗