Transport of L-4-azaleucine in Escherichia coli.

The uptake of L-4-azaleucine was examined in Escherichia coli K-12 strains to determine the systems that serve for its accumulation. L-4=Azaleucine in radio-labeled form was synthesized and resolved by the action of hog kidney N-acylamino-acid amidohydrolase (EC 3.5.1.B) on the racemic alpha-N-acetyl derivative of DL-[dimethyl-14C]4-azaleucine. L-4-Azaleucine is taken up in E. coli by energy-dependent processes that are sensitive to changes in the pH and to inhibition by leucine and the aromatic amino acids. Although a single set of kinetic parameters was obtained by kinetic experiments, other evidence indicates that transport systems for both the aromatic and the branched-chain amino acids serve for azaleucine. Azaleucine uptake in strain EO317, with a mutation leading to derepression and constitutive expression of branched-chain amino acid (LIV) transport and binding proteins, was not repressed by growth with leucine as it was in parental strain EO300. Lesions in the aromatic amino acid transport system, aroP, also led to changes in the regulation of azaleucine uptake activity when cells were grown on phenylalanine. Experiments on the specificity of azaleucine uptake and exchange experiments with leucine and phenylalanine support the hypothesis that both LIV and aroP systems transport azaleucine. The ability of external azaleucine to exchange rapidly with intracellular leucine may be an important contributor to azaleucine toxicity. We conclude from these and other studies that at least four other process may affect azaleucine sensitivity: the level of branched-chain amino acid biosynthetic enzymes; the level of leucine, isoleucine, and valine transport systems; the level of the aromatic amino acid, aroP, uptake system; and, possibly, the ability of the cell to racemize D and L amino acids. The relative importance of these processes in azaleucine sensitivity under various conditions is not known precisely.     

Uptake of 14C-α-aminoisobutyric acid by Phaseolus vulgaris

Specific uptake (S.U.) of α-aminoisobutyric acid ([1-¹⁴C]AIB), a non-metabolizable neutral amino acid analog, by dwarf bush bean plants (Phaseolus vulgaris cv Top Crop) demonstrated wide differences in active transport between various plant organs. The kinetic and timed uptake data reported were expressed as S.U. because this corrects for the diffusion of AIB which is part of the total AIB uptake process. Roots accumulated AIB to concentrations up to 18 times and leaf disks to twice those of the incubation medium. Stem tissue showed very little uptake, if any, that could not be accounted for by simple diffusion or water free space. Although initial rate kinetic studies demonstrated the presence of a normal transport system, timed uptake studies revealed greatly decreased transport by etiolated plants, suggesting a relationship between active transport and the lack of photosynthate. The reproducibility of the AIB uptake pattern by mature roots strongly supports the concept that the transport of neutral amino acids is biphasic and suggested one or more carrier systems are inducible by either low intracellular concentrations or repressed by high intracellular concentrations of the amino acid.     

Non-linear effects of temperature and urea on the thermodynamics and kinetics of folding and unfolding of hisactophilin

Extensive measurements and analysis of thermodynamic stability and kinetics of urea-induced unfolding and folding of hisactophilin are reported for 5-50 degrees C, at pH 6.7. Under these conditions hisactophilin has moderate thermodynamic stability, and equilibrium and kinetic data are well fit by a two-state transition between the native and the denatured states. Equilibrium and kinetic m values decrease with increasing temperature, and decrease with increasing denaturant concentration. The betaF values at different temperatures and urea concentrations are quite constant, however, at about 0.7. This suggests that the transition state for hisactophilin unfolding is native-like and changes little with changing solution conditions, consistent with a narrow free energy profile for the transition state. The activation enthalpy and entropy of unfolding are unusually low for hisactophilin, as is also the case for the corresponding equilibrium parameters. Conventional Arrhenius and Eyring plots for both folding and unfolding are markedly non-linear, but these plots become linear for constant DeltaG/T contours. The Gibbs free energy changes for structural changes in hisactophilin have a non-linear denaturant dependence that is comparable to non-linearities observed for many other proteins. These non-linearities can be fit for many proteins using a variation of the Tanford model, incorporating empirical quadratic denaturant dependencies for Gibbs free energies of transfer of amino acid constituents from water to urea, and changes in fractional solvent accessible surface area of protein constituents based on the known protein structures. Noteworthy exceptions that are not well fit include amyloidogenic proteins and large proteins, which may form intermediates. The model is easily implemented and should be widely applicable to analysis of urea-induced structural transitions in proteins.     

On the mechanism of substrate binding to the purine-transport system of Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae takes up adenine, guanine, hypoxanthine, and cytosine via a common energy-dependent transport system. The apparent affinity of the transport system to these and other purines and pyrimidines is correlated with their capability to be protonated to the positively charged form. Further organic molecules are competitive inhibitors when they are cationic, e.g. guanidine and octylguanidine in contrast to urea, or hexadecyltrimethylammonium in contrast to dodecylsulfate and Triton X-100. The influence of the pH on the kinetic constants of hypoxanthine transport points to a stoichiometry of one proton being associated to the transport system together with one substrate molecule. The pKa values of two ionizable groups that are involved in substrate binding are revealed; one of which (pKa = 1.8) may be attributed to the substrate, the other (pKa = 5.1) to an amino acid residue in the recognition site of the transport system. Studies with group-specific inhibitors indicate that this amino acid residue contains a carboxyl group. The results are in accordance with the assumption that a carboxyl group of the transport system, a proton and a substrate molecule arrange to an uncharged ternary complex.     

Kinetics of Neutral Amino Acid Transport Through the Blood-Brain Barrier of the Newborn Rabbit

Abstract: Since protein synthesis in the developing brain may, under certain conditions, be limited by amino acid availability, the present studies were undertaken to characterize the kinetics of large neutral amino acid transport through the blood-brain barrier (BBB) of the newborn rabbit. The K_m, V_max, and K_D of the transport of eight amino acids were determined by a nonlinear regression analysis of data obtained with the carotid injection technique. Compared with kinetic parameters observed for the adult rat, the K_m, V_max, and K_D of amino acid transport were all two- to threefold higher in the newborn. Albumin was found to bind tryptophan actively in vitro , but had no inhibitory effect on tryptophan transport through the newborn BBB. Glutamine was transported through the BBB of the newborn at rates severalfold higher than are seen in the adult rat. However, glutamine transport was not inhibited by high concentrations of N -methylaminoisobutyric acid (NMAIB), a model amino acid that is specific for the alanine-preferring or A-system present in peripheral tissues. In conclusion, these studies show that the BBB neutral amino acid transport system of the newborn rabbit has a lower affinity and higher capacity than does the BBB of the adult rat. Under conditions of high plasma amino acids, the increased capacity of the newborn transport system allows for a higher rate of amino acid transport into brain than would occur via the lower capacity system present in the adult rat brain.     

Are there both low-and high-affinity glutamate transporters in rat cortical synaptosomes?

Kinetics of sodium dependent glutamic acid transport have been studied in rat cortical synaptosomes at sufficiently high glutamic acid concentrations ([G]) to delineate the low affinity transporter. Computer optimization techniques were used to fit the data to models which account for the sodium and substrate dependence of uptake. The data fit about equally well models consisting of two carriers (Model 1) or one carrier plus a linear component (Model 2). However, the results of further studies were inconsistent with Model 1, but totally consistent with Model 2. Thus the results are incompatible with the presence of both high-and low-affinity carriers. The carrier model found in previous studies of high affinity glutamate transport predicts the effects of high [G] and [Na] observed in the present study. The biphasic effect of [Na] on velocity of uptake is the logical consequence of the operation of this model. The rate equation for this model has been utilized to define and compute kinetic parameters which characterize the transporter. These kinetic functions are remarkably similar in shape and magnitude to previous estimates from the studies of the high affinity transport (low [G]). The results of other studies by the author which corroborate and expand the predictions of the kinetic model are discussed. These have been combined with the present results to formulate a rather comprehensive model of glutamate function. This model can be used to describe function in terms of mathematical equations and to make predictions from these equations. These equations relate velocity of uptake and the kinetic parameters to sodium and substrate concentration, velocity to membrane potential, distribution ratio to the electrochemical potential, and release to time, compartment sizes, and exchange constants. Such processes as concentration in the presynaptic terminal, depolarization induced release, re-uptake following stimulus induced release, and postsynaptic depolarization are all possible consequences of the operation of this model. The wide applicability of the model to the transport of other substrates in addition to glutamate is discussed.     

Characteristics of an Uptake System for α-Aminoisobutyric Acid in Leishmania tropica Promastigotes1

ABSTRACT. Leishmania tropica promastigotes transport α-aminoisobutyric acid (AIB), the nonmetabolizable analog of neutral amino acids, against a substantial concentration gradient. AIB is not incorporated into cellular material but accumulates within the cells in an unaltered form. Intracellular AIB exchanges with external AIB. Various energy inhibitors (amytal, HOQNO, KCN, DNP, CCCP, and arsenate) and sulfhydryl reagents (NEM, pCMB, and iodoacetate) severely inhibit uptake. The uptake system is saturable with reference to AIB-and the Lineweaver-Burk plots show biphasic kinetics suggesting the involvement of two transport systems. AIB shares a common transport system with alanine, cysteine, glycine, methionine, serine, and proline. Uptake is regulated by feedback inhibition and transinhibition.     

Kinetic properties and characteristics of mouse liver mitochondrial asparagine aminotransferase

Mouse liver asparagine aminotransferase has been found to be a mixture of enzyme forms having a cytosolic component and a mitochondrial component. The molecular weight of the mitochondrial enzyme is 70,800. The mitochondrial asparagine aminotransferase is strongly inhibited by aminooxyacetate. It is less affected by D-cycloserine but a small amount of inhibition is observed. Cysteine strongly inhibits the enzyme as do several sulfhydryl modifying reagents. The activities of the cytosolic and mitochondrial aminotransferases have been separated, and the kinetic properties of the mitochondrial form determined. The mouse liver mitochondrial asparagine aminotransferase is fairly specific for asparagine, utilizing very few amino acids as alternate amino donors and none to a great extent. The keto acid specificity is very broad, but glyoxylate is one of the most active amino group acceptors. The kinetic properties of the mitochondrial enzyme are also reported here and the data indicate strong substrate and product inhibition. Abortive complex formation may account for the deviation of the double reciprocal plots from the expected pattern.     

Kinetic and equilibrium models for biosorption of Cr(VI) on chemically modified seaweed,Cystoseira indica

The biosorption data of hexavalent chromium by marine brown algae Cystoseira indica, which was chemically modified by crosslinking with epichlorohydrin (CB1, CB2), or oxidized by potassium permanganate (CB3), or only washed with distilled water (RB), has been used for kinetic studies based on fractional power, Elovich, pseudo-first order and pseudo-second order rate expressions. Five three parameter biosorption isotherm models, viz. Redlich–Peterson, Sips, Khan, Radke–Prausnitz and Toth are tested for their applicability apart from 6 two-parameter models. Non-linear curve fitting procedure was adopted for fitting the kinetic as well as equilibrium data in the kinetic and isotherm models and for the determination of parameters. The time-dependent Cr(VI) biosorption data were well-described by pseudo-second-order kinetic model. The intraparticle diffusion study revealed that film diffusion might be involved in Cr(VI) biosorption in the present case. Among the two-parameter models, the Langmuir model produces the best fit, while, among the three-parameter models, the best fit is produced by the Khan model, for the biosorption of Cr(VI) on all the four biosorbents studied.     

Lipid and temperature dependence of the kinetic and thermodynamic parameters for active amino acid transport in Escherichia coli K1060

The influence of membrane physical state on the kinetic and thermodynamic parameters for the active transport systems for two amino acids has been investigated in Escherichia coli K1060, an unsaturated fatty acid auxotrophic mutant. The apparent Michaelis constant (Km) for the uptake of L-[14C]glutamine (0.05 to 0.08 microM) or L-[14C]proline (1 microM approx.) is invariant with temperature for this mutant grown on elaidate (18:1t), palmitelaidate (16:1t), oleate (18:1c), palmitoleate (16:1c) and linoleate (18:2c,c). Arrhenius plots of the maximum velocities (Vmax) for L-glutamine transport in cells grown on 16:1t, 18:1c and 16:1c are biphasic within a limited temperature range peculiar to each UFA supplementation. Above an upper temperature limit also displayed by 18:1t and 18:2c,c-cells, Vmax decreases with temperature. A characteristic temperature (Tb) marks the point of intersection of the biphasic slope of the Arrhenius plots, and activation energy (Ea) is lower above than below Tb. Differential thermal analysis considered with membrane lipid fatty acyl profiles indicates that the upper temperature limit is governed by both membrane lipid acyl chain fluidity and heterogeneity, while Tb is governed by fluidity alone. Data on L-proline transport Vmax are similar, but the upper temperature limit and Tb are each shifted to lower temperatures relative to L-glutamine. We suggest that membrane defects related to energy-coupling and caused by abnormal fluidity and physical state are responsible for the peculiar temperature dependences of Vmax for these active transport processes.     

Methionine transport in S37 cells. Substrate-dependent function of amino acid transport system A in exchange processes.

Methionine had been observed to interact with two principal transport systems for amino acids in mammalian cells, the A and L systems. The present study of methionine transport and of exchange processes through system A arose in the course of a study to define the specificity of a transinhibition effect caused by cysteine. Methionine uptake through two transport systems in the S37 cell was confirmed by the occurrence of a biphasic double-reciprocal plot for labeled methionine uptake. Preloading cells with methionine stimulated labeled histidine uptake through systems A and L. Efflux of labeled methionine from cells was stimulated by histidine in a biphasic manner, so that bothe systems A and L can be used for exchange when methionine is the intracellular amino acid. Aminocycloheptanecarboxylic acid elicited exchange efflux of labeled methionine only through system L. ALPHA-Aminoisobutyric acid and N-methyl-alpha-aminoisobutyric acid both stimulated efflux of labeled N-methyl-alpha-aminoisobutyric acid from S37 cells. These findings are interpreted a showing that transport system A is capable of functioning as an exchange system depending upon the identity of intracellular and extracellular substrates available.     

Modeling of Cellular Arginine Uptake by More Than One Transporter

Determining the kinetic constants of arginine uptake by endothelial cells mediated by more than one transporter from linearization of data as Eadie-Hofstee plots or modeling which does not include the concentration of trace radiolabeled amino acid used to measure uptake may not be correct. The initial rate of uptake of trace [³H]l-arginine by HUVECs and ECV₃₀₄ cells in the presence of a range of unlabeled arginine and modifiers was used in nonlinear models to calculate the constants of arginine uptake using GraphPad Prism. Theoretical plots of uptake derived from constants determined from Eadie-Hofstee graphs overestimated uptake, whereas those from the nonlinear modeling approach agreed with experimental data. The contribution of uptake by individual transporters could be modeled and showed that leucine inhibited the individual transporters differently and not necessarily competitively. N-Ethylmaleimide inhibited only y⁺ transport, and BCH may be a selective inhibitor of y⁺L transport. The absence of sodium reduced arginine uptake by y⁺L transport and reduced the K _m′, whereas reducing sodium decreased arginine uptake by y⁺ transport without affecting the K _m′. The nonlinear modeling approach using raw data avoided the errors inherent in methods deriving constants from the linearization of the uptake processes following Michaelian kinetics. This study provides explanations for discrepancies in the literature and suggests that a nonlinear modeling approach better characterizes the kinetics of amino acid uptake into cells by more than one transporter.     

Evidence that cycloleucine affects the high-affinity systems of amino acid uptake in cultured human fibroblasts.

The influence of cycloleucine on kinetic parameters of uptake of L-alanine, L-proline and L-leucine into cultured human fibroblasts was examined under initial-rate conditions with substrate concentrations of 0.05-10 mM and 5 mM-cycloleucine. Kinetic data obtained by computer analysis showed that, in the absence of cycloleucine, cell uptake was heterogeneous for each amino acid. L-Alanine and L-leucine entered by two transport systems with different affinities; L-proline was taken up by one saturable transport system plus a diffusion-like process. This heterogeneity disappeared in the presence of cycloleucine, since the high-affinity systems were no longer detectable. The remaining process had the same kinetic constants as the low-affinity system for alanine and leucine and a KD similar to the diffusion constant for proline. The influence of cycloleucine on the amino acid uptake was not specific either to the amino acid concerned or to a particular transport system, since the three neutral amino acid-transport systems, A, ASC and L, were involved in these experiments. This influence was shown to be unaffected by the absence of Na+ (for leucine uptake). ATP content of the cells was identical in the presence or in the absence of cycloleucine.     

Mathematical representation of batch culture data

A generalized logistic equation is proposed for the mathematical representation of batch culture kinetic data. Properties of the equation are discussed. A computer program is used to fit the generalized equation to both artificial and actual batch culture data. The equation is shown to be capable of fitting data exhibiting lag, exponential, deceleration, stationary, and death phases, as well as diauxic growth. The fitted equation is useful for differentiation, interpolation, and other manipulations of the data, and it is a convenient means of data storage.     

On the difficulties of fitting the double Michaelis-Menten equation to kinetic data

The double Michaelis-Menten equation describes the reaction kinetics of two independent, saturable uptake mechanisms. The use of this equation to describe drug uptake has been reported several times in the literature, and several methods have been published to fit the equation to data. So far, however, confidence intervals on the fitted kinetic parameters have not been provided. We present a grid-search method for fitting the double Michaelis-Menten equation to kinetic uptake data, and a Monte-Carlo procedure for estimating confidence intervals on the fitted parameters. We show that the fitting problem is extremely ill-conditioned, and that very accurate data are required before any confidence can be placed in the fitted parameters.     

Uptake of Amino Acids and Other Organic Compounds by Lemna paucicostata Hegelm. 6746

A survey of the capacity of Lemna paucicostata to take up organic compounds such as might be present in the natural environment of this plant has identified eight discrete transport systems. Reciprocal inhibition studies defined the preferred substrates for these systems as follows: (a) neutral l-α-amino acids, (b) basic amino acids, (c) purine bases, (d) choline, (e) ethanolamine, (f) tyramine, (g) urea, and (h) aldohexoses. Each of these systems takes up its preferred substrates at high rates. At low concentrations, each Lemna frond during each minute takes up amounts which would be found in volumes ranging from 0.4 (tyramine) to 3.9 (urea) times its own volume. The two systems for amino acid transport both showed kinetics of the biphasic type, so that uptake by each can be described as the composite result of two Michaelis-Menten processes. The neutral amino acid system neither transports basic amino acids nor is inhibited by these compounds. The basic amino acid system does not transport neutral amino acids but is strongly inhibited by some, but not all, of these compounds. It is argued that the maintenance of these active, specific, and discrete systems in Lemna suggests they play important roles permitting this plant to utilize organic compounds occurring naturally in its environment.     

A kinetic analysis of the release of acidic amino acids from rat cortical synaptosomes following pre-loading with [14C]glutamic acid

The kinetics of the release of acidic amino acids have been studied in rat cortical synaptosomes. After pre-loading for 30 minutes in labeled glutamate, labeled glutamate and aspartate appear to be totally releasable. However, extra-synaptosomal Ca does not facilitate release. When elevated [K]₀ was used to depolarize, release was unaffected by removal of Ca from the incubation medium. When veratridine was used as a depolarizing agent, presence of Ca in the incubation medium inhibited release. In all solutions, semi-log plots of synaptosomal label content as a function of time were non-linear, which is incompatible with release from a single compartment. Previous studies of the effect of membrane potential on transport led to the development of a carrier model which should participate in depolarization induced release (19). Under the conditions used in the present studies, this carrier should be saturated. When the data were fitted to a two compartment model, with release from compartment A linear with compartment size and release from compartment B via a saturated carrier, an excellent fit was obtained. Under control conditions, about 90% of the labeled amino acid is in compartment B and about 70% of the total release is from this compartment. Rate of release is greatly accelerated in depolarizing solutions. Under depolarizing conditions, there is a large shift of labeled amino acid from compartment B to compartment A and release from compartment A predominates. Analysis of the results under the several depolarizing conditions used shows that the present results are consistent with the predictions of the carrier model which has been developed from previous studies of the Na and membrane potential dependence of glutamate transport.     

Kinetic and molecular properties of citraconyl-aldolase. The reversible denaturation and hybridization of the native and modified enzymes

1. The preparation of enzymically active N-citraconyl derivatives of fructose diphosphate aldolase from rabbit muscle is described. Reaction is restricted to amino groups and the derivatives are not very heterogeneous with respect to the number of substituents. 2. Linear double-reciprocal plots of enzyme velocity against substrate concentration are found up to about 15% blocking of amino groups. With more than 15% blocking, there is a marked downward curvature in the double-reciprocal plots at high substrate concentrations. 3. Over the range 0-25% blocking of amino groups the apparent V(max.) for fructose diphosphate falls to 10% that of the native enzyme, and the apparent K(m) rises from 1 to 400mum. 4. Various pieces of evidence suggest that citraconyl-aldolase is slightly distorted in structure compared with the native enzyme. However, the kinetic properties and tetrameric structure of citraconyl-aldolase can be completely recovered after denaturation in 4m-guanidine hydrochloride. 5. After removal of the citraconyl groups in acid conditions the kinetic and molecular properties of native enzyme are restored. 6. Hybrid forms of aldolase can be constructed containing native and citraconylated subunits and the suitability of these derivatives for the study of subunit interactions in the enzyme is discussed. 7. The kinetic properties of hybridized aldolase containing native and citraconylated subunits are not exactly those predicted from the kinetic properties of the two parental forms. This result is interpreted in terms of conformational changes induced in the native and modified subunits when both are present in a hybrid molecule, evidently as a result of interactions in the tetramer.     

Transport of cationic and zwitterionic amino acids in preimplantation rat conceptuses

The ability of preimplantation rat conceptuses to take up several amino acids was examined under a variety of conditions, and the characteristics of uptake were compared to those determined previously for mouse conceptuses. Mediated leucine transport in two-cell rat conceptuses is Na(+)-independent and inhibited almost completely by 2-amino-endobicyclo[2.2.1]heptane-2-carboxylic acid (BCH), so it resembles system L which predominates in two-cell mouse conceptuses. System L becomes less conspicuous than homoarginine-sensitive, Na(+)-independent leucine transport (provisionally designated system bo,+) by the time rat conceptuses develop into blastocysts, as is also the case for mouse conceptuses. In contrast to leucine transport, system bo,+ appears to be the most conspicuous transporter of cationic amino acids throughout preimplantation development of both species. A Na(+)-independent cation-preferring amino acid transport process also appears to be present in rat as well as in mouse conceptuses. Moreover, rat conceptuses resemble mouse conceptuses because Na(+)-dependent transport system Gly activity virtually disappears from them by the time they form blastocysts. Unlike mouse conceptuses, however, Na(+)-dependent system Bo,+ activity appears to be present throughout preimplantation development of rat conceptuses, whereas it has not been detected until at least the two-cell stage in the mouse. Although system Bo,+ becomes more conspicuous in mouse than in rat conceptuses by the time they form blastocysts, system Bo,+ activity appears to increase when blastocysts of both species are removed from the uterus just prior to implantation. The latter observation is consistent with the possibility that system Bo,+ activity is controlled, in part, by the uterus near the time of implantation, although further studies are needed to verify this possibility. Similarities as well as differences in the amino acid transport processes present in conceptuses of rats and mice may eventually be understood best in relation to the environments in which they develop in vitro and in situ.     

Glucagon regulation of amino acid transport in hepatocytes: Effect of cell enucleation

Glucagon and cAMP analogs stimulate amino acid transport in freshly isolated hepatocytes by inducing the synthesis of new transport proteins. The role of the cell nucleus in the glucagon regulation of amino acid transport has been studied in rat hepatocytes enucleated by centrifugation through a discontinuous Ficoll gradient in the presence of cytochalasin B. Enucleated hepatocytes take up alpha-aminoisobutyric acid (AIB) through a Na⁺-dependent transport component with kinetic properties similar to those found in intact hepatocytes. Cytoplasts prepared from glucagon-stimulated cells retain the increase AIB transport induced by the hormone in the intact cells. The direct addition of glucagon to cytoplasts has no effect on AIB transport, in spite of the fact that the cytoplasts exhibit a higher capacity to bind glucagon than their nucleated counterparts. These data indicate that the nucleus is required for the glucagon stimulation of amino acid transport in isolated hepatocytes.