scholarly journals Molecular and structural characterization of NADPH-dependent d-glycerate dehydrogenase from the enteric parasitic protist Entamoeba histolytica

2003 ◽  
Vol 375 (3) ◽  
pp. 729-736 ◽  
Author(s):  
Vahab ALI ◽  
Yasuo SHIGETA ◽  
Tomoyoshi NOZAKI
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Entamoeba Histolytica ◽  
Reductase Activity ◽  
Phylogenetic Analyses ◽  
Kinetic Studies ◽  
Biochemical Properties ◽  
Exchange Chromatography ◽  
Subunit Structure ◽  
Protein Alignment

Putative NADPH-dependent GDH (d-glycerate dehydrogenase) of the protozoan parasite Entamoeba histolytica (EhGDH) has been characterized. The EhGDH gene encodes a protein of 318 amino acids with a calculated isoelectric point of 6.29 and a molecular mass of 35.8 kDa. EhGDH showed highest identities with GDH from ε-proteobacteria. This close kinship was also supported by phylogenetic analyses, suggesting possible lateral transfer of the gene from ε-proteobacteria to E. histolytica. In contrast with the implications from protein alignment and phylogenetic analysis, kinetic studies revealed that the amoebic GDH showed biochemical properties similar to those of mammalian GDH, i.e. a preference for NADPH as cofactor and higher affinities towards NADPH and β-hydroxypyruvate than towards NADP+ and d-glycerate. Whereas the amino acids involved in nucleotide binding and catalysis are totally conserved in EhGDH, substitution of a negatively charged amino acid with a non-charged hydroxy-group-containing amino acid is probably responsible for the observed high affinity of EhGDH for NADP+/NADPH. In addition, the amoebic GDH, dissimilar to the bacterial and mammalian GDHs, lacks glyoxylate reductase activity. Native and recombinant EhGDH showed comparable subunit structure, kinetic parameters and elution profiles on anion-exchange chromatography. We propose that the GDH enzyme is likely to be involved in regulation of the intracellular concentration of serine, and, thus, also in controlling cysteine biosynthesis located downstream of serine metabolic pathways in this protist.

Effect of Deproteinating Agents (Picric Acid and Sulfosalicylic Acid) on Analysis for Free Amino Acids in Swine Blood and Tissue

1969 ◽  
Vol 52 (5) ◽  
pp. 981-984 ◽  
Author(s):  
J E Knipfel ◽  
D A Christensen ◽  
B D Owen
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Ion Exchange ◽  
Free Amino ◽  
Biological Fluids ◽  
Picric Acid ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Sulfosalicylic Acid ◽  
Coefficients Of Variation

Abstract Amino acid analyses were performed on samples of blood, liver tissue, loin muscle, and ham muscle by ion exchange chromatography after deproteination of the samples with picric acid or sulfosalicylic acid (SSA). Resolution of threonine and serine from the ion exchange column was poor when SSA was used as the deproteinating agent. Twelve of sixteen amino acids were higher (P < 0.05) in serum deproteinated with picric acid as compared to concentrations determined after SSA deproteination. Amino acid values for ham muscle tended to be higher after deproteination with picric acid; however, with liver and loin muscle samples, the values were somewhat higher after SSA deproteination. In both serum and tissue analyses, coefficients of variation were lower for niGSt amino acids when picric acid was utilized as the deproteinating agent. The latter observation, in particular, suggests that picric acid is preferable to SSA as a deproteinating agent before amino acid analyses of biological fluids. Standardization of methods of deproteination is needed to allow meaningful comparisons of data.

scholarly journals Molecular Analysis of the Rebeccamycin l-Amino Acid Oxidase from Lechevalieria aerocolonigenes ATCC 39243

2005 ◽  
Vol 187 (6) ◽  
pp. 2084-2092 ◽  
Author(s):  
Tomoyasu Nishizawa ◽  
Courtney C. Aldrich ◽  
David H. Sherman
Keyword(s):  
Amino Acid ◽  
Biochemical Characterization ◽  
Kinetic Studies ◽  
Biochemical Properties ◽  
Protein Product ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
Left Hand ◽  
Key Enzyme ◽  
Hand Region

ABSTRACT Rebeccamycin, a member of the tryptophan-derived indolocarbazole family, is produced by Lechevalieria aerocolonigenes ATCC 39243. The biosynthetic pathway that specifies biosynthesis of this important metabolite is comprised of 11 genes spanning 18 kb of DNA. A presumed early enzyme involved in elaboration of the rebeccamycin aglycone is encoded by rebO, located at the left-hand region of the reb gene cluster. The deduced protein product, RebO (51.9 kDa), is an l-amino acid oxidase (l-AAO) that has 27% identity to an l-AAO from Scomber japonicus (animal, mackerel) and is a member of the family of FAD-dependent oxidase enzymes. In order to study the biochemical properties of this key enzyme, the rebO gene was overexpressed and purified from Escherichia coli. Biochemical characterization showed that RebO is dimeric, with a molecular mass of approximately 101 kDa. Further analysis revealed that the enzyme contains a noncovalently bound FAD cofactor and is reoxidized at the expense of molecular oxygen by producing one molecule of hydrogen peroxide. Based on kinetic studies, RebO shows significant preference for 7-chloro-l-tryptophan, suggesting its likely role as the natural early pathway substrate. Furthermore, the native RebO enzyme has evident, albeit limited, flexibility as shown by bioconversion studies with unnatural substrates. This work provides the first analysis of a structural enzyme involved in construction of this important class of indolocarbazole natural products.

Recycling of an amino acid label with prolonged isotope infusion: implications for kinetic studies

1985 ◽  
Vol 248 (4) ◽  
pp. E482-E487 ◽  
Author(s):  
W. F. Schwenk ◽  
E. Tsalikian ◽  
B. Beaufrere ◽  
M. W. Haymond
Keyword(s):  
Amino Acids ◽  
Steady State ◽  
Amino Acid ◽  
Infusion Rate ◽  
Kinetic Studies ◽  
Intracellular Pool ◽  
Group I ◽  
Group Ii ◽  
Plasma Leucine ◽  
Amino Acid Label

To investigate whether recycling of a labeled amino acid would occur after 24 h of infusion, two groups of normal volunteers were infused with [3H]leucine and alpha-[14C]-ketoisocaproate for 4 h and [2H3]leucine for either 4 or 24 h (groups I and II, respectively). Entry of [2H3 )leucine at steady state into the plasma space was indistinguishable from its infusion rate for group I but 30% higher (P less than 0.001) than this rate for group II, demonstrating significant recycling of label. After discontinuation of the infusions, isotope disappearance from the plasma space was followed for 2 h. The 3H and 14C decay data for both groups suggest that plasma leucine and alpha-ketoisocaproate are derived from a single intracellular pool in the postabsorptive state. In group I, the 3H and 2H labels decayed identically; whereas, in group II, the decay of [2H3]-leucine and alpha-[2H3]ketoisocaproate was slower (P less than 0.01) than the decay of [3H]leucine and alpha-[3H]ketoisocaproate, confirming re-entry of label after a 24-h infusion. Therefore kinetic values calculated from models assuming no recycling of labeled amino acids are most likely not quantitative and must be interpreted with care when flux does not change or decreases.

Isolation of a Crosslinked Peptide from Hitman Fibrin and Development of a Radioimmunoassay

1979 ◽  
Author(s):  
R. Canfield ◽  
B. Lahiri ◽  
R. D’Alisa ◽  
V. Butler ◽  
H. Nossel ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Factor Xiiia ◽  
Cross Linking ◽  
Edman Degradation ◽  
Cnbr Cleavage ◽  
Normal Hemostasis

Factor XIIIa introduces up to six crossllnklng bonds per molecule of fibrin; the bonds between the γ chains on adjacent fibrin molecules form most rapidly. Since cross linking is essential for normal hemostasis and is likely to be important in tests to detect thrombosis, we have attempted to develop a radioimmunoassay that exhibits specificity for the γ chain crosslinks. The immunogen consisted of a 54 amino acid, crosslinked peptide, isolated from purified human γ-γ chains following CNBr cleavage, gel filtration on Sephadex G-50 and ion-exchange chromatography on SP-Sephadex. Amino acid analysis and Edman degradation through step 24 confirmed the sequence of Chen and Doolittle (Biochemistry 10: i486, 1971), and the two degradation steps that failed to liberate the expected PTH-amino acids matched the reported location of the Gin-Lys crosslinks. Antisera were obtained against this immunogen coupled either to bovine thyroglobulin or bovine serum albumin. All antisera elicited bound immunogen that was covalently coupled to ribonuclease radiolabeled with 125I as a tracer. The unlabeled γ-γ, crosslinked peptide effectively inhibited binding (0.03-0.08 picomoles for 50% inhibition), while with some antisera up to 500 times more of the 27 amino acid γ monomer peptide was required for the same degree of inhibition. Fibrinogen and fragment D also were poor Inhibitors. The results Indicate that it is possible by radioimmunoassay to distinguish the COOH-termlnal region of the γ-γ dlmer from that of uncrosslinked molecules.

Influence of amino acids and ammonium on nitrate reduction in corn seedlings

1979 ◽  
Vol 57 (17) ◽  
pp. 1824-1829 ◽  
Author(s):  
Ann Oaks ◽  
Ineke Stulen ◽  
Ingrid L. Boesel
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Nitrate Reduction ◽  
Nitrate Reductase Activity ◽  
Reductase Activity ◽  
Root Tip ◽  
Reduction Of No ◽  
End Products ◽  
Corn Seedling

Corn seedlings were transferred 48 h after imbibition to a medium containing [Formula: see text] Hoagland's salts and 5 mM nitrate (K14NO3 or K15NO3). Three treatments were used during the ensuing 6-h induction period: (i) no further additions: (ii) corn mixture of amino acids plus 10 mM glutamine and 10 mM asparagines; and (iii) 10 mM (NH4)2SO4. The shoots, mature root sections (25–35 mm from the tip), and root tip sections (0–10 mm) were then examined for nitrate reductase activity (NR) and the ability to reduce 15NO3in vivo.Both amino acid and ammonium additions resulted in less NR in the shoots. In the roots, the development of NR was inhibited slightly by the amino acids and was enhanced by ammonium additions. Treatment with either corn amino acids or (NH4)2SO4 had no influence on the incorporation of 15N into the ammonium and amino acid fraction. Thus, although the potential end products of NR have slight effects on measured in vitro levels of NR, they have no effect on the reduction of NO3− in the intact corn seedling.

Studies on Nα-acylated proteins: The N-terminal sequences of two muscle enolases

1985 ◽  
Vol 5 (10-11) ◽  
pp. 847-854 ◽  
Author(s):  
Christopher C. Q. Chin ◽  
Finn Wold
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Ion Exchange ◽  
Coho Salmon ◽  
Standard Procedure ◽  
Exchange Chromatography ◽  
Rabbit Muscle ◽  
Terminal Amino Acid ◽  
Terminal Amino ◽  
Acylated Proteins

A standard procedure for the identification of the N-terminal amino acid in Nα-acylated proteins has been developed. After exhaustive proteolysis, the amino acids with blocked α-amino groups are separated from positively charged, free amino acids by ion exchange chromatography and subjected to digestion with acylase I. Amino acid analysis before and after the acylase treatment identifies the blocked N-terminal amino acid. A survey of acylamino acid substrates showed that acytase will liberate all the common amino acids except Asp, Cys or Pro from their N-acetyl- and N-butyryl derivatives, and will also catalyze the hydrolysis of N-formyl-Met and N-myristyl-Val. Thus, the procedure cannot identify acylated Asp, Cys or Pro, nor, because of the ion exchange step, Nα-acyl-derivatives of Arg, Lys or His. Whenever the protease treatment releases free acylamino acids, the remaining amino acids should be detected. When applied to several proteins, the procedure confirmed known N-terminal acylamino acids and identified acyl-Ser in enolases from chum and coho salmon muscle and in pyruvate kinase from rabbit muscle, and acyl-Thr in phosphofructokinase from rabbit muscle. The protease-acylase assay has been used to identify blocked peptides from CNBr- or protease-treated proteins. When such peptides were treated with 1n HCl at 110° for 10 min, sufficient yields of deacylated, mostly intact, peptide were obtained to permit direct automatic sequencing. The N-terminal sequences of rabbit muscle and coho salmon enolase were determined in this way and are compared to each other and to the sequence of yeast enolase.

scholarly journals Improved Affinity of a Human Anti-Entamoeba histolytica Gal/GalNAc Lectin Fab Fragment by a Single Amino Acid Modification of the Light Chain

2004 ◽  
Vol 11 (6) ◽  
pp. 1085-1088 ◽  
Author(s):  
Hiroshi Tachibana ◽  
Naohisa Matsumoto ◽  
Xun-Jia Cheng ◽  
Hideo Tsukamoto ◽  
Eisaku Yoshihara
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Entamoeba Histolytica ◽  
Light Chain ◽  
Fluorescent Antibody ◽  
Human Monoclonal Antibody ◽  
Affinity Maturation ◽  
Single Amino Acid ◽  
Acid Modification ◽  
Fab Fragment

ABSTRACT We previously produced, in Escherichia coli, a human monoclonal antibody Fab fragment, CP33, specific for the galactose- and N-acetyl-d-galactosamine-inhibitable lectin of Entamoeba histolytica. To prepare antibodies with a higher affinity to the lectin, recombination PCR was used to exchange Ser91 and Arg96 in the third complementarity-determining region of the light chain with other amino acids. The screening of 200 clones of each exchange by an indirect fluorescent antibody test showed that 14 clones for Ser91 and nine clones for Arg96 reacted strongly with E. histolytica trophozoites. Sequence analyses revealed that the substituted amino acids at Ser91 were Ala in five clones, Gly in three clones, Pro in two clones, and Val in two clones, while the amino acid at position 96 was substituted with Leu in three clones. The remaining eight clones exhibited no amino acid change at position 91 or 96. These mutant Fab fragments were purified and subjected to a surface plasmon resonance assay to measure the affinity of these proteins to the cysteine-rich domain of lectin. Pro or Gly substitution for Ser91 caused an increased affinity of the Fab, but substitution with Ala or Val did not. The replacement of Arg96 with Leu did not affect affinity. These results demonstrate that modification of antibody genes by recombination PCR is a useful method for affinity maturation and that amino acid substitution at position 91 yields Fabs with increased affinity for the lectin.

scholarly journals Biochemical and Genetic Characterization of Mundticin KS, an Antilisterial Peptide Produced by Enterococcus mundtii NFRI 7393

2002 ◽  
Vol 68 (8) ◽  
pp. 3830-3840 ◽  
Author(s):  
Shinichi Kawamoto ◽  
Jun Shima ◽  
Rumi Sato ◽  
Tomoko Eguchi ◽  
Sadahiro Ohmomo ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Genetic Characterization ◽  
Solid Phase ◽  
Exchange Chromatography ◽  
Nucleotide Sequencing ◽  
Lactobacillus Curvatus ◽  
Amino Acid Peptide ◽  
Enterococcus Mundtii

ABSTRACT Mundticin KS, a bacteriocin produced by Enterococcus mundtii NFRI 7393 isolated from grass silage in Thailand, is active against closely related lactic acid bacteria and the food-borne pathogen Listeria monocytogenes. In this study, biochemical and genetic characterization of mundticin KS was done. Mundticin KS was purified to homogeneity by ammonium sulfate precipitation, sequential ion-exchange chromatography, and solid-phase extraction. The gene cluster (mun locus) for mundticin KS production was cloned, and DNA sequencing revealed that the mun locus consists of three genes, designated munA, munB, and munC. The munA gene encodes a 58-amino-acid mundticin KS precursor, munB encodes a protein of 674 amino acids involved in translocation and processing of the bacteriocin, and munC encodes a mundticin KS immunity protein of 98 amino acids. Amino acid and nucleotide sequencing revealed the complete, unambiguous primary structure of mundticin KS; mundticin KS comprises a 43-amino-acid peptide with an amino acid sequence similar to that of mundticin ATO6 produced by E. mundtii ATO6. Mundticin KS and mundticin ATO6 are distinguished by the inversion of the last two amino acids at their respective C termini. These two mundticins were expressed in Escherichia coli as recombinant peptides and found to be different in activity against certain Lactobacillus strains, such as Lactobacillus plantarum and Lactobacillus curvatus. Mundticin KS was successfully expressed by transformation with the recombinant plasmid containing the mun locus in heterogeneous hosts such as E. faecium, L. curvatus, and Lactococcus lactis. Based on our results, the mun locus is located on a 50-kb plasmid, pML1, of E. mundtii NFRI 7393.

scholarly journals Determination of free amino acid and biogenic amine contents of hungarian sparkling wines

2004 ◽  
Vol 22 (SI - Chem. Reactions in Foods V) ◽  
pp. S287-S289 ◽  
Author(s):  
L. Simon-Sarkadi ◽  
E. Szőke ◽  
A. Kerekes
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Biogenic Amines ◽  
Free Amino Acid ◽  
Biogenic Amine ◽  
Free Amino Acids ◽  
Free Amino ◽  
Exchange Chromatography ◽  
Sparkling Wine

Comparative study was conducted on the basis of free amino acids and biogenic amines of Hungarian sparkling wines originated from 3 producers (Törley, Hungária, Balaton Boglár). Determination of amino acids and biogenic amines was accomplished by ion-exchange chromatography using an amino acid analyser. The dominant free amino acids in sparkling wines were proline and arginine and the major biogenic amine was spermidine. Based on results of chemometric analyses, free amino acid and biogenic amine contents seemed to be closely related to quality and the technology of sparkling wine making.

scholarly journals Phylogenetic Analyses of Sites in Different Protein Structural Environments Result in Distinct Placements of the Metazoan Root

Biology ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 64 ◽  
Author(s):  
Akanksha Pandey ◽  
Edward L. Braun
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Solvent Accessibility ◽  
Phylogenetic Signal ◽  
Phylogenetic Analyses ◽  
Sister Group ◽  
Striking Difference ◽  
Relative Solvent Accessibility ◽  
Protein Datasets ◽  
The Impact

Phylogenomics, the use of large datasets to examine phylogeny, has revolutionized the study of evolutionary relationships. However, genome-scale data have not been able to resolve all relationships in the tree of life; this could reflect, at least in part, the poor-fit of the models used to analyze heterogeneous datasets. Some of the heterogeneity may reflect the different patterns of selection on proteins based on their structures. To test that hypothesis, we developed a pipeline to divide phylogenomic protein datasets into subsets based on secondary structure and relative solvent accessibility. We then tested whether amino acids in different structural environments had distinct signals for the topology of the deepest branches in the metazoan tree. We focused on a dataset that appeared to have a mixture of signals and we found that the most striking difference in phylogenetic signal reflected relative solvent accessibility. Analyses of exposed sites (residues located on the surface of proteins) yielded a tree that placed ctenophores sister to all other animals whereas sites buried inside proteins yielded a tree with a sponge+ctenophore clade. These differences in phylogenetic signal were not ameliorated when we conducted analyses using a set of maximum-likelihood profile mixture models. These models are very similar to the Bayesian CAT model, which has been used in many analyses of deep metazoan phylogeny. In contrast, analyses conducted after recoding amino acids to limit the impact of deviations from compositional stationarity increased the congruence in the estimates of phylogeny for exposed and buried sites; after recoding amino acid trees estimated using the exposed and buried site both supported placement of ctenophores sister to all other animals. Although the central conclusion of our analyses is that sites in different structural environments yield distinct trees when analyzed using models of protein evolution, our amino acid recoding analyses also have implications for metazoan evolution. Specifically, our results add to the evidence that ctenophores are the sister group of all other animals and they further suggest that the placozoa+cnidaria clade found in some other studies deserves more attention. Taken as a whole, these results provide striking evidence that it is necessary to achieve a better understanding of the constraints due to protein structure to improve phylogenetic estimation.

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