scholarly journals Quantitative study of unsaturated transport of glycerol through aquaglyceroporin that has high affinity for glycerol

RSC Advances ◽  
2020 ◽  
Vol 10 (56) ◽  
pp. 34203-34214
Author(s):  
Roberto A. Rodriguez ◽  
Ruth Chan ◽  
Huiyun Liang ◽  
Liao Y. Chen
Keyword(s):  
Quantitative Study ◽  
Transport Pathway ◽  
High Affinity ◽  
Unsaturated Transport ◽  
Substrate Concentrations

In addition to the single-glycerol mechanism for saturable kinetics, a second transport pathway becomes more significant at higher substrate concentrations, resulting in unsaturable transport characteristics of an aquaglyceroporin.

scholarly journals Quantitative study of unsaturated transport of glycerol through aquaglyceroporin that has high affinity for glycerol

2020 ◽  
Author(s):  
Roberto A. Rodriguez ◽  
Ruth Chan ◽  
Huiyun Liang ◽  
Liao Y. Chen
Keyword(s):  
Free Energy ◽  
In Silico ◽  
Energy Profile ◽  
Free Energy Profile ◽  
High Affinity ◽  
Glycerol Transport ◽  
Wide Range ◽  
Unsaturated Transport ◽  
Substrate Concentrations

Graphical AbstractABSTRACTThe structures of several aquaglyceroporins have been resolved to atomic resolution showing two or more glycerols bound inside a channel and confirming a glycerol-facilitator’s affinity for its substrate glycerol. However, the kinetics data of glycerol transport experiments all point to unsaturated transport that is characteristic of low substrate affinity in terms of the Michaelis-Menten kinetics. In this article, we present an in silico-in vitro research focused on AQP3, one of the human aquaglyceroporins that is natively expressed in the abundantly available erythrocytes. We conducted 2.1 μs in silico simulations of AQP3 embedded in a model erythrocyte membrane with intracellular-extracellular asymmetries in leaflet lipid compositions and compartment salt ions. From the equilibrium molecular dynamics (MD), we elucidated the mechanism of glycerol transport at high substrate concentrations. From the steered MD simulations, we computed the Gibbs free-energy profile throughout the AQP3 channel. From the free-energy profile, we quantified the kinetics of glycerol transport that is unsaturated due to glycerol-glycerol interaction mediated by AQP3 resulting in the concerted movement of two glycerol molecules for the transport of one glycerol molecule across the cell membrane. We conducted in vitro experiments on glycerol uptake into human erythrocytes for a wide range of substrate concentrations and various temperatures. The experimental data quantitatively validated our theoretical-computational conclusions on the unsaturated glycerol transport through AQP3 that has high affinity for glycerol.

Nanomolar Levels of Dimethylsulfoniopropionate, Dimethylsulfonioacetate, and Glycine Betaine Are Sufficient To Confer Osmoprotection to Escherichia coli

1999 ◽  
Vol 65 (8) ◽  
pp. 3304-3311 ◽  
Author(s):  
Anne Cosquer ◽  
Vianney Pichereau ◽  
Jean-Alain Pocard ◽  
Jacques Minet ◽  
Michel Cormier ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Salinity Tolerance ◽  
Glycine Betaine ◽  
Standard Laboratory ◽  
E Coli ◽  
High Affinity ◽  
Bacterial Cultures ◽  
Transport Studies ◽  
Ecological Implications ◽  
Substrate Concentrations

ABSTRACT We combined the use of low inoculation titers (300 ± 100 CFU/ml) and enumeration of culturable cells to measure the osmoprotective potentialities of dimethylsulfoniopropionate (DMSP), dimethylsulfonioacetate (DMSA), and glycine betaine (GB) for salt-stressed cultures of Escherichia coli. Dilute bacterial cultures were grown with osmoprotectant concentrations that encompassed the nanomolar levels of GB and DMSP found in nature and the millimolar levels of osmoprotectants used in standard laboratory osmoprotection bioassays. Nanomolar concentrations of DMSA, DMSP, and GB were sufficient to enhance the salinity tolerance of E. coli cells expressing only the ProU high-affinity general osmoporter. In contrast, nanomolar levels of osmoprotectants were ineffective with a mutant strain (GM50) that expressed only the low-affinity ProP osmoporter. Transport studies showed that DMSA and DMSP, like GB, were taken up via both ProU and ProP. Moreover, ProU displayed higher affinities for the three osmoprotectants than ProP displayed, and ProP, like ProU, displayed much higher affinities for GB and DMSA than for DMSP. Interestingly, ProP did not operate at substrate concentrations of 200 nM or less, whereas ProU operated at concentrations ranging from 1 nM to millimolar levels. Consequently,proU + strains of E. coli, but not the proP + strain GM50, could also scavenge nanomolar levels of GB, DMSA, and DMSP from oligotrophic seawater. The physiological and ecological implications of these observations are discussed.

Uptake of 3,5,3′-l-tri-iodothyronine in human erythrocytes

1989 ◽  
Vol 121 (3) ◽  
pp. 585-591 ◽  
Author(s):  
K. Yamauchi ◽  
R. Horiuchi ◽  
H. Takikawa
Keyword(s):  
Binding Sites ◽  
Binding Capacity ◽  
Human Erythrocytes ◽  
The Other ◽  
Transport Pathway ◽  
High Affinity ◽  
High Affinity Site ◽  
Maximum Binding Capacity ◽  
Energy Dependent ◽  
Dependent Pathway

ABSTRACT The mechanisms of 3,5,3′-l-tri-iodothyronine (T3) uptake into human erythrocytes were examined. Purified membranes of human erythrocytes were shown to have two classes of T3-binding sites with one being a high-affinity site (dissociation constant, 59·2±17·8 nmol/l; maximum binding capacity, 344·3 ± 95·5 fmol/μg protein). Furthermore, it was shown that there were two pathways for T3 uptake in human erythrocytes; one was saturable, stereospecific (T3»thyroxine > 3,5,3′-d-tri-iodothyronine), energydependent and dominant at 15 °C; the other was not displaced by unlabelled T3 and was energyindependent but did not occur by passive diffusion. The former pathway which, it is suggested, is a receptor-mediated transport pathway, was inhibited by monodansylcadaverine, phloretin or oligomycin at 15 or 37 °C, but the latter pathway was not inhibited by these inhibitors. Our results strongly suggest that uptake of T3 by the energy-independent pathway became predominant over the energy-dependent pathway at 37 °C and accounted for 83% of total T3 uptake of human erythrocytes. Journal of Endocrinology (1989) 121, 585–591

scholarly journals Passive potassium transport in LK sheep red cells. Modification by N-ethyl maleimide.

1983 ◽  
Vol 81 (6) ◽  
pp. 861-885 ◽  
Author(s):  
P Logue ◽  
C Anderson ◽  
C Kanik ◽  
B Farquharson ◽  
P Dunham
Keyword(s):  
Cell Volume ◽  
Potassium Transport ◽  
Transport Pathway ◽  
High Affinity ◽  
High Affinity Site ◽  
Free Media ◽  
Low K ◽  
Cell Volumes ◽  
K Transport ◽  
Stimulation Of

Passive K transport, as modified by N-ethyl maleimide (NEM), was studied in erythrocytes of the low-K (LK) phenotype of sheep. Brief (5-min) treatment with NEM at less than 0.5 mM caused inhibition of passive K influx; NEM at concentrations greater than 0.5 mM caused stimulation of K influx. NEM had similar effects on K efflux. The treatments with NEM did not affect cell volumes (passive K transport in LK cells is sensitive to changes in cell volume). The stimulation of K transport by high [NEM] was also not a consequence of an effect on the metabolic state of the cells. Passive K transport in LK cells is dependent on Cl (it is inhibited in Cl-free media; it may be K/Cl cotransport). NEM had no effect on K influx in Cl-free (NO3-substituted) media. Pretreatment of the cells with anti-L antiserum (L antigen is found on LK cells and not on HK cells) prevented stimulation of K influx by NEM, but did not prevent inhibition. Therefore, NEM modifies the Cl-dependent K transport pathway at two separate sites, a low-affinity site, at which it stimulates, and a high-affinity site, at which it inhibits. Anti-L antibody prevents NEM's action, but only at the low-affinity site.

Characterization of three types of calcium channel in the luminal membrane of the distal nephron

2004 ◽  
Vol 82 (1) ◽  
pp. 30-37 ◽  
Author(s):  
M G Brunette ◽  
M Leclerc ◽  
D Couchourel ◽  
J Mailloux ◽  
Y Bourgeois
Keyword(s):  
Calcium Transport ◽  
Distal Tubule ◽  
Distal Nephron ◽  
High Affinity ◽  
Luminal Membrane ◽  
Kinetics Of ◽  
Substrate Concentrations ◽  
Renal Calcium Transport ◽  
Specific Inhibitors

We previously reported a dual kinetics of Ca2+ transport by the distal tubule luminal membrane of the kidney, suggesting the presence of several types of channels. To better characterize these channels, we examined the effects of specific inhibitors (i.e., diltiazem, an L-type channel; ω-conotoxin MVIIC, a P/Q-type channel; and mibefradil, a T-type channel antagonist) on 0.1 and 0.5 mM Ca2+ uptake by rabbit nephron luminal membranes. None of these inhibitors influenced Ca2+ uptake by the proximal tubule membranes. In contrast, in the absence of sodium (Na+), the three channel antagonists decreased Ca2+ transport by the distal membranes, and their action depended on the substrate concentrations: 50 µM diltiazem decreased 0.1 mM Ca2+ uptake from 0.65 ± 0.07 to 0.48 ± 0.06 pmol·µg–1·10 s–1 (P < 0.05) without influencing 0.5 mM Ca2+ transport, whereas 100 nM ω-conotoxin MVIIC decreased 0.5 mM Ca2+ uptake from 1.02 ± 0.05 to 0.90 ± 0.05 pmol·µg–1·10 s–1 (P < 0.02) and 1 µM mibefradil decreased it from 1.13 ± 0.09 to 0.94 ± 0.09 pmol·µg–1·10 s–1 (P < 0.05); the latter two inhibitors left 0.1 mM Ca2+ transport unchanged. Diltiazem decreased the Vmax of the high-affinity channels, whereas ω-conotoxin MVIIC and mibefradil influenced exclusively the Vmax of the low-affinity channels. These results not only confirm that the distal luminal membrane is the site of Ca2+ channels, but they suggest that these channels belong to the L, P/Q, and T types.Key words: renal calcium transport, calcium channels, diltiazem, mibefradil, ω-conotoxin.

scholarly journals Isolation of a High-Affinity Functional Protein Complex between OmcA and MtrC: Two Outer Membrane Decaheme c-Type Cytochromes of Shewanella oneidensis MR-1

2006 ◽  
Vol 188 (13) ◽  
pp. 4705-4714 ◽  
Author(s):  
Liang Shi ◽  
Baowei Chen ◽  
Zheming Wang ◽  
Dwayne A. Elias ◽  
M. Uljana Mayer ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Protein Complex ◽  
Specific Activity ◽  
Reductase Activity ◽  
Shewanella Oneidensis ◽  
Stable Complex ◽  
Wild Type ◽  
Functional Protein ◽  
Transport Pathway ◽  
High Affinity

ABSTRACT Shewanella oneidensis MR-1 is a facultatively anaerobic bacterium capable of using soluble and insoluble forms of manganese [Mn(III/IV)] and iron [Fe(III)] as terminal electron acceptors during anaerobic respiration. To assess the structural association of two outer membrane-associated c-type decaheme cytochromes (i.e., OmcA [SO1779] and MtrC [SO1778]) and their ability to reduce soluble Fe(III)-nitrilotriacetic acid (NTA), we expressed these proteins with a C-terminal tag in wild-type S. oneidensis and a mutant deficient in these genes (i.e., ΔomcA mtrC). Endogenous MtrC copurified with tagged OmcA in wild-type Shewanella, suggesting a direct association. To further evaluate their possible interaction, both proteins were purified to near homogeneity following the independent expression of OmcA and MtrC in the ΔomcA mtrC mutant. Each purified cytochrome was confirmed to contain 10 hemes and exhibited Fe(III)-NTA reductase activity. To measure binding, MtrC was labeled with the multiuse affinity probe 4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein (1,2-ethanedithiol)2, which specifically associates with a tetracysteine motif engineered at the C terminus of MtrC. Upon titration with OmcA, there was a marked increase in fluorescence polarization indicating the formation of a high-affinity protein complex (Kd < 500 nM) between MtrC and OmcA whose binding was sensitive to changes in ionic strength. Following association, the OmcA-MtrC complex was observed to have enhanced Fe(III)-NTA reductase specific activity relative to either protein alone, demonstrating that OmcA and MtrC can interact directly with each other to form a stable complex that is consistent with their role in the electron transport pathway of S. oneidensis MR-1.

scholarly journals Modulation of cell migration by integrin-mediated cytoskeletal linkages and ligand-binding affinity.

1996 ◽  
Vol 134 (6) ◽  
pp. 1551-1562 ◽  
Author(s):  
A Huttenlocher ◽  
M H Ginsberg ◽  
A F Horwitz
Keyword(s):  
Cell Migration ◽  
Ligand Binding ◽  
Cho Cells ◽  
Substrate Concentration ◽  
Migration Rate ◽  
Focal Adhesions ◽  
Time Lapse ◽  
Integrin Receptors ◽  
High Affinity ◽  
Substrate Concentrations

Integrin cell surface adhesion receptors play a central role in mediating cell migration. We have developed a model system consisting of CHO cells ectopically expressing the alpha IIb beta 3 integrin to study integrin affinity and cytoskeletal interactions during cell migration. The alpha IIb beta 3 integrins are suited for study of integrin receptors during cell migration because they are well characterized with respect to ligand binding, cytoskeletal interactions, and signal transduction, and mutants with altered receptor function are available. The alpha IIb beta 3 receptor specifically mediates migration of alpha IIb beta 3-transfected CHO cells. The migration of transfected CHO cells was studied on a fibrinogen substrate both by time lapse videomicroscopy and by random and haptotactic transwell assays. Haptotactic and random transwell assays measured distinct aspects of migration, with the random transwell assay correlating most closely with time lapse videomicroscopy. Mutations in the cytoplasmic domains that increase ligand affinity or activation of the alpha IIb beta 3 receptor into a high affinity state by the LIBS6 antibody decreased the migration rate. Likewise, mutations that increase cytoskeletal organization without affecting affinity also decreased the migration rate. In contrast, truncation of the beta chain, which alters cytoskeletal associations as assayed by absence of focal adhesions, decreased haptotactic migration while increasing random migration. These effects on the migration rate were partially compensated for by altering substrate concentration, demonstrating optimum substrate concentrations that supported maximal migration. For example, cells expressing integrins locked in the high affinity state showed maximal migration at lower substrate concentrations than cells expressing low affinity receptor. Together, these results implicate the strength of adhesion between cell and substrate, as modulated by receptor affinity, organization of adhesive complexes, and substrate concentration, as important regulators of cell migration rate. Further, we demonstrate a dominant effect of high affinity integrin in inhibiting migration regardless of the organization of adhesive complexes. These observations have potential implications for tumor metastasis and its therapy.

scholarly journals ScOPT1 and AtOPT4 function as proton-coupled oligopeptide transporters with broad but distinct substrate specificities

2005 ◽  
Vol 393 (1) ◽  
pp. 267-275 ◽  
Author(s):  
Hiroki Osawa ◽  
Gary Stacey ◽  
Walter Gassmann
Keyword(s):  
Xenopus Laevis Oocytes ◽  
Transport Mechanisms ◽  
Biological Functions ◽  
Yeast Growth ◽  
Substrate Specificities ◽  
High Affinity ◽  
Electrode Voltage ◽  
Inward Currents ◽  
Derivatives Of ◽  
Substrate Concentrations

A group of OPTs (oligopeptide transporters) exclusively identified in plants and fungi are proposed to transport oligopeptides and derivatives of three to six amino acids in length, but their transport mechanisms and biological functions are poorly understood. We expressed the Saccharomyces cerevisiae (yeast) OPT ScOPT1 and five Arabidopsis thaliana AtOPTs in Xenopus laevis oocytes for two-electrode voltage-clamp studies. ScOPT1 produced inward currents in response to GSH or GSSG, the phytochelatin (PC) PC2 and oligopeptides including the tetrapeptide GGFL, but not KLGL. Inward currents were dependent on the external proton and substrate concentrations, with high affinity for both. This and the inward currents evoked by substrates with net negative charges showed that ScOPT1 is a proton-coupled transporter. ScOPT1 displayed highest apparent affinity for PC2, with small differences in the maximal current among substrates. Glutathione transport by any of the tested AtOPTs, including AtOPT6, was not detected in yeast growth complementation assays. With AtOPT4, initially only small KLGL-dependent currents were recorded in batches of oocytes showing high ScOPT1 expression. AtOPT4 expression was optimized by swapping the 5′-untranslated region with that of ScOPT1. AtOPT4 displayed a higher affinity for KLGL than ScOPT1 did for any peptide. AtOPT4-mediated KLGL transport was detectable at pH 5.0, but not at pH 6.0 or 7.0. Taken together, our results demonstrate that ScOPT1 and AtOPT4 are proton-coupled OPTs with broad but distinct substrate specificities and affinities.

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