Interactions between the thiol-group reagent N-ethylmaleimide and neutral and basic amino acid transporter-related amino acid transport

Peter, George J.; Davies, Anthony M.; Watt, Peter; Birrell, Jacqueline; Taylor, Peter M.

doi:10.1042/0264-6021:3430169

Cited by 11 publications

(23 citation statements)

References 10 publications

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“…Since glycerol uptake was also inhibited by pCMBS, a thiol-modifying reagent, cysteine residues, which have a thiol group, seem to play an important role in the function of the carrier, as suggested for Na ϩ -dependent D-glucose transporter 1 (SGLT1) 8) and several other carriers. [9][10][11][12][13] At the higher concentration of 40 mM (Tables 1, 2), glycerol uptake was not altered by NaCl substitution or DNP, consistent with the suggestion that the contribution of passive transport is predominant.…”

Section: Time-courses Of Glycerol Uptakesupporting

confidence: 66%

Functional Characterization of the Carrier-Mediated Transport System for Glycerol in Everted Sacs of the Rat Small Intestine

Kato

Hayashi

Inoue

et al. 2004

Biological & Pharmaceutical Bulletin

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The mechanism of intestinal glycerol transport was investigated by using the in vitro everted sac method involving the rat small intestine. The uptake of glycerol into everted sacs was saturable with a Michaelis constant (K m ) of 0.77 mM and a maximum transport rate (J max ) of 11.5 nmol/min/100 mg wet tissue weight (wtw), suggesting the involvement of carrier-mediated transport, and was accompanied by unsaturable transport (passive transport) with a membrane permeability clearance (CL m,d ) of 4.9 m ml/min/100 mg wtw. The carrier-mediated uptake of glycerol was inhibited by the removal of Na ؉ and also by the addition of 2,4-dinitrophenol (DNP) and sodium azide (NaN 3 ), which are metabolic inhibitors. These results suggest that the carrier-mediated glycerol transport is Na ؉ -dependent and secondary active. Since glycerol uptake was also inhibited by p-chloromercuribenzene sulfonate (pCMBS), a thiol-modifying reagent, cysteine residues, which have a thiol group, seem to play an important role in the function of the carrier. We further found that glycerol uptake was selectively inhibited by glycerol-3-phosphate, chloramphenicol and voglibose, which are alcohol-related compounds analogous to glycerol. Several other compounds that did not inhibit glycerol uptake included D-glucose and 5-fluorouracil, which are known to be transported by specific carriers, and none of the selective inhibitors of glycerol uptake inhibited the uptake of D-glucose and 5-fluorouracil. Therefore, the carriers for these two compounds do not seem to be involved in glycerol uptake. It is likely that the carrier-mediated transport system involved in glycerol uptake is specific to glycerol and, possibly, some analogous compounds with hydroxyl groups. It would be interesting to examine the possibility that the carrier-mediated glycerol transport system might be involved in drug absorption and also that it might be used for oral drug delivery.

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Section: Time-courses Of Glycerol Uptakesupporting

confidence: 66%

Functional Characterization of the Carrier-Mediated Transport System for Glycerol in Everted Sacs of the Rat Small Intestine

Kato

Hayashi

Inoue

et al. 2004

Biological & Pharmaceutical Bulletin

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“…Inhibition of L-arginine uptake in the presence of Na ϩ by L-leucine is a known phenomena of CAT family. 24 Especially, characteristic inhibition by N-ethylmaleimide (NEM), which distinguishes CAT from other L-arginine transporters such as y ϩ L, 25,26 showed that CAT is mainly responsible for transport of L-arginine into K562 cells, although incomplete inhibition by NEM suggests a minor contribution of other transporters. Thus, CAT1 could be predominantly responsible for L-arginine uptake into erythroleukemic K562 cells.…”

Section: Discussionmentioning

confidence: 99%

l-arginine import via cationic amino acid transporter CAT1 is essential for both differentiation and proliferation of erythrocytes

Shima

Maeda

Aizawa

et al. 2006

Blood

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In the present study, we examined the role in hematopoiesis of cationic amino acid transporter 1 (CAT1) , IntroductionCytokines such as interleukin-3 (IL-3) and erythropoietin play an important role in the differentiation of hematopoietic stem cells. 1,2 However, the roles of small molecules such as vitamins, nucleic acids, and amino acids in cell differentiation have not been elucidated well, though it is likely that specific transporters are involved in importing such small hydrophilic molecules into the cells. The ABC transporter Bcrp1 (abcg2) is expressed mainly in hematopoietic stem cells, 3 and more recently, efflux transporter activities in murine hematopoietic stem cells (HSCs) were found to vary according to developmental and activation status. 4 Thiamine transporter Thtr-1 (slc19a2) gene knockout mice showed abnormalities of erythroid, myeloid, and megakaryocyte lineages in bone marrow when fed a thiamine-free diet. 5 The importance of CAT1 (cationic amino acid transporter)-mediated transport was recently underscored by the production of cat1 gene knockout mice, which exhibit anemia. 6 These reports indicate that substrates of transporters expressed on hematopoietic stem cells are physiologically essential in differentiation or proliferation of the cells, or both, and the expression levels and activities of these transporters are likely to be regulated stage and lineage specifically.Cationic amino acid transporter (CAT, slc7a), known as system y ϩ , transports cationic amino acids such as L-lysine, L-histidine, L-ornithine, and L-arginine. 7-9 System y ϩ is a facilitative process that is Na ϩ -independent and pH insensitive. CAT1 (slc7a1), whose mouse ortholog was first identified as a virus receptor, 10,11 is expressed ubiquitously, including fetal liver and bone marrow, though not adult liver. 12 It is known that fetal and embryonic liver plays a significant role in hematopoiesis. 13 Furthermore, CAT1 mRNA is induced in regenerating liver in a short-lived manner and has multiple sites for regulation of gene expression, indicating that system y ϩ is tightly regulated and essential for liver cells to enter mitosis. 14,15 These findings suggest that CAT1 could be involved in hematopoietic activity.One of the substrates of CAT1, L-arginine, is a precursor of nitric oxide (NO) and polyamines. NO can induce apoptosis in megakaryocytes and platelet formation. 16,17 L-ornithine is produced by arginase and further metabolized to polyamines, which regulate the cell cycle. 18 Recently, low concentrations of L-glutamine were reported to induce functional differentiation of U937 myelomonocytic cells. 19 These facts suggest that low-molecular-weight molecules such as amino acids may be involved in hematopoiesis, as well as large-molecular cytokines such as IL-3 and erythropoietin. In the present study, we investigated the effects of hCAT1 expression level and L-arginine on differentiation and proliferation of human cord blood cells and a cloned human erythroid model, K562 cells. Materials and methods Tissue cultur...

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“…Oocytes were washed three times in uptake buffer before measuring 65 Zn 2ϩ incorporation into single oocytes as ␥ emission. Measurement of zinc efflux was based on a method described previously (13). Fifty nanoliters of 65 ZnCl 2 at a specific activity of 6.4 Ci/ml were injected into the cytoplasm of individual oocytes.…”

Section: Methodsmentioning

confidence: 99%

ZnT5 Variant B Is a Bidirectional Zinc Transporter and Mediates Zinc Uptake in Human Intestinal Caco-2 Cells

Valentine

Jackson

Christie

et al. 2007

Journal of Biological Chemistry

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101

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Zinc is an essential micronutrient, so it is important to elucidate the molecular mechanisms of zinc homeostasis, including the functional properties of zinc transporters. Mammalian zinc transporters are classified in two major families: the SLC30 (ZnT) family and the SLC39 family. The prevailing view is that SLC30 family transporters function to reduce cytosolic zinc concentration, either through efflux across the plasma membrane or through sequestration in intracellular compartments, and that SLC39 family transporters function in the opposite direction to increase cytosolic zinc concentration. We demonstrated that human ZnT5 variant B (ZnT5B (hZTL1)), an isoform expressed at the plasma membrane, operates in both the uptake and the efflux directions when expressed in Xenopus laevis oocytes. We measured increased activity of the zinc-responsive metallothionein 2a (MT2a) promoter when ZnT5b was coexpressed with an MT2a promoter-reporter plasmid construct in human intestinal Caco-2 cells, indicating increased total intracellular zinc concentration. Increased cytoplasmic zinc concentration mediated by ZnT5B, in the absence of effects on intracellular zinc sequestration by the Golgi apparatus or endoplasmic reticulum, was also confirmed by a dramatically enhanced signal from the zinc fluorophore Rhodzin-3 throughout the cytoplasm of Caco-2 cells overexpressing ZnT5B at the plasma membrane when compared with control cells. Our findings demonstrate clearly that, in addition to mediating zinc efflux, ZnT5B at the plasma membrane can function to increase cytoplasmic zinc concentration, thus indicating a need to reevaluate the current paradigm that SLC30 family zinc transporters operate exclusively to decrease cytosolic zinc concentration.

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Interactions between the thiol-group reagent N-ethylmaleimide and neutral and basic amino acid transporter-related amino acid transport

Cited by 11 publications

References 10 publications

Functional Characterization of the Carrier-Mediated Transport System for Glycerol in Everted Sacs of the Rat Small Intestine

Functional Characterization of the Carrier-Mediated Transport System for Glycerol in Everted Sacs of the Rat Small Intestine

l-arginine import via cationic amino acid transporter CAT1 is essential for both differentiation and proliferation of erythrocytes

ZnT5 Variant B Is a Bidirectional Zinc Transporter and Mediates Zinc Uptake in Human Intestinal Caco-2 Cells

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