5′-Nucleotidase activity has been described in bacteria, plant ce

5′-Nucleotidase activity has been described in bacteria, plant cells and in various vertebrate tissues (Zimmermann, 1992). Little information is available about ecto-5′-nucleotidase and extracellular free adenosine in the pathogenic processes of fungi. In this work, we identified some biochemical properties of C. parapsilosis ecto-5′-nucleotidase that could be involved in the release of free adenosine into extracellular

medium. The detection of cell surface-located AMP hydrolysis was confirmed and 5′-nucleotidase activity in supernatant was <20% of that found in intact cells (Fig. 1). In all conditions used during the incubation periods, the cells were viable, suggesting that the low 5′-AMP hydrolysis observed in the supernatant could be attributed to secreted enzymes. A phosphatase inhibitor, sodium orthovanadate (de Almeida-Amaral et selleck chemicals llc al., 2006; Kiffer-Moreira et Small molecule library al., 2007a; Leite et al., 2007; Amazonas et al., 2009), inhibited ectophosphatase

on the surface of C. parapsilosis; however, no inhibitory effect was seen in the ecto-5′-nucleotidase activity (Fig. 4). The optimum pH for this nucleotidase enzyme is in the acidic range, with maximal activity at a pH of 4.5 (Fig. 3b). Interestingly, this result is different from that observed in T. vaginalis strains, in which the optimum pH was in the neutral range (Tasca et al., 2003), and in mammalian ecto-5′-nucleotidase, DNA ligase in which maximal enzyme activity was obtained in the alkaline pH range of 7–8 (Zimmermann, 1992). This assay also rules out the possibility of 5′-AMP hydrolysis due to the action of ecto-ATPase because the activity of ecto-ATPase is primarily in the alkaline pH range (Kiffer-Moreira et al., 2010). Candida parapsilosis ecto-5′-nucleotidase activity is independent of divalent cations, but it can be activated by Ca2+ and Mg2+ (Fig. 3a). These same characteristics

were observed for 5′-nucleotidase activity in T. vaginalis (Tasca et al., 2003). The enzyme also showed a high sensitivity to ammonium molybdate, a classical nucleotidase inhibitor (Gottlieb & Dwyer, 1983; Borges et al., 2007), in which concentrations above 0.5 mM abolished the enzyme activity altogether (Fig. 5). Intact cells of C. parapsilosis were able to hydrolyze all substrate monophosphates, except 3′-AMP. As described in other cells, 5′-nucleotidases hydrolyze exclusively nucleoside 5′-monophosphates, showing no activity for 3′-monophosphates. 5′-AMP is commonly known as the most hydrolyzable nucleotide by 5′-nucleotidase (Zimmermann, 1992, 1996; Borges et al., 2007). Nevertheless, C. parapsilosis ecto-5′-nucleotidase activity seems to exhibit no significant difference in hydrolyzing 5′-AMP, 5′-UMP and 5′-IMP as substrates (Fig. 2).

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