ATP-dependent primary active transport of cysteinyl leukotrienes across liver canalicular membrane. Role of the ATP-dependent transport system for glutathione S-conjugates

The liver is the major organ which eliminates leukotriene C4 (LTC4) and other cysteinyl leukotrienes from the blood circulation into bile. Transport of LTC4 was studied using inside-out vesicles enriched in canalicular and sinusoidal membranes from rat liver. The incubation of canalicular membrane vesicles with [3H]LTC4 in the presence of ATP resulted in an uptake of LTC4 into vesicles. The initial rate of ATP-stimulated LTC4 uptake was about 40-fold higher in canalicular than in sinusoidal membrane vesicles. When liver plasma membrane vesicles were incubated in the absence of ATP, an apparent transient uptake of LTC4 was observed which was temperature-dependent and not affected by the osmolarity. This indicates that LTC4 was bound to proteins on the surface of plasma membrane vesicles. Two proteins with relative molecular weights of 17,000 and 25,000 were detected by direct photoaffinity labeling as major LTC4-binding proteins. One protein (Mr 25,000) was ascribed to subunit 1 (Ya) of glutathione S-transferase which was associated with the membrane. LTD4, LTE4, N-acetyl-LTE4, and omega-carboxy-N-acetyl-LTE4 were also transported into liver plasma membrane vesicles in an ATP-dependent manner with initial rates relative to LTC4 (1.0) of 0.46, 0.11, 0.35, and 0.22, respectively. Mutual competition between the cysteinyl leukotrienes and S-(2,4-dinitrophenyl)-glutathione for uptake indicated that they are transported by a common carrier. Apparent Km values of the transport system for LTC4, LTD4, and N-acetyl-LTE4 were 0.25, 1.5, and 5.2 microM, respectively. The ATP-dependent transport of LTC4 into vesicles was not inhibited by doxorubicin, daunorubicin, or verapamil, or by the monoclonal antibody C219, suggesting that the transport system differs from P-glycoprotein. Liver plasma membrane vesicles prepared from mutant rats deficient in the hepatobiliary excretion of cysteinyl leukotrienes lacked the ATP-dependent transport of cysteinyl leukotrienes and S-(2,4-dinitrophenyl)-glutathione. These results demonstrate that the ATP-dependent carrier system is responsible for the transport of cysteinyl leukotrienes and glutathione S-conjugates from the hepatocytes into bile.     

Characterization of an active transport system for calcium in inverted membrane vesicles of Escherichia coli.

The energy-dependent uptake of calcium by inverted membrane vesicles of Escherichia coli was investigated. Methods for preparation and storage of the vesicles were devised to allow for the maximal activity and stability of the calcium transport system. The pH and temperature optima for the reaction were observed to occur at pH 8.0 AND 30 DEGREES, RESPECTIVELY. The eft was found that the extent of the reaction depended on the presence of phosphate or oxalate. Phosphate was found to enter the vesicles at a rate slower than that of calcium. A Ca2+:Pi ratio of approximately 1.5 was found, suggesting formation of Ca3(PO4)2. Monovalent cations stimulated calcium uptake, with the order of effectiveness being K+ is greater than Na+ is greater than Li+ is greater than NH4+. Inhibition was found with certain divalent cations, but these also inhibited the electron transport chain. Of the divalent cations examined only Mg2+ and Sr2+ inhibited calcium transport without a corresponding inhibition of respiration. Calcium transport exhibited biphasic Kinetics, with a low affinity system and a high affinity system. The low affinity system showed a Km of 0.34 mM and a Vmax of 85 nmol/min/mg of protein. The kinetic constants of the high affinity system were 4.5 muM and 2 nmol/min/mg of protein. The energy for calcium transport could be derived from the electron transport chain by oxidation of NADH, D-lactate, and succinate, in order of their effectiveness. Respiration-driven calcium transport was inhibited by inhibitors of the electron transport chain and by uncouplers of oxidative phosphorylation. ATP could also be used to supply enerty for calcium transport. The ATP-driven reaction was inhibited by inhibitors of the Mg2+ATPase and by an antiserum prepared against that protein, demonstrating that that enzyme is involved in the utilization of ATP for active transport in inverted vesicles.     

Characterization of a lysine-specific active transport system in Rickettsia prowazeki.

Rickettsia prowazeki possesses an active transport system for lysine with a Kt of influx of 1 muM. Extraction and chromatographic analysis of the accumulated labeled material show the material to be lysine rather than a derivative. This intracellular lysine pool can be exchanged with external unlabeled substrates for at least 10 min; The lysine analogues L-aminoethyl cysteine, N-methyl lysine, hydroxylysine, and D-lysine competitively inhibit uptake of L-lysine, but cadaverine, diaminopimelate, arginine, ornithine, and epsilon-aminocaproate do not. Accumulation of lysine can be inhibited by the energy poisons potassium cyanide, triphenylmethyl phosphonium bromide, and 2,4-dinitrophenol. The effect of potassium cyanide, but not 2,4-dinitrophenol or triphenylmethyl phosphonium bromide, can be overcome by adenosine 5'-triphosphate. Both energy-dependent influx and energy-independent efflux are inhibited by the sulfhydryl reagents N-ethyl maleimide and p-chloromercuriphenyl sulfonic acid.     

Requirement for membrane potential in active transport of glutamine by Escherichia coli.

The effect of reducing the membrane potential on glutamine transport in cells of Escherichia coli has been investigated. Addition of valinomycin to tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetic acid-treated E. coli cells in the presence of 20 mM exogenous potassium reduced the membrane potential, as measured by the uptake of the lipophilic cation triphenylmethylphosphonium, and caused a complete inhibition of glutamine transport. Valinomycin plus potassium also caused a rapid decrease in the intracellular levels of ATP of normal E. coli cells, but had little if any effect on the ATP levels of two mutants of E. coli carrying lesions in the energy-transducing ATP complex (unc mutants). Yet both the membrane potential and the capacity to transport glutamine were depressed in the unc mutants by valinomycin and potassium. These findings are consistent with the hypothesis that both ATP and a membrane potential are essential to the active transport of glutamine by E. coli cells.     

Erythrocyte membrane sulfhydryl groups and the active transport of cations

RNA synthesis was studied by autoradiographic analysis using tritiated uridine incorporation in the Chinese hamster cell line Dede after a one-minute pulse labeling period. RNA synthesis continues during all stages of interphase and mitosis except during metaphase and anaphase. Cytoplasmic RNA was apparently synthesized in the nucleus, since no grains were observed above the background level in the sample immediately following the labeling. Nucleoli synthesize their own RNA and are not reservoirs for RNA synthesized elsewhere. Both actinomycin D and nogalamycin inhibited the RNA synthetic activity of chromatin and nucleoli. However, the nucleolar synthetic activity was more susceptible to these agents than that of chromatin. Furthermore, actinomycin D was a stronger inhibitor than nogalamycin.     

Uptake of glutamate in Corynebacterium glutamicum. 2. Evidence for a primary active transport system

The inducible glutamate uptake system in Corynebacterium glutamicum (Kr?mer, R., Lambert, C., Hoischen, C. & Ebbighausen, H., preceding paper in this journal) was characterized with respect to its mechanism and energy coupling. All possible secondary active uptake mechanisms can be excluded. Glutamate transport is not coupled to the translocation of H+, Na+ or K+ ions. Although changes in membrane potential and uptake activity cannot completely be separated, no correlation between these two parameters is observed. The uptake of glutamate resembles a primary active, ATP-dependent transport mechanism in several respects. (a) The substrate affinity is very high (1.3 microM). (b) Accumulation of glutamate reaches values of greater than 2.10(5), at least as high as those reported for binding-protein-dependent systems in Gram-negative bacteria. (c) The uptake is unidirectional. Even after complete deenergization, the accumulation ratio was not significantly reduced. (d) The rate of glutamate uptake is directly correlated to the cytosolic ATP content and also to the ATP/ADP ratio. This is shown by varying internal ATP by different procedures applying inhibitors (NaCN, dicyclohexyl carbodiimide), uncouplers (carbonyl m-chlorophenylhydrazone), ionophores (valinomycin), and even by shifting the cells to anaerobiosis. Uptake is not promoted by cytosolic ATP levels below 1.5 mM, the maximum uptake rate is reached at 4-5 mM ATP.     

Model for the cell biomembrane electric potential during active transport of various ions

A model of a stationary electrical potential on biomembrane was created. This model takes into account conformational changes in transport ATPase. N positive ions are transported simultaneously by the system of active transport. The model allows one to determine independently ion concentrations inside the cell and membrane electrical potential. It is shown that, to obtain the electrical potential, it is necessary to take into account organic negative intracellular ions. The effect of positive ions that are not transported by active transport systems on the potential value is discussed. The results obtained are in a good agreement with experimental data for various cells.     

Evolution of concentration field in a membrane system

This paper deals with the evolution of concentration field at a single membrane system. Concentration field evolution is described by concentration effect of stable boundary layers, which originate in this system. The concentration effect of boundary layers (CBLE) is studied experimentally on the basis concentration profiles obtained from computer analysis of interferometric pictures of near-membrane regions. Besides experimental results, we also report theoretical investigations and numerical calculations of this effect for two models of membranes (an infinite thin wall and the wall of thickness l). Evolution of concentration field at different distances from membrane surface describes accurately the spatio-temporal structure of the concentration boundary layers (CBLs). Results have shown that their spatial structure is fully established and these layers develop diffusively.     

Prosthecae of Asticcacaulis biprosthecum: system for the study of membrane transport.

Prosthecae removed from cells of Asticcacaulis biprosthecum were examined for their ability to accumulate proline, alanine, aspartate, glutamate, and glucose against a concentration gradient. The transport of all of these compounds into prosthecae was stimulated by the nonphysiological electron donors phenazine methosulfate and N,N,N',N'-tetramethyl-p-phenylene diamine dihydrochloride. Reduced pyridine nucleotides caused very slight stimulation of transport of proline and glucose. Other physiological electron donors did not stimulate uptake. Evidence is presented indicating that the failure of certain potential electron donors to drive respiratory chain-linked transport is due to the inabilityof these compounds to enter prosthecae rather than to the absence of enzymes for their oxidation in prosthecae. Inhibition of respiration and uncouplers of oxidative phosphorylation, with the exception of arsenate, inhibit active transport systems of prosthecae.     

Time-resolved x-ray diffraction studies of the sarcoplasmic reticulum membrane during active transport.

X-ray and neutron diffraction studies of oriented multilayers of a highly purified fraction of isolated sarcoplasmic reticulum (SR) have previously provided the separate profile structures of the lipid bilayer and the Ca2+-ATPase molecule within the membrane profile to approximately 10-A resolution. These studies used biosynthetically deuterated SR phospholipids incorporated isomorphously into the isolated SR membranes via phospholipid transfer proteins. Time-resolved x-ray diffraction studies of these oriented SR membrane multilayers have detected significant changes in the membrane profile structure associated with phosphorylation of the Ca2+-ATPase within a single turnover of the Ca2+-transport cycle. These studies used the flash photolysis of caged ATP to effectively synchronize the ensemble of Ca2+-ATPase molecules in the multilayer, synchrotron x-radiation to provide 100-500-ms data collection times, and double-beam spectrophotometry to monitor the Ca2+-transport process directly in the oriented SR membrane multilayer.     

Mechanisms of active transport in isolated bacterial membrane vesicles. XII. Active transport by a mutant of Escherichia coli uncoupled for oxidative phosphorylation

Amino acid and β-galactoside transport activity catalyzed by whole cells and membrane vesicles prepared from an Escherichia coli mutant uncoupled for oxidative phosphorylation is comparable to the activity of analogous preparations from the parent strain. Valinomycin-induced rubidium uptake is also similar in membrane vesicles prepared from wild-type and mutant cells. The properties of the transport systems in mutant vesicles are the same as those of wild-type vesicles with respect to electron donors which stimulate transport, and with respect to inhibition by anoxia, cyanide, and 2,4-dinitrophenol.Magnesium ion markedly stimulates the ATPase activity of wild-type membrane vesicles and ethylenediaminetetraacetate markedly inhibits. However, these compounds have relatively slight effects on either the initial rate or extent of transport. Dicyclohexylcarbodiimide does not inhibit respiration-dependent transport despite inhibition of the calcium, magnesium-activated ATPase activity of wild-type vesicles.These results confirm earlier observations indicating that oxidative phosphorylation is not involved in respiration-linked active transport.     

On the efficiency and reversibility of active ligand transport induced by alternating rectangular electric pulses.

The stationary-state kinetic properties of a simplified two-state electro-conformational coupling model (ECC) in the presence of alternating rectangular electric potential pulses are derived analytically. Analytic expressions for the transport flux, the rate of electric energy dissipation, and the efficiency of the transducing system are obtained as a function of the amplitude and frequency of the oscillation. These formulas clarify some fundamental concept of the ECC model and are directly applicable to the interpretation and design of experiments. Based on these formulas, the reversibility and the degree of coupling of the system can be studied quantitatively. It is found that the oscillation-induced free energy transduction is reversible and tight-coupled only when the amplitude of the oscillating electric field is infinitely large. In general, the coupling is not tight when the amplitude of the electric field is finite. Furthermore, depending on the kinetic parameters of the model, there may exist a "critical" electric field amplitude, below which free energy transduction is not reversible. That is, energy may be transduced from the electric to the chemical, but not from the chemical to the electric.     

Kinetic analysis of active membrane transport systems: equations for net velocity and isotope exchange.

Equations for the initial net velocity and for isotope exchange velocities in active membrane transport systems are presented. The equations are expressed entirely in terms of kinetic constants which are experimentally determinable from appropriate reciprocal plots, and replots of slope and intercept (for net velocity) or 1/Δslope and 1/Δintercept (for isotope exchange). The equations and plots are equally applicable to a soluble iso uni uni enzyme system.The effect of pH on sulfate transport by an ATP-sulfurylase negative mutant of Penicillium notatum was analyzed assuming that ³⁵SO₄²⁻ and H⁺ are cosubstrates of the transport system. The kinetics are consistent with an ordered addition to the carrier of one H⁺ ion followed by ³⁵SO₄²⁻, with H⁺ in equilibrium with the carrier and the carrier-H⁺ complex.