No 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.

mexicana is one of the causative agents of cutaneous leishmaniasis on the American continent. L. mexicana has been reported to interact in vitro with plasminogen. This interaction contributes to the virulence of the parasite as may be concluded from differences observed in lesion size and in the distribution pattern in the lesion between plasminogen deficient and wild type mice. In this work we looked for the molecules responsible for plasminogen binding on the surface of the parasite. Using ligand blotting we found that several molecules, with the capacity to bind plasminogen, are found in the microsomal fraction of the parasite. One of these molecules had the same molecular mass of enolase. To test binding of plasminogen to enolase, the purified recombinant enzyme was immobilized on microtitre plates. Plasminogen at different concentrations was incubated and the binding revealed with anti-plasminogen antibodies. A dose-dependent and saturable binding of plasminogen to enolase was observed. The concentration of plasminogen necessary to achieve 50% saturation (EC50) was found to be around 0.3 muM. The binding of plasminogen to control plates coated with BSA was minimal. Binding of plasminogen to immobilized enolase was inhibited up to 80% by the lysine analogue epsilon-aminocaproic acid indicating that the lysine-binding sites of plasminogen are probably involved in this interaction. Immunofluorescence studies with permeabilized and non-permeabilized parasites (promastigotes and amastigote forms) showed that enolase is found at the external face of the plasma membrane. Moreover, plasminogen binding was tested in living parasites in the presence of anti-enolase antibodies. Up to 50% inhibition of plasminogen binding was observed. Thus, enolase could function, besides in metabolism, as plasminogen receptor at the surface of the parasite.