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Antiviral and antiparasite properties of an L-amino acid oxidase from the Snake Bothrops jararaca: Cloning and identification of a complete cDNA sequence (Retracted article. See vol. 80, pg. 288, 2010)

SANT`ANA, Carolina D.; MENALDO, Danilo L.; COSTA, Tassia R.; GODOY, Harryson; MULLER, Vanessa D. M.; AQUINO, Victor H.; ALBUQUERQUE, Sergio; SAMPAIO, Suely V.; MONTEIRO, Marta C.; STABELI, Rodrigo G.; SOARES, Andreimar M.
Fonte: PERGAMON-ELSEVIER SCIENCE LTD Publicador: PERGAMON-ELSEVIER SCIENCE LTD
Tipo: Artigo de Revista Científica
ENG
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L-Amino acid oxidases (LAAOs, EC 1.4.3.2) are flavoenzymes that catalyze the stereospecific oxidative deamination of an L-amino acid substrate to the corresponding a-ketoacid with hydrogen peroxide and ammonia production. The present work describes the first report on the antiviral (Dengue virus) and antiprotozoal (trypanocidal and leishmanicide) activities of a Bothrops jararaca L-amino acid oxidase (BjarLAAO-I) and identify its cDNA sequence. Antiparasite effects were inhibited by catalase, suggesting that they are mediated by H(2)O(2) production. Cells infected with DENV-3 virus previously treated with BjarLAAO-I, showed a decrease in viral titer (13-83-fold) when compared with cells infected with untreated viruses. Untreated and treated promastigotes (T. cruzi and L. amazonensis) were observed by transmission electron microscopy with different degrees of damage. Its complete cDNA sequence, with 1452 bp, encoded an open reading frame of 484 amino acid residues with a theoretical molecular weight and pl of 54,771.8 and 5.7, respectively. The cDNA-deduced amino acid sequence of BjarLAAO shows high identity to LAAOs from other snake venoms. Further investigations will be focused on the related molecular and functional correlation of these enzymes. Such a study should provide valuable information for the therapeutic development of new generations of microbicidal drugs. (C) 2008 Elsevier Inc. All rights reserved.

Isolamento e caracterização bioquímica e funcional de L-aminoácido oxidase do veneno de 'Bothrops atrox'; Isolation and characterization of an L-amino acid oxidase from Bothrops atrox snake venom

Alves, Raquel de Melo
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 16/03/2007 PT
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75.88%
Os venenos de serpentes são ricos em proteínas, enzimas e peptídeos biologicamente ativos. Diversas enzimas tais como fosfolipases A2 (PLA2s), metaloproteases (MP), L-aminoácido oxidases (LAAOs) são responsáveis pelo quadro clínico do envenenamento ofídico. Atualmente, o isolamento e a caracterização funcional e estrutural destas enzimas têm auxiliado na compreensão do mecanismo de ação destas toxinas e nas formas alternativas de tratamento. Além disso, estas enzimas se tornaram valiosas ferramentas de aplicação biotecnológica, na busca de novos fármacos de interesse na clínica médica. O objetivo deste trabalho foi isolar e caracterizar bioquímica e funcionalmente a L-aminoácido oxidase do veneno de Bothrops atrox. O isolamento da LAAO de B. atrox (LAAOBatrox) envolveu três etapas cromatográficas: a exclusão molecular em Sephadex G-75, troca iônica em ES-502N utilizando CLAE e, por último, a cromatografia de afinidade com uso da Lentil-Lectina, conferindo um alto grau de pureza à proteína confirmado por SDS-PAGE. Após a caracterização bioquímica, a LAAOBatrox demonstrou ser uma glicoproteína com 12% de açúcar, massa molecular relativa de 67.000 e pI de 4,4 confirmando seu caráter ácido pela composição em aminoácidos...

Caracterização bioquímica, estrutural e funcional de uma L-aminoácido oxidase isolada de peçonha de Lachesis muta (Serpentes, Viperidae); Purification, biochemistry and functional characterization of a new L-amino acid oxidase from Lachesis muta venom.

Silva, Cristiane Bregge da
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 31/10/2011 PT
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76.04%
As peçonhas de serpentes contêm uma mistura complexa de substâncias farmacologicamente ativas, como metaloproteases, fosfolipases A2, serino-proteases, L-aminoácido oxidase (LAAO), além de outros importantes compostos sem ação enzimática. LAAOs são flavoproteínas que catalisam a desaminação oxidativa de L-aminoácidos e produzem o -cetoácido correspondente, com a concomitante liberação de amônia e peróxido de hidrogênio. A peçonha de Lachesis muta (L. muta) contém L-aminoácido oxidase, a qual pode contribuir com o envenenamento. Portanto, o objetivo deste trabalho é a purificação da L-amino acido oxidase de peçonha de Lachesis muta (LmLAAO) e a sua caracterização bioquímica, estrutural e funcional. Para isso, foram desenvolvidos dois protocolos distintos de purificação, os quais forneceram LmLAAO com grande pureza. No primeiro protocolo, 20 mg de peçonha bruta de L. muta foram submetidos a uma gel filtração em Sephacryl S100®. Das dez frações obtidas, a primeira fração apresentou atividade L-aminoácido oxidase e foi submetida a mais um passo cromatográfico em Mono Q®. A homogeneidade da fração com atividade L-aminoácido oxidase após a troca iônica foi comprovada por presença de banda única com 60...

Antibacterial and antiparasitic effects of Bothrops marajoensis venom and its fractions: Phospholipase A(2) and L-amino acid oxidase

Costa Torres, Alba Fabiola; Dantas, Rodrigo Tavares; Toyama, Marcos H.; Diz Filho, Eduardo; Zara, Fernando Jose; Rodrigues de Queiroz, Maria Goretti; Pinto Nogueira, Nadia Accioly; de Oliveira, Marcia Rosa; Toyama, Daniela de Oliveira; Monteiro, Helena S.
Fonte: Pergamon-Elsevier B.V. Ltd Publicador: Pergamon-Elsevier B.V. Ltd
Tipo: Artigo de Revista Científica Formato: 795-804
ENG
Relevância na Pesquisa
75.89%
Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); Some proteins present in snake venom possess enzymatic activities, such as phospholipase A(2) and L-amino acid oxidase. In this study, we verify the action of the Bothrops marajoensis venom (BmarTV), PLA(2) (BmarPLA(2)) and LAAO (BmarLAAO) on strains of bacteria, yeast, and Leishmania sp. The BmarTV was isolated by Protein Pack 5PW, and several fractions were obtained. Reverse phase HPLC showed that BmarPLA(2) was isolated from the venom, and N-terminal amino acid sequencing of sPLA(2) showed high amino acid identity with other lysine K49 sPLA(2)s isolated from Bothrops snakes. The BmarLAAO was purified to high molecular homogeneity and its N-terminal amino acid sequence demonstrated a high degree of amino acid conservation with others LAAOs. BmarLAAO was able to inhibit the growth of P. aeruginosa, C. albicans and S. aureus in a dose-dependent manner. The inhibitory effect was more significant on S. aureus, with a MIC = 50 mu g/mL and MLC = 200 mu g/mL However, the BmarTV and BmarPLA(2) did not demonstrate inhibitory capacity. BmarLAAO was able to inhibit the growth of promastigote forms of L chagasi and L amazonensis, with an IC50 = 2.55 mu g/mL and 2.86 mu g/mL for L. amazonensis and L. chagasi...

Amino acid sequence of a myotoxic Lys49-phospholipase A(2) homologue from the venom of Cerrophidion (Bothrops) godmani

de Sousa, M. V.; Morhy, L.; Arni, R. K.; Ward, R. J.; Diaz, C.; Gutierrez, J. M.
Fonte: Elsevier B.V. Publicador: Elsevier B.V.
Tipo: Artigo de Revista Científica Formato: 204-208
ENG
Relevância na Pesquisa
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The complete amino acid sequence of myotoxin II (godMT-II), a myotoxic phospholipase A( 2 )(PLA(2)) homologue from the venom of the Central American crotaline snake Cerrophidion (Bothrops) godmani, was determined by direct protein sequencing methods. GodMT-II is a class II PLA, showing a Lys instead of Asp at position 49. An additional substitution in the calcium binding loop region (Asn instead of Tyr at position 28) suggests the lack of enzymatic activity observed in this toxin is due to loss of its ability to bind the co-factor Ca2+, since the residues involved in forming the catalytic network of PLA(2)s (His-48, Tyr-52 and Asp-99) an conserved in godMT-II. This myotoxin shows highest sequence homology with other Lys-49 PLA(2)s from Bothrops, Agkistrodon and Trimeresurus species, suggesting that they constitute a conserved family of proteins, yet in contrast presents lower homology with Bothrops asper myotoxin III, a catalytically-active PLA(2). The C-terminal region of godMT-II, which is rich in cationic and hydrophobic residues, shares high sequence homology to the corresponding region in the myotoxin II from B. asper, which has been proposed to play an important role in the Ca2+-independent membrane damaging activity. (C) 1998 Elsevier B.V. B.V. All rights reserved.

Amino acid sequence and crystal structure of BaP1, a metalloproteinase from Bothrops asper snake venom that exerts multiple tissue-damaging activities

Watanabe, L.; Shannon, J. D.; Valente, R. H.; Rucavado, A.; Alape-Giron, A.; Kamiguti, A. S.; Theakston, RDG; Fox, J. W.; Gutierrez, J. M.; Arni, R. K.
Fonte: Cold Spring Harbor Lab Press, Publications Dept Publicador: Cold Spring Harbor Lab Press, Publications Dept
Tipo: Artigo de Revista Científica Formato: 2273-2281
ENG
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BaP1 is a 22.7-kD P-I-type zinc-dependent metalloproteinase isolated from the venom of the snake Bothrops asper, a medically relevant species in Central America. This enzyme exerts multiple tissue-damaging activities, including hemorrhage, myonecrosis, dermonecrosis, blistering, and edema. BaP1 is a single chain of 202 amino acids that shows highest sequence identity with metalloproteinases isolated front the venoms of snakes of the subfamily Crotalinae. It has six Cys residues involved in three disulfide bridges (Cys 117-Cys 197, Cys 159-Cys 181, Cys 157-Cys 164). It has the consensus sequence H(142)E(143)XXH(146)XXGXXH(152), as well as the sequence C164I165M166, which characterize the metzincin superfamily of metalloproteinases. The active-site cleft separates a major subdomain (residues 1-152), comprising four a-helices and a five-stranded beta-sheet, from the minor subdomain, which is formed by a single a-helix and several loops. The catalytic zinc ion is coordinated by the N-epsilon2 nitrogen atoms of His 142, His 146, and His 152, in addition to a solvent water molecule, which in turn is bound to Glu 143. Several conserved residues contribute to the formation of the hydrophobic pocket, and Met 166 serves as a hydrophobic base for the active-site groups. Sequence and structural comparisons of hemorrhagic and nonhemorrhagic P-I metalloproteinases from snake venoms revealed differences in several regions. In particular...

Eucalyptus ESTs associated with resistance to herbicide inhibitors of aromatic and branched-chain amino acid synthesis

Velini, Edivaldo Domingues; Trindade, Maria Lúcia Bueno; Alves, Elza; Catâneo, Ana Catarina; Marino, Celso Luis; de Godoy Maia, Ivan; Mori, Edson Seizo; Furtado, Edson Luiz; Guerrini, Iraê Amaral; Wilcken, Carlos Frederico
Fonte: Universidade Estadual Paulista Publicador: Universidade Estadual Paulista
Tipo: Artigo de Revista Científica Formato: 575-581
ENG
Relevância na Pesquisa
75.98%
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP); Herbicides inhibit enzymatic systems of plants. Acetolactate synthase (ALS, EC = 4.1.3.18) and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS, EC 2.5.1.19) are key enzymes for herbicide action. Hundreds of compounds inhibit ALS. This enzyme is highly variable, enabling the selective control of weeds in a number of crops. Glyphosate, the only commercial herbicide inhibiting EPSPS is widely used for non-selective control of weeds in many crops. Recently, transgenic crops resistant to glyphosate were developed and have been used by farmers. The aim of this study was the data mining of eucalypt expressed sequence tags (ESTs) in the FORESTs Genome Project database (https://forests.esalq.usp.br) related to these enzymes. Representative amino acid sequences from the NCBI database associated with ALS and EPSPS were blasted with ESTs from the FORESTs database using the tBLASTx option of the blast tool. The best blasting reads and clusters from FORESTs, represented as nucleotide sequences, were blasted back with the NCBI database to evaluate the level of similarity with available sequences from different species. One and seven clusters were identified as showing high similarity with EPSPS and ALS sequences from the literature...

Structural insights into selectivity and cofactor binding in snake venom l-amino acid oxidases

Ullah, A.; Souza, T. A C B; Abrego, J. R B; Betzel, C.; Murakami, M. T.; Arni, R. K.
Fonte: Universidade Estadual Paulista Publicador: Universidade Estadual Paulista
Tipo: Artigo de Revista Científica Formato: 124-128
ENG
Relevância na Pesquisa
75.96%
l-Amino acid oxidases (LAAOs) are flavoenzymes that catalytically deaminate l-amino acids to corresponding α-keto acids with the concomitant production of ammonia (NH 3) and hydrogen peroxide (H 2O 2). Particularly, snake venom LAAOs have been attracted much attention due to their diverse clinical and biological effects, interfering on human coagulation factors and being cytotoxic against some pathogenic bacteria and Leishmania ssp. In this work, a new LAAO from Bothrops jararacussu venom (BjsuLAAO) was purified, functionally characterized and its structure determined by X-ray crystallography at 3.1å resolution. BjsuLAAO showed high catalytic specificity for aromatic and aliphatic large side-chain amino acids. Comparative structural analysis with prokaryotic LAAOs, which exhibit low specificity, indicates the importance of the active-site volume in modulating enzyme selectivity. Surprisingly, the flavin adenine dinucleotide (FAD) cofactor was found in a different orientation canonically described for both prokaryotic and eukaryotic LAAOs. In this new conformational state, the adenosyl group is flipped towards the 62-71 loop, being stabilized by several hydrogen-bond interactions, which is equally stable to the classical binding mode. © 2012 Elsevier Inc.

N-terminal amino acid sequences of the major outer membrane proteins from a Neisseria meningitidis group B strain isolated in Brazil

De Simone,Salvatore Giovanni; Soares,Solange AF; Souza,Andre LA; Danelli,Maria GM
Fonte: Instituto Oswaldo Cruz, Ministério da Saúde Publicador: Instituto Oswaldo Cruz, Ministério da Saúde
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/02/1996 EN
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85.91%
The four dominant outer membrane proteins (46, 38, 33 and 28 kDa) were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in a semi-purified preparation of vesicle membranes of a Neisseria meningitidis (N44/89, B:4:P1.15:P5.5,7) strain isolated in Brazil. The N-terminal amino acid sequence for the 46 kDa and 28 kDa proteins matched that reported by others for class 1 and 5 proteins respectively, whereas the sequence (25 amino acids) for the 38 kDa (class 3) protein was similar to class 1 meningococcal proteins. The sequence for the 33 kDa (class 4) was unique and not homologous to any known protein.

Amino acid sequences of proteins from Leptospira serovar pomona

Alves,Selmo F; LeFebvre,Rance B; Probert,William
Fonte: Instituto Oswaldo Cruz, Ministério da Saúde Publicador: Instituto Oswaldo Cruz, Ministério da Saúde
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/08/2000 EN
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This report describes a partial amino acid sequences from three putative outer envelope proteins from Leptospira serovar pomona. In order to obtain internal fragments for protein sequencing, enzymatic and chemical digestion was performed. The enzyme clostripain was used to digest the proteins 32 and 45 kDa. In situ digestion of 40 kDa molecular weight protein was accomplished using cyanogen bromide. The 32 kDa protein generated two fragments, one of 21 kDa and another of 10 kDa that yielded five residues. A fragment of 24 kDa that yielded nineteen residues of amino acids was obtained from 45 kDa protein. A fragment with a molecular weight of 20 kDa, yielding a twenty amino acids sequence from the 40 kDa protein.

A protein with amino acid sequence homology to bovine insulin is present in the legume Vigna unguiculata (cowpea)

Venâncio,T.M.; Oliveira,A.E.A.; Silva,L.B.; Machado,O.L.T.; Fernandes,K.V.S.; Xavier-Filho,J.
Fonte: Associação Brasileira de Divulgação Científica Publicador: Associação Brasileira de Divulgação Científica
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/09/2003 EN
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75.86%
Since the discovery of bovine insulin in plants, much effort has been devoted to the characterization of these proteins and elucidation of their functions. We report here the isolation of a protein with similar molecular mass and same amino acid sequence to bovine insulin from developing fruits of cowpea (Vigna unguiculata) genotype Epace 10. Insulin was measured by ELISA using an anti-human insulin antibody and was detected both in empty pods and seed coats but not in the embryo. The highest concentrations (about 0.5 ng/µg of protein) of the protein were detected in seed coats at 16 and 18 days after pollination, and the values were 1.6 to 4.0 times higher than those found for isolated pods tested on any day. N-terminal amino acid sequencing of insulin was performed on the protein purified by C4-HPLC. The significance of the presence of insulin in these plant tissues is not fully understood but we speculate that it may be involved in the transport of carbohydrate to the fruit.

Purification and N-terminal amino acid sequence comparisons of structural proteins from retrovirus-D/Washington and Mason-Pfizer monkey virus.

Henderson, L E; Sowder, R; Smythers, G; Benveniste, R E; Oroszlan, S
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /09/1985 EN
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66.08%
A new D-type retrovirus originally designated SAIDS-D/Washington and here referred to as retrovirus-D/Washington (R-D/W) was recently isolated at the University of Washington Primate Center, Seattle, Wash., from a rhesus monkey with an acquired immunodeficiency syndrome and retroperitoneal fibromatosis. To better establish the relationship of this new D-type virus to the prototype D-type virus, Mason-Pfizer monkey virus (MPMV), we have purified and compared six structural proteins from each virus. The proteins purified from each D-type retrovirus include p4, p10, p12, p14, p27, and a phosphoprotein designated pp18 for MPMV and pp20 for R-D/W. Amino acid analysis and N-terminal amino acid sequence analysis show that the p4, p12, p14, and p27 proteins of R-D/W are distinct from the homologous proteins of MPMV but that these proteins from the two different viruses share a high degree of amino acid sequence homology. The p10 proteins from the two viruses have similar amino acid compositions, and both are blocked to N-terminal Edman degradation. The phosphoproteins from the two viruses each contain phosphoserine but are different from each other in amino acid composition, molecular weight, and N-terminal amino acid sequence. The data thus show that each of the R-D/W proteins examined is distinguishable from its MPMV homolog and that a major difference between these two D-type retroviruses is found in the viral phosphoproteins. The N-terminal amino acid sequences of D-type retroviral proteins were used to search for sequence homologies between D-type and other retroviral amino acid sequences. An unexpected amino acid sequence homology was found between R-D/W pp20 (a gag protein) and a 28-residue segment of the env precursor polyprotein of Rous sarcoma virus. The N-terminal amino acid sequences of the D-type major gag protein (p27) and the nucleic acid-binding protein (p14) show only limited amino acid sequence homology to functionally homologous proteins of C-type retroviruses.

Amino Acid Metabolic Origin as an Evolutionary Influence on Protein Sequence in Yeast

de Bivort, Benjamin Lovegren; Perlstein, Ethan O.; Kunes, Samuel M.; Schreiber, Stuart L.
Fonte: Springer-Verlag Publicador: Springer-Verlag
Tipo: Artigo de Revista Científica
EN_US
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75.96%
The metabolic cycle of Saccharomyces cerevisiae consists of alternating oxidative (respiration) and reductive (glycolysis) energy-yielding reactions. The intracellular concentrations of amino acid precursors generated by these reactions oscillate accordingly, attaining maximal concentration during the middle of their respective yeast metabolic cycle phases. Typically, the amino acids themselves are most abundant at the end of their precursor’s phase. We show that this metabolic cycling has likely biased the amino acid composition of proteins across the S. cerevisiae genome. In particular, we observed that the metabolic source of amino acids is the single most important source of variation in the amino acid compositions of functionally related proteins and that this signal appears only in (facultative) organisms using both oxidative and reductive metabolism. Periodically expressed proteins are enriched for amino acids generated in the preceding phase of the metabolic cycle. Proteins expressed during the oxidative phase contain more glycolysis-derived amino acids, whereas proteins expressed during the reductive phase contain more respiration-derived amino acids. Rare amino acids (e.g., tryptophan) are greatly overrepresented or underrepresented...

Cloning and characterization of an intracellular esterase from the wine-associated lactic acid bacterium Oenococcus oeni

Sumby, K.; Matthews, A.; Grbin, P.; Jiranek, V.
Fonte: Amer Soc Microbiology Publicador: Amer Soc Microbiology
Tipo: Artigo de Revista Científica
Publicado em //2009 EN
Relevância na Pesquisa
75.94%
We report the cloning and characterization of EstB28, the first esterase to be so characterized from the wine-associated lactic acid bacterium, Oenococcus oeni. The published sequence for O. oeni strain PSU-1 was used to identify putative esterase genes and design PCR primers in order to amplify the corresponding region from strain Ooeni28, an isolate intended for inoculation of wines. In this way a 912-bp open reading frame (ORF) encoding a putative esterase of 34.5 kDa was obtained. The amino acid sequence indicated that EstB28 is a member of family IV of lipolytic enzymes and contains the GDSAG motif common to other lactic acid bacteria. This ORF was cloned into Escherichia coli using an appropriate expression system, and the recombinant esterase was purified. Characterization of EstB28 revealed that the optimum temperature, pH, and ethanol concentration were 40°C, pH 5.0, and 28% (vol/vol), respectively. EstB28 also retained marked activity under conditions relevant to winemaking (10 to 20°C, pH 3.5, 14% [vol/vol] ethanol). Kinetic constants were determined for EstB28 with p-nitrophenyl (pNP)-linked substrates ranging in chain length from C₂to C₁₈. EstB28 exhibited greatest specificity for C₂to C₄pNP-linked substrates.; Krista M. Sumby...

Single amino acids in the carboxyl terminal domain of aquaporin-1 contribute to cGMP-dependent ion channel activation

Boassa, D.; Yool, A.
Fonte: BioMed Central Ltd. Publicador: BioMed Central Ltd.
Tipo: Artigo de Revista Científica
Publicado em //2003 EN
Relevância na Pesquisa
75.93%
BACKGROUND Aquaporin-1 (AQP1) functions as an osmotic water channel and a gated cation channel. Activation of the AQP1 ion conductance by intracellular cGMP was hypothesized to involve the carboxyl (C-) terminus, based on amino acid sequence alignments with cyclic-nucleotide-gated channels and cGMP-selective phosphodiesterases. RESULTS Voltage clamp analyses of human AQP1 channels expressed in Xenopus oocytes demonstrated that the nitric oxide donor, sodium nitroprusside (SNP; 3–14 mM) activated the ionic conductance response in a dose-dependent manner. Block of soluble guanylate cyclase prevented the response. Enzyme immunoassays confirmed a linear dose-dependent relationship between SNP and the resulting intracellular cGMP levels (up to 1700 fmol cGMP /oocyte at 14 mM SNP). Results here are the first to show that the efficacy of ion channel activation is decreased by mutations of AQP1 at conserved residues in the C-terminal domain (aspartate D237 and lysine K243). CONCLUSIONS These data support the idea that the limited amino acid sequence similarities found between three diverse classes of cGMP-binding proteins are significant to the function of AQP1 as a cGMP-gated ion channel, and provide direct evidence for the involvement of the AQP1 C-terminal domain in cGMP-mediated ion channel activation.

Amino acid similarity matrices based on force fields

Doszt�nyi, Zsuzsanna; Torda, Andrew
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
Relevância na Pesquisa
75.93%
Motivation: We propose a general method for deriving amino acid substitution matrices from low resolution force fields. Unlike current popular methods, the approach does not rely on evolutionary arguments or alignment of sequences or structures. Instead,

Functional effects of amino acid substitutions within the large binding pocket of the phosphotriesterase OpdA from Agrobacterium sp. P230

Horne, Irene; Qiu, Xinghui; Ollis, David; Russell, Robyn; Oakeshott, John Graham
Fonte: Elsevier Publicador: Elsevier
Tipo: Artigo de Revista Científica
Relevância na Pesquisa
75.87%
The phosphotriesterase OpdA from Agrobacterium sp. P230 has about 10-fold higher activity for dimethyl organophosphate (OP) insecticides, than its homologue from Flavobacterium sp. ATCC27551, organophosphate hydrolase (OPH). OpdA shows about 10% amino acid sequence divergence from OPH and also has a 20 residue C-terminal extension. Here we show that the difference in kinetics is largely explained by just two amino acid differences between the two proteins. A truncated form of OpdA demonstrated that the C-terminal extension has no effect on its preference for dimethyl organophosphate substrates. Chimeric proteins of OPH and OpdA were then analysed to show that replacement of a central region of OpdA sequence, which encodes the residues in the large subsite of the active site, with the homologous region in OPH decreased the activity of OpdA towards dimethyl OPs, to values close to those for OPH. Site-directed mutagenesis in this region identified two differences between the proteins, Y257H and F272L (with the OpdA residues first) as being responsible for this reduction. These two differences were also responsible for the increased activity of OpdA towards the diisopropyl organophosphate, diisopropyl fluorophosphate, relative to OPH. Molecular modelling of triethyl phosphate in the active site of OpdA confirmed a reduction in the size of the large subsite relative to OPH.

Evolution of HLA class II molecules: Allelic and amino acid site variability across populations

Salamon, H; Klitz, William; Easteal, Simon; Gao, Xiaojiang; Fernandez-Vina, M; Trachtenberg, Elizabeth; McWeeney, Shannon; Nelson, Mark; Thomson, Gleys
Fonte: Genetics Society of America Publicador: Genetics Society of America
Tipo: Artigo de Revista Científica
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75.76%
Analysis of the highly polymorphic β1 domains of the HLA class II molecules encoded by the DRB1, DQB1, and DPB1 loci reveals contrasting levels of diversity at the allele and amino acid site levels. Statistics of allele frequency distributions, based on

The same amino acid substitution in orthologous esterases confers organophosphate resistance on the house fly and a blowfly

Claudianos, Charles; Russell, Robyn; Oakeshott, John Graham
Fonte: Pergamon-Elsevier Ltd Publicador: Pergamon-Elsevier Ltd
Tipo: Artigo de Revista Científica
Relevância na Pesquisa
75.87%
Organophosphate (OP) insecticide resistance in certain strains of Musca domestica is associated with reduction in the carboxylesterase activity of a particular esterase isozyme. This has been attributed to a 'mutant ali- esterase hypothesis', which invokes a structural mutation to an ali-esterase resulting in the loss of its carboxylesterase activity but acquisition of OP hydrolase activity. It has been shown that the mutation in Lucilia cuprina is a Gly137 → Asp substitution in the active site of an esterase encoded by the LcαE7 gene (Newcomb, R.D., Campbell, P.M., Ollis, D.L., Cheah, E., Russell, R.J., Oakeshott, J.G., 1997. A single amino acid substitution converts a carboxylesterase to an organophosphate hydrolase and confers insecticide resistance on a blowfly. Proc. Natl. Acad. Sci. USA 94, 7464- 7468). We now report the cloning and characterisation of the orthologous M. domestica MdαE7 gene, including the sequencing of cDNAs from the OP resistant Rutgers and OP susceptible sbo and WHO strains. The MdαE7 gene has the same intron structure as LcαE7 and encodes a protein with 76% amino acid identity to LcαE7. Comparisons between susceptible and resistance alleles show resistance in M. domestica is associated with the same Gly137 → Asp mutation as in L. cuprina. Bacterial expression of the Rutgers allele shows its product has OP hydrolase activity. The data indicate identical catalytic mechanisms have evolved in orthologous MdαE7 and LcαE7 molecules to endow diazinon-type resistance on the two species of higher Diptera.

Adaptation of plasma membrane amino acid transport mechanisms to physiological demands

Broer, Stefan
Fonte: Springer Publicador: Springer
Tipo: Artigo de Revista Científica
Relevância na Pesquisa
75.92%
The molecular identification of almost all physiologically characterized amino acid transporters in recent years has facilitated the functional analysis of this important class of transport proteins. The picture that emerges from these studies is that antiport is the prevalent mode of amino acid transport rather than a combination of uniporters and cotransporters. Mainly neurotransmitters and osmolytes are transported by complex cotransport mechanisms that allow a high intracellular accumulation. Antiport mechanisms almost invariably include the nonessential amino acids alanine and glutamine, which are used as exchange substrates. The intracellular level of both amino acids is well regulated by Na+/amino acid cotransporters. Transport mechanisms are not conserved within families and may change with mutation of even a single amino acid residue in the transport protein. Thus transport mechanisms are easily adapted to physiological demands during evolution.