Purification and characterization of hexokinase from Leishmania mexicana

Miguel A. Pabón, Ana J. Cáceres, Melisa Gualdrón, Wilfredo Quiñones, Luisana Avilán, Juan L. Concepción

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Abstract

Hexokinase from Leishmania mexicana was purified to homogeneity from a glycosome-enriched fraction obtained after a differential centrifugation of promastigote form. The kinetic properties of the pure enzyme were determined and the Km values for glucose (Km = 66 microM) and ATP (Km = 303 muM) were comparable to those from hexokinase of Trypanosoma cruzi. L. mexicana hexokinase was able to use fructose (Km = 142 microM), which reflects the condition found in the insect host. In contrast with hexokinases from other trypanosomatids, the enzyme exhibited a moderate sensitivity to inhibition by glucose 6-phosphate. This inhibition was competitive with respect to both ATP and glucose, indicating that an allosteric site for glucose 6-phosphate does not exist in this enzyme. The enzyme was also inhibited by inorganic pyrophosphate, the inhibition being higher than that observed for T. cruzi enzyme. As expected, the enzyme was localized, by immunofluorescence analysis, in glycosomes and is present in both promastigotes and true amastigotes obtained from hamster lesion. Hexokinase specific activity increased with the aging of promastigote culture, and this increment was related to glucose consumption. However, the level of the hexokinase protein remains constant as determined by Western blotting. Several hypotheses are discussed to explain this result.

Original languageEnglish
Pages (from-to)803-810
Number of pages8
JournalParasitology Research
Volume100
Issue number4
Early online date24 Oct 2006
DOIs
Publication statusPublished - Mar 2007

Keywords

  • Animals
  • Diphosphates/metabolism
  • Hexokinase/antagonists & inhibitors
  • Leishmania mexicana/enzymology

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