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1.
Inflamm Bowel Dis ; 19(11): 2273-81, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23949620

RESUMO

BACKGROUND: Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract, which is currently treated with injected monoclonal antibodies specific for tumor necrosis factor (TNF). We developed and characterized AVX-470, a novel polyclonal antibody specific for human TNF. We evaluated the oral activity of AVX-470m, a surrogate antibody specific for murine TNF, in several well-accepted mouse models of IBD. METHODS: AVX-470 and AVX-470m were isolated from the colostrum of dairy cows that had been immunized with TNF. The potency, specificity, and affinity of both AVX-470 and AVX-470m were evaluated in vitro and compared with infliximab. AVX-470m was orally administered to mice either before or after induction of colitis, and activity was measured by endoscopy, histopathology, immunohistochemistry, and quantitative measurement of messenger RNA levels. Colitis was induced using either 2,4,6-trinitrobenzene sulfonate or dextran sodium sulfate. RESULTS: AVX-470 and AVX-470m were shown to be functionally comparable in vitro. Moreover, the specificity, neutralizing potency, and affinity of AVX-470 were comparable with infliximab. Orally administered AVX-470m effectively reduced disease severity in several mouse models of IBD. Activity was comparable with that of oral prednisolone or parenteral etanercept. The antibody penetrated the colonic mucosa and inhibited TNF-driven mucosal inflammation with minimal systemic exposure. CONCLUSIONS: AVX-470 is a novel polyclonal anti-TNF antibody with an in vitro activity profile comparable to that of infliximab. Oral administration of a surrogate antibody specific for mouse TNF is effective in treating mouse models of IBD, delivering the anti-TNF to the site of inflammation with minimal systemic exposure.


Assuntos
Anticorpos/administração & dosagem , Colite/tratamento farmacológico , Modelos Animais de Doenças , Imunoglobulina G/administração & dosagem , Inflamação/tratamento farmacológico , Mucosa Intestinal/efeitos dos fármacos , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Administração Oral , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Anticorpos/farmacologia , Anticorpos Monoclonais/farmacologia , Western Blotting , Bovinos , Colite/induzido quimicamente , Colite/imunologia , Sulfato de Dextrana/toxicidade , Ensaio de Imunoadsorção Enzimática , Humanos , Técnicas Imunoenzimáticas , Imunoglobulina G/farmacologia , Inflamação/etiologia , Inflamação/patologia , Infliximab , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ácido Trinitrobenzenossulfônico/toxicidade , Fator de Necrose Tumoral alfa/imunologia
2.
Biochem Biophys Res Commun ; 427(1): 212-7, 2012 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-22995310

RESUMO

Arginine kinase catalyzes the reversible transfer of a phosphoryl group between ATP and l-arginine and is a monomeric homolog of the human enzyme creatine kinase. Arginine and creatine kinases belongs to the phosphagen kinase family of enzymes, which consists of eight known members, each of which is specific for its own phosphagen. Here, the source of phosphagen specificity in arginine kinase is investigated through the use of phosphagen analogs. Crystal structures have been determined for Limulus polyphemus arginine kinase with one of four arginine analogs bound in a transition state analog complex: l-ornithine, l-citrulline, imino-l-ornithine, and d-arginine. In all complexes, the enzyme achieves a closed conformation very similar to that of the cognate transition state analog complex, but differences are observed in the configurations of bound ligands. Arginine kinase exhibits no detectable activity towards ornithine, citrulline, or imino-l-ornithine, and only trace activity towards d-arginine. The crystal structures presented here demonstrate that phosphagen specificity is derived neither from a lock-and-key mechanism nor a modulation of induced-fit conformational changes, but potentially from subtle distortions in bound substrate configurations.


Assuntos
Difosfato de Adenosina/química , Arginina Quinase/química , Arginina/química , Citrulina/química , Caranguejos Ferradura/enzimologia , Nitratos/química , Animais , Domínio Catalítico , Cristalografia por Raios X , Estrutura Terciária de Proteína
3.
J Biol Chem ; 286(11): 9338-50, 2011 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-21212263

RESUMO

Lombricine kinase is a member of the phosphagen kinase family and a homolog of creatine and arginine kinases, enzymes responsible for buffering cellular ATP levels. Structures of lombricine kinase from the marine worm Urechis caupo were determined by x-ray crystallography. One form was crystallized as a nucleotide complex, and the other was substrate-free. The two structures are similar to each other and more similar to the substrate-free forms of homologs than to the substrate-bound forms of the other phosphagen kinases. Active site specificity loop 309-317, which is disordered in substrate-free structures of homologs and is known from the NMR of arginine kinase to be inherently dynamic, is resolved in both lombricine kinase structures, providing an improved basis for understanding the loop dynamics. Phosphagen kinases undergo a segmented closing on substrate binding, but the lombricine kinase ADP complex is in the open form more typical of substrate-free homologs. Through a comparison with prior complexes of intermediate structure, a correlation was revealed between the overall enzyme conformation and the substrate interactions of His(178). Comparative modeling provides a rationale for the more relaxed specificity of these kinases, of which the natural substrates are among the largest of the phosphagen substrates.


Assuntos
Anelídeos/enzimologia , Simulação por Computador , Modelos Moleculares , Fosfotransferases (Aceptor do Grupo Nitrogenado)/química , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Animais , Domínio Catalítico , Cristalografia por Raios X , Ressonância Magnética Nuclear Biomolecular , Fosfotransferases (Aceptor do Grupo Nitrogenado)/metabolismo , Estrutura Secundária de Proteína
5.
Protein Sci ; 13(3): 575-85, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-14978299

RESUMO

Phosphagen kinases catalyze the reversible transfer of a phosphate between ATP and guanidino substrates, a reaction that is central to cellular energy homeostasis. Members of this conserved family include creatine and arginine kinases and have similar reaction mechanisms, but they have distinct specificities for different guanidino substrates. There has not been a full structural rationalization of specificity, but two loops have been implicated repeatedly. A small domain loop is of length that complements the size of the guanidino substrate, and is located where it could mediate a lock-and-key mechanism. The second loop contacts the substrate with a valine in the methyl-substituted guanidinium of creatine, and with a glutamate in the unsubstituted arginine substrate, leading to the proposal of a discriminating hydrophobic/hydrophilic minipocket. In the present work, chimeric mutants were constructed with creatine kinase loop elements inserted into arginine kinase. Contrary to the prior rationalizations of specificity, most had measurable arginine kinase activity but no creatine kinase activity or enhanced phosphocreatine binding. Guided by structure, additional mutations were introduced in each loop, recovering arginine kinase activities as high as 15% and 64% of wild type, respectively, even though little activity would be expected in the constructs if the implicated sites had dominant roles in specificity. An atomic structure of the mismatched complex of arginine kinase with creatine and ADP indicates that specificity can also be mediated by an active site that allows substrate prealignment that is optimal for reactivity only with cognate substrates and not with close homologs that bind but do not react.


Assuntos
Arginina Quinase/química , Arginina/análogos & derivados , Creatina Quinase/química , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Arginina/química , Arginina/metabolismo , Arginina Quinase/genética , Arginina Quinase/metabolismo , Sítios de Ligação/genética , Creatina Quinase/genética , Creatina Quinase/metabolismo , Cristalografia por Raios X , Bases de Dados de Proteínas , Caranguejos Ferradura/enzimologia , Ligação de Hidrogênio , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Compostos Organofosforados/química , Compostos Organofosforados/metabolismo , Fosfocreatina/química , Fosfocreatina/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Relação Estrutura-Atividade , Especificidade por Substrato
6.
J Biol Chem ; 278(29): 26952-7, 2003 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-12732621

RESUMO

Arginine kinase is a member of the phosphagen kinase family that includes creatine kinase and likely shares a common reaction mechanism in catalyzing the buffering of cellular ATP energy levels. Abstraction of a proton from the substrate guanidinium by a catalytic base has long been thought to be an early mechanistic step. The structure of arginine kinase as a transition state analog complex (Zhou, G., Somasundaram, T., Blanc, E., Parthasarathy, G., Ellington, W. R., and Chapman, M. S. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8449-8454) showed that Glu-225 and Glu-314 were the only potential catalytic residues contacting the phosphorylated nitrogen. In the present study, these residues were changed to Asp, Gln, and Val or Ala in several single and multisite mutant enzymes. These mutations had little impact on the substrate binding constants. The effect upon activity varied with reductions in kcat between 3000-fold and less than 2-fold. The retention of significant activity in some mutants contrasts with published studies of homologues and suggests that acid-base catalysis by these residues may enhance the rate but is not absolutely essential. Crystal structures of mutant enzymes E314D at 1.9 A and E225Q at 2.8 A resolution showed that the precise alignment of substrates is subtly distorted. Thus, pre-ordering of substrates might be just as important as acid-base chemistry, electrostatics, or other potential effects in the modest impact of these residues upon catalysis.


Assuntos
Arginina Quinase/química , Arginina Quinase/metabolismo , Substituição de Aminoácidos , Animais , Arginina Quinase/genética , Catálise , Domínio Catalítico/genética , Cristalografia por Raios X , Caranguejos Ferradura/enzimologia , Caranguejos Ferradura/genética , Técnicas In Vitro , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Eletricidade Estática , Especificidade por Substrato
7.
Protein Sci ; 12(1): 103-11, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12493833

RESUMO

Arginine kinase (AK) is a member of the guanidino kinase family that plays an important role in buffering ATP concentration in cells with high and fluctuating energy demands. The AK specifically catalyzes the reversible phosphoryl transfer between ATP and arginine. We have determined the crystal structure of AK from the horseshoe crab (Limulus polyphemus) in its open (substrate-free) form. The final model has been refined at 2.35 A with a final R of 22.3% (R(free) = 23.7%). The structure of the open form is compared to the previously determined structure of the transition state analog complex in the closed form. Classically, the protein would be considered two domain, but dynamic domain (DynDom) analysis shows that most of the differences between the two structures can be considered as the motion between four rigid groups of nonsequential residues. ATP binds near a cluster of positively charged residues of a fixed dynamic domain. The other three dynamic domains close the active site with separate hinge rotations relative to the fixed domain. Several residues of key importance for the induced motion are conserved within the phosphagen kinase family, including creatine kinase. Substantial conformational changes are induced in different parts of the enzyme as intimate interactions are formed with both substrates. Thus, although induced fit occurs in a number of phosphoryl transfer enzymes, the conformational changes in phosphagen kinases appear to be more complicated than in prior examples.


Assuntos
Arginina Quinase/química , Difosfato de Adenosina/metabolismo , Animais , Arginina Quinase/genética , Arginina Quinase/metabolismo , Sítios de Ligação , Clonagem Molecular , Creatina Quinase/química , Cristalografia por Raios X , Guanidinas , Caranguejos Ferradura/enzimologia , Humanos , Modelos Moleculares , Conformação Proteica , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Especificidade por Substrato
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