Phospholipid composition of the membrane of lutoids from Hevea brasiliensis latex

Intact lutoids were isolated from the latex of Hevea brasiliensis and purified on a sucrose density gradient. Lutoid membranes prepared by osmotic lysis are characterized by a high content of phosphatidic acid in which unsaturated and saturated fatty acids are present in equal proportions. Linolenic acid is absent. The results are discussed in relation to the biological role of lutoids in the latex vessels of Hevea brasiliensis.     

Proton-coupledl-lysine uptake by renal brush border membrane vesicles from mullet (Mugil cephalus)

The uptake of the basic amino acid, L-lysine, was studied in brush border membrane vesicles isolated from the kidney of the striped mullet (Mugil cephalus). The uptake of L-lysine was not significantly stimulated by a Na+ gradient and no overshoot was observed. However, when a proton gradient (pHo = 5.5; pHi = 8.3) was imposed across the membrane in the absence of Na+, uptake was transiently stimulated. When the proton gradient was short circuited by the proton ionophore, carbonylcyanide p-triflouromethoxyphenyl hydrazone, proton gradient-dependent uptake of lysine was inhibited. Kinetics of lysine uptake determined under equilibrium exchange conditions indicated that the Vmax increased as available protons increased (2.1 nmol/min/mg protein at pH 7.5 to 3.7 nmol/min/mg at pH 5.5), whereas the apparent Km (4.9 +/- 0.6 mM) was not altered appreciably. When membrane potential (inside negative) was imposed by K+ diffusion via valinomycin, a similar (but smaller) stimulation of lysine uptake was observed. When the membrane potential and the proton gradient were imposed simultaneously, a much higher stimulation in lysine uptake was shown, and the uptake of lysine was approximately the sum of the components measured separately. These results indicate that the uptake mechanism for basic amino acids is different from that of neutral or acidic amino acids and that the proton-motive force can provide the driving force for the uptake of L-lysine into the isolated brush border membrane vesicles.     

Amino acid transport by the filamentous fungus Arthrobotrys conoides

Uptake of l-valine by germinated spores of Arthrobotrys conoides has all the characteristics of a system of transport that requires an expenditure of energy by the cells. It is dependent on temperature and has an energy of activation of 16,000 cal/mole. Uptake is optimal at pH 5 to 6. l-Valine accumulated against a concentration gradient and is not lost from the cells by leakage or exchange. The process requires energy supplied by the metabolic reactions that are inhibited by catalytic amounts of 2,4-dinitrophenol and azide. The kinetics of the system are consistent with a mechanism of transport that depends on a limited number of sites on the cell surface, and the Michaelis constant for the system is 1.5 x 10(-5) to 7.5 x 10(-5)m. Modification of the amino or carboxyl group abolishes l-valine uptake. The process is competitively inhibited by d-valine, glycine, and other neutral amino acids (K(i) = 1.5 x 10(-5) to 4.0 x 10(-5)m), indicating a lack of stereospecificity, and also indicating that aliphatic side chain is not required for binding with the carrier. The transport system has less affinity for acidic amino acids (glutamic and aspartic acids) than neutral amino acids, and a greater affinity for basic amino acids (histidine, lysine, and arginine). The range of affinity is in the order of 100, as measured in terms of K(i) values for various compounds. The data presented provide suggestive evidence that the uptake by A. conoides of all amino acids except proline is mediated by a single carrier system that possesses an overall negative charge.     

Transport of basic amino acids in Riccia fluitans: Evidence for a second binding site

The transport of several amino acids with different side-chain characteristics has been investigated in the aquatic liverwort Riccia fluitans. i) The saturation of system I (neutral amino acids) by addition of excess -aminoisobutyric acid to the external medium completely eliminated the electrical effects which are usually set off by neutral amino acids. Under these conditions arginine and lysine significantly depolarized the plasmalemma. ii) L- and D-lysine/arginine were discriminated against in favour of the L-isomers. iii) Increasing the external proton concentration in the interval pH 9 to 4.5 stimulated plasmalemma depolarization, electrical net current, and uptake of [¹⁴C]-basic amino acids. iv) Uptake of [¹⁴C]-glutamic acid took place only at acidic pHs. v) [¹⁴C]-histidine uptake had an optimum between pH 6 and 5.5. vi) Overlapping of the transport of basic, neutral, and acidic amino acids was common. It is suggested that besides system I, a second system (II), specific for basic amino acids, exists in the plasmalemma of Riccia fluitans. It is concluded that the amino-acid molecule with an uncharged side chain is the substrate for system I, which also binds and transports the neutral species of acidic amino acids, whereas system II is specific for amino acids with a positively charged side chain. The possibility of system II being a proton cotransport is discussed.Abbreviation AiB -aminoisobutyric acid     

Multiphasic Uptake of Amino Acids by Barley Roots

Concentration-dependence and other characteristics of uptake of ³H-labeled l -lysine, l -methionine and l -proline by excised roots of barley (Hordeum vulgare L.) were studied. Use of relatively short uptake and wash periods and low solute concentrations ensured good estimates of influx across the plasmalemma. Uptake in the range of 10^?7M– 6.3 × 10^?3M can be precisely represented by four or five phases of single, multiphasic mechanisms. The mechanisms appear to be relatively specific as judged from the competition by unlabeled analogues. Structural requirements for interaction of a compound with the uptake site for methionine are given, as are the effects of analogues on the phase pattern for this amino acid. There is no indication of separate uptake and transition sites for methionine or lysine. i.e. phase transitions seem in this case to be caused by binding of molecule(s) to the uptake site. Uptake, but not phase patterns, was highly pH-dependent. The optima were pH 5 for lysine, pH 3–5 (a broad peak) for methionine and about pH 5.5 for proline. Uptake of the three amino acids was strongly inhibited by 2,4-dinitrophenol. sulfhydryl reagents and deoxycholate.     

Characterization of amino-acid transport in Ricinus communis roots using isolated membrane vesicles

The mechanism and specificity of amino-acid transport at the plasma membrane of Ricinus communis L. roots was investigated using membrane vesicles isolated by phase partitioning. The transport of glutamine, isoleucine, glutamic acid and aspartic acid was driven by both a pH gradient and a membrane potential (internally alkaline and negative), created artificially across the plasma membrane. This is consistent with transport via a proton symport. In contrast, the transport of the basic amino acids, lysine and arginine, was driven by a negative internal membrane potential but not by a pH gradient, suggesting that these amino acids may be taken up via a voltage-driven uniport. The energized uptake of all of the amino acids tested showed a saturable phase, consistent with carrier-mediated transport. In addition, the membrane-potential-driven transport of all the amino acids was greater at pH 5.5 than at pH 7.5, which suggests that there could be a direct pH effect on the carrier. Several amino-acid carriers could be resolved, based on competition studies: a carrier with a high affinity for a range of neutral amino acids (apart from asparagine) but with a low affinity for basic and acidic amino acids; a carrier which has a high affinity for a range of neutral amino acids except isoleucine and valine, but with a low affinity for basic and acidic amino acids; and a carrier which has a higher affinity for basic and some neutral amino acids but has a lower affinity for acidic amino acids. The existence of a separate carrier for acidic amino acids is discussed.Abbreviations PM plasma membrane - TPP⁺ tetraphenylphosphonium ion - pH pH gradient - membrane potential This work was supported by the Agricultural and Food Research Council and The Royal Society. We would like to thank Mrs. Sue Nelson for help with some of the membrane preparations.     

Postnatal amino acid uptake by the rat small intestine. Energetics of membrane transport systems for amino acids in the developing jejunum

The energetics of amino acid uptake by the developing small intestine was investigated in vitro. L-valine, L-leucine, L-phenylalanine, L-methionine, L-lysine and L-arginine were all actively transported by the newborn rat jejunum. Metabolic inhibitors (e.g. 2,4-dinitrophenol) significantly reduced uptake of all amino acids but uptake against a concentration gradient was not totally abolished. Uptake of all amino acids was reduced at low[Na+]. Inhibition of transport of neutral amino acids by reduced luminal [Na+] was greater than that of basic amino acids, and the tissue was barely able to concentrate the neutral amino acids. [Na+] affected the Michaelis constant (Km) of neutral transport systems for their substrates; for the basic amino acids Km values were unaffected by the presence or absence of Na+. Ouabain significantly inhibited neutral amino acid uptake but had no effect on L-lysine or L-arginine uptake. These results are discussed in terms of the Na+ gradient hypothesis for amino acid transport, and the site of energy input to active transport. The role of glycolysis in providing energy for intestinal transport in the neonatal rat and the efficiency of Na+ dependent and independent transport mechanisms are considered. It is concluded that the energetics of amino acid transport systems in neonatal and adult rats are essentially similar.     

Amino Acid Transport into Cultured Tobacco Cells: I. LYSINE TRANSPORT

Lysine transport into suspension-cultured Wisconsin-38 tobacco cells was observed. Uptake was linear (up to 90 minutes) with respect to time and amount of tissue only after 4 to 6 hours preincubation in calcium-containing medium. The observed cellular accumulation of lysine was against a concentration gradient and not due to exchange diffusion. Transport was stimulated by low pH and characterized by a biphasic uptake isotherm with two K(m) values for lysine. System I (K(m) approximately 5 x 10(-6) molar; V(max) approximately 180 nanomoles per gram fresh weight per hour) and system II (K(m) approximately 10(-4) molar; V(max) approximately 1900 nanomoles per gram fresh weight per hour) were inhibited by N-ethylmaleimide and a variety of respiratory inhibitors. This inhibition was not due to increased efflux. In antagonism experiments, system I was inhibited most effectively by basic amino acids, followed by the sulfur amino acids. System I was only slightly inhibited by the neutral and aromatic amino acids and was not inhibited by the acidic amino acids aspartic and glutamic acids. Transport by system II was inhibited by all of the tested amino acids (including aspartic and glutamic acids) and analogs; however, this system was not inhibited by d-arginine. Neither system was strongly inhibited by d-lysine or the lysine analog S-2-aminoethyl-l-cysteine. Arginine was shown to be a competitive inhibitor of both systems with values for K(i) similar to the respective K(m) values.These studies suggest the presence of at least two amino acid permeases in W-38 tobacco cells.     

Amino acid uptake by Lemna gibba by a mechanism with affinity to neutral L-and D-amino acids

Earlier work suggested that amino acid uptake by Lemna gibba cells is a H⁺-cotransport mechanism driven by a proton-electrochemical gradient at the plasmalemma. The present investigations of the transient membrane depolarizations elicited by amino acids and tracer-uptake experiments show that all neutral -L-amino acids, D-alanine and analogues, like -alanine and p-fluorophenylalanine, are transported by the same system. It remains to be seen if there are separate mechanisms for the uptake of acidic and basic amino acids.     

Lysine transport in two barley mutants with altered uptake of basic amino acids in the root

Amino acid uptake was examined in two barley (Hordeum vulgare L.) mutants R906 and R4402 which had been selected as resistant to the lysine analog S-(2-aminoethyl)-cysteine. The mutants were found to be allelic by crossing and examination of F₁ and F₂ progeny. The mutant genes were designated aec1a and aec1b, respectively. The uptake of the basic amino acids lysine, arginine, and ornithine from 50 micromolar solutions was strongly decreased in roots of the mutants, whereas uptake of neutral and acidic amino acids was unaffected. The pattern of uptake of lysine over the range 10⁻⁷ to 10⁻² molar was consistent with there being, principally, two uptake systems operating for basic amino acids in roots and that a low-concentration, high-affinity system is reduced or lacking in the mutants. The residual transport activity in the mutants had a different relative affinity for lysine and arginine to the wild-type system. Uptake of lysine by leaf slices was unimpaired in the mutants suggesting that the leaf uptake system is unaffected by the aec1 gene.     

The primary structure of hevamine, an enzyme with lysozyme/chitinase activity from Hevea brasiliensis latex

The primary structure of hevamine, an enzyme with lysozyme/chitinase activity from Hevea brasiliensis latex, has been determined predominantly with conventional non-automatic methods. The positions of three disulfide bridges have been determined. The sequence has about 60% identity with that of a chitinase from cucumber and 95% with the N-terminal sequence of the lysozyme/chitinase of Parthenocissus quinquefolia. The half-cystine residues in hevein and cucumber chitinase are located at identical positions. Hevamine is a basic protein from the lutoids (vacuoles) of rubber latex and may have a role in plugging the latex vessels and cessation of latex flow. The differences in cellular location, charge properties and sequence between hevamine and cucumber chitinase are similar to those between class I and class II chitinases from tobacco and other plant species.     

Amino acid transport inRhodotorula glutinis

The yeastRhodotorula glutinis was found to transport amino acids against a concentration gradient (100∶1 for 10⁻⁶ m l-lysine and 1500∶1 for 10⁻⁶ m α-aminoisobutyric acid). Anaerobically, the concentration gradients of free amino acids were occasionally higher than aerobically. The influx is saturable with an apparentK _m of 1mm forl-lysine and 2mm for α-aminoisobutyric acid. The pH optimum for AIB uptake was 5.0, the apparent activation energy between 5° and 30° was 13,200 cal/mole. Competition of an asymmetric nature among various amino acids for uptake was observed. Intracellular amino acids did not leave the cell under any conditions of incubation, short of breaking up the plasma membrane, but they showed a powerful “trans” inhibitory effect on the uptake of amino acids.     

The Latex of Hevea brasiliensis Contains High Levels of Both Chitinases and Chitinases/Lysozymes

The latex of the commercial rubber tree, Hevea brasiliensis, was fractionated by ultracentrifugation as described by G. F. J. Moir ([1959] Nature 184: 1626-1628) into a top layer of rubber particles, a cleared cytoplasm, and a pellet that contains primarily specialized vacuoles known as lutoids. The proteins in each fraction were resolved by two-dimensional gel electrophoresis. Both the pellet fraction and cleared cytoplasm contained large amounts of relatively few proteins, suggesting that laticifers serve a very specialized function in the plant. More than 75% of the total soluble protein in latex was found in the pellet fraction. Twenty-five percent of the protein in the pellet was identified as chitinases/lysozymes, which are capable of degrading the chitin component of fungal cell walls and the peptidoglycan component of bacterial cell walls. Both the chitinase and lysozyme activities were localized exclusively in the pellet or lutoid fraction. The chitinases/lysozymes were resolved into acidic and basic classes of proteins and further purified. An acidic protein (molecular mass 25.5 kD) represented 20% of the chitinase activity in latex; this protein lacked the low level of lysozyme activity that is associated with many plant chitinases. Six basic proteins, having both chitinase and lysozyme activities in various ratios and molecular mass of 27.5 or 26 kD, were resolved. Two of the basic proteins had very high lysozyme specific activities which were comparable to the specific activities reported for animal lysozymes. Like animal lysozymes, but unlike previously characterized plant chitinases/lysozymes, these basic chitinases/lysozymes were also capable of completely lysing or clearing suspensions of bacterial cell walls. These results suggest that laticifers may serve a defensive role in the plant.     

Intestinal transport of monosaccharides and amino acids during postnatal development of mink

Intestinal development is typically studied using omnivores. For comparative purposes, we examined an altricial carnivore, the mink (Mustela vison). In mink, intestinal dimensions increase up to 8 wk after birth and then remain constant (length) or decrease (mass) into maturity despite continuing gains in body mass. Rates of glucose and fructose transport decline after birth for intact tissues but increase for brush-border membrane vesicles (BBMV). Rates of absorption for five amino acids that are substrates for the acidic (aspartate), basic (lysine), neutral (leucine and methionine), and imino acid (proline) carriers increase between birth and 24 h for intact tissues before declining, but increase after 2 wk for BBMV. The proportion of BBMV amino acid uptake that is Na(+)-dependent increases during development but for aspartate is nearly 100% at all ages. Tracer uptake by BBMV can be inhibited by 100 mmol/l of unlabeled amino acid, except for lysine. BBMV uptake of the dipeptide glycyl-sarcosine does not differ between ages, is not Na(+) dependent, and is only partially inhibited by 100 mmol/l unlabeled dipeptide. Despite the ability to rapidly and efficiently digest high dietary loads of protein, rates of amino acid and peptide absorption are not markedly higher than those of other mammals.     

一种快速、高效的橡胶树胶乳总RNA 提取方法

胶乳是橡胶树(Hevea brasiliensis)乳管中特殊的细胞质, 主要由橡胶粒子、黄色体、F-W粒子和普通细胞质成分构成, 其中橡胶粒子占20%-40%, 蛋白含量高达1%-2%。由于高比例橡胶粒子和蛋白质的干扰, 目前使用的胶乳RNA提取方 法都具有步骤繁琐、胶乳需求量大、操作技巧性强不易掌握等缺点。为快速、高效地获取高质量的胶乳RNA, 我们在现有方法的基础上摸索出一套步骤简单、容易操作、快速、高效提取橡胶树胶乳总RNA的简易方法, 获得了较好的实验效果。紫外分光光度计、RT-PCR和RACE分析结果表明, 使用该方法提取的胶乳RNA质量完全能够满足相应的分子操作, 但所需时间仅为目前常用方法的50%, RNA获得率提高了2-3倍, 操作难度大大降低。     

THE EFFECTS OF PHENYLALANINE ON AMINO ACID METABOLISM AND PROTEIN SYNTHESIS IN BRAIN CELLS IN VITRO1

Incubation of brain cell suspensions with 14 mM-phenylalanine resulted in rapid alterations of amino acid metabolism and protein synthesis. Both thc rate of uptake and the final intracellular concentration of several radioactively-labelled amino acids were decreased by high concentrations oi phenylalanine. By prelabelling cells with radioactive amino acids, phenylalanine was also shown to effect a rapid loss of the labelled amino acids from brain cells. Amino acid analysis after the incubation of the cells with phenylalanine indicated that several amino acids were decreased in their intracellular concentrations with effects similar to those measured with radioisotopic experiments (large neutral > small and large basic > small neutral > acidic amino acids). Although amino acid uptake and efflux were altered by the presence of 14 mwphenylalanine, little or no alteration was detected in the resulting specific activity of the intracellular amino acids. High levels of phenylalanine did not significantly altcr cellular catabolism of either alanine, lysine, leucine or isoleucine. As determined by the isolation of labcllcd aminoacyl-tRNA from cells incubated with and without phenylalanine, there was little or no alteration in the level of this precursor for radioactive alanine and lysine. There was, however, a detectable decrease in thc labelling of aminoacyl-tRNA for leucine and isoleucine. Only aftcr correcting for the changes of the specific activity of the precursors and thcir availability to translational events, could the effects of phenylalanine on protein synthesis be established. An inhibition of the incorporation into protein for each amino acid was approximately 20%.     

Dimorphism-associated changes in amino acid transport of Candida albicans

Abstract Amino acid uptake was followed during pH-regulated dimorphism of Candida albicans . It was observed that transport activities of various amino acids differed with the morphological phenotype. The uptake rates of l-alanine , l -phenylalanine and of l -lysine were lower and those of l -methionine were higher in elongated hypha (germ tube), while the rates of glycine, l -glutamic acid and l -proline were similar in bud and hyphal phenotypes. Minimum threshold of amino acids transport activity is required at the time of phenotypic commitment in a diverging population of Candida albicans .     

Identification and estimation of some uncommon diamino acids

Application of the amino acid analyzer is described for the separation, identification, and estimation of several uncommon dibasic amino acids that are eluted with a 0.35 m, pH 5.28, sodium citrate buffer between lysine and ammonia, namely, 2,5-diaminohexanoate, threo- and erythro-3,5-diaminohexanoate, 3,6-diaminohexanoate (β-lysine) and 2,4-diaminopentanoic acid. Conditions are also given for estimating d- and l-β-lysine as their l-glutamyl peptides, and for estimating the enantiomers of threo- and erythro-3,5-diaminohexanoate as the l-glutamyl derivative of the corresponding lactams.     

Characterization of system L and system y+ amino acid transport activity in cultured vascular smooth muscle cells

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.     

Allergenic proteins of natural rubber latex

As the living cytoplasm of laticiferous cells, Hevea brasiliensis latex is a rich blend of organic substances that include a mélange of proteins. A small number of these proteins have given rise to the problem of latex allergy. The salient characteristics of H. brasiliensis latex allergens that are recognized by the International Union of Immunological Societies (IUIS) are reviewed. These are the proteins associated with the rubber particles, the cytosolic C-serum proteins and the B-serum proteins that originate mainly from the lutoids. Procedures for the isolation and purification of latex allergens are discussed, from latex collection in the field to various preparative approaches adopted in the laboratory. As interest in recombinant latex allergens increases, there is a need to validate recombinant proteins to ascertain equivalence with their native counterparts when used in immunological studies, diagnostics, and immunotherapy.