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1.
BMC Pregnancy Childbirth ; 20(1): 117, 2020 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-32075598

RESUMO

BACKGROUND: Pelvic floor muscles (PFM) and rectus abdominis muscles (RAM) of pregnant diabetic rats exhibit atrophy, co-localization of fast and slow fibers and an increased collagen type I/III ratio. However, the role of similar PFM or RAM hyperglycemic-related myopathy in women with gestational diabetes mellitus (GDM) remains poorly investigated. This study aims to assess the frequency of pelvic floor muscle disorders and pregnancy-specific urinary incontinence (PS-UI) 12 months after the Cesarean (C) section in women with GDM. Specifically, differences in PFM/RAM hyperglycemic myopathy will be evaluated. METHODS: The Diamater is an ongoing cohort study of four groups of 59 pregnant women each from the Perinatal Diabetes Research Centre (PDRC), Botucatu Medical School (FMB)-UNESP (São Paulo State University), Brazil. Diagnosis of GDM and PS-UI will be made at 24-26 weeks, with a follow-up at 34-38 weeks of gestation. Inclusion in the study will occur at the time of C-section, and patients will be followed at 24-48 h, 6 weeks and 6 and 12 months postpartum. Study groups will be classified as (1) GDM plus PS-UI; (2) GDM without PS-UI; (3) Non-GDM plus PS-UI; and (4) Non-GDM without PS-UI. We will analyze relationships between GDM, PS-UI and hyperglycemic myopathy at 12 months after C-section. The mediator variables to be evaluated include digital palpation, vaginal squeeze pressure, 3D pelvic floor ultrasound, and 3D RAM ultrasound. RAM samples obtained during C-section will be analyzed for ex-vivo contractility, morphological, molecular and OMICS profiles to further characterize the hyperglycemic myopathy. Additional variables to be evaluated include maternal age, socioeconomic status, educational level, ethnicity, body mass index, weight gain during pregnancy, quality of glycemic control and insulin therapy. DISCUSSION: To our knowledge, this will be the first study to provide data on the prevalence of PS-UI and RAM and PFM physical and biomolecular muscle profiles after C-section in mothers with GDM. The longitudinal design allows for the assessment of cause-effect relationships between GDM, PS-UI, and PFMs and RAMs myopathy. The findings may reveal previously undetermined consequences of GDM.


Assuntos
Diabetes Gestacional/fisiopatologia , Doenças Musculares/fisiopatologia , Incontinência Urinária/fisiopatologia , Adulto , Brasil , Cesárea , Estudos de Coortes , Feminino , Idade Gestacional , Ganho de Peso na Gestação , Humanos , Idade Materna , Contração Muscular/fisiologia , Força Muscular/fisiologia , Palpação , Diafragma da Pelve/fisiopatologia , Período Pós-Parto , Gravidez , Reto do Abdome/fisiopatologia , Vagina
2.
Biochim Biophys Acta ; 1861(9 Pt A): 970-979, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27233517

RESUMO

Brown spider phospholipases D from Loxosceles venoms are among the most widely studied toxins since they induce dermonecrosis, triggering inflammatory responses, increase vascular permeability, cause hemolysis, and renal failure. The catalytic (H12 and H47) and metal-ion binding (E32 and D34) residues in Loxosceles intermedia phospholipase D (LiRecDT1) were mutated to understand their roles in the observed activities. All mutants were identified using whole venom serum antibodies and a specific antibody to wild-type LiRecDT1, they were also analyzed by circular dichroism (CD) and differential scanning calorimetry (DSC). The phospholipase D activities of H12A, H47A, H12A-H47A, E32, D34 and E32A-D34A, such as vascular permeability, dermonecrosis, and hemolytic effects were inhibited. The mutant Y228A was equally detrimental to biochemical and biological effects of phospholipase D, suggesting an essential role of this residue in substrate recognition and binding. On the other hand, the mutant C53A-C201A reduced the enzyme's ability to hydrolyze phospholipids and promote dermonecrosis, hemolytic, and vascular effects. These results provide the basis understanding the importance of specific residues in the observed activities and contribute to the design of synthetic and specific inhibitors for Brown spider venom phospholipases D.


Assuntos
Domínio Catalítico/genética , Fosfolipase D/química , Fosfolipídeos/química , Venenos de Aranha/enzimologia , Animais , Aranha Marrom Reclusa/química , Aranha Marrom Reclusa/enzimologia , Permeabilidade Capilar , Dicroísmo Circular , Hemólise , Mutação , Fosfolipase D/metabolismo , Fosfolipídeos/metabolismo , Diester Fosfórico Hidrolases/química , Venenos de Aranha/química
3.
Int J Biol Macromol ; 83: 178-84, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26592780

RESUMO

Southern bean mosaic virus (SBMV) RNA purified from infected plants was used for cloning the viral genome-linked protein (VPg) and was subsequently expressed in Escherichia coli. Circular dichroism (CD), dynamic light scattering (DLS) and saturation transfer difference (STD) by nuclear magnetic resonance (NMR) measurements were employed to determine the degree of monodispersity and to investigate the conformational changes in the absence and presence of trifluoroethanol (TFE) which indicated increased helical content with increasing concentration of TFE. 8-Anilino-1-naphthalenesulfonic acid (ANS) was used as a probe to compare the unfolding regions of the protein before and after addition of TFE. The results indicated that although the TFE concentration influences VPg folding, it does not play a role in nucleotide binding and that the local solvent hydrophobicity causes significant conformational changes.


Assuntos
Fabaceae/virologia , Vírus de Plantas/genética , Vírus de Plantas/metabolismo , Trifluoretanol/metabolismo , Trifluoretanol/farmacologia , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Expressão Gênica , Histidina , Dados de Sequência Molecular , Nucleotídeos/metabolismo , Ligação Proteica , Conformação Proteica/efeitos dos fármacos , Proteínas não Estruturais Virais/química
4.
Curr Protein Pept Sci ; 16(8): 768-74, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25961401

RESUMO

Phospholipases D (PLDs), the major dermonecrotic factors from brown spider venoms, trigger a range of biological reactions both in vitro and in vivo. Despite their clinical relevance in loxoscelism, structural data is restricted to the apo-form of these enzymes, which has been instrumental in understanding the functional differences between the class I and II spider PLDs. The crystal structures of the native class II PLD from Loxosceles intermedia complexed with myo-inositol 1-phosphate and the inactive mutant H12A complexed with fatty acids indicate the existence of a strong ligand-dependent conformation change of the highly conserved aromatic residues, Tyr 223 and Trp225 indicating their roles in substrate binding. These results provided insights into the structural determinants for substrate recognition and binding by class II PLDs.


Assuntos
Fosfolipase D/química , Fosfolipase D/metabolismo , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/metabolismo , Venenos de Aranha/química , Venenos de Aranha/metabolismo , Aranhas/química , Sequência de Aminoácidos , Animais , Caprilatos/metabolismo , Cristalografia por Raios X , Fosfatos de Inositol , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Alinhamento de Sequência , Especificidade por Substrato
5.
Protein Sci ; 22(1): 128-32, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23139169

RESUMO

Snake venom serine proteinases (SVSPs) are hemostatically active toxins that perturb the maintenance and regulation of both the blood coagulation cascade and fibrinolytic feedback system at specific points, and hence, are widely used as tools in pharmacological and clinical diagnosis. The crystal structure of a thrombin-like enzyme (TLE) from Bothrops jararacussu venom (Jararacussin-I) was determined at 2.48 Å resolution. This is the first crystal structure of a TLE and allows structural comparisons with both the Agkistrodon contortrix contortrix Protein C Activator and the Trimeresurus stejnegeri plasminogen activator. Despite the highly conserved overall fold, significant differences in the amino acid compositions and three-dimensional conformations of the loops surrounding the active site significantly alter the molecular topography and charge distribution profile of the catalytic interface. In contrast to other SVSPs, the catalytic interface of Jararacussin-I is highly negatively charged, which contributes to its unique macromolecular selectivity.


Assuntos
Serina Endopeptidases/química , Venenos de Serpentes/enzimologia , Trombina/química , Trombina/economia , Venenos de Víboras/química , Animais , Biocatálise , Bothrops , Cromatografia de Afinidade , Cromatografia em Gel , Cristalografia por Raios X , Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Modelos Moleculares , Conformação Proteica , Serina Endopeptidases/isolamento & purificação , Serina Endopeptidases/metabolismo , Venenos de Víboras/isolamento & purificação , Venenos de Víboras/metabolismo
6.
Int J Biol Macromol ; 51(3): 209-14, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22584077

RESUMO

Catalytically inactive phospholipase A(2) (PLA(2)) homologues play key roles in the pathogenesis induced by snake envenomation, causing extensive tissue damage via a mechanism still unknown. Although, the amino acid residues directly involved in catalysis are conserved, the substitution of Asp49 by Arg/Lys/Gln or Ser prevents the binding of the essential calcium ion and hence these proteins are incapable of hydrolyzing phospholipids. In this work, the crystal structure of a Lys49-PLA(2) homologue from Bothrops brazili (MTX-II) was solved in two conformational states: (a) native, with Lys49 singly coordinated by the backbone oxygen atom of Val31 and (b) complexed with tetraethylene glycol (TTEG). Interestingly, the TTEG molecule was observed in two different coordination cages depending on the orientation of the nominal calcium-binding loop and of the residue Lys49. These structural observations indicate a direct role for the residue Lys49 in the functioning of a catalytically inactive PLA(2) homologue suggesting a contribution of the active site-like region in the expression of pharmacological effects such as myotoxicity and edema formation. Despite the several crystal structures of Lys49-PLA(2) homologues already determined, their biological assembly remains controversial with two possible conformations. The extended dimer with the hydrophobic channel exposed to the solvent and the compact dimer in which the active site-like region is occluded by the dimeric interface. In the MTX-II crystal packing analysis was found only the extended dimer as a possible stable quaternary arrangement.


Assuntos
Proteínas Mutantes/química , Fosfolipases A2/química , Animais , Domínio Catalítico , Etilenoglicóis/química , Ligantes , Modelos Moleculares , Proteínas Mutantes/isolamento & purificação , Fosfolipases A2/isolamento & purificação , Conformação Proteica , Multimerização Proteica , Estrutura Secundária de Proteína
7.
Biochem Biophys Res Commun ; 421(1): 124-8, 2012 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-22490662

RESUMO

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(2)O(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 3.1 Å 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.


Assuntos
Bothrops , Venenos de Crotalídeos/enzimologia , L-Aminoácido Oxidase/química , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Estabilidade Enzimática , Interações Hidrofóbicas e Hidrofílicas , Dados de Sequência Molecular , Estrutura Secundária de Proteína
8.
Toxicon ; 55(2-3): 361-8, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-19706302

RESUMO

Hemostatically active snake venom metalloproteinases (SVMPs) perturb the blood coagulation cascade at specific points and due to their potential application as thrombolytic agents, the fibrin(ogen)olytic non-hemorrhagic SVMPs have been employed as biochemical tools in coagulation research and diagnosis. Structural studies complemented by the design of metalloproteinase inhibitors have been instrumental in understanding their stereo specificity and action mechanism. We present here, details of the crystal structure of BmooMPalpha-I, a 22.6 kDa non-hemorrhagic P-I class SVMP isolated from Bothrops moojeni venom, determined at 1.76 A resolution. In this structure, the catalytic zinc ion displays an unusual octahedral coordination formed by the three canonical histidines (His(142), His(146) and His(152)) and additionally, by three solvent molecules. Comparative sequence and structural studies indicate that the motif comprising amino acid segments 153-164 and 167-176 adjacent to the methionine-turn is a salient feature that differentiates both non and hemorrhagic P-I class SVMPs and could directly be involved in the development of the hemorrhagic activity.


Assuntos
Bothrops/fisiologia , Metaloproteases/química , Venenos de Víboras/enzimologia , Sequência de Aminoácidos , Animais , Cristalização , Eletroforese em Gel de Poliacrilamida , Hemorragia/induzido quimicamente , Metaloproteases/antagonistas & inibidores , Metaloproteases/farmacologia , Modelos Moleculares , Dados de Sequência Molecular , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Ligação Proteica , Conformação Proteica , Espectrometria de Massas por Ionização por Electrospray , Relação Estrutura-Atividade , Especificidade por Substrato , Venenos de Víboras/farmacologia , Difração de Raios X , Zinco/química
9.
10.
Protein Pept Lett ; 15(7): 724-30, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18782069

RESUMO

Miliin, a new thiol-dependent serine protease purified from the latex of Euphorbia milii possesses a molecular weight of 79 kDa, an isoelectric point of 4.3 and is optimally active at 60 degrees C in the pH range of and 7.5-11.0. Activity tests indicate that milliin is a thiol-dependent serine protease.


Assuntos
Euphorbia/enzimologia , Serina Endopeptidases/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Concentração de Íons de Hidrogênio , Focalização Isoelétrica , Ponto Isoelétrico , Cinética , Látex/química , Peso Molecular , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Temperatura
11.
Artigo em Inglês | MEDLINE | ID: mdl-17401196

RESUMO

Crotoxin, a potent neurotoxin from the venom of the South American rattlesnake Crotalus durissus terrificus, exists as a heterodimer formed between a phospholipase A(2) and a catalytically inactive acidic phospholipase A(2) analogue (crotapotin). Large single crystals of the crotoxin complex and of the isolated subunits have been obtained. The crotoxin complex crystal belongs to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 38.2, b = 68.7, c = 84.2 A, and diffracted to 1.75 A resolution. The crystal of the phospholipase A(2) domain belongs to the hexagonal space group P6(1)22 (or its enantiomorph P6(5)22), with unit-cell parameters a = b = 38.7, c = 286.7 A, and diffracted to 2.6 A resolution. The crotapotin crystal diffracted to 2.3 A resolution; however, the highly diffuse diffraction pattern did not permit unambiguous assignment of the unit-cell parameters.


Assuntos
Crotoxina/química , Fosfolipases A/química , Cristalização , Cristalografia por Raios X , Dimerização , Fosfolipases A2 , Conformação Proteica
12.
J Mol Biol ; 366(2): 602-10, 2007 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-17173931

RESUMO

NAPc2, an anticoagulant protein from the hematophagous nematode Ancylostoma caninum evaluated in phase-II/IIa clinical trials, inhibits the extrinsic blood coagulation pathway by a two step mechanism, initially interacting with the hitherto uncharacterized factor Xa exosite involved in macromolecular recognition and subsequently inhibiting factor VIIa (K(i)=8.4 pM) of the factor VIIa/tissue factor complex. NAPc2 is highly flexible, becoming partially ordered and undergoing significant structural changes in the C terminus upon binding to the factor Xa exosite. In the crystal structure of the ternary factor Xa/NAPc2/selectide complex, the binding interface consists of an intermolecular antiparallel beta-sheet formed by the segment of the polypeptide chain consisting of residues 74-80 of NAPc2 with the residues 86-93 of factor Xa that is additional maintained by contacts between the short helical segment (residues 67-73) and a turn (residues 26-29) of NAPc2 with the short C-terminal helix of factor Xa (residues 233-243). This exosite is physiologically highly relevant for the recognition and inhibition of factor X/Xa by macromolecular substrates and provides a structural motif for the development of a new class of inhibitors for the treatment of deep vein thrombosis and angioplasty.


Assuntos
Ancylostoma/química , Fator Xa/química , Proteínas de Helminto/química , Animais , Anticoagulantes/farmacologia , Sítios de Ligação , Bovinos , Fator VIIa/química , Fator VIIa/metabolismo , Fator Xa/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Tromboplastina/química , Tromboplastina/metabolismo
13.
Biochimie ; 88(5): 543-9, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16376474

RESUMO

The electrophile Ca(2+) is an essential multifunctional co-factor in the phospholipase A(2) mediated hydrolysis of phospholipids. Crystal structures of an acidic phospholipase A(2) from the venom of Bothrops jararacussu have been determined both in the Ca(2+) free and bound states at 0.97 and 1.60 A resolutions, respectively. In the Ca(2+) bound state, the Ca(2+) ion is penta-coordinated by a distorted pyramidal cage of oxygen and nitrogen atoms that is significantly different to that observed in structures of other Group I/II phospholipases A(2). In the absence of Ca(2+), a water molecule occupies the position of the Ca(2+) ion and the side chain of Asp49 and the calcium-binding loop adopts a different conformation.


Assuntos
Cálcio/metabolismo , Fosfolipases A/química , Fosfolipases A/metabolismo , Animais , Sítios de Ligação , Bothrops/metabolismo , Venenos de Crotalídeos/enzimologia , Cristalização , Cristalografia por Raios X/métodos , Fosfolipases A2 do Grupo IV , Ligação de Hidrogênio , Modelos Moleculares , Fosfolipases A2 , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
14.
Artigo em Inglês | MEDLINE | ID: mdl-16510999

RESUMO

Xylanases have been the focus of research owing to their industrial potential in animal feed production, food processing and pulp and paper processes. In order to obtain insight into the structural stability of family 11 xylanases, the mesophilic family 11 xylanase (beta-1,4-xylan xylanohydrolase; EC 3.2.1.8) from Bacillus subtilis 1A1 has been crystallized and diffraction data have been collected to 1.7 A. The crystals belong to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 50.93, b = 70.50, c = 40.05 A. The structure has been determined by molecular replacement, resulting in a crystallographic residual of 36.4% after rigid-body refinement.


Assuntos
Bacillus subtilis/enzimologia , Endo-1,4-beta-Xilanases/química , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Cristalografia por Raios X , Endo-1,4-beta-Xilanases/isolamento & purificação , Conformação Proteica , Difração de Raios X
15.
Acta Crystallogr D Biol Crystallogr ; 60(Pt 6): 1112-4, 2004 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15159572

RESUMO

SMase I, a 32 kDa sphingomyelinase found in Loxosceles laeta venom, is responsible for the major pathological effects of spider envenomation. This toxin has been cloned and functionally expressed as a fusion protein containing a 6 x His tag at its N-terminus to yield a 33 kDa protein [Fernandes-Pedrosa et al. (2002), Biochem. Biophys. Res. Commun. 298, 638-645]. The recombinant protein possesses all the biological properties ascribed to the whole L. laeta venom, including dermonecrotic and complement-dependent haemolytic activities. Dynamic light-scattering experiments conducted at 291 K demonstrate that the sample possesses a monomodal distribution, with a hydrodynamic radius of 3.57 nm. L. laeta SMase I was crystallized by the hanging-drop vapour-diffusion technique using the sparse-matrix method. Single crystals were obtained using a buffer solution consisting of 0.08 M HEPES and 0.9 M trisodium citrate, which was titrated to pH 7.5 using 0.25 M sodium hydroxide. Complete three-dimensional diffraction data were collected to 1.8 angstroms at the Laboratório Nacional de Luz Síncrotron (LNLS, Campinas, Brazil). The crystals belong to the hexagonal system (space group P6(1) or P6(5)), with unit-cell parameters a = b = 140.6, c = 113.6 angstroms. A search for heavy-atom derivatives has been initiated and elucidation of the crystal structure is currently in progress.


Assuntos
Esfingomielina Fosfodiesterase/química , Venenos de Aranha/química , Animais , Citratos/farmacologia , Cristalografia por Raios X , DNA Complementar/metabolismo , Luz , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Espalhamento de Radiação , Citrato de Sódio , Aranhas , Temperatura
16.
Biochimie ; 85(10): 983-91, 2003 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-14644553

RESUMO

Acidic phospholipase A(2) (PLA(2)) isoforms in snake venoms, particularly those from Bothrops jararacussu, have not been characterized. This article reports the isolation and partial biochemical, functional and structural characterization of four acidic PLA(2)s (designated SIIISPIIA, SIIISPIIB, SIIISPIIIA and SIIISPIIIB) from this venom. The single chain purified proteins contained 122 amino acid residues and seven disulfide bonds with approximate molecular masses of 15 kDa and isoelectric points of 5.3. The respective N-terminal sequences were: SIIISPIIA-SLWQFGKMIDYVMGEEGAKS; SIIISPIIB-SLWQFGKMIFYTGKNEPVLS; SIIISPIIIA-SLWQFGKMILYVMGGEGVKQ and SIIISPIIIB-SLWQFGKMIFYEMTGEGVL. Crystals of the acidic protein SIIISPIIB diffracted beyond 1.8 A resolution. These crystals are monoclinic with unit cell dimensions of a = 40.1 A, b = 54.2 A and c = 90.7 A. The crystal structure has been refined to a crystallographic residual of 16.1% (R(free) = 22.9%). Specific catalytic activity (U/mg) of the isolated acidic PLA(2)s were SIIISPIIA = 290.3 U/mg; SIIISPIIB = 279.0 U/mg; SIIISPIIIA = 270.7 U/mg and SIIISPIIIB = 96.5 U/mg. Although their myotoxic activity was low, SIIISPIIA, SIIISPIIB and SIIISPIIIA showed significant anticoagulant activity. However, there was no indirect hemolytic activity. SIIISPIIIB revealed no anticoagulant, but presented indirect hemolytic activity. With the exception of SIIISPIIB, which inhibited platelet aggregation, all the others were capable of inducing time-independent edema. Chemical modification with 4-bromophenacyl bromide did not inhibit the induction of edema, but did suppress other activities.


Assuntos
Bothrops , Venenos de Crotalídeos/enzimologia , Fosfolipases A/química , Sequência de Aminoácidos , Animais , Creatina Quinase/metabolismo , Venenos de Crotalídeos/química , Venenos de Crotalídeos/toxicidade , Cristalografia por Raios X , Edema/induzido quimicamente , Técnicas In Vitro , Isoenzimas/química , Isoenzimas/isolamento & purificação , Isoenzimas/farmacologia , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Músculos/efeitos dos fármacos , Fosfolipases A/isolamento & purificação , Fosfolipases A/farmacologia , Fosfolipases A/toxicidade , Fosfolipases A2 , Inibidores da Agregação Plaquetária/química , Inibidores da Agregação Plaquetária/isolamento & purificação , Inibidores da Agregação Plaquetária/farmacologia , Conformação Proteica
17.
Biochem Biophys Res Commun ; 310(2): 478-82, 2003 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-14521935

RESUMO

Convulxin (CVX), a C-type lectin, isolated from the venom of the South American rattlesnake Crotalus durissus terrificus, causes cardiovascular and respiratory disturbances and is a potent platelet activator which binds to platelet glycoprotein GPVI. The structure of CVX has been solved at 2.4A resolution to a crystallographic residual of 18.6% (R(free)=26.4%). CVX is a disulfide linked heterodimer consisting of homologous alpha and beta chains. The heterodimers are additionally linked by disulfide bridges to form cyclic alpha(4)beta(4)heterotetramers. These domains exhibit significant homology to the carbohydrate-binding domains of C-type lectins, to the factor IX-binding protein (IX-bp), and to flavocetin-A (Fl-A) but sequence and structural differences are observed in both the domains in the putative Ca(2+)and carbohydrate binding regions.


Assuntos
Venenos de Crotalídeos/química , Crotalus , Lectinas Tipo C/química , Modelos Moleculares , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cálcio/metabolismo , Venenos de Crotalídeos/metabolismo , Cristalografia por Raios X , Dissulfetos/química , Lectinas Tipo C/metabolismo , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Subunidades Proteicas , Alinhamento de Sequência
18.
Protein Pept Lett ; 10(5): 525-30, 2003 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-14561144

RESUMO

Lys49-Phospholipase A2 (Lys49-PLA(2) - EC 3.1.1.4) homologues damage membranes by a Ca2+-independent mechanism which does not involve catalytic activity. Both MjTX-II from Bothrops moojeni and BthTX-I from Bothrops jararacussu are dimeric in solution and in the crystalline states, and a model for the Ca2+-independent membrane damaging mechanism has been suggested in which flexibility at the dimer interface region permits quaternary structural transitions between "open" and "closed" membrane bound dimer conformations which results in the perturbation of membrane phospholipids and disruption of the bilayer structure. With the aim of gaining insights into the structural determinants involved in protein/lipid association, we report here the crystallization and preliminary X-ray analysis of the (i) MjTX-II/SDS complex at a resolution of 2.78A, (ii) MjTX-II/STE complex at a resolution of 1.8 A and (iii) BthTX-I/DMPC complex at 2.72A. These complexes were crystallized by the hanging drop vapour-diffusion technique in (i) HEPES buffer (pH 7.5) 1.8M ammonium sulfate with 2% (w/v) polyethyleneglycol 400, in (ii) 0.6-0.8 M sodium citrate as the precipitant (pH 6.0-6.5) and in (iii) sodium citrate buffer (pH 5.8) and PEG 4000 and 20% isopropanol, respectively. Single crystals of these complexes have been obtained and X-ray diffraction data have been collected at room temperature using a R-AXIS IV imaging plate system and graphite monochromated Cu Kalpha X-ray radiation generated by a Rigaku RU300 rotating anode generator for (i) and (iii) and using using a Synchrotron Radiation Source (Laboratório Nacional de Luz Sincrotron, LNLS, Campinas, Brazil) for (ii).


Assuntos
Venenos de Crotalídeos/química , Fosfolipases A/química , Animais , Cristalização , Dimiristoilfosfatidilcolina/química , Fosfolipases A2 , Dodecilsulfato de Sódio/química , Ácidos Esteáricos/química , Difração de Raios X/estatística & dados numéricos
19.
Acta Crystallogr D Biol Crystallogr ; 59(Pt 10): 1813-5, 2003 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-14501123

RESUMO

Convulxin, an alphabeta C-type lectin, is a potent platelet activator isolated from the venom of the South American rattlesnake Crotalus durissus terrificus. It is a 26.5 kDa alphabeta heterodimer consisting of two homologous disulfide-linked chains. The crystals belong to space group I4, with unit-cell parameters a = b = 131.61, c = 121.85 A, and diffraction data were collected to 2.7 A. The structure was solved by molecular replacement and the asymmetric unit contains two alphabeta heterodimers, each of which forms a disulfide-linked cyclic alpha(4)beta(4) tetramer in the unit cell. These alpha(4)beta(4) tetramers are stacked to form a large solvent channel.


Assuntos
Venenos de Crotalídeos/química , Lectinas Tipo C/química , Animais , Crotalus , Cristalização , Cristalografia por Raios X , Dissulfetos/química , Modelos Moleculares , Estrutura Quaternária de Proteína , Subunidades Proteicas
20.
Acta Crystallogr D Biol Crystallogr ; 59(Pt 8): 1454-8, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12876349

RESUMO

2-Keto-3-deoxy-6-phosphogluconate (KDPG) aldolase from Pseudomonas putida is a key enzyme in the Entner-Doudoroff pathway which catalyses the cleavage of KDPG via a class I Schiff-base mechanism. The crystal structure of this enzyme has been refined to a crystallographic residual R = 17.1% (R(free) = 21.4%). The N-terminal helix caps one side of the torus of the (betaalpha)(8)-barrel and the active site is located on the opposite, carboxylic side of the barrel. The Schiff-base-forming Lys145 is coordinated by a sulfate (or phosphate) ion and two solvent water molecules. The interactions that stabilize the trimer are predominantly hydrophobic, with the exception of the cyclically permuted bonds formed between Glu132 OE1 of one molecule and Thr129 OG1 of a symmetry-equivalent molecule. Except for the N-terminal helix, the structure of KDPG aldolase from P. putida closely resembles the structure of the homologous enzyme from Escherichia coli.


Assuntos
Aldeído Liases/química , Pseudomonas putida/enzimologia , Sítios de Ligação , Cristalografia por Raios X , Escherichia coli/metabolismo , Lisina/química , Modelos Moleculares , Fosfatos/química , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
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