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

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.