Active transport of l-aspartic acid in Neurospora crassa

Transport of l-aspartic acid in Neurospora crassa conidia is shown to be mediated by neutral and general amino acid transport systems. The transport activity is dependent on pH and results in accumulation of l-aspartic acid against a gradient. Mutants deficient in transport of l-aspartic acid are described.     

Minicells of Bacillus subtilis a unique system for transport studies

Ultrasound-purified minicells produced by Bacillus subtilis mutant div IV-Bl have been studied for their ability to transport and incorporate into macromolecules a variety of amino acids, uracil and thymine. Minicells transport all 12 amino acids examined, but are unable to incorporate them into macromolecules. No significant differences were found in the initial uptake rates of glutamic acid, aspartic acid, and alanine by minicells and parental cells. The uptake of methionine and proline by minicells was shown to be inhibited by metabolic poisons, indicating an energy-metabolism requirement for transport in this system. The proline pool in minicells was found to be readily exchangeable with exogenous proline. In contrast metabolically poisoned minicells only slowly lose their pool proline, indicating an energy requirement for pool maintenance. Packed-cell experiments reveal that minicells accumulate proline against a concentration gradient.In addition to amino acids, minicells are able to transport uracil but cannot incorporate uracil into acid-precipitable material (RNA). Neither thymine transport nor its incorporation into macromolecules can be demonstrated in minicells.Minicells appear to be a new system, therefore, in which transport may be studied in the absence of macromolecular biosynthesis.     

α-Methyl-l-glutamic acid uptake by high affinity dicarboxylic amino acid transport system in Streptococcus faecalis

The transport of α-methyl-l-glutamic acid was studied in Streptococcus faecalis. Energy-dependent uptake against substantial concentration gradients was observed. Kinetic experiments indicated that, in contrast to l-glutamic acid, only a single catalytic component (high affinity) and a diffusion controlled process participated in α-methyl-l-glutamic acid uptake. At concentrations up to 10 mM, α-methylglutamate transport was almost completely abolished in a mutant strain lacking a high affinity dicarboxylic amino acid transport system. In competition experiments, α-methylglutamic acid antagonized glutamate uptake via the high affinity system, and only slightly via the low affinity system. Column chromatography of cell extracts showed that very little (approx. 5%) of the accumulated amino acid was converted to metabolites during short term incubations. These studies indicate that, at concentrations up to 3–5 mM, α-methyl-l-glutamic acid can be used as a specific, relatively metabolically inert substrate of the high affinity dicarboxylic amino acid transport system in S. faecalis.     

Mechanism of proline uptake by Chlorella vulgaris

An amino acid uptake system specific for glycine, alanine, serine and proline was induced by glucose in Chlorella vulgaris. The uptake system translocated the zwitterionic form of the amino acid. There was more than 100-fold accumulation which indicated a coupling to metabolic energy. The depolarization of the membrane potential during proline uptake and the sensitivity of its uptake rate to the membrane potential point to coupling with an ion flow. Inhibitors of plasmalemma-bound H⁺-ATPase inhibit proline uptake. These data are interpreted to mean that proline is taken up as a proton symport. In some Chlorella strains the proline-coupled H⁺ uptake could be measured with electrodes, but not in Chlorella vulgaris. There is evidence that the transport of amino acids rapidly stimulates the proton-translocating ATPase of Chlorella vulgaris, so that the proline-coupled proton uptake is immediately neutralized.     

Catabolite repression in Bacillus subtilis

The d-gluconate transport system of Bacillus subtilis is optimally induced by exposure of cells for 2 h to 5 mM d-gluconate in the growth medium. d-gluconate transport is subject to catabolite repression, as distinct from inducer exclusion or catabolite inhibition, in a manner parallel to the repression of inducible histidase synthesis, suggesting that the repression is not specific to this transport system. Maximum repression with the repressing carbon source (10 mM) added to cells grown in either casein hydrolysate or amino acid medium is achieved within two doubling times. Urea, the only non-carbon source tested for a repressing effect, was found to act solely by inducer exclusion. The ability of a sugar carbon source to evoke catabolite repression appears to be unrelated to its suitability as a substrate for the sugar: phosphoenolpyruvate phosphotransferase system but nonetheless the conversion to a phosphorylated derivative of the sugar seems essential. Repressed cells fail to synthesize, or do so to a more limited extent, an as yet unidentified phosphorylated compound (probably a highly phosphorylated nucleotide) which is accumulated in the medium of non-repressed cells. Mutant studies imply that inosinic acid synthesis is necessary for catabolite repression whereas the adenosine highly phosphorylated nucleotides required for spurulation are not.     

Neutral amino acid transport in Leishmania promastigotes

Neutral amino acid transport was investigated in Leishmania promastigotes. Proline and alanine transport occur against their concentration gradient although there is a very rapid (40% at 30 min) conversion of proline to alanine. Uptake of these amino acids occurs by a sodium-independent route which is completely eliminated by addition of CCCP or KCN. $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values for proline and alanine are 80 μM and 63 μM with $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ values of 6.4 and 7.2 nmol/min per mg dry weight, respectively. Countertransport of proline, alanine and phenylalanine was measured by loading the cells with a variety of neutral amino acids and proline analogs, followed by CCCP addition. The effect of aminooxyacetic acid, an inhibitor of alanine aminotransferase (EC 2.6.1.2), on proline and alanine countertransport was also examined. The results obtained are consistent with the presence of at least two systems for neutral amino acid transport in Leishmania promastigotes.     

Ureidosuccinic acid permeation in Saccharomyces cerevisiae

Some strains of Saccharomyces cerevisiae exhibit a specific transport system for ureidosuccinic acid, which is regulated by nitrogen metabolism. Ureidosuccinic acid uptake occurs with proline but with ammonium sulfate as nitrogen source it is inhibited. The V for transport is 20–25 μmol/ml cell water per min. The apparent K_m is 3 · 10^-5. For the urep1 mutant (ureidosuccinic acid permease less) the internal concentration never exceeds the external one.In the permease plus strain ureidosuccinic acid can be concentrated up to 10 000 fold and the accumulated compound remains unchanged in the cells. Energy poisons such as dinitrophenol, carbonyl cyanide-m-chlorophenyl-drazone (CCCP) or NaN₃ inhibit the uptake. No significant efflux of the accumulated compound occurs even in the presence of these drugs.The specificity of the permease is very strict, only amino acids carrying an α-N-carbamyl group are strongly competitive inhibitors.The high concentration capacity of the cells and the lack of active exit of the accumulated compound support the hypothesis of a carrier mediated active transport system.     

Variability for protein and oil quality in Lupinus albus

Amino acid composition and fatty acid composition were determined on seed samples of a range of white lupin (Lupinus albus) cultivars and accessions grown in either of two environments.Variability between genotypes was found for lysine, arginine and glutamic acid content, but not for the concentrations of other amino acids. The deficiency in sulphurcontaining amino acids, typical of legume proteins, was evident, with methionine and cyst(e)ine totalling only 2.2% of the protein. Variability was limited, indicating that improvement by breeding would be impracticable. Lupinus albus differed slightly from other lupin species in amino acid composition, having higher levels of threonine, tyrosine and isoleucine, but a lower level of glutamic acid than both L. angustifolius and L. luteus. Four low-alkaloid lines of L albus each had higher lysine content than the high-alkaloid line, but ‘Kiev Mutant’, despite earlier claims, had a lysine level no higher than the other three low-alkaloid lines.Fatty acid composition of the seed oil varied considerably between genotypes. Oleic acid ranged from 43.6 to 54.4% and linolenic acid from 6.7 to 15.2%, these two fatty acids being negatively correlated at one site. Linoleic acid content varied between 17.2 and 26.9% and was not correlated with other fatty acids. Total oil content averaged 9.6% with little variability between lines.It is concluded that, relative to other lupin species, L. albus has a more favourable amino acid profile for its utilisation in cereal-based diets for animals, particularly if the energy source is wheat, which is deficient in threonine. The higher oil content would be an important energy benefit to such diets and may allow their protein/energy balance to be maintained at higher levels of incorporation of L. albus seed meal than is possible with other lupin species.     

Etude chimiotaxonomique dans les genres Macrotyloma, Dolichos et Pseudovigna

The variations of free, protein and total amino acid contents have been studied in seeds of Macrotyloma, Dolichos and Pseudovigna. The concentrations of free amino acids and of γ-glutamylphenylalanine seem to be characteristic of some species and also of some genera.     

Leucine transport by the larval midgut of the parasitoid Aphidius ervi (Hymenoptera)

The larval midgut of the hymenopteran parasitoid Aphidius ervi accomplishes a large transport of nutrients from the lumen to the haemocoel, providing most of the organic molecules necessary for rapid insect development. l-amino acids in general, and leucine in particular, are efficiently accumulated in the larval body. We show here that the intact midgut of early 3rd instar larvae incubated in vitro can take up [³H]l-leucine from the basolateral side of the epithelium by transporters insensitive to the presence of monovalent cations. When the midgut is opened and the apical membrane of the absorbing epithelial cells is exposed to the medium containing radiolabelled leucine, a sodium-dependent uptake of the amino acid becomes apparent, disclosing the presence of a symport mechanism. Inhibition experiments of leucine uptake by a 100-fold excess of different amino acids, selected according to the properties of their side chain, revealed that this apical sodium-dependent mechanism is a broad spectrum transport system with a specialization for the absorption of aliphatic amino acids, that can also transfer glutamine and proline, but not phenylalanine, lysine and arginine. Altogether the experimental results obtained with intact- and open-gut preparations suggest that leucine transport across the basolateral membrane is mediated by both an uniporter and an obligatory amino acid exchange mechanism.     

Cotugnia digonopora: Transport of leucine

The uptake of leucine through the tegument of Cotugnia digonopora, a cestode found in the fowl intestine, occurs by a process of active transport. The K_t of transport is 0.87 mM and the V_max is 0.223 μmol/min/g. Uptake of the amino acid is competitively inhibited by valine (K_t = 1.30 mM). Potassium cyanide and 2,4-dinitrophenol do not completely block the entry of leucine into the parasite.     

Heterogeneous elevation of amino acid transport rates in pantothenate-and lipid-deficient Lactobacillus plantarum

The effect of a pantothenic acid deficiency in Lactobacillus plantarum on the initial rate of amino acid transport was investigated. Although the steady-state accumulation capacity for all amino acids was markedly reduced in pantothenate-deficient cells, initial rates of uptake either were not changed (asparagine, alanine, lysine) or were increased (glutamic acid, aspartic acid, leucine). The findings suggest that a reduction in membrane lipid content heterogeneously affects the operation and/or synthesis of amino acid transport catalysts.     

Methionine transport in the malaria parasite Plasmodium falciparum

The intraerythrocytic malaria parasite, Plasmodium falciparum, derives amino acids from the digestion of host cell haemoglobin. However, it also takes up amino acids from the extracellular medium. Isoleucine is absent from adult human haemoglobin and an exogenous source of isoleucine is essential for parasite growth. An extracellular source of methionine is also important for the normal growth of at least some parasite strains. In this study we have characterised the uptake of methionine by P. falciparum-infected human erythrocytes, and by parasites functionally isolated from their host cells by saponin-permeabilization of the erythrocyte membrane. Infected erythrocytes take up methionine much faster than uninfected erythrocytes, with the increase attributable to the flux of this amino acid via the New Permeability Pathways induced by the parasite in the erythrocyte membrane. Having entered the infected cell, methionine is taken up by the intracellular parasite via a saturable, temperature-dependent process that is independent of ATP, Na⁺ and H⁺. Substrate competition studies, and comparison of the transport of methionine with that of isoleucine and leucine, yielded results consistent with the hypothesis that the parasite has at its surface one or more transporters which mediate the flux into and out of the parasite of a broad range of neutral amino acids. These transporters function most efficiently when exchanging one neutral amino acid for another, thus providing a mechanism whereby the parasite is able to import important exogenous amino acids in exchange for surplus neutral amino acids liberated from the digestion of host cell haemoglobin.     

Avt5p is required for vacuolar uptake of amino acids in the fission yeast Schizosaccharomycespombe

We identified SPBC1685.07c of Schizosaccharomyces pombe as a novel vacuolar protein, Avt5p, with similarity to vacuolar amino acid transporters Avt5p from Saccharomyces cerevisiae. Avt5p localizes to the vacuolar membrane and upon disruption of avt5, uptake of histidine, glutamate, tyrosine, arginine, lysine or serine was impaired. During nitrogen starvation, the transient increase of vacuolar lysine transport observed for wild-type cells still occurred in the mutant cells, however, uptake of glutamate did not significantly increase in response to nitrogen starvation. Our results show that under diverse growth conditions Avt5p is involved in vacuolar transport of a selective set of amino acids.     

Chloroplast Import Signals: The Length Requirement for Translocation In Vitro and In Vivo

Protein translocation of cytosolically synthesized proteins requires signals for both targeting of precursor proteins to the surface of the respective compartment and their transfer across its membrane. In contrast to signals for peroxisomal and endoplasmic reticulum translocation, the signals for mitochondrial and chloroplast transport are less well defined with respect to length and amino acid requirements. To study the properties of signals required for translocation into chloroplasts in vitro and in vivo, we used fusion proteins composed of transit peptides and the Ig-like module of the muscle protein titin as passenger. We observed that about 60 amino acids—longer than the transit peptide length of many experimentally confirmed chloroplast proteins—are required for efficient translocation. However, within native chloroplast precursor proteins with transit peptides shorter than 60 amino acids, extension appears to be present as they are efficiently imported into organelles. In addition, the interaction of an unfolded polypeptide stretch of 60 or more amino acids with receptors at the chloroplast surface results in the unidirectionality of protein translocation into chloroplasts even in the presence of a competing C-terminal peroxisomal targeting signal. These findings prove the existing ideas that initial targeting is defined by the N-terminal signal and that the C-terminal signal is sensed only subsequently.     

Kinetics of neutral amino-acid transport by isolated gill tissue of the bivalve Mya arenaria (L.)

An in vitro technique was used to examine the absorption by the gill of Mya arenaria (L.) of six neutral l-amino acids chosen for differences in their side chains, viz., short chain — glycine and alanine, long chain — leucine, sulphur containing — methionine, aromatic — phenylalanine, hydroxylic — serine. The uptake of all these substrates was active and carrier-mediated, and was analysed by Michaelis-Menten kinetics. Values of K_t, the transport constant, decreased with increasing length of the side chain for glycine, l-serine, l-alanine, l-methionine, l-phenylalanine, and l-leucine, while values for V_max, the maximum velocity of uptake, decreased as chain length increased, except in the case of l-serine. Inhibition experiments suggested that at least one transport locus was common to all the neutral amino acids examined, but homogeneity of transport was only demonstrated in the case of methionine and leucine. The transport of the basic amino acid l-lysine overlapped with several of the-neutral amino acids. These results emphasize the need to consider the mutual inhibitory effects between amino acids absorbed from sea water, when calculations are made of the value of this source of nutrition to marine invertebrates.     

三角鲂和长春鳊肌肉营养成分分析与品质评价

用常规方法测定、分析了三角鲂(Megalobrama tarminalis)和长春鳊(Parabramis pekinensis肌肉中营养成分组成与含量.结果显示,三角鲂肌肉蛋白质、脂肪含量分别为18.19％和3.06％,长春鳊肌肉蛋白质、脂肪含量分别为19.38％和2.89％.三角鲂和长春鳊肌肉中均检测出18种氨基酸,其中包括了8种人体必需氨基酸.三角鲂肌肉中氨基酸总量为76.27％,其中,8种人体必需氨基酸含量为32.17％,占氨基酸总量的42.18％;长春鳊肌肉中氨基酸总量为77.60％,其中,8种人体必需氨基酸含量为31.70％,占氨基酸总量的40.85％.必需氨基酸的构成比例基本符合FAO/WHO的标准.三角鲂肌肉中限制性氨基酸主要为甲硫氨酸加胱氨酸,必需氨基酸指数为63.55,4种呈味氨基酸为氨基酸总量的32.81％;长春鳊肌肉中限制性氨基酸主要为色氨酸,必需氨基酸指数为66.81,4种呈味氨基酸为氨基酸总量的33.80％.脂肪酸中二十碳五烯酸(EPA)与二十二碳六烯酸(DHA)含量均较高,三角鲂为7.96％,长春鳊为3.11％.矿物元素比值合理.以上分析表明,三角鲂和长春鳊均为营养价值、经济价值都较高的优质鱼类,相比而言,三角鲂肌肉脂肪、脂肪酸含量和质量更优,而长春鳊肌肉在蛋白质、氨基酸组成与含量方面更优.     

Molecular characterization and expression analysis of a GTP-binding protein (MiRab5) in Mangifera indica

The Rab family, the largest branch of Ras small GTPases, plays a crucial role in the vesicular transport in plants. The members of Rab family act as molecular switches that regulate the fusion of vesicles with target membranes through conformational changes. However, little is known about the Rab5 gene involved in fruit ripening and stress response. In this study, the MiRab5 gene was isolated from stress-induced Mangifera indica. The full-length cDNA sequence was 984 bp and contained an open reading frame of 600 bp, which encoded a 200 amino acid protein with a molecular weight of 21.83 kDa and a theoretical isoelectric point of 6.99. The deduced amino acid sequence exhibited high homology with tomato (91% similarity) and contains all five characteristic Rab motifs. Real-time quantitative RT-PCR analysis demonstrated that MiRab5 was ubiquitously expressed in various mango tree tissues at different levels. The expression of MiRab5 was up-regulated during later stages of fruit ripening. Moreover, MiRab5 was generally up-regulated in response to various abiotic stresses (cold, salinity, and PEG treatments). Recombinant MiRab5 protein was successfully expressed and purified. SDS-PAGE and western blot analysis indicated that the expressed protein was recognized by the anti-6-His antibody. These results provide insights into the role of the MiRab5 gene family in fruit ripening and stress responses in the mango plant.     

Peptide utilization in yeast studies on methionine and lysine auxotrophs ofSaccharomyces cerevisiae

A study was made of the growth response and cellular peptidase activity of several amino acid auxotrophs ofSaccharomyces cerevisiae to peptides containing the required amino acid. A methionine-requiring auxotroph grew on, and contained intra-cellular peptidase activity toward Met-Met, Met-Met-Met, and Met-Gly-Met-Met. In contrast, Gly-Met-Gly did not support the growth of this mutant nor did three lysine-requiring strains utilize any lysine-containing peptides tested, although cell-free extracts from the respective mutants contained the necessary peptidase activity. The absence of a transport system of relatively high affinity for these peptides is suggested as the reason for their inability to satisfy the nutritional requirements of the cells.     

Effects of colicin A and staphylococcin 1580 on amino acid uptake into membrane vesicles of Escherichia coli and Staphylococcus aureus

Staphylococcin 1580 increased the relative amount of diphosphatidylglycerol and decreased the amount of phosphatidylglycerol in cells of Staphlococcus aureus, while the amounts of lysylphosphatidylglycerol, phosphatidic acid and total phospholipid remained constant.Treatment of cells of Escherichia coli and S. aureus with colicin A and staphylococcin 1580, respectively, did not affect proton impermeability but subsequent addition of carbonylcyanide-m-chlorophenylhydrazone resulted in a rapid influx of protons into the cells.Bacteriocin-resistant and -tolerant mutants of E. coli and S. aureus were isolated. The bacteriocins caused leakage of amino acids preaccumulated into membrane vesicles of resistant mutants and had no significant effect on membrane vesicles of tolerant mutants.The uptake of amino acids into membrane vesicles was inhibited by both bacteriocins, irrespective of the electron donors applied. The bacteriocin inhibition was noncompetitive. The bacteriocins did not affect oxygen consumption and dehydrogenases in membrane vesicles.Both bacteriocins suppressed the decrease in the fluorescence of 1-anilino-8-naphthalene sulfonate caused by d-lactate or α-glycerol phosphate when added to membrane vesicles.It is concluded that the bacteriocins uncouple the transport function from the electron transport system.