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E M Barnes

Publications and source records attributed to E M Barnes.

At least 55 records · Page 3Linked to original sources
PubMed1985

Characterization of ammonium (methylammonium)/potassium antiport in Escherichia coli.

The energetics of ammonium ion transport by Escherichia coli have been studied using [14C]methylammonium as a substrate. Rapid assays for uptake allowed kinetic parameters (CH3NH3+ Km = 36 microM; Vmax = 4 nmol X s-1 X mg-1 to be determined in the absence of CH3NH3+ metabolism. Cells cultured in media containing 1 mM NH4+ failed to express CH3NH3+ transport activity. Methylammonium accumulated at levels which were 100-fold higher than those of the medium. This accumulation was dependent upon the addition of glucose or pyruvate. The entry of CH3NH3+ supported by glucose oxidation in an F1F0-ATPase-deficient mutant was blocked by uncoupler. Transport by wild-type cells under similar conditions was significantly inhibited by arsenate. Thus, CH3NH3+ uptake requires both ATP and an electrochemical H+ gradient. This transport activity was lost upon exposure of E. coli to osmotic shock, but could be recovered by incubation of shocked cells with boiled shock fluid or with glucose plus K+ in the presence of chloramphenicol. Similar reconstitution was observed in K+-depleted parental strains, but not in a mutant defective in K+ transport, demonstrating a requirement for internal K+. However, external K+ proved to be a noncompetitive inhibitor (Ki = 1 mM) of CH3NH3+ uptake by K+ -replete bacteria. External Na+ had no effect on transport. The addition of NH4+ or CH3NH3+ induced a rapid exodus of intracellular 86Rb+, an analog which was able to substitute for K+. The molar ratio of CH3NH3+ uptake to Rb+ exit was 1.12 +/- 0.11. These findings support a mechanism for CH3NH3+ (NH4+) accumulation which requires K+ antiport (exchange) and is driven by the electrochemical K+ gradient.

Antiporters↗
PubMed1985

Multiple [35S]t-butylbicyclophosphorothionate binding sites in rat and chicken cerebral hemispheres.

Specific binding of [35S]t-butylbicyclophosphorothionate (TBPS) to membranes from cerebral hemispheres of adult rat and chicken was determined over a range of radioligand concentrations from 0.25 to 500 nM. Scatchard plots of these data were curvilinear and nonlinear regression analysis indicated binding to two sites that differ in affinity. For rat cerebrum, KD(1) = 1.15 nM, Bmax(1) = 0.085 pmol/mg; KD(2) = 232 nM, Bmax(2) = 16.9 pmol/mg. For chicken cerebrum, KD(1) = 1.39 nM, Bmax(1) = 0.111 pmol/mg; KD(2) = 166 nM, Bmax(2) = 17.6 pmol/mg. This multiplicity of [35S]TBPS binding was further confirmed when unlabeled TBPS or picrotoxinin displaced radioligand. The displacement curves were biphasic and yielded Hill coefficients from 0.65 to 0.70. These displacement curves were also resolved into two components with distinct IC50 values for unlabeled TBPS (rat, 1.55 and 271 nM; chicken, 2.40 and 224 nM). The IC50 values were similar to the dissociation constants obtained from equilibrium binding measurements.

Animals↗
PubMed1985

The classification and differentiation of the anaerobic gram-negative rods.

Within the family Bacteroidaceae differentiation of the genera Leptotrichia, Fusobacterium and Bacteroides still depends largely on the accumulation of certain fermentation products. The application of newer techniques, both genetical and chemical is leading to a reappraisal of the current classification. So far attention has focussed on the genus Bacteroides and has been concerned with the status of B. fragilis and related organisms and the description of a number of new species within the B. ruminicola/oralis group. The differentiation of pigment-producing strains, all known originally as B. melaninogenicus, has led to the establishment of at least 9 species. The transfer of certain Bacteroides species to new genera is also being considered.

Animals↗
PubMed1984

The role of glutamine in regulation of ammonium transport in Azotobacter vinelandii.

Under N2-fixing conditions, Azotobacter vinelandii expresses a specific transport system for methylammonium (ammonium) [E. M. Barnes, Jr. and P. Zimniak (1981) J. Bacteriol. 146, 512-516]. This activity is decreased markedly by culture of cells in the presence of 10 mM ammonium or 2 mM methylammonium; in both cases, the Vmax values for methylammonium uptake were 25% of those of N2-fixing cells. Mixing experiments with assay medium indicate that transport activity is controlled by intracellular rather than extracellular metabolites. Glutamine synthetase activity of cells cultured with ammonium was 33% that of N2-fixing cultures, but activity was unaffected by incubation with methylammonium. Thus ammonium transport and ammonium fixation are regulated independently. When ammonium was removed from the medium, cells recovered over 90% of the initial transport activity after 1 h; this recovery was not affected by addition of chloramphenicol. The loss of uptake activity in cells incubated with ammonium or methylammonium correlated with over sixfold increases in intracellular levels of glutamine and gamma-glutamylmethylamide, respectively. Recovery of transport was accompanied by similar reductions in pools of these compounds. Over one-half of methylammonium transport activity could be blocked by direct addition of 10 mM glutamine or gamma-glutamylmethylamide to transport assays; these concentrations were similar to those observed in vivo. The glutamine analog, 6-diazo-5-oxo-L-norleucine, was the most potent inhibitor found (68% inhibition at 10 microM). These results indicate that the regulation of ammonium transport by ammonium and methylammonium is due to inhibition of the transporter by intracellular gamma-glutamyl amides rather than by repression of transporter synthesis.

Amino Acids↗
PubMed1984

gamma-Aminobutyric acid-gated chloride channels in cultured cerebral neurons.

gamma-Aminobutyric acid (GABA), the most common inhibitory neurotransmitter in the vertebrate brain, acts by increasing the conductance of the neuronal membrane to chloride ions. The addition of GABA to monolayer cultures of chick cerebral neurons produced a 3-fold increase in the uptake of 36Cl-. This stimulation was maximal during the first 20 s after GABA addition but declined rapidly thereafter. The GABA-dependent uptake activity was doubled by increasing the external K+ concentration from 5.5 to 40 mM. The dependence of the 36Cl- entry rate on the external concentrations of GABA (K0.5 = 6 microM; Vmax = 4.4 nmol/mg of cell protein/s) and Cl-(Km = 105 mM; Vmax = 9.9 nmol mg-1 s-1) followed Michaelis-Menten kinetics. The GABA analog, muscimol, produced a similar response (K0.5 = 8 microM; Vmax = 5.2 nmol mg-1 s-1). While 50 microM 3-aminopropane sulfonate also stimulated 36Cl- uptake, 2,4-diaminobutyrate, taurine, and glycine were without effect. Bicuculline (Ki = 3.5 microM) was a noncompetitive inhibitor of GABA-dependent Cl- entry, while the inhibition by picrotoxin (Ki = 1.0 microM) was uncompetitive with GABA. Nearly one-half of the basal activity, observed in the absence of GABA, was blocked by the anion transport inhibitors, furosemide or 4-acetamido-4'-isothiocyano-2,2'-stilbene sulfonate, but these compounds gave no significant inhibition of the GABA-dependent activity. These results indicate that the basal route for 36Cl- entry into cerebral neurons involves electroneutral processes while the GABA-dependent influx occurs via specific ligand-gated Cl-channels.

Animals↗
PubMed1983

Adenosine transport by cultured glial cells from chick embryo brain.

The transport of adenosine was studied in pure cultures of glial cells from chick embryo brain. In order to avoid complications in uptake measurements due to adenosine metabolism, cultures were depleted of ATP by incubation with cyanide and iodoacetate prior to addition of [3H]adenosine. Under the 5- to 25-s periods used for the transport assay, no adenosine metabolism could be detected. Initial rates of adenosine transport under these conditions obeyed the Michaelis-Menten relationship with Km = 370 microM and Vmax = 10.3 nmol/min/mg cell protein. ATP depletion or elimination of Na+ from the assay medium had no significant effect on initial rates of adenosine uptake. However, when assays were carried out under conditions of significant adenosine metabolism (10-min uptake in the absence of metabolic inhibitors), a high-affinity incorporation process could be demonstrated in the glial cells (Km = 12 microM; Vmax = 0.34 nmol/min/mg protein). The transport activity expressed in ATP-depleted glial cells was most sensitive to inhibition by nitrobenzylthioinosine, dipyridamole, and N6-benzyladenosine. In decreasing order of potency, N6-methyladenosine, 2-chloroadenosine, inosine, and thymidine also blocked adenosine translocation in glial cultures. Thus, adenosine transport by cultured glial cells occurs by means of a low-affinity, facilitated diffusion system which is similar to the nucleoside transporter in cells of nonneural origin.

Adenosine↗
PubMed1983

A filtration method for measuring cellular uptake of [14C]methylamine and other highly permeant solutes.

A filtration technique has been developed for study of the uptake of [14C]methylamine by Azotobacter vinelandii. This dual filter arrangement requires a precision microporous polycarbonate film which overlays a paper filter. Cellular uptake of radioactivity is terminated by vacuum filtration of the reaction mixture onto the polycarbonate filter without dilution or washing. Filtration was complete in 0.7 s with retention of less than 0.2% of the extracellular radioactivity. The dual filter method gave 20-fold higher levels for intracellular methylamine than filtration followed by washing. Without washing, nitrocellulose filters retained 18 times more extracellular [3H]sorbitol and 80 times more extracellular [14C]methylamine than polycarbonate filters. Use of an underlying paper filter did not significantly improve the performance of nitrocellulose filters. However, addition of a paper filter reduced extracellular methylamine and sorbitol retention on polycarbonate filters by 77 and 86%, respectively. This method is generally applicable to measurement of the uptake of highly permeant molecules by cells and subcellular organelles.

Azotobacter↗
PubMed1983

Reconstitution of calcium transporters from Azotobacter vinelandii membranes.

Plasma membranes from Azotobacter vinelandii contain two Ca2+ transport activities: an electrophoretic uniporter and an electroneutral Ca2+/2H+ exchanger (P. Zimniak and E. M. Barnes, Jr. J. Biol. Chem. 255, 10,140 (1980)). Both activities were reconstituted by the freeze-thaw technique of M. Kasahara and P. C. Hinkle (J. Biol. Chem. 252, 7384 (1977)) using phosphatidylcholine/phosphatidylethanolamine (1:1) at a lipid-to-protein ratio of 40. Reconstitution was evidenced both by expansion of the intravesicular volume accessible to Ca2+ and by transfer of the transport activities to vesicles with a buoyant density less than that of native membranes. The Ca2+ transporters, reconstituted into K+-filled proteoliposomes, retained their dependence on the membrane potential or delta pH induced by the addition of valinomycin or nigericin, respectively. The kinetic parameters of the reconstituted activities were similar to those in native membranes, as was their sensitivity to inhibitors. The sensitivities of the electrophoretic Ca2+ transporter to ruthenium red, morpholinoethanesulfonate, and external K+ and of the Ca2+/2H+ antiporter to Sr2+ and heat treatment were also retained by the reconstituted system.

Azotobacter↗
PubMed1983

Adenosine transport by primary cultures of neurons from chick embryo brain.

The transport of adenosine was studied in pure cultures of neurons from chick embryo brain. In order to avoid complications due to adenosine metabolism, the cells were depleted of ATP by treatment with cyanide and iodoacetate prior to incubation with [3H]adenosine. During the 5-25-s periods used for transport assays, no significant adenosine metabolism was detectable. ATP depletion reduced the initial rate of adenosine entry by less than 10%, but blocked over 90% of the radioactivity accumulated by untreated cells after 15 min. Elimination of sodium or chloride from the uptake medium had no effect on adenosine transport activity. The kinetics of adenosine entry into ATP depleted neurons obeyed the Michaelis-Menten relationship and yielded a Km of 13 microM and Vmax of 0.15 nmol/min/mg protein. The neuronal transport system has apparent selectivity for adenosine, since thymidine, inosine, or guanosine gave significant inhibition only at levels 10-100-fold higher than [3H]adenosine. Adenosine derivatives (N6-cyclohexyl-, N6-benzyl-, N6-methyl-, and 2-chloroadenosine) were more effective inhibitors; p-nitrobenzylthioinosine and dipyridamole were the most potent compounds found. These results describe a high-affinity, facilitated diffusion system for adenosine in cerebral neurons, which could participate in terminating regulatory actions of this compound in the nervous system.

Adenosine↗
PubMed1983

Role of glutamine synthetase in the uptake and metabolism of methylammonium by Azotobacter vinelandii.

Methylammonium is a substrate for the ammonium transport system of Azotobacter vinelandii. During cellular uptake methylammonium is rapidly converted to a less polar metabolite (E. M. Barnes, Jr., and P. Zimniak, J. Bacteriol. 146:512-516, 1981). This metabolite has been isolated from A. vinelandii and identified as gamma-glutamylmethylamide by mass spectroscopy, 1H nuclear magnetic resonance spectroscopy, and cochromatography with the authentic compound. Escherichia coli also accumulated gamma-glutamylmethylamide during methylammonium uptake. The biosynthesis of gamma-glutamylmethylamide in vitro required methylammonium, ATP, L-glutamate, and a soluble cell extract from A. vinelandii. The enzyme responsible for gamma-glutamylmethylamide synthesis was glutamine synthetase. In a crude extract, L-methionine-DL-sulfoximine was equipotent in inhibiting the activities for gamma-glutamyltransferase and for the synthesis of glutamine and gamma-glutamylmethylamide. Likewise, an antiserum against the glutamine synthetase of E. coli precipitated the transferase and both synthetic activities at similar titers. During repression by growth of cells on ammonium medium, the synthesis of glutamine and gamma-glutamylmethylamide in vitro was also inhibited coordinately. A partially purified preparation of glutamine synthetase from A. vinelandii utilized methylammonium as substrate (Km = 78 mM, Vmax = 0.30 mumol/min per mg), although less efficiently than ammonium (Km = 0.089 mM, Vmax = 1.1 mumol/min per mg). The kinetic properties of glutamine synthetase with methylammonium as substrate as well as the insensitivity of this activity to inhibition by T1+ were strikingly different from methylammonium translocation. Thus, methylammonium (ammonium) translocation and intracellular trapping as glutamylamides are experimentally distinguishable processes.

Azotobacter↗
PubMed1981

Calcium transport by primary cultured neuronal and glial cells from chick embryo brain.

The uptake of calcium was examined in primary cultures of pure neurons and of glial cells from dissociated hemispheres of chick embryo brain. Neuronal cultures took up calcium at a rate of 2.0 nmol per min per mg cell protein at medium concentrations of 1.2 mM-Ca2+ and 5.4 mM-K+. The rate of calcium entry into neurons was increased 2.7-fold by elevating medium potassium to 60 mM. The effect of high external potassium was to increase the Vmax value for calcium transport from 5.5 to 13 nmol per min per mg; the Michaelis constant for calcium, 1.2 mM, was unchanged. The potassium-dependent component of calcium entry into the neuronal cultures was eliminated by addition of 0.1 mM-D-600 (a verapamil derivative) or by 1 mM-CoCl2, but 0.5 microM-tetrodotoxin had no significant effect. When choline replaced potassium in uptake medium no change in calcium transport was detected in neurons, nor was the entry of calcium increased when choline replaced sodium. Glial cultures took up calcium at 20% of the basal rate for neuronal cultures on a weight-of-protein basis. Uptake was not increased by potassium; during depolarization by potassium the calcium transport activity of glia was less than 10% that of neurons. It was concluded that cultured neurons contain a depolarization-sensitive, calcium-specific channel. A similar calcium transport activity was not detected in cultured glial cells.

Animals↗
PubMed1981

Transport of ammonium and methylammonium ions by Azotobacter vinelandii.

Ammonium and methylammonium are rapidly taken up by cultures of Azotobacter vinelandii respiring in the presence of succinate. The rate of methylamine uptake increased with external pH from 5.5 to 7.5 but increasing the pH further to 8.5 had little effect on activity, indicating that methylammonium cation rather than uncharged methylamine is the permeant species. The kinetics of methylammonium entry followed the Michaelis-Menten relationship, yielding a K(m) of 25 muM and a V(max) of 3.8 nmol/min per mg of cell protein. At saturating concentrations ammonium was taken up at rates 30-fold higher than those for methylammonium. Ammonium was a competitive inhibitor of methylammonium uptake and gave an inhibition constant of 1 muM. Ammonium derivatives were inhibitors of methylammonium entry in order of effectiveness: hydrazine > methylhydrazine > formamidine > guanidine > dimethylamine > ethylamine; amides and amino acids did not block uptake. Likewise, metal cations inhibited in the order Tl(+) > Cs(+) > Rb(+), whereas Na(+), K(+), and Li(+) produced no significant effect. Methylammonium uptake was blocked in cells exposed to an uncoupler, p-trifluorome-thoxycarbonyl cyanide-phenyl hydrazone or gramicidin D, but not with dicyclo-hexylcarbodiimide or arsenate. Valinomycin stimulated methylammonium entry into cells in a K(+)-free medium but prevented entry in the presence of 10 mM K(+). Monensin and nigericin had little effect on transport. These results indicate that methylammonium and ammonium ions enter A. vinelandii electrogenically via a specific transporter.

Azotobacter↗
PubMed1980

Characterization of a calcium/proton antiporter and an electrogenic calcium transporter in membrane vesicles from Azotobacter vinelandii.

Ca2+ transport across the membrane of vesicles derived from Azotobacter vinelandii was studied in the absence of respiration or functioning ATPase. Two facilitated diffusion systems were found. One, an electroneutral Ca2+/2H+ antiporter, responded to an artificially imposed deltapH, was heat-labile, and was insensitive to low concentrations of ruthenium red and lanthanides. The second, an electrogenic transporter, responded to an electrical membrane potential, was heat-stable, was inhibited by ruthenium red, lanthanides, monovalent cations, and certain anions. In vivo, when coupled to the protonmotive force, the systems would provide for the cell: (i) a mechanism to keep intracellular Ca2+ concentration low (Ca2+/2H+ antiporter); (ii) a mechanism for Ca2+ entry (electrogenic transporter).

Antiporters↗