Molecular analysis of aminopeptidase pumAPE from Ustilago maydis encoded by APEum gene: enzyme purification and differential expression
Fecha
2006Autor
Versión
Acceso abierto / Sarbide irekia
Tipo
Contribución a congreso / Biltzarrerako ekarpena
Versión
Versión publicada / Argitaratu den bertsioa
Impacto
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nodoi-noplumx
|
Resumen
Heterobasidiomycete Ustilago maydis is a dimorphic phytopathogenic fungus,
causal agent of corn smut, a widespread disease. Recently, proteolytic
system of this fungus was described and an aminopeptidase activity, probably
involved in pathogenicity, was detected. The aminopeptidase pumAPE was
purified from the haploid phase of U. maydis FB1 strain. The purification
procedure consisted of ammonium ...
[++]
Heterobasidiomycete Ustilago maydis is a dimorphic phytopathogenic fungus,
causal agent of corn smut, a widespread disease. Recently, proteolytic
system of this fungus was described and an aminopeptidase activity, probably
involved in pathogenicity, was detected. The aminopeptidase pumAPE was
purified from the haploid phase of U. maydis FB1 strain. The purification
procedure consisted of ammonium sulphate fractionation and three chromatographic
steps, resulting in a 23% recovery. The molecular mass of the
dimeric enzyme was estimated to be 110 kDa and 58 kDa by gel filtration
chromatography and SDS-PAGE respectively. Enzymatic activity was optimal
at pH 7.0 and 35 ºC toward Lys-pNA and the pI was determined to be
5.1. The enzyme was inhibited by EDTA-Na2, 1,10-phenanthroline,
bestantin, PMSF and several divalent cations (Cu2+, Hg2+ and Zn2+). The
aminopeptidase exhibited a higher specificity for substrates with lysine and
arginine in the N-position. The Km value was 54.4 μM and the Vmax value
was 408 μmol min-1 mg-1 for Lys-pNA. A pair of primers was designed in order
to amplify the gene APEum encoding this activity. In order to determine
the number of copies in the genome, a APEum gene fragment was used as
probe in a Southern blot. Only one copy of the gene by genome was detected.
Also, differential expression of APEum was assessed under different physiological
conditions. In brief, high expression levels were detected on media
supplemented with corn infusion, proline, and ammonium. [--]
Materias
Ustilago maydis,
pumAPE,
APEum gene
Editor
Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Publicado en
Antonio G. Pisabarro and Lucía Ramírez (eds.): VI Meeting on Genetics and Cellular Biology of Basidiomycetes (GCBB-VI). Pamplona: Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006.
Notas
Resumen del poster presentado al VI Meeting on Genetics and Cellular Biology of Basidiomycetes (GCBB-VI), organizado por y celebrado en la Universidad Pública de Navarra el 3-6 de junio de 2005.