scholarly journals Characterization of the high-affinity monocarboxylate transporter MCT2 in Xenopus laevis oocytes

1999 ◽  
Vol 341 (3) ◽  
pp. 529-535 ◽  
Author(s):  
Stefan BRÖER ◽  
Angelika BRÖER ◽  
Hans-Peter SCHNEIDER ◽  
Carola STEGEN ◽  
Andrew P. HALESTRAP ◽  
...  
Keyword(s):  
Ketone Bodies ◽  
Anion Channel ◽  
Monocarboxylate Transporter ◽  
Xenopus Laevis Oocytes ◽  
Lactate Transport ◽  
High Affinity ◽  
Monocarboxylate Transport ◽  
Km Value ◽  
Decreasing Ph

Observations on lactate transport in brain cells and cardiac myocytes indicate the presence of a high-affinity monocarboxylate transporter. The rat monocarboxylate transporter isoform MCT2 was analysed by expression in Xenopus laevisoocytes and the results were compared with the known characteristics of lactate transport in heart and brain. Monocarboxylate transport via MCT2 was driven by the H+ gradient over the plasma membrane. Uptake of lactate strongly increased with decreasing pH, showing half-maximal stimulation at pH 7.2. A wide variety of monocarboxylates and ketone bodies, including lactate, pyruvate, β-hydroxybutyrate, acetoacetate, 2-oxoisovalerate and 2-oxoisohexanoate, were substrates of MCT2. All substrates had a high affinity for MCT2. For lactate a Km value of 0.74±0.07 mM was determined at pH 7.0. For the other substrates, Ki values between 100 μM and 1 mM were measured for inhibition of lactate transport, which is about one-tenth of the corresponding values for the ubiquitously expressed monocarboxylate transporter isoform MCT1. Monocarboxylate transport via MCT2 could be inhibited by α-cyano-4-hydroxycinnamate, anion-channel inhibitors and flavonoids. It is suggested that cells which express MCT2 preferentially use lactate and ketone bodies as energy sources.

scholarly journals Characterization of the monocarboxylate transporter 1 expressed in Xenopus laevis oocytes by changes in cytosolic pH

1998 ◽  
Vol 333 (1) ◽  
pp. 167-174 ◽  
Author(s):  
Stefan BRÖER ◽  
Hans-Peter SCHNEIDER ◽  
Angelika BRÖER ◽  
Basim RAHMAN ◽  
Bernd HAMPRECHT ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Intracellular Ph ◽  
Expression System ◽  
Lactate Concentration ◽  
Monocarboxylate Transporter ◽  
Kinetic Properties ◽  
Xenopus Laevis Oocytes ◽  
Lactate Transport ◽  
Monocarboxylate Transporter 1

Several laboratories have investigated monocarboxylate transport in a variety of cell types. The characterization of the cloned transporter isoforms in a suitable expression system is nevertheless still lacking. H+/monocarboxylate co-transport was therefore investigated in monocarboxylate transporter 1 (MCT1)-expressing Xenopus laevis oocytes by using pH-sensitive microelectrodes and [14C]lactate. Superfusion with lactate resulted in intracellular acidification of MCT1-expressing oocytes, but not in non-injected control oocytes. The basic kinetic properties of lactate transport in MCT1-expressing oocytes were determined by analysing the rates of intracellular pH changes under different conditions. The results were in agreement with the known properties of the transporter, with respect to both the dependence on the lactate concentration and the external pH value. Besides lactate, MCT1 mediated the reversible transport of a wide variety of monocarboxylic acids including pyruvate, d,l-3-hydroxybutyrate, acetoacetate, α-oxoisohexanoate and α-oxoisovalerate, but not of dicarboxylic and tricarboxylic acids. The inhibitor α-cyano-4-hydroxycinnamate bound strongly to the transporter without being translocated, but could be displaced by the addition of lactate. In addition to changes in the intracellular pH, lactate transport also induced deviations from the resting membrane potential.

An endogenous monocarboxylate transport inXenopus laevisoocytes

2000 ◽  
Vol 278 (5) ◽  
pp. R1190-R1195 ◽  
Author(s):  
M. Tosco ◽  
M. N. Orsenigo ◽  
G. Gastaldi ◽  
A. Faelli
Keyword(s):  
Xenopus Laevis ◽  
Monocarboxylate Transporter ◽  
Monocarboxylate Transporters ◽  
Xenopus Laevis Oocytes ◽  
Lactate Transport ◽  
Ph Gradients ◽  
Monocarboxylate Transport ◽  
Functional Features ◽  
Lactate Uptake ◽  
Uptake Studies

We investigated the existence of an endogenous system for lactate transport in Xenopus laevis oocytes.36Cl-uptake studies excluded the involvement of a DIDS-sensitive anion antiporter as a possible pathway for lactate movement.l-[14C]lactate uptake was unaffected by superimposed pH gradients, stimulated by the presence of Na+in the incubating solution, and severely reduced by the monocarboxylate transporter inhibitor p-chloromercuribenzenesulphonate (pCMBS). Transport exhibited a broad cation specificity and was cis inhibited by other monocarboxylates, mostly by pyruvate. These results suggest that lactate uptake is mediated mainly by a transporter and that the preferred anion is pyruvate. [14C]pyruvate uptake exhibited the same pattern of functional properties evidenced for l-lactate. Kinetic parameters were calculated for both monocarboxylates, and a higher affinity for pyruvate was revealed. Various inhibitors of monocarboxylate transporters reduced significantly pyruvate uptake. These studies demonstrate that Xenopus laevis oocytes possess a monocarboxylate transport system that shares some functional features with the members of the mammalian monocarboxylate cotransporters family, but, in the meanwhile, exhibits some particular properties, mainly concerning cation specificity.

scholarly journals The low-affinity monocarboxylate transporter MCT4 is adapted to the export of lactate in highly glycolytic cells

2000 ◽  
Vol 350 (1) ◽  
pp. 219-227 ◽  
Author(s):  
Kai-Stefan DIMMER ◽  
Björn FRIEDRICH ◽  
Florian LANG ◽  
Joachim W. DEITMER ◽  
Stefan BRÖER
Keyword(s):  
Mammalian Cells ◽  
Short Chain Fatty Acids ◽  
Monocarboxylate Transporter ◽  
Xenopus Laevis Oocytes ◽  
Cytosolic Ph ◽  
Monocarboxylate Transport ◽  
Thiol Reagent ◽  
Km Value ◽  
Flux Measurements ◽  
Disulphonic Acid

Transport of lactate and other monocarboxylates in mammalian cells is mediated by a family of transporters, designated monocarboxylate transporters (MCTs). The MCT4 member of this family has recently been identified as the major isoform of white muscle cells, mediating lactate efflux out of glycolytically active myocytes [Wilson, Jackson, Heddle, Price, Pilegaard, Juel, Bonen, Montgomery, Hutter and Halestrap (1998) J. Biol. Chem. 273, 15920–15926]. To analyse the functional properties of this transporter, rat MCT4 was expressed in Xenopus laevis oocytes and transport activity was monitored by flux measurements with radioactive tracers and by changes of the cytosolic pH using pH-sensitive microelectrodes. Similar to other members of this family, monocarboxylate transport via MCT4 is accompanied by the transport of H+ across the plasma membrane. Uptake of lactate strongly increased with decreasing extracellular pH, which resulted from a concomitant drop in the Km value. MCT4 could be distinguished from the other isoforms mainly in two respects. First, MCT4 is a low-affinity MCT: for l-lactate Km values of 17±3mM (pH-electrode) and 34±5mM (flux measurements with l-[U-14C]lactate) were determined. Secondly, lactate is the preferred substrate of MCT4. Km values of other monocarboxylates were either similar to the Km value for lactate (pyruvate, 2-oxoisohexanoate, 2-oxoisopentanoate, acetoacetate) or displayed much lower affinity for the transporter (β-hydroxybutyrate and short-chain fatty acids). Under physiological conditions, rat MCT will therefore preferentially transport lactate. Monocarboxylate transport via MCT4 could be competitively inhibited by α-cyano-4-hydroxycinnamate, phloretin and partly by 4,4´-di-isothiocyanostilbene-2,2´-disulphonic acid. Similar to MCT1, monocarboxylate transport via MCT4 was sensitive to inhibition by the thiol reagent p-chloromercuribenzoesulphonic acid.

scholarly journals Characterization of the high-affinity monocarboxylate transporter MCT2 in Xenopus laevis oocytes

1999 ◽  
Vol 341 (3) ◽  
pp. 529 ◽  
Author(s):  
Stefan BRÖER ◽  
Angelika BRÖER ◽  
Hans-Peter SCHNEIDER ◽  
Carola STEGEN ◽  
Andrew P. HALESTRAP ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Monocarboxylate Transporter ◽  
Xenopus Laevis Oocytes ◽  
High Affinity

scholarly journals The loop between helix 4 and helix 5 in the monocarboxylate transporter MCT1 is important for substrate selection and protein stability

2003 ◽  
Vol 376 (2) ◽  
pp. 413-422 ◽  
Author(s):  
Sandra GALIĆ ◽  
Hans-Peter SCHNEIDER ◽  
Angelika BRÖER ◽  
Joachim W. DEITMER ◽  
Stefan BRÖER
Keyword(s):  
Mammalian Cells ◽  
Ketone Bodies ◽  
Monocarboxylate Transporter ◽  
Substrate Selection ◽  
Transport Activity ◽  
Complete Inactivation ◽  
Transporter Family ◽  
Monocarboxylate Transport ◽  
Critical Residues ◽  
Signature Sequence

Transport of lactate, pyruvate and the ketone bodies acetoacetate and β-hydroxybutyrate, is mediated in most mammalian cells by members of the monocarboxylate transporter family (SLC16). A conserved signature sequence has been identified in this family, which is located in the loop between helix 4 and helix 5 and extends into helix 5. We have mutated residues in this signature sequence in the rat monocarboxylate transporter (MCT1) to elucidate the significance of this region for monocarboxylate transport. Mutation of R143 and G153 resulted in complete inactivation of the transporter. For the MCT1(G153V) mutant this was explained by a failure to reach the plasma membrane. The lack of transport activity of MCT1(R143Q) could be partially rescued by the conservative exchange R143H. The resulting mutant transporter displayed reduced stability, a decreased Vmax of lactate transport but not of acetate transport, and an increased stereoselectivity. Mutation of K137, K141 and K142 indicated that only K142 played a significant role in the transport mechanism. Mutation of K142 to glutamine resulted in an increase of the Km for lactate from 5 mM to 12 mM. In contrast with MCT1(R143H), MCT1(K142Q) was less stereoselective than the wild-type. A mechanism is proposed that includes all critical residues.

scholarly journals Substrate and inhibitor specificity of monocarboxylate transport into heart cells and erythrocytes. Further evidence for the existence of two distinct carriers

1990 ◽  
Vol 269 (3) ◽  
pp. 827-829 ◽  
Author(s):  
R C Poole ◽  
S L Cranmer ◽  
A P Halestrap ◽  
A J Levi
Keyword(s):  
Guinea Pig ◽  
Cardiac Myocytes ◽  
Ketone Bodies ◽  
Heart Cell ◽  
Heart Cells ◽  
Rat Erythrocytes ◽  
High Affinity ◽  
Cell Carrier ◽  
Monocarboxylate Transport ◽  
Lactate And Pyruvate

A range of short-chain aliphatic monocarboxylates, both unsubstituted and substituted with hydroxy, chloro and keto groups, were shown to inhibit transport of L-lactate and pyruvate into both guinea-pig cardiac myocytes and rat erythrocytes. The carrier of heart cells exhibited a higher affinity (approx. 10-fold) for most of the monocarboxylates than did the erythrocyte carrier. A notable exception was L-lactate, whose Km for both carriers was similar. The K1 values of the two carriers for inhibitors such as phenylpyruvate and alpha-cyanocinnamate derivatives were also different. The high affinity of the heart cell carrier for ketone bodies and acetate may be physiologically important, since these substrates are used as fuels by the heart.

scholarly journals First Dye-Decolorizing Peroxidase from an Ascomycetous Fungus Secreted by Xylaria grammica

Biomolecules ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 1391
Author(s):  
Virginia Kimani ◽  
René Ullrich ◽  
Enrico Büttner ◽  
Robert Herzog ◽  
Harald Kellner ◽  
...  
Keyword(s):  
Amino Acids ◽  
Physiological Function ◽  
Aromatic Amino Acids ◽  
Veratryl Alcohol ◽  
Lignocellulose Degradation ◽  
High Affinity ◽  
Acidic Amino Acids ◽  
Km Value ◽  
Ascomycetous Fungus

Background: Fungal DyP-type peroxidases have so far been described exclusively for basidiomycetes. Moreover, peroxidases from ascomycetes that oxidize Mn2+ ions are yet not known. Methods: We describe here the physicochemical, biocatalytic, and molecular characterization of a DyP-type peroxidase (DyP, EC 1.11.1.19) from an ascomycetous fungus. Results: The enzyme oxidizes classic peroxidase substrates such as 2,6-DMP but also veratryl alcohol and notably Mn2+ to Mn3+ ions, suggesting a physiological function of this DyP in lignin modification. The KM value (49 µM) indicates that Mn2+ ions bind with high affinity to the XgrDyP protein but their subsequent oxidation into reactive Mn3+ proceeds with moderate efficiency compared to MnPs and VPs. Mn2+ oxidation was most effective at an acidic pH (between 4.0 and 5.0) and a hypothetical surface exposed an Mn2+ binding site comprising three acidic amino acids (two aspartates and one glutamate) could be localized within the hypothetical XgrDyP structure. The oxidation of Mn2+ ions is seemingly supported by four aromatic amino acids that mediate an electron transfer from the surface to the heme center. Conclusions: Our findings shed new light on the possible involvement of DyP-type peroxidases in lignocellulose degradation, especially by fungi that lack prototypical ligninolytic class II peroxidases.

Characterization of a Mode of Specific Binding of Fibrin Monomer through its Amino-Terminal Domain by Macrophages and Macrophage Cell-Lines

1990 ◽  
Vol 63 (02) ◽  
pp. 193-203 ◽  
Author(s):  
John R Shainoff ◽  
Deborah J Stearns ◽  
Patricia M DiBello ◽  
Youko Hishikawa-Itoh
Keyword(s):  
Peritoneal Macrophages ◽  
Affinity Binding ◽  
Macrophage Cell ◽  
High Affinity Binding ◽  
Amino Terminal ◽  
High Affinity ◽  
Terminal Domain ◽  
Weak Binding ◽  
Amino Terminal Domain

SummaryThe studies reported here probe the existence of a receptor-mediated mode of fibrin-binding by macrophages that is associated with the chemical change underlying the fibrinogen-fibrin conversion (the release of fibrinopeptides from the amino-terminal domain) without depending on fibrin-aggregation. The question is pursued by 1) characterization of binding in relation to fibrinopeptide content of both the intact protein and the CNBr-fragment comprising the amino-terminal domain known as the NDSK of the protein, 2) tests of competition for binding sites, and 3) photo-affinity labeling of macrophage surface proteins. The binding of intact monomers of types lacking either fibrinopeptide A alone (α-fibrin) or both fibrinopeptides A and B (αβ-fibrin) by peritoneal macrophages is characterized as proceeding through both a fibrin-specific low density/high affinity (BMAX ≃ 200–800 molecules/cell, KD ≃ 10−12 M) interaction that is not duplicated with fibrinogen, and a non-specific high density/low affinity (BMAX ≥ 105 molecules/cell, KD ≥ 10−6 M) interaction equivalent to the weak binding of fibrinogen. Similar binding characteristics are displayed by monocyte/macrophage cell lines (J774A.1 and U937) as well as peritoneal macrophages towards the NDSK preparations of these proteins, except for a slightly weaker (KD ≃ 10−10 M) high-affinity binding. The high affinity binding of intact monomer is inhibitable by fibrin-NDSK, but not fibrinogen-NDSK. This binding appears principally dependent on release of fibrinopeptide-A, because a species of fibrin (β-fibrin) lacking fibrinopeptide-B alone undergoes only weak binding similar to that of fibrinogen. Synthetic Gly-Pro-Arg and Gly-His-Arg-Pro corresponding to the N-termini of to the α- and the β-chains of fibrin both inhibit the high affinity binding of the fibrin-NDSKs, and the cell-adhesion peptide Arg-Gly-Asp does not. Photoaffinity-labeling experiments indicate that polypeptides with elec-trophoretically estimated masses of 124 and 187 kDa are the principal membrane components associated with specifically bound fibrin-NDSK. The binding could not be up-regulated with either phorbol myristyl acetate, interferon gamma or ADP, but was abolished by EDTA and by lipopolysaccharide. Because of the low BMAX, it is suggested that the high-affinity mode of binding characterized here would be too limited to function by itself in scavenging much fibrin, but may act cooperatively with other, less limited modes of fibrin binding.

Acid-Sensing Ion Channels

2020 ◽  
Author(s):  
Stefan Gründer
Keyword(s):  
Nervous System ◽  
Ion Channels ◽  
Excitatory Synapses ◽  
Detailed Characterization ◽  
High Affinity ◽  
Acid Sensing Ion Channels ◽  
Long Time ◽  
Pathological Pain

Acid-sensing ion channels (ASICs) are proton-gated Na+ channels. Being almost ubiquitously present in neurons of the vertebrate nervous system, their precise function remained obscure for a long time. Various animal toxins that bind to ASICs with high affinity and specificity have been tremendously helpful in uncovering the role of ASICs. We now know that they contribute to synaptic transmission at excitatory synapses as well as to sensing metabolic acidosis and nociception. Moreover, detailed characterization of mouse models uncovered an unanticipated role of ASICs in disorders of the nervous system like stroke, multiple sclerosis, and pathological pain. This review provides an overview on the expression, structure, and pharmacology of ASICs plus a summary of what is known and what is still unknown about their physiological functions and their roles in diseases.

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