Effects Of Amino Acid Analogues Research Articles

Behavioral effects of alpha-alkylated amino acid analogs in the C57BL/6J mouse

Although a series of amino acid analogs have been shown to modulate brain function, information on the pharmacology of alpha-alkylated amino acids (AAAA) is limited. In particular there is no information on the effect of these amino acid analogs (AAA) on the elevated plus maze, the tail suspension test and the forced swim test. It was therefore the aim of the study to test a series of AAAA in these paradigms in order to explore behavioral activities of this compound class.10 male mice per group aged between 10 and 14 weeks were used. Vehicle-treated controls were used in addition to intraperitoneal injections of 1, 10 and 100mg/kg body weight of each, alpha-amino-isobutyic acid (AIB), isovaline (IVA), alpha-propyl-alanine (APA), alpha-butyl-alanine (ABA), alpha-pentyl-alanine (APnA), alpha-ethylphenylglycine (AEPG) and alpha-methyl-valine (AMV). The elevated plus maze (EPM), the tail suspension test (TST) and forced swim test (FST) were used for behavioral testing.There were dose-dependent results: all compounds increased time and pathlength in the open arm of the EPM at least at one dose administered. In the TST and in the FST only the 100mg dose was showing an effect.The results show pharmacological activity modifying the EPM in low doses suggesting the use in treatment of behavioral traits and symptoms represented by or linked to the EPM including anxiety-related behavior including depression. Compounds acting at higher doses may be used to induce behavioral changes and thus serve as neurobiological-neuropharmacological tools.

Cloning and expression analysis of heat shock cognate 70 gene promoter in tiger shrimp ( Penaeus monodon)

Heat shock cognate 70 (HSC70) functions as a molecular chaperon and plays an important role in protein folding. HSC70 cDNA of tiger shrimp ( Penaeus monodon) was cloned and characterized in our previous study. After shrimps were treated with the 1-hr heat shock, the HSC70 mRNA level in hemocytes increased (∼ 8 fold) using real-time quantitative PCR. An hsc70 clone was obtained from genomic library screening. The gene contains 2 exons separated by a 1557-bp intron. The 5′-flanking region sequence (∼ 1 kb) ahead of the hsc70 gene contains a putative core promoter region and transcription elements including perfect heat shock element (HSE), imperfect HSE, CAAT elements, SP1, NF-κB and GC box. In insect Sf21 cells, the region could drive expression of the enhanced green fluorescent protein (EGFP) and luciferase gene to verify its promoter function. In the luciferase assay system, the effects of serial deletions on the hsc70 promoter were elucidated. Autographa californica multiple nuclear polyhedrosis virus infection (MOI = 0.1) on Sf21 cells significantly increased the hsc70 promoter activity. In addition, the effects of amino acid analogs and arsenic acid incubation with the cells on the hsc70 promoter activity were examined.

Substrate Structure And Amino Acid/K+ Symport In Brush-Border Membrane Vesicles From Larval Manduca Sexta Midgut

The effects of amino acid sidechain length, substituent position and c chirality on amino acid/K+ symport have been examined in rapid filtration experiments on brush-border membrane vesicles prepared from larval Manduca sexta midgut. Cis-inhibition and trans-stimulation protocols were used to examine the effects of amino acid analogs on the uptake of alanine, phenylalanine, leucine and lysine, which are cotransported with K+ by a zwitterionic symporter at the high pH characteristic of the midgut in vivo. The symporter was found to translocate both L- and D-stereoisomers of alanine, leucine and lysine, but only the L-form of phenylalanine. Alterations to substrate structure that leave the charge distribution unchanged do not affect symport. Thus, moving the methyl group from C-3 to C-5 in the sequence isoleucine, leucine and norleucine has no effect on their ability to inhibit leucine symport. Increasing sidechain length among alanine homologs has little effect on their ability to inhibit alanine uptake, but increasing the sidechain length of lysine homologs from 1 to 3 methylene groups enhances cis-inhibition and trans-stimulation of lysine symport. The substantial difference in molecular charge distribution among aminobutanoic acid isomers has a large impact on alanine symport with only alpha- (or 2-) aminobutanoic acid functioning as an alanine analog. Only those changes in substrate structure that are coupled to the molecular charge distribution seem to affect symport. The tolerance of the symporter may reflect a balance mandated by the conflicting demands of selectivity and throughput.

Effect of amino acid analogs on the development of thermotolerance and on thermotolerant cells

Exposure of HA-1 Chinese hamster fibroblasts to amino acid analogs has been shown to have a heat-sensitizing effect as well as inducing the heat shock response (Li and Laszlo, 1985a). In this study, we have examined the effect of amino acid analogs on the development of thermotolerance after a brief heat shock or exposure to sodium arsenite and the effect of amino acid analogs on cells that are already thermotolerant. Exposure of HA-1 cells to amino acid analogs inhibited the development of thermotolerance following a mild heat shock or treatment with sodium arsenite. However, cells that were already thermotolerant were resistant to the sensitizing action of amino acid analogs. The refractoriness of thermotolerant cells to amino acid analog treatment developed in parallel with thermotolerance. The uptake of the arginine analog, canavanine, and its incorporation into proteins was not altered in the thermotolerant cells. Furthermore, another biological consequence of exposure to amino acid analogs, sensitization to ionizing radiation, also was not altered in the thermotolerant cells. The inhibition of the development of thermotolerance by amino acid analogs and the refractoriness of thermotolerant cells to the heat-sensitizing action of amino acid analogs lend further support the role of heat-shock proteins in the phenomenon of thermotolerance.

Structural determinants in substrate recognition by proton-amino acid symports in plasma membrane vesicles isolated from sugar beet leaves

Amino acids are actively transported across the plasma membrane of plant cells by proton-coupled symports. Previously, we identified four amino acid symports in isolated plasma membrane vesicles, including two porters for the neutral amino acids. Here we investigated the effect of amino acid analogues on neutral amino acid transport to identify structural features that are important in molecular recognition by Neutral System I (isoleucine) and Neutral System II (alanine and leucine). d-Isomers of alanine and isoleucine were not effective transport antagonists of the l-isomers. These data are characteristic of stereospecificity and suggest that the positional relationship between the α-amino and car☐yl groups is an important parameter in substrate recognition. This conclusion was supported by the observation that β-alanine and analogues with methylation at the α-carbon, at the car☐yl group, or at the β-amino group were not effective transport inhibitors. Specific binding reactions were also implicated in these experiments because substitution of the α-amino group with a space filling methyl or hydroxyl group eliminated transport inhibition. In contrast, analogues with various substitutions at the distal end of the amino acid were potent antagonists. Moreover, the relative activity of several analogues was influenced by the location of sidechain branches and Neutral Systems I and II were resolved based on differential sensitivity to branching at the β-carbon. The kinetics of azaserine and p-nitrophenylalanine inhibition of leucine transport were competitive. We conclude that the binding site for the car☐yl end of the amino acid is a well-defined space that is characterized by compact, asymmetric positional relationships and specific ligand interactions. Although the molecular interactions associated with the distal portion of the amino acid were less restrictive, this component of the enzyme-substrate complex is also important in substrate recognition because the neutral amino acid symports are able to discriminate between specific neutral amino acids and exclude the acidic and basic amino acids.

Post-transcriptional regulation of S-adenosylmethionine synthetase from its stored mRNA in germinated wheat embryos

About 2-3-fold stimulation of S-adenosylmethionine synthetase was witnessed in germinated wheat embryos (48 h). The enhancement of enzyme activity was significantly inhibited by cycloheximide and amino acid analogues. Simultaneous addition of corresponding amino acids alleviated the inhibitory effect of amino acid analogues. Conclusive proof for the de novo synthesis of S-adenosylmethionine synthetase was obtained by labelling this enzyme with [35SO4]2- in vivo. Thus de novo enzyme synthesis seemed necessary for the rise in activity of AdoMet synthetase in wheat embryos. Curiously, blocking of transcription with cordycepin failed to repress the de novo synthesis of AdoMet synthetase in germinated wheat embryos. We envisage the presence of stored mRNA for AdoMet synthetase in wheat embryos. Thus the regulation of this enzyme occurs at the post-transcriptional level. L-Methionine, which is one of the substrates of AdoMet synthetase, stimulated the enzyme activity (2-2.4-fold) over that observed in control germinated embryos. L-Methionine promotes increased de novo synthesis of AdoMet synthetase. Preincubation of enzyme fraction with L-Methionine failed to activate or stabilize the activity of AdoMet synthetase. Three isozymes of AdoMet synthetase were physically separated by DE-52 ion-exchange chromatography. One of the isozymes of AdoMet synthetase has been purified (1529-fold) to electrophoretic homogeneity by resorting to phenyl Sepharose and ATP Sepharose affinity chromatography. The purified enzyme catalyzed the synthesis of S-adenosylmethionine and also exhibited tripolyphosphatase activity. The reaction product of the purified enzyme was chemically and enzymatically characterized as S-adenosylmethionine. The molecular weight of the native enzyme is 174,000 and that of its subunit is 84,000 as determined on SDS-PAGE. Thus the native enzyme seems to be dimeric in nature.

Biosynthesis of aromatic amino acids in Nocardia sp. 239: effects of amino acid analogues on growth and regulatory enzymes

Further steps required for overproduction of aromatic amino acids by a mutant strain of Nocardia sp. 239 (Noc 87-13), unable to grow on l-phenylalanine as a sole carbon and energy source, were investigated. A number of analogues of the aromatic amino acids displayed severe inhibitory effects on the activities of regulatory enzymes in the biosynthetic pathway and growth of the organism in glucose mineral medium. l-Tryptophane analogues strongly inhibited 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase activity. l-Tyrosine analogues especially inhibited DAHP synthase and chorismate mutase, whereas l-phenylalanine analogues strongly inhibited chorismate mutase and prephenate dehydratase activity. Addition of the aromatic amino acids and their precursors chorismate, 4-hydroxyphenylpyruvate, phenylpyruvate and anthranilate, to the medium counteracted the growth inhibitory effect of specific analogues. The data indicate that ortho- (OFP) and para-fluoro-d,l-phenylalanine (PFP), and l-phenylalanine amide, are the most suitable analogues for the isolation of feedback-inhibition-insensitive prephenate dehydratase mutants. Attempts to isolate l-tyrosine and l-trytophane auxotrophic mutants were only successful in the latter case, resulting in the selection of a stable anthranilate synthase-negative mutant (Noc 87-13-14). Uptake of aromatic amino acids in Nocardia sp. 239 most likely involves a common transport system. This necessitates the use of anthranilate, rather than l-trytophane, as a supplement during the isolation of l-tyrosine auxotrophic and OFP- and/or PFP-resistant mutant derivative strains of Noc 87-13-14.

Effect of amino acid analogs on the processing of the pancreatic polypeptide precursor in primary cell cultures.

Amino acid analogs, which can be incorporated into nascent peptide chains were used in cultures of endocrine cells from canine pancreas to study the effect on processing of the metabolically labeled precursor for pancreatic polypeptide. Analogs for basic amino acids, canavanine, and aminoethylcysteine prevented the di-basic processing of the prohormone. The polar leucine analog, beta-hydroxyleucine, only partially perturbed the function and cleavage of the signal peptide but efficiently and unexpectedly blocked the dibasic cleavage of the prohormone. Other nonbasic amino acid analogs, beta-hydroxynorvaline and azetidine-2-carboxylic acid, which only could be incorporated into the prohormone at a distance from the processing site, also prevented dibasic cleavage of the prohormone. Although there are no phenylalanine residues in the prohormone, analogs for this amino acid, fluoro-phenylalanine and particularly phenylserine, could also block the processing of the prohormone at the dibasic site. This effect was prevented by addition of a small quantity of phenylalanine. It is concluded that amino acid analogs can interfere with precursor processing through altering both the primary and the secondary structure of the precursor but also through incorporation into cosynthesized protein(s) which are necessary for the precursor processing.

61] Applications of amino acid analogs for studying co- and posttranslational modifications of proteins

This chapter presents general considerations of applications of amino acid analogs for studying co- and post-translational modifications of proteins. This approach involves selection of amino acid analogs, selection of analog concentration, demonstration of amino acid analog incorporation into protein, and analysis of the effects of amino acid analogs on protein processing. When a particular analog is applied to a new system, it is necessary to assess critically whether the analog is incorporated into protein and to establish its effective concentration. Selection of amino acids for incorporation into protein depends on the selectivity of aminoacyl-tRNA synthetases, which possess an extraordinary ability to discriminate among structurally similar amino acid substrates. However, determination of the appropriate analog concentration for use with intact cells presents greater difficulty owing to the added complications of uptake into cells, amino acid metabolism, and endogenous amino acid pools.

Immunochemical analysis of the turnover of ubiquitin-protein conjugates in intact cells. Relationship to the breakdown of abnormal proteins.

Previous studies in a cell-free proteolytic system from reticulocytes indicated that the conjugation of ubiquitin with proteins plays a role in protein breakdown. To examine some of the physiological functions of the ubiquitin conjugation system, and immunochemical method was developed for the isolation of ubiquitin-protein conjugates from intact cells. A specific antiserum was raised against ubiquitin and purified by affinity chromatography on ubiquitin-Sepharose. When cells are labeled with tryptophan (which is missing from ubiquitin), labeled immunoreactive material isolated by the antibody is derived from the protein moiety of ubiquitin-protein conjugates. There is a marked increase in the labeling of ubiquitin-protein conjugates during the formation of abnormal proteins in reticulocytes (induced by the incorporation of amino acid analogs), suggesting that proteins with abnormal structure are more readily conjugated to ubiquitin than most normal proteins. Essentially similar, although less marked, effects of amino acid analogs were observed in Ehrlich ascites cells. When further protein synthesis was blocked with cycloheximide, ubiquitin conjugates decayed more extensively than the corresponding average labeled cellular proteins. This is consistent with the interpretation that a considerable part of ubiquitin conjugates is derived from a pool of rapidly degradable proteins.

Identification, synthesis, and modifications of simian rotavirus SA11 polypeptides in infected cells.

The synthesis and processing of simian rotavirus SA11 polypeptides was investigated after infection of MA104 cells. [35S]methionine- or 3H-amino acid-labeled cell extracts were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Viral protein synthesis was maximal 3 to 5 h postinfection, and 12 major viral polypeptides were detected. Immunoprecipitation and peptide mapping experiments, demonstrated five viral structural proteins (125,000 daltons [125K], 94K, 88K, 41K, and 38K). Three proteins (53K, 35K, and 34K) were identified as nonstructural by comparison of their partial proteolysis maps with those from polypeptides of similar molecular weight synthesized in vitro from viral RNA transcripts. Assignment as to structural or nonstructural status of two other primary gene products (26K and 20K) remains tentative. Pulse-chase experiments and tunicamycin blockage of glycosylation revealed cotranslational or post-translational modifications (or both) and precursor-product relationships of several of the polypeptides. Tunicamycin inhibition of glycosylation identified a 35.5K polypeptide which was proven to be the precursor to the 38K structural glycoprotein by immunoprecipitation and peptide mapping analyses. Tunicamycin treatment of infected cells also resulted in the disappearance of other glycoprotein species (23K to 29K) and in the concomitant build-up of an unglycosylated 20K polypeptide, suggesting a precursor-product relationship between those polypeptides. Labeling with [3H]glucosamine or [3H]mannose suggested that the rotavirus glycoproteins contained high mannose oligosaccharides. The effects of amino acid analogs on rotavirus polypeptide synthesis and processing were also investigated.

Effects of amino acid analogues on protein degradation in isolated rat hepatocytes

Analogues and derivatives of six of the amino acids which most effectively inhibit protein degradation in isolated rat hepatocytes (leucine, asparagine, glutamine, histidine, phenylalanine and tryptophan) were investigated to see if they could antagonize or mimic the effect of the parent compound. No antagonists were found. Amino alcohols and amino acid amides tended to inhibit protein degradation strongly, apparently by a direct lysosomotropic effect as indicated by their ability to cause lysosomal vacuolation. Amino acid alkyl esters and dipeptides inhibited degradation to approximately the same extent as did their parent amino acids, possibly by being converted to free amino acids intracellularly. Of several leucine analogues tested, four (L-norleucine, L-norvaline, D-norleucine and L- allo-isoleucine) were found to be as effective as leucine in inhibiting protein degradation. None of the analogues had any effect on protein synthesis. Since leucine appears to play a unique role as a regulator of bulk autophagy in hepatocytes, the availability of active leucine agonists may help tj elucidate the biochemical mechanism for control of this important process.

Differential effects of amino acid analogs on growth and heterocyst differentiation in two nitrogen-fixing blue-green algae.

Effects of a few amino acid analogs on growth and heterocyst differentiation have been studied in two nitrogen-fixing species ofAnabaena. All the analogs except α-methyl-DL-aspartic acid inhibited growth. Exposure ofAnabaena doliolum, toDL-5-fluorotryptophan andDL-p-fluorophenylalanine caused pronounced fragmentation of filaments into single cells. At low concentrations (0.01 mM), α-methyl-DL-aspartic acid stimulated growth of the strain ofA. doliolum as well as the strain of the second (unidentified)Anabaena species. Ethionine,DL-p-fluorophenylalanine,DL-5-fluorotryptophan, and canavanine blocked heterocyst differentiation, whereas α-methyl-DL-aspartic acid, α-methyl-DL-methionine,N-o-nitrophenylsulfenyl-L-tryptophan, norleucine, andS-2-aminoethyl-L-cysteine did not show any significant effect. Treatment with 7-azatryptophan,DL-β-hydroxynorvaline,L-methionine-DL-sulfoximine,L-methionine sulfone, and β-2-thienyl-DL-alanine led to a twofold increase in heterocyst frequency. Possible modes of action of the analogs in growth inhibition and changes in heterocyst frequency are discussed.

The effect of amino acid analogues and heat shock on gene expression in chicken embryo fibroblasts

The addition of certain amino acid analogues (canavanine, hydroxynorvaline, o-methylthreonine) or a mild heat shock at 45°C caused chicken embryo fibroblasts to increase rapidly the synthesis of three proteins (molecular weights 22,000, 76,000 and 95,000 daltons) to levels which dominate the cells biosynthetic capacity and exceed the level of synthesis of the major cell structural proteins. Actinomycin D blocked the increased synthesis of p22, p76 and p95 in both analogue and heat shock-treated cells, while cycloheximide addition during the “induction” period blocked formation of these proteins only in analogue-treated cells. The elevated levels of synthesis for this set of proteins began to decrease shortly after restoration of the normal amino acid or normal temperature, and the normal pattern of cell protein synthesis was found 8 hr later. Induction of a similar set of proteins was detected in mouse L cells and baby hamster kidney cells after treatment with amino acid analogues or heat shock. Several laboratories have reported synthesis of proteins with similar molecular weights in cells subjected to conditions that alter glucose metabolism, and we speculate that these proteins may be associated with a hexose transport system.

Effects of amino acid analogues on protein synthesis and degradation in isolated cells

1. Naturally-occurring and synthetic analogues of phenylalanine, tyrosine, histidine, arginine, proline, tryptophan and the sulphur amino acids have beeen tested in rat reticulocytes and in the Reuber H35 hepatoma for effects on protein synthesis and protein degradation and on the heat lability of phosphoenolpyruvate carboxykinase (EC 4.1.1.32) in the hepatoma cells. The experiments were designed to test whether the analogues could be incorporated into mammalian proteins and whether the resultant proteins would be degraded at an accelerated rate. 2. Several analogues, including thiazolylanine, triazolalanine and selenocystine both stimulated protein synthesis and produced labile protein in reticulocytes. Other analogues, such as dihydroxyphenylalanine, thioproline and pipecolic acid accelerated protein breakdown but probably indirectly via an inhibition of protein synthesis. Azetidine-2-carboxylic acid had the largest effect on protein breakdown in reticulocytes. 3. Labile protein was produced in hepatoma cells incubated in the presence of azetidine-2-carboxylic acid, canavanine, indospicine, triazolalanine, 2-, 3- and 4-fluorophenylalanine. These same analogues, together with 3,4-dehydroproline, beta-2-thienylalanine, dihydroxyphenylalanine, histidinol, 5- and 6-fluorotryptophan, selenocystine and selenomethionine produced heat-labile phosphoenolpyruvate carboxykinase. Enzyme induced in the presence of selenomethionine or indospicine showed the largest increases in heat lability, and for these analogues equimolar concentrations of methionine and arginine respectively were needed to nullify the enzyme abnormality. 4. The toxicity of the same naturally-occurring analogues has been discussed in terms of their ability to be incorporated into cell proteins.

The Effect of Amino Acid Analogues on the Aminoacylation of Transfer Ribonucleic Acid

The Effect of Amino Acid Analogues on the Aminoacylation of Transfer Ribonucleic Acid

Induction of mutation by 5-fluorouracil and amino acid analogues in Ustilago maydis

The effect of amino acid analogues and 5-fluorouracil on both forward and reverse mutation rates of Ustilago maydis has been examined. 5-Fluorouracil and para-fluorophenylalanine were found to be quite strongly mutagenic, whilst ethionine, although effective in increasing the reversion frequency, did not produce many forward mutations. It was found that there was some correlation between the mutagenicity of a compound and the extent to which it altered protein conformation, as judged by increased thermosensitivity of the enzyme glutamic dehydrogenase. Canavanine was found to induce mutation and modification in protein only when an arginine-requiring auxotroph was used.

An effect of amino acid analogues on extension growth in pea root segments under aseptic conditions.

An effect of amino acid analogues on extension growth in pea root segments under aseptic conditions.

The Effect of Amino Acid Analogues on Alkaline Phosphatase Formation in Escherichia coli K-12: IV. Substitution of canavanine for arginine

The substitution of canavanine for arginine in an arginine auxotroph of Escherichia coli K-12 allowed protein synthesis to continue but inhibited the formation of the alkaline phosphatase. Sonically disrupted extracts of cells incubated with 14C-canavanine contained 14C-protein that precipitated with antibody specific to alkaline phosphatase subunits and a fraction of 14C-active enzyme that was distinct from native alkaline phosphatase. This radioactive enzyme was partially purified and shown to have many properties in common with native enzyme. Of the 20 to 22 arginines in alkaline phosphatase, at least 13 and possibly 18 or 19 of these were replaced by canavanine. Prelabeling of cells with 3H-arginine prior to replacement of canavanine in the medium showed that there were probably 1 to 3 arginines in the enzyme. About one-fourth of the alkaline phosphatase subunits formed in the presence of canavanine were found in the active dimer, and the remainder accumulated as inactive subunits in the cell's periplasmic space. The possible differences between the two classes of subunits are discussed.

The Effect of Amino Acid Analogues on Alkaline Phosphatase Formation in Escherichia coli K-12: III. Substitution of 2-methylhistidine for histidine

SUMMARY The substitution of canavanine for arginine in an arginine auxotroph of Escherichia coli K-12 allowed protein synthesis to continue but inhibited the formation of the alkaline phosphatase. Sonically disrupted extracts of cells incubated with 4 C-canavanine contained 4 C-protein that precipitated with antibody specific to alkaline phosphatase subunits and a fraction of 14 C-active enzyme that was distinct from native alkaline phosphatase. This radioactive enzyme was partially purified and shown to have many properties in common with native enzyme. Of the 20 to 22 arginines in alkaline phosphatase, at least 13 and possibly 18 or 19 of these were replaced by canavanine. Prelabeling of cells with H-arginine prior to replacement of canavanine in the medium showed that there were probably 1 to 3 arginines in the enzyme. About one-fourth of the alkaline phosphatase subunits formed in the presence of canavanine were found in the active dimer, and the remainder accumulated as inactive subunits in the cell's periplasmic space. The possible differences between the two classes of subunits are discussed.