Purification and characterization of aminopeptidase (pumAPE) from Ustilago maydis

Abstract

The aminopeptidase pumAPE was purified from the haploid fungus Ustilago maydis FB1 strain. The purification procedure consisted of ammonium sulfate fractionation and three chromatographic steps, which included anion-exchange, hydrophobic interaction, and gel filtration chromatography, 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 at 35 degrees C 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 showed a preference for lysine and arginine in the N-position. The K(m) value was 54.4 microM and the Vmax value was 408 micromolmin(-1)mg(-1) for Lys-pNA.

The intracellular proteolytic system of Yarrowia lipolytica and characterization of an aminopeptidase

“…This inhibitory effect has been observed in other aminopeptidases isolated from yeast, such as AP‐Y from Saccharomyces cerevisiae (Yasuhara et al , 1994), aminopeptidase I from Schizosaccharomyces pombe (Arbesú et al , 1993) and lysine aminopeptidase from K. marxianus (Ramírez‐Zavala et al , 2004). Bestatin, an inhibitor of metalloenzymes, had a strong effect on the aminopeptidase from Y. lipolytica , similar to that observed on aminopeptidases from Saccharomyces cerevisiae (Yasuhara et al , 1994), Schizosaccharomyces pombe (Arbesú et al , 1993), K. marxianus (Ramírez‐Zavala et al , 2004) and Ustilago maydis (Mercado‐Flores et al , 2004). The presence of blocking agents from serine and cysteine proteases, such as pefabloc, PMSF, leupeptin and E‐64, inhibited the activity, suggesting that serine residues and thiol groups might be participating in the catalysis of this enzyme.…”

“…The aminopeptidase has a preference for substrates with N‐position basic amino acids, such as lysine. This substrate preference suggests that yylAPE is a lysine aminopeptidase similar to aminopeptidases from Aspergillus niger (Basten et al , 2001), Schizosaccharomyces pombe (Arbesú, et al , 1993), K. marxianus (Ramírez‐Zavala et al , 2004) and U. maydis (Mercado‐Flores et al , 2004).…”

“…Protein determinations were performed according to the Bradford (1976) method. The relative activities of yylAPE against several aminoacyl-4-nitroanilide substrates were determined according to Mercado-Flores et al (2004).…”

Water Stress in Biological, Chemical, Pharmaceutical and Food Systems

“…The colorimetric p-nitroanilide substrate (Bachem, Bubendorf, Switzerland) alanine-proline-p-nitroanilide (Ala-Pro-pNA) at 10 mM was selected for analyzing Xaa-Pro-DAP activity during 15 days of germination process (Mercado- Flores et al 2004;Sánchez-Mundo et al 2010). One unit of enzyme (U ) was defined as the amount of enzyme that liberates 1 μmol of p-nitroaniline per minute at 37 C.…”

Recombinant methionine aminopeptidase protein of Babesia microti: immunobiochemical characterization as a vaccine candidate against human babesiosis

“…Methionine aminopeptidases (MetAPs), a family of aminopeptidases, play an important role in the N-terminal excision of methionine from polypeptides during protein synthesis, and they have been identified in numerous microorganisms, plants, vertebrates, and invertebrates (Lowther and Matthews 2000;Addlagatta et al 2005). In addition to their involvement in N-terminal methionine excision, MetAPs are involved in the general metabolism of amino acids and proteins, the activation and inactivation of biologically active peptides, and antigen processing to be presented to the major histocompatibility system (Mercado-Flores et al 2004).…”