Binding site amino acid residues of jack fruit (artocarpus integrifolia) seed lectin: chemical modification and protein difference spectral studies

The effect of chemical modification of amino acid residues essential for sugar binding in the α-D-galactoside specific jack fruit (Artocarpus integrifolia) seed lectin and the protection of the residues by specific sugar from modification were studied. Citraconylation or maleylation of 75 % of its lysyl residues or acetylation of 70 % of the tyrosyl residues completely abolished sugar binding and agglutination without dissociation of subunits. 1-O-methyl α-D-galactoside could protect its essential lysyl and tyrosyl groups from modification. Tryptophan could not be detected in the protein. Difference absorption spectra on binding of the above sugar confirmed the role of tyrosine residues and showed an association constantK = 0.4 × 10³ M⁻¹. Data suggests that the lectin could be immobilized without any loss of sugar binding activity     

Conversion of cysteinyl residues to unnatural amino acid analogs. Examination in a model system

Improved and efficient techniques have led to an explosive growth in the application of site-directed mutagenesis to the study of enzymes. However, the limited availability of only those 20 amino acids that are translated by the genetic code has prevented the systematic variation of an amino acid's properties in order to define more precisely its role in the catalytic mechanism of an enzyme. An approach is being examined that combines the high specificity of site-directed mutagenesis with the flexibility of chemical modification to overcome these limitations. A set of reagents has been synthesized and reacted with a cysteine model to produce a series of amino acid structural analogs at appreciable rates and in good overall yields. The selective incorporation of these analogs in place of important functional amino acids in a protein will allow a more detailed examination of the role of that amino acid.     

Conformational study reveals amino acid residues essential for hemagglutinating and anti-proliferative activities of Clematis montana lectin

Clematis montana lectin （CML）, a novel mannose-binding lectin purified from C. montana Buch.-Ham stem （Ranunculaceae）, has been proved to have hemagglutinat- ing activity in rabbit erythrocytes and apoptosis-inducing activity in tumor cells. However, the biochemical properties of CML have not revealed and its structural information still needs to be elucidated. In this study, it was found that CML possessed quite good thermostability and alkaline re- sistance, and its hemagglutinating activity was bivalent metal cation dependent. In addition, hemagglutination test and fluorescence spectroscopy proved that GuHCI, urea, and sodium dodecyl sulfate could change the conformation of CML and further caused the loss of hemagglutination activity. Moreover, the changes of fluorescence spectrum indicated that the tryptophan （Trp） microenvironment conversion might be related to the conformation and bioactivities of CML. In addition, it was also found that Trp residues, arginine （Arg） residues, and sulfhydryl were important for the hemagglutinating activity of CML, but only Trp was proved to be crucial for the CML con- formation. Furthermore, the Trp, Arg, and sulfhydryl- modified CML exhibited 97.17%, 76.99%, and 49.64% loss of its anti-proliferative activity, respectively, which was consistent with the alterations of its hemagglutinating activity. Given these findings, Trp residues on the surface of CML are essential for the active center to form sub- strate-accessible conformation and suitable environment for carbohydrate binding.     

Protein content and amino acid composition of cassava leaf

On the basis of leaf dry wt, the protein content of six varieties of cassava varied from 29.3 to 38.6% and the estimated leaf protein production ranged from 242 to 953 kg per ha. On the basis of fr. wt of leaf, the total amino acids ranged from 8.42 to 9.4% while the essential amino acids averaged 4.21% and the sulphur-containing amino acids only 0.25%. The amino acid composition profiles for the six varieties was similar.     

Modification of hyaluronic acid with aromatic amino acids

Hyaluronic acid was modified with aromatic amino acids (5-aminosalicylic, 4-aminosalicylic, anthranilic, and p-aminobenzoic) in the presence of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. The modified glycans contained 9–43% of arylamide groups and 10–33% of isoureidocarbonyl groups depending on the nature of the amino acid. Reduction with sodium borohydride allowed the conversion of isoureidocarbonyl groups into hydroxymethyl groups.Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 1, 2005, pp. 90–95.Original Russian Text Copyright © 2005 by Ponedelkina, Odinokov, Vakhrusheva, Golikova, Khalilov, Dzhemilev.     

Protein restriction and branched‐chain amino acid restriction promote geroprotective shifts in metabolism

The proportion of humans suffering from age‐related diseases is increasing around the world, and creative solutions are needed to promote healthy longevity. Recent work has clearly shown that a calorie is not just a calorie—and that low protein diets are associated with reduced mortality in humans and promote metabolic health and extended lifespan in rodents. Many of the benefits of protein restriction on metabolism and aging are the result of decreased consumption of the three branched‐chain amino acids (BCAAs), leucine, isoleucine, and valine. Here, we discuss the emerging evidence that BCAAs are critical modulators of healthy metabolism and longevity in rodents and humans, as well as the physiological and molecular mechanisms that may drive the benefits of BCAA restriction. Our results illustrate that protein quality—the specific composition of dietary protein—may be a previously unappreciated driver of metabolic dysfunction and that reducing dietary BCAAs may be a promising new approach to delay and prevent diseases of aging.     

Effects of denaturation and amino acid modification on fluorescence spectrum and hemagglutinating activity of Hericium erinaceum Lectin

In a broad sense, lectins are proteins or glycoproteins ofnon-immune origin that bind specifically to carbohydrates[1]. But most lectins are usually multivalent, which meansthey have more than one carbohydrate-binding site in onemolecule, a property that enables them to agglutinate eryth-rocytes and other cells [2,3]. Some lectins exhibit blood-group specificity [4] and can be used in blood grouping;some agglutinate transformed cells better than the normalones [5]. Therefore, clinical research…     

SDS-subtilisin catalyzed synthesis of tetra-peptides containing multifunctional amino acid residues in ethanol

The role of acyl donor structure on the course of peptide bond formation catalyzed by SDS-subtilisin in ethanol was investigated. In the reaction Z---Ala---Ala---Leu---OR+H---Phe---pNA→Z---Ala---Ala---Leu---Phe---pNA, nearly quantitative product yields were observed after 2 h, regardless of whether an activated (R=CH₃, p-C₆H₅Cl) or non-activated (R=H) acyl donor was used. It was found that the enzyme can accept as acyl donors N-protected tri-peptides containing basic or acidic amino acid residues in the P₁-position. Tetra-peptides of general formula Z---Ala---Ala---P₁---P₁′---pNA, where P₁=Glu, Asp, Lys, Arg or His and P₁′=Phe, Arg or Glu have been obtained in good yield.     

Analysis of the genomic organization of the human cationic amino acid transporters CAT-1, CAT-2 and CAT-4

Summary. By screening nucleotide databases, sequences containing the complete genes of the human cationic amino acid transporters (hCATs) 1, 2 and 4 were identified. Analysis of the genomic organization revealed that hCAT-2 consists of 12 translated exons and most likely of 2 untranslated exons. The splice variants hCAT-2A and hCAT-2B use exon 7 and 6, respectively. The hCAT-2 gene structure is closely related to the structure of hCAT-1, suggesting that they belong to a common gene family. hCAT-4 consists of only 4 translated exons and 3 short introns. Exons of identical size and highly homologous to exon 3 of hCAT-4 are present in hCAT-1 and hCAT-2. Received September 8, 2000 Accepted January 8, 2001     

Kinetic resolution of amino acid esters catalyzed by lipases

Racemic amino acids were resolved by lipase via hydrolysis of their esters. Lipases (Pseudomonas lipase from Amano PS, Rhizopus lipase from Serva, and porcine pancrease lipase from Sigma) could selectively hydrolyze the L-amino acid esters in aqueous solution with high reactivities and selectivities. The effect of the structural changes in the ester moiety on the stereoselectivity of the lipases was also investigated using D ,L -homophenylalanine as a model. Procedures were developed for the resolution of natural and unnatural amino acids. © 1996 Wiley-Liss, Inc.     

Regulation of the plasma amino acid profile by leucine via the system L amino acid transporter

Plasma concentrations of amino acids reflect the intracellular amino acid pool in mammals. However, the regulatory mechanism requires clarification. In this study, we examined the effect of leucine administration on plasma amino acid profiles in mice with and without the treatment of 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) or rapamycin as an inhibitor of system L or mammalian target of rapamycin complex 1, respectively. The elevation of plasma leucine concentration after leucine administration was associated with a significant decrease in the plasma concentrations of isoleucine, valine, methionine, phenylalanine, and tyrosine; BCH treatment almost completely blocked the leucine-induced decrease in plasma amino acid concentrations. Rapamycin treatment had much less effects on the actions of leucine than BCH treatment. These results suggest that leucine regulates the plasma concentrations of branched-chain amino acids, methionine, phenylalanine, and tyrosine, and that system L amino acid transporters are involved in the leucine action.     

Structure of the genetic code suggested by the hydropathy correlation between anticodons and amino acid residues

The correlation between hydropathies of anticodons and amino acids, detected by other authors utilizing scales of amino acid molecules in solution, was improved with the utilization of scales of amino acid residues in proteins. Three partitions were discerned in the correlation plot with the principal dinucleotides of anticodons (pDiN, excluding the wobble position). (a) The set of outliers of the correlation: Gly-CC, Pro-GG, Ser-GA and Ser-CU. The amino acids are consistently small, hydro-apathetic, stabilizers of protein N-ends, preferred in aperiodic protein conformations and belong to synthetases class II. The pDiN sequences are representative of the homogeneous sector (triplets NRR and NYY), distinguished from the mixed sector (triplets NRY and NYR), that depict a 70% correspondence to the synthetases class II and I, respectively. The triplet pairs proposed to be responsible for the coherence in the set of outliers are of the palindromic kind, where the lateral bases are the same, CCC: GGG and AGA: UCU. This suggests that UCU previously belonged to Ser, adding to other indications that the attribution of Arg to YCU was due to an expansion of the Arg-tRNA synthetase specificity. The other attributions produced two correlation sets. (b) One corresponds to the remaining pDiN of the homogeneous sector, containing both synthetase classes; its regression line overlapped the one formed by the remaining attributions to class II. (c) The other contains the pDiN of the mixed sector and produced steeper slopes, especially with the class I attributions. It is suggested that the correlation was established when the amino acid composition of the protein synthetases became progressively enriched and that the set of outliers were the earliest to have been fixed.     

Study of the conformational profile of selected unnatural amino acid residues derived from l‐phenylalanine

The present work reports the results of a conformational study performed on seven unnatural amino acid residues and on its natural precursor, investigated by means of computational methods at the molecular mechanics level. Amino acid residues selected for the present study are derivatives of l ‐phenylalanine substituted at the α and/or β carbons. This series is composed of different linear analogs, including α‐methyl, β‐methyl and β‐phenyl substituted with different stereochemistry. Analysis of the Ramachandran maps of the corresponding dipeptides in vacuo reveals their conformational preferences, to be used as guidance for the synthesis of constrained peptide analogs with desired conformational propensities. The available conformational space for every dipeptide is also analysed. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Unnatural amino acid packing mutants of Escherichia coli thioredoxin produced by combined mutagenesis/chemical modification techniques.

We have produced several mutants of Escherichia coli thioredoxin (Trx) using a combined mutagenesis/chemical modification technique. The protein C32S, C35S, L78C Trx was produced using standard mutagenesis procedures. After unfolding the protein with guanidine hydrochloride (GdmCl), the normally buried cysteine residue was modified with a series of straight chain aliphatic thiosulfonates, which produced cysteine disulfides to methane, ethane, 1-n-propane, 1-n-butane, and 1-n-pentane thiols. These mutants all show native-like CD spectra and the ability to activate T7 gene 5 protein DNA polymerase activity. In addition, all mutants show normal unfolding transitions in GdmCl solutions. However, the midpoint of the transition, [GdmCl]1/2, and the free energy of unfolding at zero denaturant concentration, delta G(H2O), give inverse orders of stability. This effect is due to changes in m, the dependence of delta G0 unfolding on the GdmCl concentration. The method described here may be used to produce unnatural amino acids in the hydrophobic cores of proteins.     

Primary structure and chemical modification of some amino acid residues of bifunctional alginate lyase from a marine bacterium Pseudoalteromonas sp. strain no. 272

The entire amino acid sequence of bifunctional alginate lyase from Pseudoalteromonas sp. strain No. 272 were determined by two approaches, Edman degradation of the peptides obtained from protease digestion of the enzyme protein and analysis of PCR products of the structural gene. The former resulted in incomplete amino acid sequence in the entire sequence, due to lacking of the proper peptides from the protease digestion. To compensate for this lack of sequences we applied the method of PCR of the structural gene that was initially elucidated from the primers designed from N- and C-terminal amino acid sequences of the enzyme. The results of the amino acid sequences from these two approaches showed good agreement. The enzyme consisted of 233 amino acid residues with a molecular mass of 25,549.5, including the sole W and cystine residue. The sequence homology search among the other alginate lyases from different origins indicated that they were very weakly homologous, with the exception of the sequence homology (80.3%) of Pseudoalteromonas elyakovii alginate lyase. The consensus sequence, YFKhG + Y-Q (Wong, T. Y., Preston, L. A., and Schiller, N. L. 2000. Annu. Rev. Microbiol. 54: 289–340) in the C-terminal regions was conserved. The kinetic analyses of chemical modification of some amino acid residues of the enzyme showed that W, K, and Y appeared to be important in the enzyme function.     

Recognition of specific patterns of amino acid inhibition of growth in higher plants, uncomplicated by glutamine-reversible `general amino acid inhibition''

The complexity of the regulatory mechanisms that govern amino acid biosynthesis, particularly in multibranched pathways, frequently results in sensitivity to growth inhibition by exogenous amino acids. Usually the inhibition caused by a given amino acid(s) is relieved by another amino acid(s), thus indicating the cause of inhibition to be a specific interference with endogenous formation of the latter amino acid(s). We recently summarized the evidence that Nicotiana silvestris (and probably most higher plants), in suspension culture, exhibits a separate phenomenon of amino acid mediated growth inhibition called general amino acid inhibition. Every amino acid provokes general amino acid inhibition except for

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-glutamine. In fact,

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-glutamine completely overcomes general amino acid inhibition. We have now demonstrated that specific amino acid inhibition can be recognized and characterized at the level of growth inhibition without interference caused by general amino acid inhibition by the simple provision of exogenous

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-glutamine. Several examples of specific amino acid inhibition of growth were demonstrated in N. silvestris. In one case,

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-threonine inhibits growth partially in the presence of

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-glutamine. The residual amino acid inhibition was overcome by the additional presence of

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-lysine and

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-methionine, indicating that exogenous

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-threonine specifically inhibits the biosynthesis of both

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-lysine and

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-methionine. As a second example, the

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-valine-mediated inhibition of growth that persisted in the presence of

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-glutamine was overcome by

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-isoleucine, indicating that exogenous

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-valine inhibits

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-isoleucine biosynthesis. The use of amino acid analogs as experimental tools for biochemical-genetic studies in higher plants is also complicated by general amino acid inhibition. Conditions were demonstrated under which p-fluorophenylalanine and m-fluorotyrosine could be used as specific antimetabolites of

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-phenylalanine and

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-tyrosine biosynthesis without interference from general amino acid inhibition. We thus present a rigorous basis for recognition of specific relationships between metabolic branches that can guide detailed enzymological analyses.     

Nature of amino acid substitutions in homologous proteins during evolution

Replacement substitutions of mitochondrial cytochrome $\text{c}$ and α- and β-chains of haemoglobin have been studied by considering the structural similarity among amino acid residues at the secondary and tertiary structural levels. Secondary structural similarity explains ～70% while tertiary structural similarity explains ～50% of observed replacements for most of the cases. These structural similarities could not account for all the replacement substitutions. The study was extended to consider the composition of codons, and the chemical nature and polarity of the replacing and replaced residues. These also could not individually account for all the affected replacements. In general, no property of amino acid residues is conserved for substitutions occurring at any single position during evolution of proteins.     

Synthesis of linear and cyclic opioid‐based peptide analogs containing multiple N‐methylated amino acid residues

A series of six novel opioid peptide analogs containing one to three N‐methylamino acid residues, and six cyclic counterparts of these peptides were prepared by the solid‐phase method. Introduction of two consecutive N‐methylated amino acids, as well as cyclization of such conformationally constrained sequences, turned out to be challenging. The use of a recently reported triazine‐based coupling reagent, 4‐(4,6‐dimethoxy‐1,3,5‐triazin‐2‐yl)‐4‐methylmorpholinium toluene‐4‐sulfonate, enabled the synthesis and cyclization of the designed analogs in acceptable yields and with a lesser amount of by‐products than observed with the standard coupling reagents such as TBTU or HATU.Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Porcine pancreatic lipase-catalized enantioselective hydrolysis of N-protected amino acid methyl-esters

Summary A preparative-scale enantioselective hydrolysis of racemic methyl esters of several N-protected amino acid has been carried out by using crude porcine pancreatic lipase (Triacylglycerol lipase, EC 3.1.1.3) PPL as a hydrolytic enzyme. In all cases 50% of the racemic methyl ester was hydrolysed to the N-protected L-amino acid with high yield and high optical purity.Hydrolysis rates were very close related not only to the amino acid structure but also to the steric and/or electronic nature of the ester and N-protecting groups. Thus, the very convenient ester methyl group can be enantioselectively hydrolysed with PPL when N-protecting group is a carbonyl derivative, as it is the usual benzoyl group.     

Effects of palmitate on astrocyte amino acid contents

The effects of palmitate on intracellular and extracellular amino acid concentrations of cultured astrocytes was studied. Exposure of astrocytes to either 0.72 mM or 0.36 mM palmitate was associated with a significant reduction in the intracellular pool of glutamine and taurine. In contrast, the intracellular concentration of histidine, glycine, citrulline, isoleucine and leucine were increased in the presence of 0.72 mM palmitate. Comparable changes in the extracellular amino acid pool were not observed. The data suggest that palmitic acid, which accumulates in the brain during periods of anoxia, alters the metabolism of several amino acids in cultured astrocytes. These changes may be of significance in terms of the pathophysiology of a stress such as anoxia.Special issue dedicated to Dr. Elling Kvamme