Km For Transport Research Articles

Geochemistry of Inorganic Nitrogen in Waters Released from Coal-Bed Natural Gas Production Wells in the Powder River Basin, Wyoming

Water originating from coal-bed natural gas (CBNG) production wells typically contains ammonium and is often disposed via discharge to ephemeral channels. A study conducted in the Powder River Basin, Wyoming, documented downstream changes in CBNG water composition, emphasizing nitrogen-cycling processes and the fate of ammonium. Dissolved ammonium concentrations from 19 CBNG discharge points ranged from 95 to 527 microM. Within specific channels, ammonium concentrations decreased with transport distance, with subsequent increases in nitrite and nitrate concentrations. Removal efficiency, or uptake, oftotal dissolved inorganic nitrogen (DIN) varied between channel types. DIN uptake was greater in the gentle-sloped, vegetated channel as compared to the incised, steep, and sparsely vegetated channel and was highly correlated with diel patterns of incident light and dissolved oxygen concentration. In a larger main channel with multiple discharge inputs (n=13), DIN concentrations were >300 microM, with pH > 8.5, after 5 km of transport. Ammonium represented 25-30% of the large-channel DIN, and ammonium concentrations remained relatively constant with time, with only a weak diel pattern evident. In July 2003, the average daily large-channel DIN load was 23 kg N day(-1) entering the Powder River, an amount which substantially increased the total Powder River DIN load after the channel confluence. These results suggest that CBNG discharge may be an important source of DIN to western watersheds, at least at certain times of the year, and that net oxidation and/or removal is dependent upon the extent of contact with sediment and biomass, type of drainage channel, and time of day.

α-Isopropylmalate, a Leucine Biosynthesis Intermediate in Yeast, Is Transported by the Mitochondrial Oxalacetate Carrier

In Saccharomyces cerevisiae, alpha-isopropylmalate (alpha-IPM), which is produced in mitochondria, must be exported to the cytosol where it is required for leucine biosynthesis. Recombinant and reconstituted mitochondrial oxalacetate carrier (Oac1p) efficiently transported alpha-IPM in addition to its known substrates oxalacetate, sulfate, and malonate and in contrast to other di- and tricarboxylate transporters as well as the previously proposed alpha-IPM transporter. Transport was saturable with a half-saturation constant of 75 +/- 4 microm for alpha-IPM and 0.31 +/- 0.04 mm for beta-IPM and was inhibited by the substrates of Oac1p. Though not transported, alpha-ketoisocaproate, the immediate precursor of leucine in the biosynthetic pathway, inhibited Oac1p activity competitively. In contrast, leucine, alpha-ketoisovalerate, valine, and isoleucine neither inhibited nor were transported by Oac1p. Consistent with the function of Oac1p as an alpha-IPM transporter, cells lacking the gene for this carrier required leucine for optimal growth on fermentable carbon sources. Single deletions of other mitochondrial carrier genes or of LEU4, which is the only other enzyme that can provide the cytosol with alpha-IPM (in addition to Oac1p) exhibited no growth defect, whereas the double mutant DeltaOAC1DeltaLEU4 did not grow at all on fermentable substrates in the absence of leucine. The lack of growth of DeltaOAC1DeltaLEU4 cells was partially restored by adding the leucine biosynthetic cytosolic intermediates alpha-ketoisocaproate and alpha-IPM to these cells as well as by complementing them with one of the two unknown human mitochondrial carriers SLC25A34 and SLC25A35. Oac1p is important for leucine biosynthesis on fermentable carbon sources catalyzing the export of alpha-IPM, probably in exchange for oxalacetate.

Redefining the Facilitated Transport of Mannose in Human Cells: Absence of a Glucose-Insensitive, High-Affinity Facilitated Mannose Transport System

Current evidence suggests that extracellular mannose can be transported intracellularly and utilized for glycoprotein synthesis; however, the identity and the functional characteristics of the transporters of mannose are controversial. Although the glucose transporters are capable of transporting mannose, it has been postulated that the entry of mannose in mammalian cells is mediated by a transporter that is insensitive to glucose [Panneerselvam, K., and Freeze, H. (1996) J. Biol. Chem. 271, 9417-9421] or by a transporter induced by cell treatment with metformin [Shang, J., and Lehrman, M. A. (2004) J. Biol. Chem. 279, 9703-9712]. We performed a detailed analysis of the uptake of mannose in normal human erythrocytes and in leukemia cell line HL-60. Short uptake assays allowed the identification of a single functional activity involved in mannose uptake in both cell types, with a K(m) for transport of 6 mM. Transport was inhibited in a competitive manner by classical glucose transporter substrates. Similarly, the glucose transporter inhibitors cytochalasin B, genistein, and myricetin inhibited mannose transport by 100%. Using long uptake experiments, we identified a second, high-affinity component associated with the intracellular trapping of mannose in the HL-60 cells that is not directly involved in the transport of mannose via the glucose transporters. Thus, the transport of mannose via glucose transporters is a process which is kinetically and biologically separable from its intracellular trapping. A general survey of human cells revealed that mannose uptake was entirely blocked by concentrations of cytochalasin B that obliterates the activity of the glucose transporters. The transport and inhibition data demonstrate that extracellular mannose, whose physiological concentration is in the micromolar range, enters cells in the presence of physiological concentrations of glucose. Overall, our data indicate that transport through the glucose transporter is the main mechanism by which human cells acquire mannose.

Specificity of the rat vesicular acetylcholine transporter.

The protein kinase A-deficient PC12 cell line PC12A123.7 lacks both choline acetyltransferase and the vesicular acetylcholine transporter. This cell line has been used to establish a stably transfected cell line expressing recombinant rat vesicular acetylcholine transporter that is appropriately trafficked to small synaptic vesicles. Acetylcholine is transported by the rat vesicular acetylcholine transporter at a maximal rate of 1.45 nmol acetylcholine/min/mg protein and exhibits a Km for transport of 2.5 mM. The transporter binds vesamicol with a Kd of 7.5 nM. The ability of structural analogs of acetylcholine to inhibit both acetylcholine uptake and vesamicol binding was measured. The results demonstrate that like Torpedo vesicular acetylcholine transporter, the mammalian transporter can bind a diverse group of acetylcholine analogs.

Activation of GLUT1 by metabolic and osmotic stress: potential involvement of AMP-activated protein kinase (AMPK).

In the rat liver epithelial cell line Clone 9, the V(max) for glucose uptake is acutely increased by inhibition of oxidative phosphorylation and by osmotic stress. By using a membrane-impermeant photoaffinity labelling reagent together with an isoform-specific antibody, we have, for the first time, provided direct evidence for the involvement of the GLUT1 glucose transporter isoform in this response. Transport stimulation was found to be associated with enhanced accessibility of GLUT1 to its substrate and with photolabelling of formerly 'cryptic' exofacial substrate binding sites in GLUT1 molecules. The total amount of cell surface GLUT1 remained constant. The precise mechanism for this binding site 'unmasking' is unclear but appears to involve AMP-activated protein kinase: in the current study, osmotic and metabolic stresses were found to result in activation of the alpha 1 isoform of AMP-activated protein kinase, and transport stimulation could be mimicked both by 5-aminoimidazole-4-carboxamide ribonucleoside and by infection of cells with a recombinant adenovirus encoding constitutively active AMP-activated protein kinase. The effect of 5-aminoimidazole-4-carboxamide ribonucleoside, as for metabolic stress, was on the V(max) rather than on the K(m) for transport and did not affect the cell-surface concentration of GLUT1. The relevant downstream target(s) of AMP-activated protein kinase have not yet been identified, but stimulation of transport by inhibition of oxidative phosphorylation or by 5-aminoimidazole-4-carboxamide ribonucleoside was not prevented by either inhibitors of conventional and novel protein kinase C isoforms or inhibitors of nitric oxide synthase. These enzymes, which have been implicated in stress-regulated pathways in other cell types, are therefore unlikely to play a role in transport regulation by stress in Clone 9 cells.

Textural and compositional variation of sand along the Manahara-Bagmati river basin, Kathmandu Valley

The Manahara-Bagmati river basin is located in a humid climate and has a 51 km long low gradient profile. It collects sediments from metamorphic, metasedimentary, and fluvio-lacustrine sources. The sediment samples collected from 17 localities were studied texturally and compositionally to infer how sediment modifies due course of transport and whether the trends are interfered by mixing, recycling, and weathering. For this purpose, two size grades (i.e., 0-1ϕ and 2-3ϕ) were studied. Between them, the trends were more conspicuous in the 0-1ϕ sand grade. Mean (M2) and median (M6) grain sizes increase downstream exponentially. Sorting (σ1) also improves along the river course. Quartz content gradually increases downstream, whilst feldspars and rock fragments decrease remarkably along the river course. Rapid dilution of feldspars and rock fragments (from coarser size grade) occurs after 27 km of transport. The Modified Maturity Index, which ranges from 19.8 to 214, increases from the Manahara to Bagmati River sands. The mica content in the Bagmati River is from 2 to 8 times higher than in the Manahara River.
 Within the studied river course, the mean projection sphericity (ψp) of quartz remains high (0.80 in average) but does not show any consistent trend. The mean roundness (ρ) of the 0-1ϕ sand grade increases conspicuously against the distance of transport. The majority of samples belong to submature class. Local influx of sediments and recycling are profound from Sankhu to Sanothimi and from Chobhar to Hasdol. Rapid weathering of labile grains is inferred in the middle reach of the Manahara- Bagmati river system (i.e., between Sanothimi and Chobhar).

Mathematical Treatment of Transport Data of Bacterial Transport Systems to Estimate Limitation in Diffusion through the Outer Membrane

Bacterial transport systems are traditionally treated as enzymes exhibiting a saturable binding site giving rise to an apparent Kmof transport, whereas the maximal rate of transport is regarded equivalent to the Vmaxof enzymatic reactions. Thus, the Michaelis–Menten theory is usually applied in the analysis of transport data and Kmand Vmaxare derived from the treatment of data obtained from the rate of transport at varying substrate concentrations. Such an analysis tacitly assumes that the substrate recognition site of the transport system is freely accessible to substrate. However, this is not always the case. In systems endowed with high affinity in the μ M range or those recognizing large substrates or those exhibiting high Vmax, the diffusion through the outer membrane may become rate determining, particularly at low external substrate concentrations. In such a situation the dependence of the overall rate of transport (from the medium into the cytoplasm) on the substrate concentration in the medium will no longer follow Michaelis–Menten kinetics. By analysing the deviation of transport data from the corresponding ideal Michaelis–Menten plot we developed a method that allows us to determine diffusion limitation through the outer membrane. The method allows us to find the correctKm of the transport system functioning at the inner membrane even under conditions of strong diffusion limitation through the outer membrane. The model was tested and validified with the Escherichia coli binding protein-dependent ABC transporter for maltose. The corresponding systems for sn -glycerol-3-phospate of Escherichia coli and the α -cyclodextrin transport of Klebsiella oxitoca were used as test systems.

Colony-Stimulating Factors Signal for Increased Transport of Vitamin C in Human Host Defense Cells

Although serum concentrations of ascorbic acid seldom exceed 150 μmol/L, mature neutrophils and mononuclear phagocytes accumulate millimolar concentrations of vitamin C. Relatively little is known about the mechanisms regulating this process. The colony-stimulating factors (CSFs), which are central modulators of the production, maturation, and function of human granulocytes and mononuclear phagocytes, are known to stimulate increased glucose uptake in target cells. We show here that vitamin C uptake in neutrophils, monocytes, and a neutrophilic HL-60 cell line is enhanced by the CSFs. Hexose uptake studies and competition analyses showed that dehydroascorbic acid is taken up by these cells through facilitative glucose transporters. Human monocytes were found to have a greater capacity to take up dehydroascorbic acid than neutrophils, related to more facilitative glucose transporters on the monocyte cell membrane. Ascorbic acid was not transported by these myeloid cells, indicating that they do not express a sodium-ascorbate cotransporter. Granulocyte (G)- and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulated increased uptake of vitamin C in human neutrophils, monocytes, and HL-60 neutrophils. In HL-60 neutrophils, GM-CSF increased both the transport of dehydroascorbic acid and the intracellular accumulation of ascorbic acid. The increase in transport was related to a decrease in Km for transport of dehydroascorbic acid without a change in Vmax. Increased ascorbic acid accumulation was a secondary effect of increased transport. Triggering the neutrophils with the peptide fMetLeuPhe led to enhanced vitamin C uptake by increasing the oxidation of ascorbic acid to the transportable moiety dehydroascorbic acid, and this effect was increased by priming the cells with GM-CSF. Thus, the CSFs act at least at two distinct functional loci to increase cellular vitamin C uptake: conversion of ascorbic acid to dehydroascorbic acid by enhanced oxidation in the pericellular milieu and increased transport of DHA through the facilitative glucose transporters at the cell membrane. These results link the regulated uptake of vitamin C in human host defense cells to the action of CSFs.

Colony-Stimulating Factors Signal for Increased Transport of Vitamin C in Human Host Defense Cells

AbstractAlthough serum concentrations of ascorbic acid seldom exceed 150 μmol/L, mature neutrophils and mononuclear phagocytes accumulate millimolar concentrations of vitamin C. Relatively little is known about the mechanisms regulating this process. The colony-stimulating factors (CSFs), which are central modulators of the production, maturation, and function of human granulocytes and mononuclear phagocytes, are known to stimulate increased glucose uptake in target cells. We show here that vitamin C uptake in neutrophils, monocytes, and a neutrophilic HL-60 cell line is enhanced by the CSFs. Hexose uptake studies and competition analyses showed that dehydroascorbic acid is taken up by these cells through facilitative glucose transporters. Human monocytes were found to have a greater capacity to take up dehydroascorbic acid than neutrophils, related to more facilitative glucose transporters on the monocyte cell membrane. Ascorbic acid was not transported by these myeloid cells, indicating that they do not express a sodium-ascorbate cotransporter. Granulocyte (G)- and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulated increased uptake of vitamin C in human neutrophils, monocytes, and HL-60 neutrophils. In HL-60 neutrophils, GM-CSF increased both the transport of dehydroascorbic acid and the intracellular accumulation of ascorbic acid. The increase in transport was related to a decrease in Km for transport of dehydroascorbic acid without a change in Vmax. Increased ascorbic acid accumulation was a secondary effect of increased transport. Triggering the neutrophils with the peptide fMetLeuPhe led to enhanced vitamin C uptake by increasing the oxidation of ascorbic acid to the transportable moiety dehydroascorbic acid, and this effect was increased by priming the cells with GM-CSF. Thus, the CSFs act at least at two distinct functional loci to increase cellular vitamin C uptake: conversion of ascorbic acid to dehydroascorbic acid by enhanced oxidation in the pericellular milieu and increased transport of DHA through the facilitative glucose transporters at the cell membrane. These results link the regulated uptake of vitamin C in human host defense cells to the action of CSFs.

Metabolism of catecholamines by catechol-O-methyltransferase in cells expressing recombinant catecholamine transporters.

To determine if catechol-O-methyltransferase (COMT) metabolizes catecholamines within cell lines used for heterologous expression of plasmalemmal transporters and alters the measured characteristics of 3H-substrate transport, the uptake of monoamine transporter substrates was assessed in three cell lines (C6 glioma, L-M fibroblast, and HEK293 cells) that had been transfected with the recombinant human transporters. Uptake and cellular retention of 3H-catecholamines was increased by up to fourfold by two COMT inhibitors, tropolone and Ro 41-0960, with potencies similar to those for inhibition of COMT activity, whereas the uptake of two transporter substrates that are not substrates for COMT, [3H]serotonin and [3H]MPP+, was unaffected. Direct measurement of monoamine substrates by HPLC confirmed that tropolone (1 mM) increased the retention of the catecholamines dopamine and norepinephrine, but not the retention of serotonin in HEK293 cells. Saturation analysis of the uptake of [3H]dopamine by C6 cells expressing the dopamine transporter demonstrated that tropolone (1 mM) decreased the apparent Km of transport from 0.61 microM to 0.34 microM without significantly altering the maximal velocity of transport. These data suggest that endogenous COMT activity in mammalian cells may alter neurotransmitter deposition and thus the apparent kinetic characteristics of transport.

Structural domains that contribute to substrate specificity in facilitated glucose transporters are distinct from those involved in kinetic function: studies with GLUT-1/GLUT-2 chimeras.

GLUT-2 differs from other members of the facilitated glucose transporter family because it transports a wider range of substrates and exhibits a higher Km for transport of glucose analogs such as 2-deoxyglucose (2-DOG). In order to investigate the structural determinants of the unique substrate specificity and kinetic function of GLUT-2, recombinant adenoviruses were used to express native, mutant, and chimeric glucose transporters in the kidney cell line CV-1, yielding the following key observations. (1) A chimera consisting of GLUT-1 with the C-terminal tail of GLUT-2 had a Km for 2-DOG of 9.9 +/- 1.5 that was intermediate between that of native GLUT-1 (3.7 +/- 0.4) and native GLUT-2 (26.3 +/- 3.3). In contrast to the effect of the GLUT-2 C terminus on Km for 2-DOG, this substitution did not confer enhanced uptake of three alternative substrates (fructose, arabinose, or streptozotocin) which are transported efficiently by native GLUT-2 but not by GLUT-1. (2) A chimera consisting of GLUT-2 with the N-terminal 87 amino acids of GLUT-1 exhibited no change in Km for 2-DOG relative to native GLUT-2 but exhibited a significant reduction in capacity for transport of the three alternative substrates. (3) Mutation of asparagine 62 in GLUT-2 to glutamine produced a transporter lacking its N-linked oligosaccharide that exhibited a 2.5-fold increase in Km for 2-DOG but equally efficient transport of the three alternative substrates relative to native GLUT-2. These data provide insight into structural domains that affect substrate specificity in facilitated glucose transporters and demonstrate that they are distinct from elements involved in glucose transport kinetics.

Gentamicin inhibits carrier-mediated dipeptide transport in kidney.

The effect of gentamicin on transport of pyroglutamylhistidine (pGlu-His) was examined in rabbit renal brush-border membrane vesicles (BBMV). Gentamicin, an aminoglycoside antibiotic, is limited in its usage because of nephrotoxicity characterized in part by transport defects in the proximal tubule. Since there is no information regarding the effects of gentamicin on renal peptide carriers, uptake of [3H]pGlu-His was measured in BBMV following either in vivo or in vitro exposure to the antibiotic. One hour after in vivo administration, the maximal rate (Vmax) for pGlu-His transport was significantly reduced in isolated membrane vesicles washed free of the drug, but the apparent Michaelis constant (Km) was unaltered. Coincubation of membranes with gentamicin during measurements of pGlu-His uptake had a similar effect, causing a significant decrease in the Vmax but not the Km of transport. The addition of 5 mM magnesium to the uptake medium prevented the in vitro but not the in vivo effect. The data indicate that high doses of gentamicin inhibit the capacity but not the affinity of dipeptide transport in the kidney, prior to morphological changes which typify acute tubular necrosis. The in vitro effect is rapid and involves a direct action of gentamicin on the brush-border membrane. The in vivo experiments show that toxicity may be prolonged and remains following removal of the drug from the renal brush border.

NO3- transport across the plasma membrane of Arabidopsis thaliana root hairs: kinetic control by pH and membrane voltage.

High-affinity nitrate transport was examined in intact root hair cells of Arabidopsis thaliana using electrophysiological recordings to characterise the response of the plasma membrane to NO3- challenge and to quantify transport activity. The NO3(-)-associated membrane current was determined using a three-electrode voltage clamp to bring membrane voltage under experimental control and to compensate for current dissipation along the longitudinal cell axis. Nitrate transport was evident in the roots of seedlings grown in the absence of a nitrogen source, but only 4-6 days postgermination. In 6-day-old seedlings, additions of 5-100 microM NO3- to the bathing medium resulted in membrane depolarizations of 8-43 mV, and membrane voltage (Vm) recovered on washing NO3- from the bath. Voltage clamp measurements carried out immediately before and following NO3- additions showed that the NO3(-)-evoked depolarizations were the consequence of an inward-directed current that appeared across the entire range of accessible voltages (-300 to +50 mV). Both membrane depolarizations and NO3(-)-evoked currents recorded at the free-running voltage displayed quasi-Michaelian kinetics, with apparent values for Km of 23 +/- 6 and 44 +/- 11 microM, respectively and, for the current, a maximum of 5.1 +/- 0.9 muA cm-2. The NO3- current showed a pronounced voltage sensitivity within the normal physiological range between -250 and -100 mV, as could be demonstrated under voltage clamp, and increasing the bathing pH from 6.1 to 7.4-8.0 reduced the current and the associated membrane depolarizations 3- to 8-fold. Analyses showed a well-defined interaction between the kinetic variables of membrane voltage, pHo and [NO3-]o. At a constant pHo of 6.1, depolarization from -250 to -150 mV resulted in an approximate 3-fold reduction in the maximum current but a 10% rise in the apparent affinity for NO3-. By contrast, the same depolarization effected an approximate 20% fall in the Km for transport as a function in [H+]o. These, and additional characteristics of the transport current implicate a carrier cycle in which NO3- binding is kinetically isolated from the rate-limiting step of membrane charge transit, and they indicate a charge-coupling stoichiometry of 2(H+) per NO3- anion transported across the membrane. The results concur with previous studies showing a high-affinity NO3- transport system in Arabidopsis that is inducible following a period of nitrogen-limiting growth, but they underline the importance of voltage as a kinetic factor controlling NO3- transport at the plant plasma membrane.

Effect of Pyridoxal 5′-Phosphate on the Function of the Purified Mitochondrial Tricarboxylate Transport Protein

The effect of pyridoxal 5′-phosphate (PLP), a lysine-selective reagent, on the function of the purified and reconstituted mitochondrial tricarboxylate transport protein has been studied. PLP caused a concentration-dependent inhibition of citrate transport with an IC50 value of 0.09 mM. At 10 mM PLP virtually complete inhibition of transport was observed (i.e., 97%), thereby indicating that modification of a residue(s) whose participation is essential to the translocation mechanism had occurred. Substrate protection studies demonstrated that substrates for the tricarboarylate transporter (i.e., citrate, isocitrate, phosphoenolpyruvate, and malate) effectively protected against PLP inhibition, whereas other organic anions which are not transported by the tricarboxylate carrier (i.e,, malonate, α-ketoglutarate, phosphate, and succinate) afforded considerably less protection. Kinetic studies in which the transport rate was measured at varying citrate and PLP concentrations indicated that PLP caused an increase in the apparent Km of transport with little change in the Vmax, thereby resulting in a primarily competitive inhibition pattern. In combination, the above findings indicate that PLP interacts with the tricarboxylate transporter at a site(s) (i,e., a lysine residue(s) and/or the amino-terminal alanine residue) that is important in the translocation mechanism and may reside within or near the substrate binding site.

Functional expression of two glucose transporter isoforms from the parasitic protozoan Leishmania enriettii.

The parasitic protozoan Leishmania enriettii contains a family of tandemly repeated genes, designated Pro-1, that encode proteins with significant sequence similarity to mammalian facilitative glucose transporters. Pro-1 mRNAs are expressed almost exclusively in the promastigote or insect stage of the parasite life cycle. The Pro-1 tandem repeat encodes two isoforms of the putative transporter, iso-1 and iso-2, which have identical predicted amino acid sequences except for their NH2-terminal hydrophilic domains. We have now expressed both iso-1 and iso-2 by microinjecting their RNAs into Xenopus oocytes and assaying these oocytes for transport of various radiolabeled ligands. Both iso-1 and iso-2 transport [3H]2-deoxy-D-glucose, confirming that each protein is a bona fide glucose transporter. Each isoform also transports fructose and, to a much lesser degree, mannose. Compounds which inhibit 2-deoxy-D-glucose transport in L. enriettii promastigotes also inhibit transport in the microinjected oocytes expressing each isoform, indicating that the substrate specificities and pharmacological properties of each isoform are similar to those measured for 2-deoxy-D-glucose transport in intact parasites. The Km for transport of 2-deoxyglucose in oocytes expressing iso-1 is similar to that for oocytes expressing iso-2. These results reveal that both transporter isoforms have closely related functional properties and that the difference in their structures may serve some other purpose such as differential subcellular localization.

Adenosine triphosphate-dependent transport of doxorubicin, daunomycin, and vinblastine in human tissues by a mechanism distinct from the P-glycoprotein.

Previous studies have demonstrated that a human glutathione conjugate transporter, designated as dinitrophenyl-S-glutathione ATPase (DNP-SG ATPase), catalyzed ATP hydrolysis in the presence of several amphiphilic compounds other than glutathione conjugates (Singhal, S. S., R. Sharma, S. Gupta, H. Ahmad, P. Zimniak, A. Radominska, R. Lester, and Y. C. Awasthi. 1991. FEBS [Fed. Eur. Biochem. Soc.] Lett. 281:255-257). We now demonstrate that DNP-SG ATPase purified from human lung and erythrocyte membranes catalyzed the hydrolysis of ATP in the presence of doxorubicin and its metabolites. Doxorubicin-stimulated ATP hydrolysis by DNP-SG ATPase was saturable with respect to doxorubicin (Km 1.2 and 2.8 microM for the lung and erythrocyte enzymes, respectively). Antibodies against DNP-SG ATPase immunoprecipitated the ATP hydrolyzing activity stimulated by doxorubicin, its metabolites, and glutathione conjugates. Inside our vesicles prepared from erythrocyte membranes took up doxorubicin, daunomycin, and vinblastine in an ATP-dependent manner. The uptake was linear with respect to time and vesicle protein, was dependent on ATP and magnesium, was inhibited by heavy metal salts or by heating the vesicles, and was sensitive to both osmolarity and orientation of the vesicles. The transport had an activation energy of 13 kcal/mol, was saturable with respect to both doxorubicin and ATP (Km values of 1.8 microM and 1.9 mM, respectively), and was competitively inhibited by glutathione conjugates as well as by a number of amphiphiles such as daunomycin or vinblastine. Transport was diminished upon coating the vesicles with antibodies against DNP-SG ATPase. Incorporation of increasing amounts of purified DNP-SG ATPase into the vesicles resulted in a linear increase in transport of doxorubicin. These studies demonstrated for the first time that a membrane protein that catalyzed the transport of anionic amphiphilic molecules such as glutathione conjugates could also mediate the transport of weakly cationic antitumor antibiotic, doxorubicin. Notably, the Km of transport was in the range of doxorubicin concentration achievable in human serum after intravenous dosing of doxorubicin.

Displacement of the Lewis thrust sheet in southwestern Canada: New evidence from seismic reflection data

Research Article| September 01, 1994 Displacement of the Lewis thrust sheet in southwestern Canada: New evidence from seismic reflection data Arie J. van der Velden; Arie J. van der Velden 1Department of Geology and Geophysics, University of Calgary, Calgary, Alberta T2N 1N4, Canada Search for other works by this author on: GSW Google Scholar Frederick A. Cook Frederick A. Cook 1Department of Geology and Geophysics, University of Calgary, Calgary, Alberta T2N 1N4, Canada Search for other works by this author on: GSW Google Scholar Author and Article Information Arie J. van der Velden 1Department of Geology and Geophysics, University of Calgary, Calgary, Alberta T2N 1N4, Canada Frederick A. Cook 1Department of Geology and Geophysics, University of Calgary, Calgary, Alberta T2N 1N4, Canada Publisher: Geological Society of America First Online: 02 Jun 2017 Online ISSN: 1943-2682 Print ISSN: 0091-7613 Geological Society of America Geology (1994) 22 (9): 819–822. https://doi.org/10.1130/0091-7613(1994)022<0819:DOTLTS>2.3.CO;2 Article history First Online: 02 Jun 2017 Cite View This Citation Add to Citation Manager Share Icon Share Facebook Twitter LinkedIn MailTo Tools Icon Tools Get Permissions Search Site Citation Arie J. van der Velden, Frederick A. Cook; Displacement of the Lewis thrust sheet in southwestern Canada: New evidence from seismic reflection data. Geology 1994;; 22 (9): 819–822. doi: https://doi.org/10.1130/0091-7613(1994)022<0819:DOTLTS>2.3.CO;2 Download citation file: Ris (Zotero) Refmanager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentBy SocietyGeology Search Advanced Search Abstract Seismic reflection data across the Rocky Mountain trench in the southern Canadian Cordillera reveal prominent reflections from Mesoproterozoic (Aldridge Formation) sills that allow a direct stratigraphic correlation across the trench when 10 km of extension is restored. Trun cated reflections at ∼11-15 km depth on the west side of the trench are interpreted as a footwall cutoff of the Lewis thrust in Belt-Purcell strata that lies ∼115 km west of the leading edge of the Lewis thrust, exposed in the Waterton area; retrodeformation of the Lewis sheet requires a minimum of 75 km of transport on the Lewis thrust and 40 km of transport by the development of footwall-domain duplexes. The trench is located above a major change in slope of the top of the North American basement that coincides with a pronounced increase in thickness of lower Belt-Purcell strata in their retrodeformed position. This transition may represent part of an ancient margin that was initially established in Paleoproterozoic time. This content is PDF only. Please click on the PDF icon to access. First Page Preview Close Modal You do not have access to this content, please speak to your institutional administrator if you feel you should have access.

Geochemistry of the Obey River Basin, north-central Tennessee: a case of acid mine water in a karst drainage system

The East Fork of the Obey River, a tributary of the Cumberland River, drains a 523 km 2 basin along the western margin of the Cumberland Plateau. The upper basin lies on clastic rocks that include coals that were mined earlier in this century. The lower basin is drained through a deep gorge where the East Fork has cut downward into the Mississippian carbonate rocks. The lower basin is karstic, with a 9 km length of the river and most of its tributaries underdrained by conduit systems. Many of the upper basin tributaries of the East Fork are contaminated by acid mine drainage and have high levels of acidity, sulfate, iron, and aluminum. As the acid mine waters sink in the limestone portions of the basin, they are buffered but acidity is not rapidly reduced and acid waters appear at a large spring deep in the karst having survived 5 km of transport. Sulfate waters gradually convert to bicarbonate waters as more tributaries from carbonate rocks enter the system. Aluminum in solution decreases with increasing pH, and precipitates as colloidal sized particles. Although alkalinity increases in the lower reaches of the basin, most of the waters remain highly undersaturated with respect to calcite.

Binding-protein-dependent lactose transport in Agrobacterium radiobacter

Agrobacterium radiobacter NCIB 11883 was grown in lactose-limited continuous culture at a dilution rate of 0.045/h. Washed cells transported [14C]lactose and [methyl-14C]beta-D-thiogalactoside, a nonmetabolisable analog of lactose, at similar rates and with similar affinities (Km for transport, less than 1 microM). Transport was inhibited to various extents by the uncoupling agent carbonyl cyanide p-trifluoromethoxyphenylhydrazone, by unlabeled beta-galactosides and D-galactose, and by osmotic shock. The accumulation ratio for methyl-beta-D-thiogalactoside was greater than or equal to 4,100. An abundant protein (molecular weight, 41,000) was purified from osmotic-shock fluid and shown by equilibrium dialysis to bind lactose and methyl-beta-D-thiogalactoside, the former with very high affinity (binding constant, 0.14 microM). The N-terminal amino acid sequence of this lactose-binding protein exhibited some homology with several other sugar-binding proteins from bacteria. Antiserum raised against the lactose-binding protein did not cross-react with two glucose-binding proteins from A. radiobacter or with extracts of other bacteria grown under lactose limitation. Lactose transport and beta-galactosidase were induced in batch cultures by lactose, melibiose [O-alpha-D-galactoside-(1----6)alpha-D-glucose], and isopropyl-beta-D-thiogalactoside and were subject to catabolite repression by glucose, galactose, and succinate which was not alleviated by cyclic AMP. We conclude that lactose is transported into A. radiobacter via a binding protein-dependent active transport system (in contrast to the H+ symport and phosphotransferase systems found in other bacteria) and that the expression of this transport system is closely linked to that of beta-galactosidase.

The kinetics of transport of lactate and pyruvate into isolated cardiac myocytes from guinea pig. Kinetic evidence for the presence of a carrier distinct from that in erythrocytes and hepatocytes

1. Time courses for the uptake of L-lactate, D-lactate and pyruvate into isolated cardiac ventricular myocytes from guinea pig were determined at 11 degrees C or 0 degrees C (for pyruvate) in a citrate-based buffer by using a silicone-oil-filtration technique. These conditions enabled initial rates of transport to be measured without interference from metabolism of the substrates. 2. At a concentration of 0.5 mM, transport of all these substrates was inhibited by approx. 90% by 5 mM-alpha-cyano-4-hydroxycinnamate; at 10 mM-L-lactate a considerable portion of transport could not be inhibited. 3. Initial rates of L-lactate and pyruvate uptake in the presence of 5 mM-alpha-cyano-4-hydroxycinnamate were linearly related to the concentration of the monocarboxylate and probably represented diffusion of the free acid. The inhibitor-sensitive component of uptake obeyed Michaelis-Menten kinetics, with Km values for L-lactate and pyruvate of 2.3 and 0.066 mM respectively. 4. Pyruvate and D-lactate inhibited the transport of L-lactate, with Ki values (competitive) of 0.077 and 6.6 mM respectively; the Ki for pyruvate was very similar to its Km for transport. The Ki for alpha-cyano-4-hydroxycinnamate as a non-competitive inhibitor was 0.042 mM. 5. These results indicate that L-lactate, D-lactate and pyruvate share a common carrier in guinea-pig cardiac myocytes; the low stereoselectivity for L-lactate over D-lactate and the high affinity for pyruvate distinguish it from the carrier in erythrocytes and hepatocytes. The metabolic roles for this novel carrier in heart are discussed.