Stereospecificity and electrogenicity of amino acid transport in Riccia fluitans

In the aquatic liverwort Riccia fluitans, membrane depolarization (_m), change in membrane conductance (g_m), and current-voltage (I-V) characteristics in the presence of different amino acids as well as the uptake of ¹⁴C-labeled amino acids were measured. L-isomers of the tested amino acids generate larger electrical effects (_m, g_m) than D-isomers, and the I-V characteristics show that the positive electrical inward-current of 20 mA m^-2 generated by 0.5 mM D-serine is only about 50% of the current generated by adding 0.5 mM L-serine. Whereas - and -amino acids rapidly depolarize the membrane to the same extend, with -aminobutyric acid (-AB) and dipeptides no significant electrical effects have been measured. The uptake kinetics of ¹⁴C-labeled amino acids display three components: (I) A saturable high-affinity component with K_s-values of 48 M D-alanine, 12 M -aminoisobutyric acid (AIB), 9 M L-alanine, 8 M L-proline, and 6 M L-serine, respectively; (2) an apparently linear low-affinity component, and (3) an also linear but unspecific component at concentrations >20 times the given K_s-value. Uptake of ¹⁴C-labeled AIB can be inhibited competitively by all tested neutral amino acids, the L-isomers being more effective than the D-isomers, as well as by ammonium or methylamine. Vice versa, AIB competitively inhibits uptake of L-serine and L-alanine. It is concluded that an uncharged stereospecific carrier for the investigated amino acids exists in the plasmalemma of Riccia fluitans. Accumulation ratios of about 50 suggest secondary active transport driven by a transmembrane electro-chemical gradient (mainly _m) which is generated by the electrogenic proton pump. It is suggested that this carrier binds to the amino group forming either a charged binary complex with positively charged amines (Felle 1980), or an uncharged complex with -AB or dipeptides, whereas electrogenic transport of - and -amino acids is mediated by a ternary carrier complex, probably charged by a proton.Symbols and Abbreviations _m membrane potential (mV) - E_co equilibrium potential (mV) of the transport system - g_m membrane (slope) conductance (Sm^-2) - g_m change in g_m - I-V curve current-voltage curve - AIB -aminoisobutytric acid - -AB -aminobutyric acid     

Transformation of some hydroxy amino acids to other amino acids

It has been observed that -hydroxy--amino acids are transformed into other amino acids, when heated in dilute solutions with phosphorous acid, phosphoric acid or their ammonium salts. It has been shown that as in the case of previously reported glycine-aldehyde reactions, glycine also reacts with acetone to give -hydroxyvaline under prebiologically feasible conditions. It is suggested, therefore, that the formation of -hydroxy--amino acids and their transformation to other amino acids may have been a pathway for the synthesis of amino acids under primitive earth conditions.     

Neutral changes during divergent evolution of hemoglobins

Summary A comparison of the mRNAs for rabbit and human-hemoglobins shows that synonymous changes in codons have accumulated three times as rapidly as nucleotide replacements that produced changes in amino acids. This agrees with predictions based on the so-called neutral theory. In addition, seven codon changes that appear to be single-base changes (according to maximum parsimony) are actually two-base changes. This indicates that the construction of primordial sequences is of limited significance when based on inferences that assume minimum base changes for amino acid replacements.     

Design of superactive and selective integrin receptor antagonists containing the RGD sequence

Summary Integrins play a major role in cell-cell and cell-matrix interactions. The majority of the different types of integrins recognize the tripeptide sequence arginine-glycine-aspartic acid (RGD). To explore the spatial requirements of the pharmacophore for receptor selectivity and high activity, a new procedure, spatial screening, was used. The procedure is based on the experience that the conformation of small cyclic peptides is mainly determined by the chirality of the amino acids (and glycine or proline). For example, cyclic pentapeptides with one d and four l amino acids prefer a II'/ conformation. The sequence RGDFV was shifted around this spatial II'/ template by synthesis of five peptides in which one of the amino acids was used in d-configuration. It turned out that cyclo(-RGDfV-) is a selective inhibitor for the _v3 integrin, which is strongly expressed in cancer cells. Systematic variations with different turn mimetics, retro-inverso structures, modified peptide bonds and sugar amino acids are discussed.     

Rapid microbial metabolism of non-protein amino acids in the sea

The non-protein amino acids, -alanine and -aminobutyric acid, frequently dominate the amino acid composition of deep-sea sediments. This accumulation is most likely due to the slower decomposition of non-protein amino acids by microorganisms or to the preferential adsorption of non-protein amino acids by clay minerals. We investigated relative rates of heterotrophic uptake of alanine, -ala, and -aba in sea water to see if there were different rates of microbial assimilation and respiration between these protein and non-protein amino acids. Heterotrophic uptake was rapid for all three amino acids with turnover times of hours in productive coastal waters and days in more oligotrophic open-ocean waters. Uptake of the non-protein amino acids was significantly slower than uptake of alanine, particularly in anoxic waters. However, the difference in uptake rates is probably not great enough to cause significant preferential accumulation of non-protein amino acids.     

Characteristics of Cl−-dependentl-[35S]cysteic acid transport into rat brain synaptic membrane vesicles

Uptake ofl-[³⁵S]cysteic acid (L-CA) in rat synaptic membrane vesicles was investigated. Preincubation with either 10 mMl-glutamic acid (L-Glu), 25 mM L-CA, 10 mMdl-homocysteic acid, or 25 mMdl-2-amino-4-phosphonobutyrate on membrane vesicles enhanced L-[³⁵S]CA and L-[³H]Glu uptake. Na⁺ (5 mM) and omission of Cl^– from the assay medium decreased L-[³⁵S]CA uptake into both 10 mM L-Glu-loaded and non-loaded membrane vesicles. The anion transport blockers, 4-acetamide-4-isothiocyano-2,2-disulfonic acid stibene (SITS) and 4,4-diisothiocyano-2,2-disulfonic acid stilbene (DIDS), inhibited L-[³⁵S]CA uptake in a dose-dependent manner. The maximal uptake rate for L-[³⁵S]CA was decreased by 50 M SITS, while the apparent Km value of L-CA was not changed. SITS increased the EC₅₀ value of Cl^– for L-[³⁵S]CA uptake from 5 mM to 10 mM with reduction of the maximal effect. These results suggested that L-[³⁵S]CA uptake into synaptic membrane vesicles was mediated by a SITS-sensitive hetero-exchange transport with non-labeled substrates.Abbreviations SITS 4-Acetamide-4-isothiocyano-2,2-disulfonic acid stilbene - DIDS 4,4-Diisothiocyano-2,2-disulfonic acid stilbene - CA Cysteic acid - APB 2-Amino-4-phosphonobutyrate - CSA Cysteine sulfinic acid - EGTA Ethyleneglycol bis(aminoethylether) tetraacetate - GABA -Aminobutyric acid     

Secondary transport of amino acids by membrane vesicles derived from lactic acid bacteria

Lactococci are fastidious bacteria which require an external source of amino acids and many other nutrients. These compounds have to pass the membrane. However, detailed analysis of transport processes in membrane vesicles has been hampered by the lack of a suitable protonmotive force (pmf)-generating system in these model systems. A membrane-fusion procedure has been developed by which pmf-generating systems can be functionally incorporated into the bacterial membrane. This improved model system has been used to analyze the properties of amino acid transport systems in lactococci. Detailed studies have been made of the specificity and kinetics of amino acid transport and also of the interaction of the transport systems with their lipid environment. The properties of a pmf-independent, arginine-catabolism specific transport system in lactococci will be discussed.Abbreviations pmf protonmotive force - transmembrane electrical potential - pH transmembrane pH gradient - PE phosphatidylethanolamine - PC phosphatidylcholine Paper adapted from a treatise Secondary Transport of Amino Acids by Membrane Vesicles Derived from Lactic Acid Bacteria and awarded the Kluyver Prize 1988 by the Netherlands Society of Microbiology.     

Identification of beta subunit of the rhesus monkey chorionic gonadotropin (rmCG)

Chorionic gonadotropin (CG) is a placental derived hormone that plays a crucial role in successful implantation and establishment of early pregnancy in the primates. The rhesus monkey was chosen as a model to understand the feasibility of developing human DNA immuno-contraceptive. The coding region of rhesus monkey CG -subunit (rmCG) was isolated by the TDRT-PCR method. The nucleotide sequence including the leader peptide was 499 nucleotide long and encoded 166 amino acids. In comparing with the previous known primates CG -subunits, the rmCG was the highest degree of homology with baboon CG -subunit at the deduced amino acid sequence (94%), 79.5% homology with human CG -subunit and 70.4% homology with marmoset monkey CG -subunit. The eukaryotic expression vector pCMV4-rmCG inserted full-coding cDNA sequence of rmCG was constructed, and the expression of rmCG -subunit in HeLa cells transient expressing system in vitro and BALB/c mice in vivo was determined. The results demonstrated that the recombinant PCMV4-rmCG eukaryotic expression vector could express rmCG -subunit in vitro and in vivo.     

Purification and properties of gammagamma-enolase from pig brain

Isoelectric focusing revealed three enolase isoforms in pig brain, which were designated as - (pI = 6.5), - (pI = 5.6), and -enolase (pI = 5.2). The pI of purified -enolase was also 5.2. The -enolase isoform of enolase was purified from pig brain by a purification protocol involving heating to 55°C for 3 min, acetone precipitation, ammonium sulfate precipitation (40%–80%), DEAE Sephadex ion-exchange chromatography (pH 6.2), and Sephadex G200 gel filtration. The final specific activity was 82 units/mg protein. As with other vertebrate enolases, -enolase from pig proved to be a dimer with a native mass of 85 kDa and a subunit mass of 45 kDa. The pH optimum for the reaction in the glycolytic direction is 7.2. The K _m values for 2-PGA, PEP, and Mg²⁺ were determined to be 0.05, 0.25, and 0.50 mM, respectively, similar to K _m values of other vertebrate enolases. The amino acid composition of pig -enolase, as determined by amino acid analysis, shows strong similarity to the compositions of -enolases from rat, human, and mouse, as determined from their amino acid sequences. Despite the differences seen with some residues, and considering the ways that the compositions were obtained, it is assumed that pig -enolase is more similar than the composition data would indicate. Moreover, it is likely that the sequences of pig -enolase and the other -enolases are almost identical. Li⁺ proved to be a noncompetitive inhibitor with either 2-PGA or Mg²⁺ as the variable substrate. This enolase crystallized in the monoclinic space group P2, or P2₁. An R _symm <5% was obtained for data between 50 and 3.65 Å, but was a disappointing 30% for data between 3.65 and 3.10 Å, indicating crystal disorder.     

Isolation and characterization of a cDNA encoding the zebra finch myelin proteolipid protein

A cDNA was isolated from a zebra finch telencephalon cDNA library that encodes the myelin proteolipid protein. The clone was 2874 nucleotides long containing an open reading frame of 831 nucleotides that encoded a 277 amino acid myelin proteolipid protein. The 5-and 3 untranslated regions were 112 and 1931 nucleotides, respectively. In Northern blots the clone hybridized to 3 bands of 3.5, 2.4 and 1.5 Kb in mouse brain RNA, but to only a single band of 3.0 kb in zebra finch brain RNA, suggesting the lack of alternative polyadenylation sites within the 3 untranslated region of the zebra finch PLP mRNAs. There was a small degree of homology between the zebra finch and chicken PLP 5 untranslated regions, but relatively little homology of the 5 untranslated regions of the zebra finch PLP cDNA clone with the homologous regions of PLP cDNAs of many mammalian species. Except for a small stretch of considerable homology, there was little overall homology with the 3 untranslated regions of mammalian PLP mRNAs. Approximately 10% (i.e. 28) of the amino acids in the zebra finch PLP differed from mammalian PLP, with most of these changes located within exon 3. There were 16 amino acid changes between zebra finch and chicken, suggesting that greater sequence variation in PLP structure is tolerated among avian species than among mammalian species.Abbreviations DM20 25 kDa proteolipid protein in myelin - PLP classic 30 kDa myelin proteolipid protein Special issue dedicated to Dr. Marjorie B. Lees.     

Identification and DNA sequence analysis of a γ-type gliadin cDNA plasmid from winter wheat

Summary Recombinant cDNA plasmids possessing the coding sequences for the -type gliadins were isolated from a cDNA library prepared from wheat seed poly (A⁺) RNA. One of these plasmids, pGliB48, specifically hybridizes to poly (A⁺) RNA molecules 1 400–1 500 bases in length that direct the synthesis of polypeptides at 38 Kd and 46 Kd, the latter size characteristic of the -type gliadins. The cDNA sequence of pGliB48 was determined and encompasses the 3 untranslated region as well as 245 amino acids from the C-terminus of the -type gliadin polypeptide. The 5-end of the DNA coding sequence consists of a tandem repeat unit composed of eight amino acids. Localized regions of homology are observed for the /-type and -type gliadin cDNA sequences.     

Na+/L+ selectivity in transport systemA: Effects of substrate structure

Summary The effect of amino acid structure on the selectivity between Na and Li as co-substrates for transport SystemA in the Ehrlich cell has been explored to localize relative binding positions. By various tests the relative effectiveness of the two cations varies over fivefold. Changes in structure of the amino acid that lower its response to Na tend to decrease its selectivity for Na over Li, but with many exceptions. The higher the Li level required to half-maximize amino acid entry, the slower tends to be the entry attainable for both Li and amino acid. Our attention fell on strong departures from these trends. An atypically fast uptake is produced by Li in the presence of a second amino group pK₂<8.5, in exceptional association with the known fast uptake in Na. The hydroxyl group of serine yields exceptionally strong uptake, whereas hydroxyl groups in restrained orientation (as in threonine and hydroxyprolines) sharply limit co-substrate interaction. Despite the absence of a sidechain, glycine shows unexceptional relative co-substrate responses. A sidechain in the ₂ position, as ind-alanine, lowers tolerance for both ions, an aberration largely corrected by the insertion of a second (₂) methyl group, and surprisingly, even by an N-methyl group. Forl-alanine, an N-methyl group has in contrast unfavorable effects on co-substrate interaction. These factors point to disturbance by the ₂ methyl group of the position taken by the amino acid at the site, largely rectifiable by balancing effects of a second methyl group. They also point to a position of the alkali ion quite close to the -carbon and far from the position taken in SystemASC. Addition of an ethylene bridge between the -methyl groups of (methylamino)-isobutyric acid leads to the strongest discrimination seen against Li⁺ relative to Na⁺, suggesting through crowding of the area that the alkali ion adjoins the three methyl groups of this analog.     

High D-glucose does not affect binding of α-tocopherol to human erythrocytes

The role of -tocopherol uptake system in human erythrocyte in the uptake of plasma -tocopherol has been suggested. However no information is available on -tocopherol uptake activity of human erythrocytes in the presence of high levels of D-glucose which is known to lead to pathological alterations in different cells including human erythrocytes. Therefore, in order to examine the effect of D-glucose on the binding of -tocopherol to human erythrocytes, the binding characteristics of -tocopherol to these cells were established first. Binding of [3H]-tocopherol to human erythrocytes was both saturable and specific. Scatchard analysis of -tocopherol binding to these cells showed the presence of two independent classes of binding sites with widely different affinities. The high affinity binding sites had a dissociation constant (Kd1) of 90 nM with a binding capacity (n1) of 900 sites per cell, whereas the low affinity binding sites had a dissociation constant (Kd2) of 5.2 M and a binding capacity (n2) of 105,400 sites per cell. Trypsin treatment abolished all the -tocopherol binding activity. Competition for the binding of -tocopherol to human erythrocytes was effective with other homologues of -tocopherol (-tocopherol, -tocopherol and -tocopherol) and their potency was almost equal to -tocopherol itself. The order of preference was -tocopherol > -tocopherol -tocopherol -tocopherol. Incubation of human erythrocytes with various concentrations of D-glucose did not affect -tocopherol uptake activity. Our data demonstrate the presence of an -tocopherol uptake system in human erythrocytes and that the -tocopherol uptake activity is not modulated by the presence of D-glucose.     

β-Dl-Methylene-aspartate,an inhibitor of aspartate aminotransferase,potently inhibitsl-glutamate uptake into astrocytes

[³H]Glutamate uptake into astrocytes in primary culture was potently inhibited by the aspartate analoguesl- andd-aspartic acid,Dl-threo--hydroxy-aspartic acid,l-aspartic acid--hydroxymate (IC50's: 136, 259, 168, and 560 M, respectively) and by -Dl-methylene-aspartate, a suicide inhibitor of asparate aminotransferase (IC50: 524 M), and by the endogenous sulphur-containing amino acidl-cysteinesulfinic acid (IC50: 114 M). [³H]Glutamate uptake was not significantly affected by either N-methyl-d-aspartate orDl-homocysteine thiolactone. These results demonstrate that other excitatory amino acids including aspartate andl-cysteinesulfinic acid (but excludingl-homocysteic acid) interact with the glutamate transport system of astrocytes. Inhibition of glutamate uptake may significantly increase the level of neuronal excitability.     

PC12 variants deficient in norepinephrine transporter mRNA have wild type activities of several other related transporters

Wild type PC12 pheochromocytoma cells express a Na⁺-dependent norepinephrine transporter that operates in the uptake of catecholamines. In addition to the previously described Na⁺-dependent system A for the uptake of -amino-isobutyric acid and system Gly for glycine, we have identified two other Na⁺-dependent transporter systems for amino acid uptake in these cells: 1) system for -alanine and taurine; and 2) a system for creatine. Uptake of -amino-isobutyric acid, glycine, -alanine, and creatine is not affected in some PC12 variants that were previously shown to be deficient in catecholamine uptake and to have decreased levels of norepinephrine transporter mRNA. We have isolated two PC12 cDNA clones that are essentially identical in sequence to recently reported cDNAs for rat brain taurine and creatine transporters, respectively, and a third cDNA that appears to code for a novel transporter. mRNAs for these three transporters are present at wild type levels in those variants that express no or little norepinephrine transporter mRNA. These results support the notion that the expression of catecholamine reuptake transporters may be particularly susceptible to down-regulation.     

Auxin uptake and action of N-1-naphthylphthalamic acid in corn coleoptiles

The validity of a chemiosmotic hypothesis for uptake of weak acids as an explanation for the accumulation of auxin by cells has been explored further by comparing the uptake of indole-3-acetic acid (IAA) by 1-mm segments of corn (Zea mays L.) coleoptiles with that of benzoic acid and two neutral indoles, indoleethanol and indoleacetonitrile, which do not ionize. These substances, while structurally related to IAA lack both auxin activity and polar transport. Uptake of IAA and benzoic acid increase with decreasing external pH, whereas the uptake of the two neutral indoles is independent of external pH.Although metabolism of IAA, during 90 min or less, is minimal and without significant effect on its uptake, metabolism of benzoic acid appears responsible for the apparent saturation of benzoic acid uptake at high concentrations. An inhibitor of auxin transport, N-1-naphthylphathalamic acid (NPA), stimulates uptake of IAA but has no effect on uptake of either benzoic acid or the two neutral indoles. Thus, NPA does not affect the driving forces for accumulation of weak acids but probably specifically decreases the flux of the auxin anions relative to undissociated auxin. Since the electrochemical potential of auxin anions is usually higher in than outside cells, blocking the anion flux with NPA would enhance auxin uptake. Azide, which abolishes accumulation of both IAA and benzoic acid, may simply collapse the pH gradient across the plasma membrane.In the absence of NPA, increasing concentrations of auxins or the analogoue -naphthaleneacetic acid (-NAA) exert two opposing effects on the uptake of IAA-depression and stimulation. Stimulation results from saturating the anion flux. With uptake fully stimulated by NPA, however, increasing concentrations of auxins or analogues only depress uptake of [³H]IAA. These results are consistent with more than one path for auxin transport each with a different dependence on concentration. In depressing NPA-stimulated IAA uptake, the effectiveness of -NAAIAA-NAA benzoic acid, a specificity similar to that of an auxin binding site in vitro that has been implicated by others in auxin transport. The results support the general hypothesis that cellular auxin uptake and polar transport through tissues are chemiosmotically coupled to the electrochemical potential of auxin and protons.Abbreviations IAA indole-3-acetic acid - -NAA -naphthaleneacetic acid - -NAA -naphthaleneacetic acid - NPA N-1-naphthylphthalamic acid     

Isoform-Specific Membrane Translocation of Protein Kinase C After Ischemic Preconditioning

Mild cerebral anoxic/ischemic/stress insults promote tolerance and thereby protect the brain from subsequent lethal anoxic/ischemic insults. We examined whether specific activation of PKC , , , or isoforms is associated with ischemic preconditioning (IPC) in rat brain. IPC was produced by a 2-minute global cerebral ischemia. Membrane and cytosolic fractions of the hippocampi were immunoblotted using specific antibodies for PKC, , , and . PKC showed a significant translocation to the membrane fraction from 30 min to 4 h and PKC at 4 h following IPC. In contrast, the membrane/cytosol ratio of PKC showed a tendency to decrease at 30 min and 8 h, and the membrane/cytosol ratio of PKC was significantly decreased from 30 min to 24 h following IPC. These findings indicate PKC isoform-specific membrane translocations in the hippocampus after brief global brain ischemia and suggest that activation of PKC and PKC may be associated with IPC-induced tolerance in the rat hippocampus.     

Destruction of catecholamine-containing neurons by 6-hydroxydopa, an endogenous amine oxidase cofactor

Summary The amino acid, 6-hydroxydopa (6-OHDOPA), found at the active site of amine oxidases, exists as a keto-enol. Exogenously administered 6-OHDOPA is an excitotoxin like-N-oxalylamino-L-alanine (BOAA) and-N-methylamino-L-alanine (BMAA), acting at the non-N-methyl-D-aspartate (non-NMDA)-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor. BMAA and BOAA are causal factors of neurolathyrism in humans. Much exogenously administered 6-OHDOPA is biotransformed by aminoacid decarboxylase (AADC) to the highly potent and catecholamine (CA) selective neurotoxin, 6-hydroxydopamine (6-OHDA). 6-OHDOPA destroys locus coeruleus noradrenergic perikarya and produces associated denervation of brain by norepinephrine-(NE) containing fibers. Opiopeptides and opioids enhance neurotoxic effects of 6-OHDOPA on noradrenergic nerves, by a naloxone-reversible process. An understanding of mechanisms underlying neurotoxic effects of 6-OHDOPA can be helpful in defining actions of known and newfound amino acids and for investigating their potential neurotoxic properties.Abbreviations AADC aminoacid decarboxylase - AMPA -amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid - BOAA -N-oxalylamino-Lalanine - CA catecholamine - L-DOPA L-dihydroxyphenylalanine - DA dopamine - EAA excitatory amino acid - 6-OHDOPA 6-hydroxydopa - 6-OHDA 6-hydroxydopamine - NE norepinephrine - BMAA -N-methylamino-L-alanine - NMDA N-methyl-D-aspartate - S.E.M. standard error of the mean     

An Analysis of the Amino Acid Clustering Pattern in (α/β)8 Barrel Proteins

The (/)₈ barrel proteins, in spite of having a common fold, do not show any sequence similarity. In order to understand the factors which are responsible for maintaining the common fold, the three-dimensional structures of 36 (/)₈ barrel proteins are analyzed for the presence of identical amino acid clusters or physicochemically similar clusters. The results reveal 14 identical amino acid clusters and a large number of physicochemically similar clusters. Further analysis of the similar clusters points to the conservation of secondary structures, the presence of pairs of residues occupying topologically equivalent secondary structures, and the presence of certain key residues which may play a vital role in directing and stabilizing the (/)₈ barrel fold.     

D-Amino Acids as Putative Neurotransmitters: Focus on D-Serine

Of the twenty amino acids in the mammalian body, only serine and aspartate occur in D-configuration as well as L-configuration in significant amount. D-serine is selectively concentrated in the brain, localized to protoplasmic astrocytes that ensheath synapses and distributed similarly to N-methyl-D-aspartate (NMDA) subtype of glutamate receptors. D-serine has been found to function as an endogenous ligand for the glycine site of the NMDA receptor. Evidences for this include the greater potency of D-serine to activate this site than glycine, and D-amino acid oxidase, which degrades D-serine as well as other neutral D-amino acids, markedly attenuates NMDA neurotransmission. D-serine is also formed by serine racemase, a recently cloned enzyme that converts L-serine to D-serine. Thus, in many ways D-serine fulfills criteria for defining its functionality as a neurotransmitter and challenges the dogma relating to neurotransmission, for it is the unnatural isomeric form of an amino acid derived from glia rather than neurons.