Biochemical and biophysical properties of a highly active recombinant arginase from Leishmania (Leishmania) amazonensis and subcellular localization of native enzyme

Biochemical and biophysical properties of a highly active recombinant arginase from Leishmania (Leishmania) amazonensis and subcellular localization of native enzyme

Autor Silva, Edson Roberto da Google Scholar
Laranjeira da Silva, Maria Fernanda Google Scholar
Fischer, Hannes Google Scholar
Mortara, Renato A. Autor UNIFESP Google Scholar
Mayer, Mario Gustavo Google Scholar
Framesqui, Karine Google Scholar
Silber, Ariel Mariano Google Scholar
Floeter-Winter, Lucile Maria Google Scholar
Instituição Universidade de São Paulo (USP)
Univ Luterana Brasil
Universidade Federal de São Paulo (UNIFESP)
Inst Butantan Serv Genet
Resumo Arginase (L-arginine amidinohydrolase, E.C. 3.5.3.1) is a metalloenzyme that catalyses the hydrolysis of L-arginine to L-ornithine and urea. in Leishmania spp., the biological role of the enzyme may be involved in modulating NO production upon macrophage infection. Previously, we cloned and characterized the arginase gene from Leishmania (Leishmania) amazonensis. in the present work, we successfully expressed the recombinant enzyme in E. coli and performed biochemical and biophysical characterization of both the native and recombinant enzymes. We obtained K-M and V-max. values of 23.9(+/- 0.96) mM and 192.3 mu mol/min mg protein (+/- 14.3), respectively, for the native enzyme. for the recombinant counterpart, K-M was 21.5(+/- 0.90) mM and V-max was 144.9(+/- 8.9) mu mol/min mg. Antibody against the recombinant protein confirmed a glycosomal cellular localization of the enzyme in promastigotes. Data from light scattering and small angle X-ray scattering showed that a trimeric state is the active form of the protein. We determined empirically that a manganese wash at room temperature is the best condition to purify active enzyme. the interaction of the recombinant protein with the immobilized nickel also allowed us to confirm the structural disposition of histidine at positions 3 and 324. the determined structural parameters provide substantial data to facilitate the search for selective inhibitors of parasitic sources of arginase, which could subsequently point to a candidate for leishmaniasis therapy. (c) 2008 Elsevier B.V. All rights reserved.
Palavra-chave protein structure
manganese activation
immunolocalization
drug design
Idioma Inglês
Data de publicação 2008-06-01
Publicado em Molecular and Biochemical Parasitology. Amsterdam: Elsevier B.V., v. 159, n. 2, p. 104-111, 2008.
ISSN 0166-6851 (Sherpa/Romeo, fator de impacto)
Publicador Elsevier B.V.
Extensão 104-111
Fonte http://dx.doi.org/10.1016/j.molbiopara.2008.02.011
Direito de acesso Acesso restrito
Tipo Artigo
Web of Science WOS:000256198000004
Endereço permanente http://repositorio.unifesp.br/handle/11600/30704

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