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1.
Membrane vesicles were prepared from Micrococcus denitrificans by osmotic shock of lysozyme spheroplasts. These vesicles concentrated 4 amino acids via two systems; one for glycine-alanine and the other for asparagine-glutamine. Amino acid transport was coupled to the membrane-bound electron transport system and involved interactions of the primary dehydrogenases, cytochromes, cytochrome oxidase and oxygen. After transport the amino acids were recovered unchanged from the vesicles. The substrates of the membrane-bound electron transport system d-lactate, l-lactate, formate, succinate, NADH, glucose-6-phosphate and α-glycerolphosphate all stimulated transport at least 2-fold. Both oxygen and nitrate could serve as terminal electron acceptors with vesicles prepared from cells grown anaerobically with nitrate. Anaerobic transport in the presence of nitrate was not inhibited by cyanide but was inhibited by nitrite. A system stimulated by substrates of the electron transport system but independent of added terminal electron acceptors was found also in the vesicles prepared from anaerobically grown cells. Addition of one combination of two substrates for electron transport produced an amino acid uptake 12 to 15% greater than the sum of the rates for each substrate added singly. Additions of other combinations gave rates of transport less than the sum of the rates of each added alone. Both the dehydrogenase activities and the coupling of electron transport to amino acid uptake were modified by changing the growth conditions and differences between the effectiveness of each substrate for each of the two transport systems could be detected. The efficiency of the vesicles per protoheme, the prosthetic group of the membrane-bound cytochrome b, with d-lactate as substrate was 27% for glutamine and 6% for glycine of the rates of transport of these two amino acids in intact cells when driven by endogenous respiration. Assuming one amino acid transported per electron, the transport of glycine utilized 1% of the respiratory capacity with glucose-6-phosphate as substrate. The coupling to the electron transport with the other substrates was less efficient. It appeared that a small portion of the total capacity of the electron transport system was coupled to amino acid transport and the coupling to respiration, as well as the primary dehydrogenase activities and terminal cytochrome oxidase, were modified in response to the conditions of growth.  相似文献   

2.
The initiation of growth of a polyaromatic auxotrophic mutant of Saccharomyces cerevisiae was inhibited by several amino acids, whereas growth of the parent prototroph was unaffected. A comparative investigation of amino acid transport in the two strains employing (14)C-labeled amino acids revealed that the transport of amino acids in S. cerevisiae was mediated by a general transport system responsible for the uptake of all neutral as well as basic amino acids. Both auxotrophic and prototrophic strains exhibited stereospecificity for l-amino acids and a K(m) ranging from 1.5 x 10(-5) to 5.0 x 10(-5) M. Optimal transport activity occurred at pH 5.7. Cycloheximide had no effect on amino acid uptake, indicating that protein synthesis was not a direct requirement for amino acid transport. Regulation of amino acid transport was subject to the concentration of amino acids in the free amino acid pool. Amino acid inhibition of the uptake of the aromatic amino acids by the aromatic auxotroph did not correlate directly with the effect of amino acids on the initiation of growth of the auxotroph but provides a partial explanation of this effect.  相似文献   

3.
Na+-stimulated amino acid transport was investigated in MDCK kidney epithelial cell monolayers and in isolated membrane vesicles. When transport polarity was assessed in confluent polarized epithelial cell monolayers cultured on Nucleopore filters and mounted between two lucite chambers, Na+-stimulated transport of 2-(methylamino)isobutyric acid (MeAIB), a substrate specific for the A system, was predominantly localized on the basolateral membrane. Na+-stimulated amino acid transport activity was maximal in subconfluent cultures, and was substantially reduced after confluence. A membrane vesicle preparation was isolated from confluent MDCK cell cultures which was enriched in Na+-stimulated MeAIB transport activity and Na+,K+,ATPase activity, a basolateral marker, but was not enriched in apical marker enzyme activities or significantly contaminated by mitochondria. Na+-coupled amino acid transport activity assayed in vesicles exhibited a marked dependence on external pH, with an optimum at pH 7.4. The pattern of competitive interactions among neutral amino acids was characteristic of A system transport. Na+-coupled MeAIB and AIB transport in vesicles was electrogenic, stimulated by creation of an interior-negative membrane potential. The Na+ dependence of amino acid transport in vesicles suggested a Na+ symport mechanism with a 1:1 stoichiometry between Na+ and amino acid.  相似文献   

4.
The aim of this work was to investigate the relationship between amino acid requirements and peptidase enzyme systems in three Streptococcus salivarius subsp. thermophilus strains. A synthetic medium without nitrogen components and a milk (RD milk) without its non-protein nitrogen fraction were prepared with different mixtures of amino acids. The strains showed different amino acid requirements. Some amino acids proved to be essential, some were required, while others did not affect growth. In the synthetic medium, only leucine and glutamic acid were essential for growth. In RD milk, the amino acid requirements were found to be lower, with only the absence of glutamic acid causing complete inhibition of growth. Relationships between aminopeptidase activities of the strains and their amino acid requirements were observed. Strains with higher amino acid requirements were also found to express a wider range of peptidases.  相似文献   

5.
The pool of phenylalanine, tyrosine, and tryptophan is formed in Escherichia coli K-12 by a general aromatic transport system [Michaelis constant (K(m)) for each amino acid approximately 5 x 10(-7)m] and three further transport systems each specific for a single aromatic amino acid (K(m) for each amino acid approximately 2 x 10(-6)m, reference 3). When the external concentration of a particular aromatic amino acid is saturating for both classes of transport system, the free amino acid pool is supplied with external amino acid by both systems. Blocking the general transport system reduces the pool size by 80 to 90% but does not interfere with the supply of the amino acid to protein synthesis. If, however, the external concentration is too low to saturate specific transport, blocking general transport inhibits the incorporation of external amino acid into protein by about 75%. It is concluded that the amino acids transported by either class of transport system can be used for protein synthesis. Dilution of the external amino acid or deprivation of energy causes efflux of the aromatic pool. These results and rapid exchange observed between pool amino acid and external amino acids indicate that the aromatic pool circulates rapidly between the inside and the outside of the cell. Evidence is presented that this exchange is mediated by the aromatic transport systems. Mutation of aroP (a gene specifying general aromatic transport) inhibits exit and exchange of the small pool generated by specific transport. These findings are discussed and a simple physiological model of aromatic pool formation, and exchange, is proposed.  相似文献   

6.
Uptake of 16 amino acids by the filamentous, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 was characterized with regard to kinetic parameters of transport, intracellular accumulation of the transported amino acids, and sensitivity of the transport process to energy metabolism inhibitors. Mutants resistant to certain toxic analogs of some amino acids were isolated that were impaired in amino acid transport. Results obtained in this study, together with those reported previously (A. Herrero and E. Flores, J. Biol. Chem. 265:3931-3935, 1990), suggest that there are at least five amino acid transport systems in strain PCC 7120: one high-affinity, active system for basic amino acids; one low-affinity, passive system for basic amino acids; two high-affinity, active systems with overlapping, but not identical, specificities for neutral amino acids; and one putative system for acidic amino acids. Some of the amino acid transport mutants were impaired in diazotrophic growth. These mutants were unable to develop a normal percentage of heterocysts and normal nitrogenase activity in response to nitrogen stepdown. Putative roles for the amino acid transport systems in uptake of extracellular amino acids, recapture of amino acids that have leaked from the cells, and intercellular transfer of amino acids in the filaments of Anabaena sp. strain PCC 7120 are discussed.  相似文献   

7.
We report here on the cloning and functional characterization of the third subtype of amino acid transport system A, designated ATA3 (amino acid transporter A3), from a human liver cell line. This transporter consists of 547 amino acids and is structurally related to the members of the glutamine transporter family. The human ATA3 (hATA3) exhibits 88% identity in amino acid sequence with rat ATA3. The gene coding for hATA3 contains 16 exons and is located on human chromosome 12q13. It is expressed almost exclusively in the liver. hATA3 mediates the transport of neutral amino acids including α-(methylamino)isobutyric acid (MeAIB), the model substrate for system A, in a Na+-coupled manner and the transport of cationic amino acids in a Na+-independent manner. The affinity of hATA3 for cationic amino acids is higher than for neutral amino acids. The transport function of hATA3 is thus similar to that of system y+L. The ability of hATA3 to transport cationic amino acids with high affinity is unique among the members of the glutamine transporter family. hATA1 and hATA2, the other two known members of the system A subfamily, show little affinity toward cationic amino acids. hATA3 also differs from hATA1 and hATA2 in exhibiting low affinity for MeAIB. Since liver does not express any of the previously known high-affinity cationic amino acid transporters, ATA3 is likely to provide the major route for the uptake of arginine in this tissue.  相似文献   

8.
Experiments were carried out to determine whether the addition of a mixture of indispensable amino acids (IAA) lacking in threonine, phenylalanine or histidine, respectively, to a nutritionally complete diet would increase the hepatic activities of the rate-limiting enzymes for catabolism of threonine, phenylalanine or histidine and prevent the adverse effects of the amino acid on growth when the dietary level of the amino acid is excessive. Week old Leghorn chicks were fed semi-purified diets containing 19% crude protein to which were added no IAA supplement or 10% crude protein from an IAA mix and 5 graded levels of either L-threonine, L-phenylalanine or L-histidine in a 2 x 5 factorial arrangement of treatments. Each amino acid was investigated in a separate experiment involving four replicate pens (seven chicks each) per diet. Weight gains and feed consumptions were determined on the fourteenth day of each experiment. The groups receiving no excess, and 1.0% or 2.0% excesses of amino acids were sampled on the fifteenth day for enzyme activities and plasma amino acid concentrations. Weight gain and/or feed consumption were lower, and plasma concentrations of threonine, phenylalanine and histidine were higher, in chicks receiving 1.5 to 2.0% dietary additions of threonine, phenylalanine, and histidine, respectively, than in chicks that did not receive these amino acids. Chicks that received the amino acids in diets that also contained the IAA supplement had better growth and feed consumption, lower plasma concentrations of threonine, phenylalanine or histidine, higher plasma concentrations of other indispensable amino acids, and higher activities of threonine dehydrogenase, phenylalanine hydroxylase, and histidase than chicks receiving excess amino acids in the absence of IAA supplements. We conclude that the dietary level of protein, not the dietary level of individual amino acids, is the primary determinant of the activity of amino acid degrading enzymes in liver. The increased activity of these enzymes may be the mechanism by which dietary protein alleviates the adverse effects of excessive levels of individual amino acids.  相似文献   

9.
Transport of Aromatic Amino Acids by Pseudomonas aeruginosa   总被引:9,自引:5,他引:4       下载免费PDF全文
Kinetic studies of the transport of aromatic amino acids by Pseudomonas aeruginosa revealed the existence of two high-affinity transport systems which recognized the three aromatic amino acids. From competition data and studies on the exchange of preformed aromatic amino acid pools, the first transport system was found to be functional with phenylalanine, tyrosine, and tryptophan (in order of decreasing activity), whereas the second system was active with tryptophan, phenylalanine, and tyrosine. The two systems also transported a number of aromatic amino acid analogues but not other amino acids. Mutants defective in each of the two and in both transport systems were isolated and described. When the amino acids were added at low external concentrations to cells growing logarithmically in glucose minimal medium, the tryptophan pool very quickly became saturated. Under identical conditions, phenylalanine and tyrosine each accumulated in the intracellular pool of P. aeruginosa at a concentration which was 10 times greater than that of tryptophan.  相似文献   

10.
Abstract: Transport of representative neutral α-amino acids was measured in brain slices after injecting thy-roxine into donor rats of various ages from 1 to 23 days old. The hormone did not alter uptake in slices from 1-day-old rats even when treatment was begun on pregnant rats as much as 10 days before delivery. Injecting thy-roxine until age 6 days, however, decreased the activity of transport system A (the major sodium-dependent system in most mammalian cells) and caused appearance of a new transport system used by the model amino acids, 2-aminoisobutyric acid and 2-(methylamino)isobutyric acid. Uptake at 6 days was similar to that found in slices from older, untreated rats (e.g., those 14 days old). These results strongly suggest that one action of thyroxine is to accelerate the development of neutral α-amino acid transport systems of brain over the first six days after birth. Thyroxine treatment of rats from birth to age 14 days also appears to increase the activities of both system A and the second transport system used by the two model amino acids in brains from 14-day-old rats.  相似文献   

11.
Preimplantation mouse blastocysts were found to contain at least three mediated components of Na+-independent amino acid transport. The two less conspicuous components seemed to be selective for either cationic or zwitterionic substrates but were not characterized further or examined for multiple transport activities. L-Leucine and L-lysine competed strongly for uptake by the most conspicuous Na+-independent transport process detected in these conceptuses (referred to as component b0,+), and no further heterogeneity of transport activities was found within this component. A series of inhibitors of various strengths had about the same effect on component b0,+ when either leucine or lysine was the substrate, and uptake of each substrate was not affected significantly by changes in the pH between 6.3 and 8.0. Furthermore, the Ki values for mutually competitive inhibition of transport between leucine and lysine and their Km values for transport via component b0,+ were all on the order of about 100 microM. In addition, the Ki values for competitive inhibition of leucine or lysine uptake by valine were approximately 5 mM in both cases, and alanine appeared to be a similarly weak competitive inhibitor of leucine transport. Based on these results, component b0,+ prefers to interact with bulky amino acids that do not branch at the beta-carbon. Moreover, amino acids that branch at the alpha-carbon, such as the leucine analog 3-amino-endo-bicyclo[3.2.1]octane-3-carboxylic acid, were virtually excluded by this component. The substrate reactivity of component b0,+ is more limited than the Na+-dependent transport system B0,+ in blastocysts which accepts both these branched species and less bulky amino acids relatively well as substrates. Thus, mediated amino acid transport in the mouse trophoblast is clearly distinguishable from that in most other mammalian tissues that have been studied. Not only do component b0,+ and system B0,+ and system B0,+ fail to discriminate strongly between basic and zwitterionic substrates, but their relative reactivity with bicyclic amino acids, such as 3-amino-endo-bicyclo[3.2.1]octane-3-carboxylic acid, is the reverse of transport processes in other cell types where these amino acids react strongly with Na+-independent, but not Na+-dependent, systems.  相似文献   

12.
A method for the isolation of amino acid auxotrophs of Thiobacillus thioparus is described. Characterization of a leucine auxotroph indicated that leucine biosynthesis in T. thioparus was not different from that of heterotrophic bacteria. T. thioparus cells accumulated amino acids via an active mechanism. Kt values of amino acid transport were between 15 and 330 microM, and Vmax values were 200 to 350 pmol min-1 mg of protein-1. Amino acid transport was carried out by a limited number of systems, each responsible for the uptake of several amino acids. Amino acid auxotrophs of T. thioparus exhibited transport and growth properties similar to those of transport-deficient mutants of heterotrophs which lost the high-affinity, but retained the low-affinity, amino acid transport systems.  相似文献   

13.
We studied the uptake of leucine, phenylalanine, and the amino acid analog, 2-aminonorborane-2-carboxylic acid, by rat hepatoma cells in tissue culture. The uptake of these amino acids was partially mediated by a plasma membrane transport system similar to the L agency described in other cell types in that it does not require extracellular sodium and is subject to trans-stimulation. Initial rates of sodium-independent transport of these amino acids were calculated using mathematical transformations of the uptake time course curves. The glucocorticoid dexamethasone inhibits the activity of this transport system; the initial rates of sodium-independent uptake of leucine, phenylalanine, and 2-aminonorborane-2-carboxylic acid are decreased by approximately one-third (average = 30%, n = 19) after incubation of HTC cells with 0.1 microM dexamethasone. This inhibition requires at least 15 h, reaching a maximum at 24 h of exposure of the cells to the hormone. Dexamethasone has an asymmetrical effect on sodium-independent amino acid transport in that exposure of the cells to the hormone does not inhibit the rates of outflow of leucine or phenylalanine from preloaded cells into medium without sodium. Inhibition of uptake is blocked by 0.1 mM cycloheximide and 4 microM actinomycin D, indicating the need for continuous protein synthesis for dexamethasone action. Insulin, which is known to partially reverse the inhibitory effect of dexamethasone on the A amino acid transport system in HTC cells, does not alter the action of dexamethasone on the L system. Previous investigations have demonstrated inhibition by dexamethasone of at least two distinct sodium-dependent amino acid transport activities in HTC cells. The data presented here, showing inhibition by the glucocorticoid of a sodium-independent transport activity, indicate that the effect of the hormone is independent of the energy source of the amino acid transport systems affected.  相似文献   

14.
We compared the inhibitory action of individual amino acids in vitro on the activities of alanyl-, arginyl-, leucyl- and pyroglutamyl aminopeptidases purified from human and porcine skeletal muscle tissues. The range of susceptibility to inhibition by individual amino acids (<25 mM) for different aminopeptidase types broadly paralleled that for the respective substrate specificities (in terms of relative rates of hydrolysis of amino acyl-AMC derivatives) for these enzymes. Thus, alanyl aminopeptidase (which hydrolyses a broad range of aminoacyl-AMC substrates) was inhibited by a correspondingly broad range of amino acids (although the respective ranking order of amino acids was not identical in each case), whereas pyroglutamyl aminopeptidase (which hydrolyses only pyroglutamyl AMC as substrate) was inhibited by pyroglutamic acid only. The mode of inhibition (competitive/non-competitive) varied for different enzyme types, both within and between each species. For enzymes purified from human muscle, alanyl, arginyl and leucyl aminopeptidases were inhibited by amino acids via the non-competitive mode (pyroglutamyl aminopeptidase via the competitive mode), whereas corresponding enzymes purified from porcine muscle were inhibited via the competitive mode. The data obtained indicate that the same aminopeptidase types are present in human and porcine skeletal muscle tissues, with corresponding enzymes having broadly similar assay characteristics and susceptibilities to inhibition by amino acids (although the mode of inhibition for corresponding enzymes may differ in each species). Such data obtained in vitro may prove of value in devising experimental strategies to manipulate protein turnover/muscle deposition in vivo, via inhibition of aminopeptidase action after administration of an appropriate admixture of amino acids.  相似文献   

15.
Our understanding of the interaction of carbon (C) metabolism with nitrogen (N) metabolism and growth is based mainly on studies of responses to environmental treatments, and studies of mutants and transformants. Here, we investigate which metabolic parameters vary and which parameters change in a coordinated manner in 24 genetically diverse Arabidopsis (Arabidopsis thaliana) accessions, grown in C-limited conditions. The accessions were grown in short days, moderate light, and high nitrate, and analyzed for rosette biomass, levels of structural components (protein, chlorophyll), total phenols and major metabolic intermediates (sugars, starch, nitrate, amino acids), and the activities of seven representative enzymes from central C and N metabolism. The largest variation was found for plant weight, reducing sugars, starch at the end of the night, and several enzyme activities. High levels of one sugar correlated with high levels of other sugars and starch, and a trend to increased amino acids, slightly lower nitrate, and higher protein. The activities of enzymes at the interface of C and N metabolism correlated with each other, but were unrelated to carbohydrates, amino acid levels, and total protein. Rosette weight was unrelated or showed a weak negative trend to sugar and amino acid contents at the end of the day in most of the accessions, and was negatively correlated with starch at the end of the night. Rosette weight was positively correlated with several enzyme activities. We propose that growth is not related to the absolute levels of starch, sugars, and amino acids; instead, it is related to flux, which is indicated by the enzymatic capacity to use these central resources.  相似文献   

16.
Although a considerable amount of information is available on the ecology, genetics, and physiology of life-history traits, much more limited data are available on the biochemical and genetic correlates of life-history variation within species. Specific activities of five enzymes of lipid biosynthesis and two enzymes of amino acid catabolism were compared among lines selected for flight-capable (LW[f]) versus flightless (SW) morphs of the cricket Gryllus firmus. These morphs, which exist in natural populations, differ genetically in ovarian growth (100-400% higher in SW) and aspects of flight capability including the size of wings and flight muscles, and the concentration of triglyceride flight fuel (40% greater in LW[f]). Consistently higher activity of each enzyme in LW(f) versus SW-selected lines, and strong co-segregation between morph and enzyme activity, demonstrated genetically based co-variance between wing morph and enzyme activity. Developmental profiles of enzyme activities strongly paralleled profiles of triglyceride accumulation during adulthood and previous measures of in vivo lipid biosynthesis. These data strongly imply that genetically based elevation in activities of lipogenic enzymes, and enzymes controlling the conversion of amino acids into lipids, is an important cause underlying the elevated accumulation of triglyceride in the LW(f) morph, a key biochemical component of the trade-off between elevated early fecundity and flight capability. Global changes in lipid and amino-acid metabolism appear to have resulted from microevolutionary alteration of regulators of metabolism. Finally, strong genotype x environment (diet) interactions were observed for most enzyme activities. Future progress in understanding the functional causes of life-history evolution requires a more detailed synthesis of the fields of life-history evolution and metabolic biochemistry. Wing polymorphism is a powerful experimental model in such integrative studies.  相似文献   

17.
The activities of several selected Na(+)-dependent amino acid transporters were identified in human liver plasma membrane vesicles by testing for Na(+)-dependent uptake of several naturally occurring neutral amino acids or their analogs. Alanine, 2-(methylamino)isobutyric acid, and 2-aminoisobutyric acid were shown to be almost exclusively transported by the same carrier, system A. Kinetic analysis of 2-(methylamino)isobutyric acid uptake by the human hepatic system A transporter revealed an apparent Km of 0.15 mM and a Vmax of 540 pmol.mg-1 protein.min-1. Human hepatic system A accepts a broad range of neutral amino acids including cysteine, glutamine, and histidine, which have been shown in other species to be transported mainly by disparate carriers. Inhibition analysis of Na(+)-dependent cysteine transport revealed that the portion of uptake not mediated by system A included at least two saturable carriers, system ASC and one other that has yet to be characterized. Most of the glutamine and histidine uptake was Na(+)-dependent, and the component not mediated by system A constituted system N. The largest portion of glycine transport was mediated through system A and the remainder by system ASC with no evidence for system Gly activity. Our examination of Na(+)-dependent amino acid transport documents the presence of several transport systems analogous to those described previously but with some notable differences in their functional activity. Most importantly, the results demonstrate that liver plasma membrane vesicles are a valuable resource for transport analysis of human tissue.  相似文献   

18.
We report here on the cloning and functional characterization of the protein responsible for the system A amino acid transport activity that is known to be expressed in most mammalian tissues. This transporter, designated ATA2 for amino acid transporter A2, was cloned from rat skeletal muscle. It is distinct from the neuron-specific glutamine transporter (GlnT/ATA1). Rat ATA2 consists of 504 amino acids and bears significant homology to GlnT/ATA1 and system N (SN1). ATA2-specific mRNA is ubiquitously expressed in rat tissues. When expressed in mammalian cells, ATA2 mediates Na(+)-dependent transport of alpha-(methylamino)isobutyric acid, a specific model substrate for system A. The transporter is specific for neutral amino acids. It is pH-sensitive and Li(+)-intolerant. The Na(+):amino acid stoichiometry is 1:1. When expressed in Xenopus laevis oocytes, transport of neutral amino acids via ATA2 is associated with inward currents. The substrate-induced current is Na(+)-dependent and pH-sensitive. The amino acid transport system A is particularly known for its adaptive and hormonal regulation, and therefore the successful cloning of the protein responsible for this transport activity represents a significant step toward understanding the function and expression of this transporter in various physiological and pathological states.  相似文献   

19.
Aromatic amino acid transport in Yersinia pestis.   总被引:2,自引:2,他引:0       下载免费PDF全文
The uptake and concentration of aromatic amino acids by Yersinia pestis TJW was investigated using endogenously metabolizing cells. Transport activity did not depend on either protein synthesis or exogenously added energy sources such as glucose. Aromatic amino acids remained as the free, unaltered amino acid in the pool fraction. Phenylalanine and tryptophan transport obeyed Michaelis-Menten-like kinetics with apparent Km values of 6 x 10(-7) to 7.5 x 10(-7) and 2 x 10(-6) M, respectively. Tyrosine transport showed biphasic concentration-dependent kinetics that indicated a diffusion-like process above external tyrosine concentrations of 2 x 10(-6) M. Transport of each aromatic amino acid showed different pH and temperature optima. The pH (7.5 TO8) and temperature (27 C) optima for phenylalanine transport were similar to those for growth. Transport of each aromatic amino acid was characterized by Q10 values of approximately 2. Cross inhibition and exchange experiments between the aromatic amino acids and selected aromatic amino acid analogues revealed the existence of three transport systems: (i) tryptophan specific, (ii) phenylalanine specific with limited transport activity for tyrosine and tryptophan, and (iii) general aromatic system with some specificity for tyrosine. Analogue studies also showed that the minimal stereo and structural features for phenylalanine recognition were: (i) the L isomer, (ii) intact alpha amino and carboxy group, and (iii) unsubstituted aromatic ring. Aromatic amino acid transport was differentially inhibited by various sulfhydryl blocking reagents and energy inhibitors. Phenylalanine and tyrosine transport was inhibited by 2,4-dinitrophenol, potassium cyanide, and sodium azide. Phenylalanine transport showed greater sensitivity to inhibition by sulfhydryl blocking reagents, particularly N-ethylmaleimide, than did tyrosine transport. Tryptophan transport was not inhibited by either sulfhydryl reagents or sodium azide. The results on the selective inhibition of aromatic amino acid transport provide additional evidence for multiple transport systems . These results further suggest both specific mechanisms for carrier-mediated active transport and coupling to metabolic energy.  相似文献   

20.
Transport of gamma-glutamyl amino acids, a step in the proposed glutathione-gamma-glutamyl transpeptidase-mediated amino acid transport pathway, was examined in mouse kidney. The transport of gamma-glutamyl amino acids was demonstrated in vitro in studies on kidney slices. Transport was followed by measuring uptake of 35S after incubation of the slices in media containing gamma-glutamyl methionine [35S]sulfone. The experimental complication associated with extracellular conversion of the gamma-glutamyl amino acid to amino acid and uptake of the latter by slices was overcome by using 5-oxoproline formation (catalyzed by intracellular gamma-glutamyl-cyclotransferase) as an indicator of gamma-glutamyl amino acid transport. This method was also successfully applied to studies on transport of gamma-glutamyl amino acids in vivo. Transport of gamma-glutamyl amino acids in vitro and in vivo is inhibited by several inhibitors of gamma-glutamyl transpeptidase and also by high extracellular levels of glutathione. This seems to explain urinary excretion of gamma-glutamylcystine by humans with gamma-glutamyl transpeptidase deficiency and by mice treated with inhibitors of this enzyme. Mice depleted of glutathione by treatment with buthionine sulfoximine (which inhibits glutathione synthesis) or by treatment with 2,6-dimethyl-2,5-heptadiene-4-one (which effectively interacts with tissue glutathione) exhibited significantly less transport of gamma-glutamyl amino acids than did untreated controls. The findings suggest that intracellular glutathione functions in transport of gamma-glutamyl amino acids. Evidence was also obtained for transport of gamma-glutamyl gamma-glutamylphenylalanine into kidney slices.  相似文献   

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