Specificity of the tyrosine-phenylalanine transport system in Bacillus subtilis

l-Tyrosine and l-phenylalanine enter cells of Bacillus subtilis via a system of active transport that exhibits complex kinetic behavior. The specificity of the transport system was characterized both at low concentrations of transport substrate (where affinity for l-tyrosine or l-phenylalanine is high but capacity is low) and at high concentrations (where affinity is low but capacity is high). Specificity was not found to differ significantly as a function of either l-tyrosine or l-phenylalanine concentration. Kinetic analysis showed that the relationship between the uptake of l-phenylalanine and l-tyrosine is strictly competitive. Neither l-tyrosine nor l-phenylalanine uptake was competitively inhibited by other naturally occurring l-amino acids, indicating the importance of the phenyl side chain to uptake specificity. Hence, it is concluded that l-tyrosine and l-phenylalanine are transported by a common system that is specific for these two amino acids. The abilities of analogue derivatives of l-tyrosine and l-phenylalanine to inhibit the uptake of l-[(14)C]tyrosine and l-[(14)C]phenylalanine competitively were determined throughout a wide range of substrate and inhibitor concentrations. In this manner, the contributions of the side chain, the alpha-amino group and the carboxyl group to uptake specificity were established. It is concluded that the positively charged alpha-amino group contributes more significantly to uptake specificity than does the negatively charged carboxyl group. The recognition of a phenyl ring is an essential feature of specificity; other amino acids with aromatic side chains, such as the indole and imidazole rings of l-tryptophan and l-histidine, do not compete with l-tyrosine and l-phenylalanine for uptake. The presence of the p-hydroxy substitutent in the side chain (as in l-tyrosine) enhances the uptake of the aryl amino acid analogues investigated.     

Substrate recognition by the mammalian proton-dependent amino acid transporter PAT1

The PAT family of proton-dependent amino acid transporters has recently been identified at the molecular level. This paper describes the structural requirements in substrates for their interaction with the cloned murine intestinal proton/amino acid cotransporter (PAT1). By using Xenopus laevis oocytes as an expression system and by combining the two-electrode voltage clamp technique with radiotracer flux studies, it was demonstrated that the aliphatic side chain of L-α-amino acids substrates can consist maximally of only one CH₂-unit for high affinity interaction with PAT1. With respect to the maximal separation between the amino and carboxyl groups, only two CH₂-units, as in γ-aminobutyric acid (GABA), are tolerated. PAT1 displays no or even a reversed stereoselectivity, tolerating serine and cysteine only in the form of the D-enantiomers. A methyl-substitution of the carboxyl group (e.g. O-methyl-glycine) markedly diminishes substrate affinity and transport rates, whereas methyl-substitutions at the amino group (e.g. sarcosine or betaine) have only minor effects on substrate interaction with the transporter binding site. Furthermore, it has been shown (by kinetic analysis of radiolabelled betaine influx and inhibition studies) that the endogenous PAT system of human Caco-2 cells has very similar transport characteristics to mouse PAT1. In summary, one has defined the structural requirements and limitations that determine the substrate specificity of PAT1. A critical recognition criterion of PAT1 is the backbone charge separation distance and side chain size, whereas substitutions on the amino group are well tolerated.     

Substrate recognition by the mammalian proton-dependent amino acid transporter PAT1

The PAT family of proton-dependent amino acid transporters has recently been identified at the molecular level. This paper describes the structural requirements in substrates for their interaction with the cloned murine intestinal proton/amino acid cotransporter (PAT1). By using the Xenopus laevis oocytes as an expression system and by combining the two-electron voltage clamp technique with radiotracer flux studies, it was demonstrated that the aliphatic side chain of L-alpha-amino acids substrates can consist maximally of only one CH2-unit for high affinity interaction with PAT1. With respect to the maximal separation between the amino and carboxyl groups, only two CH2-units, as in gamma-aminobutyric acid (GABA), are tolerated. PAT1 displays no or even a reversed stereoselectivity, tolerating serine and cystein only in the form of D-enantiomers. A methyl-substitution of the carboxyl group (e.g. O-methyl-glycine) markedly diminishes substrate affinity and transport rates, whereas methyl-substitutions at the amino group (e.g. sarcosine or betaine) have only minor effects on substrate interaction with the transporter binding site. Furthermore, it has been shown (by kinetic analyses of radiolabelled betaine influx and inhibition studies) that the endogenous PAT system of human Caco-2 cells has very similar transport characteristics to mouse PAT1. In summary, one has defined the structural requirements and limitations thet determine the substrate specificity of PAT1. A critical recognition criterion of PAT1 is the backbone charge separation distance and the side chain size, whereas substitutions on the amino group are well tolerated.     

Stereoselectivity and structural determinants in molecular recognition by the ACC transport system in isolated maize mesophyll vacuoles

The ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), is actively transported across the tonoplast of plant cells, impacting cellular compartmentation of ACC and ethylene biosynthesis. In the present study, the effects of ACC and amino acid analogs on ACC uptake into isolated maize (Zea mays L. cv. Golden Cross Bantam) mesophyll vacuoles were investigated to identify the stereospecific and structural features that are important in molecular recognition by the ACC transport system. Of the four stereoisomers of l-amino-2-ethylcyclopropane-l-carboxylic acid (AEC), (1S, 2R)-(–)-AEC having a configuration corresponding to an L-amino acid was the preferred substrate for the ACC transport system, competitively inhibiting ACC transport with a Ki of 18 μM. Of 11 neutral amino acid stereoisomers, L-isomers were stronger inhibitors of ACC transport than corresponding D-isomers. Neutral L-amino acids with nonpolar side chains generally were more inhibitory than those with polar side chains, whereas several cationic and anionic L-amino acids were ineffective antagonists of ACC transport. These observations suggest that the ACC transport system is stereospecific for relatively nonpolar, neutral L-amino acids. This conclusion was supported by the observation that group additions, substitutions, or deletions at the carboxyl. α-amino and the Pro- (R) methylene or hydrogen moieties (analogous to D-amino acids) of ACC and other neutral amino acids and analogs essentially eliminated transport inhibition. In contrast, L-amino acid analogs with variable substitutions at the distal end of the molecule remained antagonists. The relative activity of analogs was influenced by the length and degree of unsaturation of the side chain and by the location of side chain branching. Increasing the ring size of ACC analogs reduced antagonism whereas incorporating the α-amino group into the ring structure as an L-amino acid increased antagonism. The kinetics of L-methoxyvinylglycine, L-methionine. p-nitro-L-phenylalanine and 1-aminocyclobutane-l-carboxylic acid were competitive with Ki values of 3, 13, 16 and 19 μM, respectively. These results indicate that the ACC transport system can be classifie as a neutral L-amino acid carrier having a relatively high affinity for ACC and other nonpolar amino acids. The results also suggest that the carrier interacts with the carboxyl, α-amino and Pro-(R) groups and with other less restricted side chain substituents of substrate amino acids.     

Some Characteristics of Hydrolysis of Synthetic Substrates and Proteins by the Alkaline Proteinase from Aspergillus sojae

The specificity of the alkaline proteinase from Aspergillus sojae was investigated. In the specificity studies with synthetic substrates, the enzyme hydrolyzed the peptide linkages involving the carboxyl group of leucine, tyrosine, phenylalanine, arginine and lysine. In the hydrolysis of natural proteins, the enzyme liberated relatively large peptides and traces of free amino acids, suggesting that the enzyme is of a typical endo-type.

N- and C-Terminal amino acid residues appearing during time course digestion of various proteins were determined. Considering the influence of amino acid composition of substrates on the frequencies of appearance of the terminal amino acids, it was estimated that the susceptibility of peptide bonds of substrate to the enzyme depends mainly on the carboxyl side residues, and, to far less extent, on the amino side residues of the peptide bonds. The enzyme showed relatively high specificity for lysine, tyrosine, histidine, arginine and phenylalanine residues at the carboxyl side of the susceptible linkages.     

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.     

Phenylalanine transport at the human blood-brain barrier. Studies with isolated human brain capillaries

The exquisite sensitivity of brain amino acid availability to changes in plasma amino acid composition arises from the uniquely high affinity (low Km) of blood-brain barrier transport sites as compared to cell membrane transport systems in nonbrain tissues. The extension of this paradigm from rats to man assumes that the Km of blood-brain barrier amino acid transport in the human is low as in the rat. This hypothesis is tested in the present studies wherein isolated human brain capillaries are used as a model system for the human blood-brain barrier. Capillaries were obtained from autopsy brain between 20 and 45 h after death and were isolated in high yield and free of adjoining brain tissue. [3H]Phenylalanine transport into the isolated human, rabbit, or rat brain capillary was characterized by two saturable transport systems and a nonsaturable component. The Km values of phenylalanine transport into brain capillaries via the two saturable systems averaged 0.26 +/- 0.08 and 22.3 +/- 7.1 microM for five human subjects. These studies provide the first evidence for a very high affinity (Km = 0.26 microM) neutral amino acid transport system at the blood-brain barrier, and it is hypothesized that this system is selectively localized to the brain side of the blood-brain barrier. The results also show that the transport Km values for phenylalanine transport are virtually identical at both the rat and human blood-brain barrier.     

STRUCTURE ACTIVITY RELATIONS FOR THE INHIBITION OF 5-HT UPTAKE INTO RAT HYPOTHALAMIC HOMOGENATES BY SEROTONIN AND TRYPTAMINE ANALOGUES

Abstract— Various 5-HT and tryptamine analogues have been examined as inhibitors of [³H]5-HT uptake into rat hypothalamic homogenates. Acetylation of the terminal amino group or methylation of the hydroxyl group of 5-HT resulted in compounds having a reduced affinity for the serotonin uptake site. This also occurred when the hydroxyl group of 5-HT was substituted in other positions on the benzene ring. Substitution of the tryptamine side chain in the a-position by methyl or ethyl groups, but not by a carboxyl function, enhanced the affinity for the 5-HT uptake site. Increasing the tryptamine side chain by one carbon atom also resulted in a more potent compound. Several of the compounds tested are known to be either hallucinogens or antidepressants.     

Mechanisms and specificity of amino acid transport in Taenia crassiceps larvae (Cestoda)

The absorption of lysine, arginine, phenylalanine and methionine by Taenia crassiceps larvae is linear with respect to time for at least 2 min. Arginine uptake occurs by a mediated system and diffusion, and arginine, lysine and ornithine (in order of decreasing affinity) are completely competitive inhibitors of arginine uptake. The basic amino acid transport system has a higher affinity for l-amino acids than d-amino acids, and blocking the α-amino group of an amino acid destroys its inhibitory action. Phenylalanine uptake by T. crassiceps larvae is inhibited in a completely competitive fashion by serine, leucine, alanine, methionine, histidine, phenylalanine, tyrosine and tryptophan (in order of increasing affinity). Methionine apparently binds non-productively to the phenylalanine (aromatic amino acid-preferring) transport system. l-methionine uptake by larvae is inhibited more by d-alanine and d-valine than by their respective l-isomers, while d- and l-methionine inhibit l-methionine uptake equally well. The presence of an unsubstituted α-amino group is essential for an inhibitor to have a high affinity for the methionine transport system. Uptake of arginine, phenylalanine and methionine is Na⁺-insensitive, and both phenylalanine and methionine are accumulated by larvae against a concentration difference in the presence or absence of Na⁺. Arginine accumulation is precluded by its rapid metabolism to proline, ornithine and an unidentified compound.     

Identification of the LIV-I/LS system as the third phenylalanine transporter in Escherichia coli K-12

In Escherichia coli, the active transport of phenylalanine is considered to be performed by two different systems, AroP and PheP. However, a low level of accumulation of phenylalanine was observed in an aromatic amino acid transporter-deficient E. coli strain (DeltaaroP DeltapheP Deltamtr Deltatna DeltatyrP). The uptake of phenylalanine by this strain was significantly inhibited in the presence of branched-chain amino acids. Genetic analysis and transport studies revealed that the LIV-I/LS system, which is a branched-chain amino acid transporter consisting of two periplasmic binding proteins, the LIV-binding protein (LIV-I system) and LS-binding protein (LS system), and membrane components, LivHMGF, is involved in phenylalanine accumulation in E. coli cells. The K(m) values for phenylalanine in the LIV-I and LS systems were determined to be 19 and 30 micro M, respectively. Competitive inhibition of phenylalanine uptake by isoleucine, leucine, and valine was observed for the LIV-I system and, surprisingly, also for the LS system, which has been assumed to be leucine specific on the basis of the results of binding studies with the purified LS-binding protein. We found that the LS system is capable of transporting isoleucine and valine with affinity comparable to that for leucine and that the LIV-I system is able to transport tyrosine with affinity lower than that seen with other substrates. The physiological importance of the LIV-I/LS system for phenylalanine accumulation was revealed in the growth of phenylalanine-auxotrophic E. coli strains under various conditions.     

Author index for volume 171

Kinetic analyses of the irreversible inhibition of l-tyrosine and l-phenylalanine transport in Bacillus subtilis by phenylalanine chloromethyl ketone revealed that the inhibition was due to an affinity labeling process. Phenylalanine chloromethyl ketone is a competetive inhibitor of l-tyrosine and l-phenylalanine transport. The K_i values for irreversible inhibition of l-tyrosine and l-phenylalanine transport were 194 and 177 μm, respectively, and the first order rate constants for the alkylation reaction leading to inactivation of transport of l-tyrosine and l-phenylalanine were 0.016 and 0.012 min^?1, respectively. The similarity of these constants are consistent with the involvement of the same functional site for l-phenylalanine and l-tyrosine transport. A second effect of phenylalanine chloromethyl ketone was inhibition of the uptake of neutral, aliphatic amino acids; transport of basic and acidic amino acids was unaffected by it. Since high concentrations of any amino acid did not reduce the inhibitory effects of phenylalanine chloromethyl ketone on transport of neutral, aliphatic amino acids, an independent effect, not due to an affinity labeling process was inferred. A procedure for selective labeling of the l-tyrosine/l-phenylalanine transport system was demonstrated that should be applicable to the introduction of a radioactive label into the transport protein(s).     

The transport of amino acids,amino acid derivatives and ions across ion-exchange membranes

The passage of inorganic salts, glucose, amino acids and peptides across polystyrene-backed double membranes (negative-positive fixed-charge junctions) was studied in a two-compartment cell and compared to a known cellular system, the Ehrlich-Lettre ascites carcinoma. It was concluded that passage proceeds by a 1 : 1 exchange of diffusing ions in the membrane. The more rapidly transported systems reflected an increased probability of exchange in all cases, as evidenced both by a saturation effect and by the degree to which the space charge was perturbed at the membrane. The amino group (as NH₃⁺) of the amino acid involved in the exchange process was vital for transport. The presence of a second amino group, either ionized or as -NH₂, accelerated the exchange. The presence of electron-attracting groups on the side chain or of a methyl group on the alpha carbon also facilitated passage. Cation dependence was seen. Passage was hindered by a second carboxyl group, an alcoholic group, or a lengthened side chain. Use of double membranes permits experimental electrodic transport modelling and may facilitate design of a drug delivery system.     

Substrate specificity and transport mode of the proton-dependent amino acid transporter mPAT2.

The PAT2 transporter has been shown to act as an electrogenic proton/amino acid symporter. The PAT2 cDNA has been cloned from various human, mouse and rat tissues and belongs to a group of four genes (pat1 to pat4) with PAT3 and PAT4 still resembling orphan transporters. The first immunolocalization studies demonstrated that the PAT2 protein is found in the murine central nervous system in neuronal cells with a proposed role in the intra and/or intercellular amino acid transport. Here we provide a detailed analysis of the transport mode and substrate specificity of the murine PAT2 transporter after expression in Xenopus laevis oocytes, by electrophysiological techniques and flux studies. The structural requirements to the PAT2 substrates - when considering both low and high affinity type substrates - are similar to those reported for the PAT1 protein with the essential features of a free carboxy group and a small side chain. For high affinity binding, however, PAT2 requires the amino group to be located in an alpha-position, tolerates only one methyl function attached to the amino group and is highly selective for the L-enantiomers. Electrophysiological analysis revealed pronounced effects of membrane potential on proton binding affinity, but substrate affinities and maximal transport currents only modestly respond to changes in membrane voltage. Whereas substrate affinity is dependent on extracellular pH, proton binding affinity to PAT2 is substrate-independent, favouring a sequential binding of proton followed by substrate. Maximal transport currents are substrate-dependent which suggests that the translocation of the loaded carrier to the internal side is the rate-limiting step.     

The specific guanine binding site of the ribonuclease T1 family enzymes and of G-proteins is modeled in the cocrystal formed by 7-methylguanosine-5'-phosphate and phenylalanine

In the cocrystal formed by 7-methylguanosine-5'-phosphate.phenylalanine.6H2O, the interactions between guanine and phenylalanine are similar to those observed in the complex of ribonuclease T1 with 2'-guanylic acids, and those of the two G-proteins, Elongation Factor-Tu and ras oncogene p21, with GDP. They are similar in the following three points: (a) guanine N(1)H and N(2)H donate cyclic N-H...O hydrogen bonds to the carboxylate group of phenylalanine in the former cocrystal and to the side chain carboxylate group of Asp or Glu in the latter proteins, (b) O(6) of guanine accepts hydrogen bond(s) from main-chain NH group(s), and (c) the purine moiety is sandwiched between aromatic (or hydrophobic) amino acid side chains.     

Structural requirements of amino acids and related compounds for stimulation of receptors in crayfish walking leg

Summary Previous recordings from single afferent neurons in the walking legs of the crayfish demonstrated the presence of amino acid sensitivity; several characteristics of this extracellularly recorded impulse discharge, such as responses to mixtures of effective amino acids and cross adaptation experiments, indicate that all such units represent a single type of receptor. Here the effectiveness of more than 100 systematically varying amino acids and analog substances were tested in eliciting a response of the cell. Molecules lacking the amino or carboxyl group were found to be ineffective. The amino group must be unsubstituted and bear an ionic charge; modifications of the carboxyl group, including removal of charge, are tolerated with some loss of effectiveness. Replacement of the-hydrogen by a methyl group reduced effectiveness by about 1 decade. Amino acids not in the L configuration, or the amino group of which was not in the position, were still less effective. The affinity of the amino acids for the receptor was related to the number of C atoms in the side chain, with a maximum at 3. Molecules with voluminous, hydrophobic or negatively charged side chains had little effect. For high effectiveness, the side chain should be short and bear a hydrophilic group, but there is relatively little restriction on its chemical properties. These findings suggest the presence of three subunits in the receptive area. One of the proposed subunits would be involved in ionic binding to the amino group and the two others, in the formation of hydrogen bridges to the carboxyl group and the side chain. Their orientation with respect to one another would be expected to meet certain steric requirements.     

Amino acid transport during development of Neurospora crassa conidia: Substrate repression

The increasing amino acid transport activity which occurs during germination of Neurospora crassa is repressed by substrate amino acid. This repression acts on the transport systems similarly to competition in that amino acids within a specific transport class (e.g., basic) repress that system. Repression of the other system (neutral-aromatic) by that amino acid is shown to be repression of the general transport system. The level of repression and the rate of derepression after removal of the amino acid appear to depend on the nonrepressed level and rate. The extent of repression caused by increasing the concentration of the amino acid is shown to be different for two amino acids. A mutant deficient in developmental transport for arginine and phenylalanine contains two mutations. The mutation affecting phenylalanine transport maps on linkage group III and results in an accumulation of phenylalanine in the medium, thus repressing the development of this transport activity.This work was supported in part by a National Institutes of Health, U.S. Public Health Service Traineeship in Genetics (2-T01-GM1316).     

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.     

Stepwise synthesis of oligopeptides with N-carboxy α-amino acid anhydrides. II. Oligopeptides with some polar side chains

The reaction of NCA's with some amino acids having a nucleophilic functional group on the side chain was studied in a heterogeneous reaction medium (acetonitrile-water). Glutamic acid and aspartic acid, having a free carboxyl group on the side chain, were successfully used to synthesize oligopeptides without interactions of the γ- and β-carboxyl group with NCA's. Two products were obtained by the reaction of NCA with L -lysine, which contains a free amino group on the side chain. ε-Protected lysine was used to prepare α-peptides as a nucleophile in the reaction. No racemization was observed in the synthesis of peptides by the NCA method in the heterogeneous solvent system. Oligopeptides with some polar side chains were synthesized by the NCA method.     

A Phosphate-Bond-Driven Dipeptide Transport System in Streptococcus cremoris Is Regulated by the Internal pH

The uptake of amino acids and peptides by Streptococcus cremoris is mediated by different highly specific transport systems. The leucine transport system has a high affinity only for leucine, isoleucine, and valine and no affinity for leucyl-peptides. The transport system for leucyl-leucine is strongly inhibited by several dipeptides with hydrophobic, neutral, N-terminal amino acids but not by leucine. The leucyl-leucine transport system has a high affinity for dipeptides containing β-methyl groups in the side chain; the C terminus of the dipeptide affects the affinity to a much lower extent. Leucyl-leucine transport in whole cells was studied as a function of the internal pH at different external pH values in the presence and absence of nigericin. The internal pH was shown to be an important controlling factor in leucyl-leucine uptake, but the ΔpH was not involved as a driving force. At increasing external pH values, the affinity of the transport system for leucyl-leucine decreased. Uptake of leucyl-leucine was also studied in the presence of arsenate, which inhibited ATP synthesis by substrate-level phosphorylation. The rate of leucyl-leucine transport appeared to be dependent on the intracellular ATP concentrations. These results indicate that the energy for the leucyl-leucine transport is directly supplied by ATP.