Biochemical Characterization of Branched Chain Amino Acids Uptake in Trypanosoma cruzi

Trypanosoma cruzi is the etiological agent of Chagas disease. During its life cycle, it alternates among vertebrate and invertebrate hosts. Metabolic flexibility is a main biochemical characteristic of this parasite, which is able to obtain energy by oxidizing a variety of nutrients that can be transported from the extracellular medium. Moreover, several of these metabolites, more specifically amino acids, have a variety of functions beyond being sources of energy. Branched chain amino acids (BCAA), beyond their role in ATP production, are involved in sterol biosynthesis; for example, leucine is involved as a negative regulator of the parasite differentiation process occurring in the insect midgut. BCAA are essential metabolites in most nonphotosynthetic eukaryotes, including trypanosomes. In view of this, the metabolism of BCAA in T. cruzi depends mainly on their transport into the cell. In this work, we kinetically characterized the BCAA transport in T. cruzi epimastigotes. Our data point to BCAA as being transported by a single saturable transport system able to recognize leucine, isoleucine and valine. In view of this, we used leucine to further characterize this system. The transport increased linearly with temperature from 10 to 45 °C, allowing the calculation of an activation energy of 51.30 kJ/mol. Leucine uptake was an active process depending on ATP production and a H⁺ gradient, but not on a Na⁺ or K⁺ gradient at the cytoplasmic membrane level.     

Insight into the stability of the hydrophobic binding proteins of Escherichia coli: assessing the proteins for use as biosensors

Spectroscopic methods were used to monitor the unfolding of the leucine specific (LS) and the leucine-isoleucine-valine (LIV) binding proteins. Our studies indicate that ligand-free protein undergoes a simple two-state unfolding, whereas the protein-ligand complex undergoes a three-state unfolding model. Ligand binding causes significant stabilization of both proteins. There is correlation between ligand hydrophobicity and protein stabilization: the most hydrophobic ligand, isoleucine, causes the most significant stabilization of LIV protein. A disulfide bond present in N-domain of both proteins makes a large contribution to the protein stability of these periplasmic binding receptors.     

Structural basis for substrate recognition by the editing domain of isoleucyl-tRNA synthetase

In isoleucyl-tRNA synthetase (IleRS), the "editing" domain contributes to accurate aminoacylation by hydrolyzing the mis-synthesized intermediate, valyl-adenylate, in the "pre-transfer" editing mode and the incorrect final product, valyl-tRNA(Ile), in the "post-transfer" editing mode. In the present study, we determined the crystal structures of the Thermus thermophilus IleRS editing domain complexed with the substrate analogues in the pre and post-transfer modes, both at 1.7 A resolution. The active site accommodates the two analogues differently, with the valine side-chain rotated by about 120 degrees and the adenosine moiety oriented upside down. The substrate-binding pocket adjusts to the adenosine-monophosphate and adenosine moieties in the pre and post-transfer modes, respectively, by flipping the Trp227 side-chain by about 180 degrees . The substrate recognition mechanisms of IleRS are characterized by the active-site rearrangement between the two editing modes, and therefore differ from those of the homologous valyl and leucyl-tRNA synthetases from T.thermophilus, in which the post-transfer mode is predominant. Both modes of editing activities were reduced by replacements of Trp227 with Ala, Val, Leu, and His, but not by those with Phe and Tyr, indicating that the aromatic ring of Trp227 is important for the substrate recognition. In both editing modes, Thr233 and His319 recognize the substrate valine side-chain, regardless of the valine side-chain rotation, and reject the isoleucine side-chain. The T233A and H319A mutants have detectable editing activities against the cognate isoleucine.     

L-arabinose transport and the L-arabinose binding protein of escherichia coli

The active accumulation of L-arabinose by arabinose induced cultures of Escherichia coli is mediated by 2 independent transport mechanisms. One, specified by the gene locus araE, is membrane bound and possesses a relatively “low affinity.” The other, specified in part by the genetic locus araF, contains as a functional component the L-arabinose binding protein and functions with a “high affinity” for the substrate. The L-arabinose binding protein has been purified, partially characterized, crystallized, and sequenced.     

The leucine binding proteins of Escherichia coli as models for studying the relationships between protein structure and function

The genes encoding the leucine binding proteins in E coli have been cloned and their DNA sequences have been determined. One of the binding proteins (LIV-BP) binds leucine, isoleucine, valine, threonine, and alanine, whereas the other (LS-BP) binds only the D- and L-isomers of leucine. These proteins bind their solutes as they enter the periplasm, then interact with three membrane components, livH, livG, and livM, to achieve the translocation of the solute across the bacterial cell membrane. Another feature of the binding proteins is that they must be secreted into the periplasmic space where they carry out their function. The amino acid sequence of the two binding proteins is 80% homologous, indicating that they are the products of an ancestral gene duplication. Because of these characteristics of the leucine binding proteins, we are using them as models for studying the relationships between protein structure and function.     

Characterization of Trypanosoma cruzil-cysteine transport mechanisms and their adaptive regulation

l -Cysteine and methionine are unique amino acids that act as sulfur donors in all organisms. In the specific case of Trypanosomatids, l -cysteine is particularly relevant as a substrate in the synthesis of trypanothione. Although it can be synthesized de novo , l -cysteine is actively transported in Trypanosoma cruzi epimastigote cells. l -Cysteine uptake is highly specific; none of the amino acids assayed yield significant differences in terms of transport rates. l -Cysteine is transported by epimastigote cells with a calculated apparent K _m of 49.5 μM and a V _max of about 13 pmol min⁻¹ per 10⁷ cells. This transport is finely regulated by amino acid starvation, extracellular pH, and between the parasite growth phases. In addition, l -cysteine is incorporated post-translationally into proteins, suggesting its role in iron–sulfur core formation. Finally, the metabolic fates of l -cysteine were predicted in silico .     

Control of amino acid transport in the mammary gland of the pregnant mouse

The regulation of the uptake of the amino acid analog α-aminoisobutyric acid was studied in diced mammary glands from pregnant mice. Stimulation of uptake by insulin was not prevented by inhibitors of protein synthesis; protein synthesis inhibitors decreased uptake by 20%; this response occurred more promptly in insulintreated tissues. Elimination of extracellular amino acids led to a substantial increase in transport which was not abolished by inhibitors of protein synthesis. These results indicate that insulin does not increase amino acid transport in this system by altering synthesis and degradation of transport protein. They are consistent with a model in which the activity of the existing amino acid transport protein is subject to negative feedback regulation from the intracellular amino acid pool.     

Crystal structure of the dipeptide binding protein from Escherichia coli involved in active transport and chemotaxis.

The Escherichia coli periplasmic dipeptide binding protein functions in both peptide transport and taxis toward peptides. The structure of the dipeptide binding protein in complex with Gly-Leu (glycyl-L-leucine) has been determined at 3.2 A resolution. The binding site for dipeptides is designed to recognize the ligand's backbone while providing space to accommodate a variety of side chains. Some repositioning of protein side chains lining the binding site must occur when the dipeptide's second residue is larger than leucine. The protein's fold is very similar to that of the Salmonella typhimurium oligopeptide binding protein, and a comparison of the structures reveals the structural basis for the dipeptide binding protein's preference for shorter peptides.     

Altered rates of protein transport in Arabidopsis mutants deficient in chloroplast membrane unsaturation

Protein transfer across membranes is mediated by protein machinery embedded in the membrane. The complement of different lipid classes within a membrane is known to influence the efficiency of some protein translocation processes, but very little is known about whether the fatty acid composition of the membrane bilayer also affects protein transport. We investigated this issue using three mutants of Arabidopsis, fad6, fad5, and fad3 fad7 fad8, that have reduced levels of fatty acid unsaturation in their thylakoid membranes. Interestingly, the effect of reduced unsaturation was different for three distinct pathways of protein transport. In thylakoids from all three mutants, transport of the OE17 protein on the DeltapH/Tat pathway was reduced by up to 50% relative to wild-type controls, when assays were run at 10, 20 or 30 degrees C. By contrast, transport of the OE33 protein on the Sec pathway was substantially increased in all the mutants at the three temperatures. Transport of the CF(O)II protein (ATPg) on the 'spontaneous' pathway was largely unaffected by reduced unsaturation of the thylakoid membranes. Experiments with intact chloroplasts from wild-type Arabidopsis and the three mutants confirmed these changes in thylakoid transport reactions and also demonstrated an increased rate of protein import across the chloroplast envelope in each of the mutants. This increased capacity of chloroplast protein import may partially compensate for the reduced capacity of thylakoid transport by the DeltapH/Tat pathway. The fad5, fad6 and fad3 fad7 fad8 mutants used in this study grow normally at 15-20 degrees C, but exhibit reduced photosynthesis and growth, relative to wild-type controls, at low temperatures (4 degrees C). The results reported here indicate that protein transport and chloroplast biogenesis may well contribute to these low-temperature phenotypes.     

Role of acylCoA binding protein in acylCoA transport,metabolism and cell signaling

Long chain acylCoA esters (LCAs) act both as substrates and intermediates in intermediary metabolism and as regulators in various intracellular functions. AcylCoA binding protein (ACBP) binds LCAs with high affinity and is believed to play an important role in intracellular acylCoA transport and pool formation and therefore also for the function of LCAs as metabolites and regulators of cellular functions [1]. The major factors controlling the free concentration of cytosol long chain acylCoA ester (LCA) include ACBP [2], sterol carrier protein 2 (SCP2) [3] and fatty acid binding protein (FABP) [4]. Additional factors affecting the concentration of free LCA include feed back inhibition of the acylCoA synthetase [5], binding to acylCoA receptors (LCA-regulated molecules and enzymes), binding to membranes and the activity of acylCoA hydrolases [6].     

Differential effects of visible light on active transport in E. coli

Light of wavelengths above 400 nm inactivated several active transport systems in E. coli ML 308. Rates of inactivation for uptake of threonine, glycine, leucine and methionine were similar and differed from those for methyl thio-β-D-galactoside and phenylalanine. These differential effects indicate that inactivation of the threonine, glycine, leucine and methionine systems is linked to a common photochemical lesion differing from that involved in the inactivation of the methyl thio-β-D-galactoside and phenylalanine systems. These lesions may serve as labels to identify molecules involved in transport or energy coupling processes.     

Sulphydryl groups in the maintenance of the activity of binding protein for N-1-naphthylphthalamic acid fromAcer pseudoplatanus cells

Binding protein for N-1-naphthylphthalamic acid (NPA), an auxin transport inhibitor, was studied by analysis of the effects of reactions which modify particular amino acid side chains upon their binding activity. Na₂SO₃, N-ethylmaleimide (NEM) and dithiobisnitrobenzoic acid all inhibited the specific binding of NPA to its binding protein fromAcer pseudoplatanus L. cells. The presence of 10^-6 M Na₂SO₃ in the binding assay reduced the affinity of the binding protein to NPA from K_d of 1.5 ￡ 10^-8 M to K_d of 2.1 ￡ 10_-8 M, while concentration of the binding protein was not significantly changed. When the same analysis was applied to NPA binding to the NEM-treated membrane particles, it was found that NEM inactivated binding without changing the affinity for NPA. This study revealed the importance of sulphydryl group(s) in the maintenance of NPA binding protein activity.     

Hormonal regulation of hepatic amino acid transport

The transport of 2-aminoisobutyric acid (AIB) into liver tissue was increased by both insulin and glucagon. We have now shown that these hormones do not stimulate the same transport system. Glucagon, possibly via cAMP, increased the hepatic uptake of AIB by a mechanism which resembled system A. This glucagon-sensitive system could be monitored by the use of the model amino acid MeAIB. In contrast, the insulin-stimulated system exhibited little or no affinity for MeAIB and will be referred to as system B. On the basis of other reports that the hepatic transport of AIB is almost entirely Na⁺ dependent and the present finding that the uptake of 2-aminobicyclo [2,2,1] heptane-2-carboxylic acid (BCH) was not stimulated by either hormone, we conclude that system B is Na⁺ dependent. Furthermore, insulin added to the perfusate of livers from glucagon-pretreated donors suppressed the increase in AIB or MeAIB uptake. Depending upon the specificities of systems A and B, both of which are unknown for liver tissue, the insulin/glucagon ratio may alter the composition of the intracellular pool of amino acids.     

Role of specific activators of intestinal amino acid transport in Bombyx mori larval growth and nutrition.

Nutrient absorption and its modulation are critical for animal growth. In this paper, we demonstrate that leucine methyl ester (Leu-OMe) can greatly increase the activity of the transport system responsible for the absorption of most essential amino acids in the larval midgut of the silkworm Bombyx mori. We investigated leucine uptake activation by Leu-OMe in brush border membrane vesicles and in the apical membrane of epithelial cells in the midgut incubated in vitro. Moreover, the addition of this strong activator of amino acid absorption to diet significantly affected larval growth. Silkworms fed on artificial diet supplemented with Leu-OMe reached maximum body weight 12-18 h before control larvae, and produced cocoon shells up to 20% heavier than those of controls. The activation of amino acid absorption plays an essential role in larval development so that larval growth and cocoon production similar to controls reared on an artificial diet with 25% of dry mulberry leaf powder were observed in silkworms fed on an artificial diet with only 5% of mulberry powder. Arch.     

Hexose regulation of sodium-hexose transport in LLC-PK1 epithelia: The nature of the signal

Summary We have shown previously that the concentration of glucose in the growth medium regulates sodium-coupled hexose transport in epithelia formed by the porcine renal cell line LLC-PK₁. Assayed in physiological salt solution, the ratio of the concentration of -methyl glucoside (AMG) accumulated inside the cell at steady state to its concentration outside, and the number of glucose transporters, as measured by phlorizin binding, was inversely related to the glucose concentration in the growth medium. In this study, using a cloned line of LLC-PK₁ cells, we provide evidence that the difference in AMG concentrating capacity is the result of a regulatory signal and not simply due to a selection process where the growth of cells with enhanced glucose transport is favored by low glucose medium or vice-versa. By adding glucose to conditioned medium (collected after 48 hr incubation with cells and therefore containing less than 0.1mm glucose), we demonstrate that the signal in the growth medium is indeed the concentration of glucose rather than another factor secreted into or depleted from the medium. Fructose and mannose, two sugars not transported by the sodium-dependent glucose transporter, can substitute for glucose as a carbohydrate source in the growth medium and have a modest glucose-like effect on the transporter. Growth in medium containing AMG does not affect the transporter, indicating that the regulatory signal is not a direct effect of the hexose on its carrier but involves hexose metabolism.     

Genetic control of amino acid transport in sheep erythrocytes

An inherited amino acid transport deficiency results in low concentrations of glutathione (GSH) in the erythrocytes of certain sheep. Earlier studies based on phenotyping according to GSH concentrations indicated that the gene Tr ^H, which controls normal levels of GSH, behaves as if dominant or incompletely dominant to the allele Tr ^h, which controls the GSH deficiency. The present paper shows that when sheep are classified according to amino acid transport activity, the Tr ^H gene behaves as if codominant to Tr ^h. Erythrocytes from sheep homozygous for the Tr ^H gene exhibit rapid saturable l-alanine influx (apparent K _m ,21.6mm; V _max, 22.4 mmol/liter cells/hr). Cells from sheep homozygous for the Tr ^h gene exhibit slow nonsaturable l-alanine uptake (0.55 mmol/liter cells/hr at 50mm extracellular l-alanine). Cells from heterozygous sheep show saturable l-alanine uptake with a diminished V _max (apparent K _m, 19.1mm; V _max, 12.7 mmol/liter cells/hr). These erythrocytes have a significantly lower GSH concentration than cells from Tr ^H, Tr^H sheep but similar intracellular levels of dibasic amino acids.The authors are grateful to the M.R.C. for a Project Grant.     

A defined medium for and the effect of insulin on the growth,amino acid transport,and morphology of chinese hamster ovary cells,CHO-K1 (CCL 61) and the isolation of insulin “independent” mutants

Summary Insulin, FeSO₄, or transferrin are major requirements together with HEPES buffer and selenium for the growth of CHO-K1 (CCL 61) in a modified F12 medium (M-F12). Insulin stimulates growth at 1 ng/ml to 10 μg/ml. In the defined medium minus insulin, CHO-K1 grows slowly as elongated, elliptical cells in parallel arrays typical of normal diploid fibroblasts in contrast to round-to-cuboid cells in loosely overlapping arrays in the presence of serum or insulin. During prolonged incubation in the absence of insulin the cells gather up into a large spherical cluster of viable cells. Insulin “independent” mutants have been isolated whose growth rate during exponential phase in the absence of insulin (48 h to 84 or 96 hrs) is 2.7 to 3.6 times that of the parental culture. Insulin stimulates the growth of these variants only during the first 48 h and is inhibitory at 50 to 500 ng/ml during the exponential phase. Insulin induction of the A system of amino acid transport occurs in about 8 h and requires both protein and RNA synthesis. This work was supported in part by National Science Foundation Grant PCM 7903242 and by the University of California.