Radiation inactivation analysis of amino acid transport systems in Neurospora crassa

Radiation inactivation analysis of amino acid transport systems in Neurospora crass

Pathways of L-glutamic acid transport in cultured human fibroblasts.

The transport of L-glutamic acid has been studied in skin-derived diploid human fibroblasts. Competition analysis in the presence and absence of Na+ and mathematical discrimination by nonlinear regression indicated that L-glutamic acid enters the cell by at least three transport systems: 1) a high affinity Na+-dependent system which has been found to be identical to the previously described system for anionic amino acids (Gazzola, G. C., Dall'Asta, V., Bussolati, O., Makowske, M., and Christensen, H. N. (1981) J. Biol. Chem. 256, 6054-6059) and which is provisionally designated as System X-AG; this route was shared by L-aspartic acid; 2) a low affinity Na+-dependent system resembling the ASC System for neutral amino acids (Franchi-Gazzola, R., Gazzola, G. C., Dall'Asta, V., and Guidotti, G. G. (1982) J. Biol. Chem. 257, 9582-9587); its reactivity toward L-glutamic acid was strongly inhibited by L-serine, but not by 2-(methyl-amino)isobutyric acid; and 3) a Na+-independent system similar to System XC- described in fetal human lung fibroblasts (Bannai, S., and Kitamura, E. (1980) J. Biol. Chem. 255, 2372-2376). The XC- system served for L-glutamic acid and L-cystine, the latter amino acid behaving as a potent inhibitor of L-glutamic acid uptake. Amino acid starvation did not change the uptake of L-glutamic acid by the two Na+-dependent systems, but enhanced the activity of System XC- by increasing its Vmax. L-Glutamic acid transport was also affected by the density of the culture. An increased cell density lowered the uptake of the amino acid by Systems ASC and XC- and promoted the uptake by System X-AG. All these variations were dependent upon changes in Vmax

Functional characterization of L-tryptophan transport across mammalian cornea

In last few years transporter targeted drug delivery has drawn attention of research to identify and explore various nutrient transport systems including amino acid transporters for better drug delivery. The aim of present research work is to investigate the transport characteristics of L-tryptophan (L-try) across goat cornea. Transport of L-try was investigated using a glass diffusion cell for effect of concentration, pH, presence of other amino acids or metabolic inhibitor or dipeptide and tripeptide. The amount of L-try transported increased as the pH of L-try aqueous solution increased from 5 to 9. Inhibition was observed in L-try transport in absence of sodium ions where L-try solution was made isotonic with dextrose. Amino acids like L-histidine, L-arginine, L-lysine (cationic), L-glutamic acid, L-aspartic acid (anionic), glycine and L-proline (neutral) inhibited the L-try transport as compared to control (L-try alone). In presence of sodium azide and Ouabain the inhibition in L-try transport across goat cornea was observed while no marked inhibition was observed on L-try transport across goat cornea in presence of aspartame and glutathione. The L-try transport was favored up to concentration 1% w/v and at higher pH in presence of sodium ions through excised goat cornea. Functional presence of a sodium dependent L-try transport system as inhibited by ouabain having affinity to cationic and neutral amino acid is evident on goat cornea.Keywords: Cornea; Amino acid; Tryptophan; Transpor

Functional characterization of L-tryptophan transport across mammalian cornea

In last few years transporter targeted drug delivery has drawn attention of research to identify and explore various nutrient transport systems including amino acid transporters for better drug delivery. The aim of present research work is to investigate the transport characteristics of L-tryptophan (L-try) across goat cornea. Transport of L-try was investigated using a glass diffusion cell for effect of concentration, pH, presence of other amino acids or metabolic inhibitor or dipeptide and tripeptide. The amount of L-try transported increased as the pH of L-try aqueous solution increased from 5 to 9. Inhibition was observed in L-try transport in absence of sodium ions where L-try solution was made isotonic with dextrose. Amino acids like L-histidine, L-arginine, L-lysine (cationic), L-glutamic acid, L-aspartic acid (anionic), glycine and L-proline (neutral) inhibited the L-try transport as compared to control (L-try alone). In presence of sodium azide and Ouabain the inhibition in L-try transport across goat cornea was observed while no marked inhibition was observed on L-try transport across goat cornea in presence of aspartame and glutathione. The L-try transport was favored up to concentration 1% w/v and at higher pH in presence of sodium ions through excised goat cornea. Functional presence of a sodium dependent L-try transport system as inhibited by ouabain having affinity to cationic and neutral amino acid is evident on goat cornea.Keywords: Cornea; Amino acid; Tryptophan; Transpor

Isolation and characterization of PTR3 : a novel gene required for amino acid-responsive signal transduction in Saccharomyces cerevisiae

Specific proteins present in virtually all cells examined to date facilitate the transport of small peptides across the plasma membrane in an energy- dependent fashion. The physiology of peptide transport systems has been studied extensively in procaryotic cells and, in many cases, the genes responsible for this phenomenon have been isolated. However, only recently have the genes responsible for peptide transport in eukaryotic cells begun to be identified. Genetic analyses of the model eukaryote Saccharomyces cerevisiae have indicated that at least three genes are required for amino acid-inducible Peptide TRansport: PTR1, PTR2, and PTR3. The major goal of my project was to isolate and characterize one of these genes required for peptide transport in yeast, namely PTR3. Once identified, PTR3 was found to encode a novel gene that exhibited no similarity to any other protein in the database. Deletion of the PTR3 open reading frame had a pleiotropic phenotype; it simultaneously reduced the sensitivity to toxic peptides and amino acid analogues. Initial rates of radiolabeled dipeptide uptake demonstrated that elimination of PTR3 resulted in the loss of amino acid-inducible peptide transport activity. Northern analyses revealed that PTR3 was required for amino acid-induced expression of the PTRl, the gene encoding the di- tripeptide permease of S. cerevisiae. Further studies indicated that PTR3 also regulated the expression of at least two amino acid permease genes. These discoveries provided a molecular basis for the observed pleiotropic phenotype associated with ptr3 alleles, and indicated that the cellular role of PTR3 was the regulation of gene expression in response to small amounts of extracellular amino acids. The completion of additional genetic studies provided evidence that PTR3 functions within a novel regulatory pathway that coordinates amino acid-responsive regulation of a number of genes. Finally, it is hypothesized that PTR3 functions as a component of a novel amino acid-sensing signal transduction pathway

Developmental Changes in the Neutral Α-Amino Acid Transport Systems of Rat Brain Over the First Three Weeks After Birth

Transport of seven different amino acids into brain slices increased as donor rats aged from 1 to 6 days. Uptakes of 2-aminoisobutyric acid, 2-(methyl-amino)isobutyric acid, and L-alanine then decreased by day 14, while uptakes of other amino acids continued to increase or remained fairly constant. Neutral Α-amino acid transport systems were characterized by measuring inhibition of uptakes and kinetics for representative amino acids at different ages. Results indicate that 2-ami-noisobutyrate and 2-(methylamino)isobutyrate used only one (and the same) system in brain slices from 6-day-old rats, with characteristics of system A (the major sodium-dependent system in most mammalian cells). They used at least two systems at ages 1, 14, and 23 days, but, of these, only at 1 day did they use the same systems in the same proportions. Alanine and leucine used more than one system at all four ages, and somewhat different combinations than used by each other or by 2-aminoisobu-tyrate or 2-(methylamino)isobutyrate. Their transport characteristics showed they used mostly system ASC (a sodium-dependent system distinguished from A) and/or system L (sodium-independent). We conclude that system A increases as the brain ages from 1 to 6 days and declines thereafter. System L probably increases with aging from 1 to 23 days.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66235/1/j.1471-4159.1984.tb02780.x.pd

Amino acid transporters & amino acid metabolism in Trypanosoma brucei brucei

The development of new drugs against Human African Trypanosomiasis is much needed due to toxicity, efficacy and availability problems with current drug treatments for this resurgent parasitic disease. Delivery of drugs into cells is an important determinant of therapeutic efficacy of drugs. An effective means of selective drug delivery is to use plasma membrane transport systems to mediate the entry of drugs into the cell. Some amino acid transporters fulfil the criteria needed for successful exploitation of nutrient transport systems for drug delivery. The Trypanosoma brucei genomic database was screened to identify the full gene repertoire of amino acid transporters. From this, candidate genes were selected and functional genetic approaches were employed to characterise candidate amino acid transporter genes. Further characterisation of TbAATP1, a RNAi cell line shown to be a transporter of small neutral amino acids (serine, glycine, cysteine, asparagine and alanine), showed a role in threonine uptake. Amino acid analogues were tested for trypanocidal activity. Of the 96 tested, two (Azaserine and Levodopa) were investigated in more detail, paying special attention to the nature of their trypanocidal action and possible route of entry through an amino acid transporter. Azaserine showed a trypanostatic action as well multiple routes of entry into the protozoan interior (as shown by inhibition of glutamine, phenylalanine and tyrosine uptake). The trypanocidal Levodopa showed entry through a tyrosine specific transporter. However, it is possible that Levodopa’s trypanocidal activity may not be as a result of the analogue itself, but secondary products of the analogue. Amino acids are important for protozoa as energy sources as well as forming pools of soluble osmolites. Amino acid usage in trypanosomes was investigated. Upregulation of proline transport and catabolism in response to reduced glucose availability was exhibited by the genome strain of T. brucei. Moreover, this metabolic shift could be mimicked by addition of GlcNAc to the medium, which blocks the hexose transporter limiting glucose entry to the cell. Systems biology approaches were initiated to investigate the undergoing metabolic changes. More specifically, mass spectrometry methodologies were employed to investigate underlying metabolite changes in procyclic form trypanosomes grown in differing medium

Thyroxine-Induced Changes in the Development of Neutral Α-Amino Acid Transport Systems of Rat Brain

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.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66252/1/j.1471-4159.1984.tb02781.x.pd