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1.
The transport of glycine and L-lysine into murine P388 leukemia cells has been examined. Glycine transport appears to be shared by both systems A and ASC in P388 cells. Glycine transport is Na+-dependent and is effectively blocked by alpha-(methylamino)isobutyric acid, threonine and alanine but only a marginal reduction in transport is seen with 100-fold excess cold 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid. System gly is not expressed in P388 cells. Lysine is largely transported by a Na+-independent, pH-insensitive system with a Km of 0.079 mM. Lysine transport is relatively unaffected by the addition of 100-fold excess cold alpha-(methylamino)isobutyric acid, 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid and the anionic amino acids, L-glutamate and L-aspartate. A partial inhibition of lysine transport was observed with L-threonine and L-leucine while L-arginine and L-histidine radically decreased lysine transport. Lysine appears to be transported by a system similar to the system y+ seen in cultured human fibroblasts, Ehrlich ascites cells, and hepatoma cell lines. 相似文献
2.
L M García-Segura J A Ferragut A V Ferrer-Montiel P V Escriba J M Gonzalez-Ros 《Biochimica et biophysica acta》1990,1029(1):191-195
Freeze-fracture studies of daunomycin-sensitive and daunomycin-resistant P388 cell lines, reveal a significant increase in the numerical density of intramembrane particles at both, the protoplasmic and the exoplasmic leaflets of the plasma membrane from the drug-resistant cells. Such change in plasma membrane architecture is not accompanied by overexpression of P-glycoproteins. Furthermore, drug-sensitive cells exhibited an increased number of exo-endocytotic images when compared to drug-resistant cells. Our observations suggest that there are global changes in the structural organization of the plasma membrane, which are related to the acquisition of the cellular drug-resistant phenotype. 相似文献
3.
4.
The uptake of L-leucine and L-lysine into vascular smooth muscle cells cultured from the aortas of rats has been investigated. Both amino acids are taken up by saturable systems that are independent of the presence of a ·Na+ gradient and can be stimulated in trans by neutral bulky amino acids for leucine and cationic amino acids for lysine. Leucine uptake is inhibited competitively in cis by several neutral amino acids, whereas lysine uptake is inhibited strongly by other cationic amino acids but also significantly by neutral amino acids such as leucine. The leucine inhibition is noncompetitive. Cells preloaded with leucine and lysine could also export these amino acids and the rate of efflux was stimulated by the presence of appropriate amino acids in trans. These data are all consistent with leucine being transported largely if not entirely by System L and lysine by the System y+ transporter. © 1993 Wiley-Liss, Inc. 相似文献
5.
Waud WR Gilbert KS Parker WB Secrist JA 《Nucleosides, nucleotides & nucleic acids》2011,30(11):826-838
A murine P388 leukemia line fully resistant to clofarabine was obtained after only two courses of intraperitoneal treatment (three times a day for nine consecutive days). The resistance was stable for at least 13 weeks without treatment. The subline was as sensitive to 5-fluorouracil, methotrexate, cyclophosphamide, cisplatin, melphalan, BCNU, doxorubicin, etoposide, irinotecan, vincristine, and docetaxel as was the parental P388/0 line but was cross-resistant to five antimetabolites [palmO-ara-C, 4'-thio-ara-C, fludarabine phosphate, cladribine, and gemcitabine-all of which require deoxycytidine kinase for activation] and paclitaxel. The subline had less than 1% of the deoxycytidine kinase activity in comparison to P388/0. 相似文献
6.
The regulation of amino acid transport in L6 muscle cells by amino acid deprivation was investigated. Proline uptake was Na+-dependent, saturable and concentrative, and was predominantly through system A. Proline uptake was inhibited by alanine, α-amino isobutyric acid (AIB), and by α-methylamino isobutyric acid, but not by lysine or valine. At 25°C, Km of proline uptake was 0.5 mM. Amino acid-deprivation resulted in a progressive increase in the rate of proline uptake, reaching up to 6-fold stimulation after 6 hours. The basal and stimulated transport were equally Na+-dependent, and both were inhibited by competition with the same amino acids. Kinetic analysis showed that Km decreased by a factor of 2.4 and Vmax increased 1.9-fold in deprived cells. Amino acid-deprivation did not stimulate amino acid uptake through systems other than system A. This suggests that the higher Km in proline-supplemented cells is not due to release of intracellular amino acids into unstirred layers surrounding the cells. The presence of amino acids which are substrates of system A (including AIB) during proline-deprivation, prevented stimulation of proline uptake, whereas those transported by systems Ly+ or L exclusively were ineffective. The stimulation of the transport-rate in deprived cells could be reversed by subsequent exposure to proline or other substrates of system A. L6 cells, deprived of proline for 6 hours, retained the stimulation of transport after detachment from the monolayers with trypsin. Uptake rates were comparable in suspended and attached cells in monolayer culture. Thus, amino acid-depreivation of L6 cells results in an adaptive increase in proline uptake, which is not due to unstirred layers but appears to be mediated by other mechanisms of selective transport regulation. 相似文献
7.
Amino acid starvation causes an adaptive increase in the initial rate of transport of selected neutral amino acids in an established line of rat hepatoma cells in tissue culture. After a lag of 30 min, the initial rate of transport of alpha-aminoisobutyric acid (AIB) increases to a maximum after 4 to 6 h starvation of 2 to 3 times that seen in control cells. The increased rate of transport is accompanied by an increase in the Vmax and a modest decrease in the Km for this transport system, and is reversed by readdition of amino acids. The enhancement is specific for amino acids transported by the A or alanine-preferring system (AIB, glycine, proline); uptake of amino acids transported by the L or leucine-preferring system (threonine, phenylalanine, tyrosine, leucine) or the Ly+ system for dibasci amino acids (lysine) is decreased under these conditions. Amino acids which compete with AIB for transport also prevent the starvation-induced increase in AIB transport; amino acids which do not compete fail to prevent the enhancement. Paradoxically threonine, phenylalanine, tryptophan, and tyrosine, which do not compete with AIB for transport, block the enhancement of transport upon amino acid starvation. The starvation-induced enhancement of amino acid transport does not appear to be the result of a release from transinhibition. After 30 min of amino acid starvation, AIB transport is either unchanged or slightly decreased even though amino acid pools are already depleted. Furthermore, loading cells with high concentrations of a single amino acid following a period of amino acid starvation fails to prevent the enhancement of AIB transport, whereas incubation of the cells with the single amino acid for the entire duration of amino acid starvation prevents the enhancement; intracellular amino acid pools are similar under both conditions. The enhancement of amino acid transport requires concomitant RNA and protein synthesis, consistent with the view that the adaptive increase reflects an increased amount of a rate-limiting protein involved in the transport process. Dexamethasone, which dramatically inhibits AIB transport in cells incubated in amino acid-containing medium, both blocks the starvation-induced increase in AIB transport, and causes a time-dependent decrease in transport velocity in cells whose transport has previously been enhanced by starvation. 相似文献
8.
N M Shanbaky T S Samy R Rubin A Krishan 《International journal of peptide and protein research》1986,27(4):414-420
We have isolated an 84-fold adriamycin resistant subline, P388/R84, from mouse leukemia P388 cells by serial cultivation in methylcellulose in the presence of increasing drug concentrations. Electrophoresis of detergent soluble fractions of radiolabeled sensitive and resistant cells suggested marked alterations in the protein fractions of 160, 100, 60, 45, and 30 kd. In resistant clones labeled with 125I an increase in 160 and 100 kd proteins was accompanied by concomitant reduction in the 60, 45, and 30 kd proteins. In 35S methionine-labeled resistant cells, similar increases in the 160 and 100 kd components were observed but in contrast to 125I-labeled cells the 30 kd component was also higher. Alterations in surface proteins were confirmed in experiments where the cell extracts were adsorbed to concanavalin A polymers and extracted with 0.26 M methyl-alpha-D-mannopyranoside. Our data confirm earlier reported observations on cell-surface protein changes in cells resistant to anthracyclines and alkaloids. 相似文献
9.
The transport of neutral amino acids in marine pseudomonad B-16 (ATCC 19855) has been investigated. From patterns of competitive inhibition, mutant analysis, and kinetic data, two active transport systems with overlapping substrate specificities were distinguished and characterized. One system (DAG) served glycine, D-alanine, D-serine, and alpha-aminoisobutyric acid (AIB) and, to a lesser extent, L-alanine and possibly other related neutral D- and L-amino acids. The other system (LIV) showed high stereospecificity for neutral amino acids with the L configuration and served primarily to transport L-leucine, L-isoleucine, L-valine, and L-alanine. This system exhibited low affinity for alpha-aminoisobutyric acid. Neither system was able to recognize structural analogues with modified alpha-amino or alpha-carboxyl groups. The kinetic parameters for L-alanine transport by the DAG and LIV systems were determined with appropriate mutants defective in either system. For L-alanine, Kt values of 4.6 X 10(-5) and 1.9 X 10(-4) M and Vmax values of 6.9 and 20.8 nmol/min per mg of cell dry weight were obtained for transport via the DAG and LIV systems respectively. alpha-Aminoisobutyric acid transport heterogeneity was also resolved with the mutants, and Kt values of 2.8 X 10(-5) and 1.4 X 10(-3) M AIB were obtained for transport via the DAG and LIV systems, respectively. Both systems required Na+ for activity (0.3 M Na+ optimal) and in this regard are distinguished from systems of similar substrate specificity reported in nonmarine bacteria. 相似文献
10.
Characterization of a polyamine transport system in murine embryonic palate mesenchymal cells 总被引:2,自引:0,他引:2
Polyamines (putrescine, spermidine, and spermine) are normal cellular constituents able to modulate cellular proliferation and differentiation in a number of developing systems. Ornithine decarboxylase (ODC), the rate-limiting enzyme in the polyamine biosynthetic pathway, has been shown to be causally related to an increase in glycosaminoglycan synthesis in murine embryonic palatal mesenchyme cells (MEPM). In order to understand other mechanisms that exist to regulate polyamine levels in cells derived from the developing craniofacial area, the present study investigated the capacity of MEPM cells to accumulate exogenous putrescine and tests the hypothesis that polyamine transport can serve as an adaptational response of MEPM cells to a change in their ability to synthesize polyamines. Transport was initiated in confluent cultures of MEPM cells by the addition of 0.1 microCi/ml of 14C-putrescine. The rate of transport, monitored for 20-120 minutes, was found to be a time-dependent saturable process. The rate of initial transport, determined by incubating MEPM cells for 15 minutes in the presence of different concentrations (1.0-20.0 microM) of 14C-putrescine, was also found to be saturable, suggesting a carrier-mediated event. Lineweaver-Burk analysis of these data revealed an apparent Km of 5.78 microM and a Vmax of 2.63 nmol/mg protein/15 minutes. Transport measured either at 4 degrees C or in the presence of 2-4 DNP was dramatically inhibited. Thus, putrescine transport is an active process, dependent upon metabolic energy. Conditions in which 1) NaCl was iso-osmotically replaced with choline chloride or 2) the Na+-electrochemical gradient was dissipated with Na+, K+-specific ionophores resulted in a decreased rate of transport indicating that putrescine transport in these cells is Na+ dependent. Noncompetitive inhibition assays utilizing sulfhydryl reagents that blocked sulfhydryl groups inhibited putrescine transport, suggesting that sulfhydryl groups are important for putrescine uptake. Competitive inhibition assays demonstrated that while spermidine and spermine inhibited putrescine uptake, ornithine did not inhibit transport. Spermidine, spermine, and putrescine thus appear to share a common transport system that is separate from that for ornithine. Putrescine transport is subject to adaptive regulation in both exponentially growing and confluent cultures of MEPM cells.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
11.
Amino acid transport systems for alanine and leucine have been reconstituted into artificial lipid vesicles. Purified plasma membrane vesicles from Ehrlich ascites cells were dissolved in 2% sodium cholate, 1 mM dithiothreitol, 0.5 mM EDTA, a mixture which solubilized approximately 50% of the membrane protein. This solubilized protein fraction was further purified by a combination of ammonium sulfate precipitations, gel filtration, and DEAE-cellulose chromatography. A fraction containing approximately 15 Coomassie blue staining bands on sodium dodecyl sulfate gels was obtained. This material was reconstituted into liposomes, and preliminary results demonstrated transport of alanine and leucine dependent on a sodium gradient. In addition, an electrogenic gradient mediated by valinomycin-induced potassium diffusion seemed to stimulate alanine uptake further. 相似文献
12.
Harringtonine showed cross resistance in adriamycin-resistant murine leukemia P388 (P388/ADM) and human leukemia K562 (K562/ADM) cells. The relative resistance of the P388/ADM and K562/ADM cells to harringtonine was about 7 and 40, respectively. Calcium influx blockers, diltiazem and the biscoclaurine alkaloid cepharanthine enhanced the cytotoxicity of harringtonine in P388/ADM and K562/ADM cells. The extent of enhancement was different for the two drugs, and up to a 9- to 10-fold increase in harringtonine cytotoxicity occurred in P388/ADM cells, and 14- to 22-fold enhancement in K562/ADM cells with diltiazem or cepharanthine. Harringtonine resistance of P388/ADM was circumvented completely, and the resistance of K562/ADM was circumvented partially, by diltiazem or cepharanthine. The mechanism of enhanced cytotoxicity by diltiazem and cepharanthine is probably inhibition of active efflux of harringtonine in P388/ADM and K562/ADM cells. 相似文献
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14.
Methionine had been observed to interact with two principal transport systems for amino acids in mammalian cells, the A and L systems. The present study of methionine transport and of exchange processes through system A arose in the course of a study to define the specificity of a transinhibition effect caused by cysteine. Methionine uptake through two transport systems in the S37 cell was confirmed by the occurrence of a biphasic double-reciprocal plot for labeled methionine uptake. Preloading cells with methionine stimulated labeled histidine uptake through systems A and L. Efflux of labeled methionine from cells was stimulated by histidine in a biphasic manner, so that bothe systems A and L can be used for exchange when methionine is the intracellular amino acid. Aminocycloheptanecarboxylic acid elicited exchange efflux of labeled methionine only through system L. ALPHA-Aminoisobutyric acid and N-methyl-alpha-aminoisobutyric acid both stimulated efflux of labeled N-methyl-alpha-aminoisobutyric acid from S37 cells. These findings are interpreted a showing that transport system A is capable of functioning as an exchange system depending upon the identity of intracellular and extracellular substrates available. 相似文献
15.
The transport of phenylalanine by the general aromatic transport system in spheroplasts of Escherichia coli 9723 has been found to be stimulated by exogenous tRNA. Neither periodate-treated tRNA nor phenylalanine-charged tRNA stimulated, and the latter inhibited, phenylalanine uptake. Among preparations of specific tRNAs, tRNAPhe and tRNATyr were effective in stimulating the uptake of phenylalanine and tyrosine, respectively, and tRNAGlu and tRNAVal gave no detectable stimulation of phenylalanine or tyrosine transport. The preparation of tRNATyr was 10 times as active as unfractionated tRNA and gave as much as 167% stimulation of tyrosine transport. Correspondingly, the preparation of tRNAPhe was at least 3.5 times as active as the unfractionated tRNA and 2.5 times as active as the preparation of tRNATyr in stimulation of phenylalanine transport. Preliminary results in fractionation of the active component of tRNA for stimulating phenylalanine uptake show that the major activity resides in minor isoacceptor(s) tRNAPhe rather than the major component tRNAPhe, and the slight activity of preparations of tRNATyr is probably due to a contamination of the active tRNAPhe. Other preliminary results indicate that this type of stimulation occurs with uptake of other amino acids and their tRNA. 相似文献
16.
The regulation of amino acid transport in animal cells. 总被引:32,自引:0,他引:32
17.
Methionine had been observed to interact with two principal transport systems for amino acids in mammalian cells, the A and L systems. The present study of methionine transport and of exchange processes through system A arose in the course of a study to define the specificity of a transinhibition effect caused by cysteine.Methionine uptake through two transport systems in the S37 cell was confirmed by the occurrence of a biphasic double-reciprocal plot for labeled methionine uptake. Preloading cells with methionine stimulated labeled histidine uptake through both systems A and L. Efflux of labeled methionine from cells was stimulated by histidine in a biphasic manner, so that both systems A and L can be used for exchange when methionine is the intracellular amino acid. Aminocycloheptanecarboxylic acid elicited exchange efflux of labeled methionine only through system L. α-Aminoisobutyric acid and both stimulated efflux of labeled from S37 cells. These findings are interpreted a showing that transport system A is capable of functioning as an exchange system depending upon the identity of intracellular and extracellular substrates available. 相似文献
18.
A V Ferrer-Montiel J M Gonzalez-Ros J A Ferragut 《Biochimica et biophysica acta》1992,1104(1):111-116
When the anthracycline daunomycin (DNM) is incorporated into isolated plasma membranes from P388 murine leukemia cells, the drug partitions between 'deep' and 'surface' membrane domains. Such domains have been characterized on the basis of: (1) fluorescence resonance energy transfer between 1,6-diphenylhexa-1,3,5-triene or 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene as energy donors, which are well known in their positioning within the membrane, and daunomycin as the energy acceptor, and (2) quenching of the fluorescence of the membrane-associated drug by the water-soluble quencher iodide. The distribution of DNM between the two plasma membrane domains is different depending on the cellular phenotype. Thus, in membranes from drug-sensitive cells, DNM is preferentially confined to 'surface' domains, while in membranes from drug-resistant cells, the drug distributes more homogeneously between 'surface' and 'deep' domains. Experiments using artificial lipid vesicles suggest that differences in the relative levels of certain lipids in the plasma membranes from drug-sensitive and drug-resistant cells, namely phosphatidylserine and cholesterol, are partly responsible for the observed differences in the distribution of DNM. Since drug-membrane interactions are important in anthracycline cytotoxicity, it is possible that our observations on a different membrane distribution of daunomycin, may be related to the different sensitivity to the drug exhibited by these cells. 相似文献
19.
M1 cells derived from mouse myeloid leukemia have been reported to differentiate to macrophage-like cells upon treatment with substances such as lipopolysaccharide. Previously we found that in mouse peritoneal macrophages most of the neutral amino acids were taken up through a unique Na+-independent system. In this paper we have investigated the neutral amino acid transport in M1 cells and in those treated with lipopolysaccharide. In M1 cells serine, alanine and proline were taken up mainly by Na+-dependent transport systems, and leucine was largely transported by a Na+-independent system. By treating the cells with lipopolysaccharide, the activities of the Na+-dependent systems markedly decreased, whereas the activity of the Na+-independent system was little affected. The amino acid concentrations in the cells and the culture medium were measured. As a whole, the intracellular to extracellular distribution ratios for neutral amino acids that are preferred substrates for Na+-dependent systems were decreased on lipopolysaccharide treatment, whereas those for amino acids that are mainly transported by a Na+-independent system were slightly increased. From these results we conclude that M1 cells treated with lipopolysaccharide tend to differentiate to macrophage-like cells with respect to the neutral amino acid transport. 相似文献
20.
The main purpose of the present investigation was to study the effect of cloturin on aerobic glycolysis, endogenous and exogenous respiration and the level of ATP in both Ehrlich ascites carcinoma (EAC) and P388 murine leukaemia cells incubated in vitro. Also its effect on the level of total (T-SH) and non-protein (NP-SH) thiol groups was investigated. A significant inhibition of aerobic glycolysis was found only in P388 cells after 60 min of cloturin action. Cloturin inhibited both endogenous and exogenous respiration of EAC with succinate as substrate. Cloturin decreased the level of ATP after 2 h incubation in both types of tumour cell. The level of NP-SH was decreased more than that of T-SH in both types of cell. 相似文献