Molecular analysis of two acidic proteinases pumAe and pumAi and aminopeptidase pumAPE from Ustilago maydis: enzymes purification and differential expression

Date

2006

Authors

Noriega Reyes, M.Y.
Miramón Martínez, P.
Mercado Flores, Y.
Ramírez Zavala, B.
Hernández Rodríguez, C.
Villa Tanaca, L.

Director

Publisher

Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Acceso abierto / Sarbide irekia
Contribución a congreso / Biltzarrerako ekarpena
Versión publicada / Argitaratu den bertsioa

Project identifier

Abstract

Proteolytic system of Ustilago maydis was recently partially described (Mercado-Flores et al., 2003). Two acidic proteinases pumAe (extracellular) and pumAi (intracellular) and aminopeptidase pumAPE were detected and purified from the haploid phase of U. maydis. Purification consisted of ammonium sulphate fractionation and different chromatographic steps. Molecular masses were estimated: 58 kDa for pumAPE, 72 kDa for pumAe and 35.3 kDa for pumAi. Enzymatic activity was optimal at pH 7.0 and 35 ºC for pumAPE and 4.0 for the two proteinases. pumAPE was inhibited by EDTA-Na2, 1,10-phenanthroline, bestantin, PMSF and several divalent cations, while proteinase pumAi was inhibited by pepstatine A, also finding that yeast-to-mycelium transition was inhibited by Pepstatine A in the culture medium. Primers were designed in order to amplify the gene APEum encoding pumAPE and PRAum gene encoding pumAi, and they were used as probes in a Southern blot. One copy of each gene was detected by genome in several strains. Differential expression of APEum was assessed under different physiological conditions, detecting high expression levels on media supplemented with corn infusion, proline, peptone and ammonium sulphate. PRAum is expressed when cells are exposed to corn infusion and ammonium sulphate.

Description

Comunicación presentada 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.

Keywords

Ustilago maydis, Molecular analysis

Department

Faculty/School

Degree

Doctorate program

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