Permeabilization of Trypanosoma cruzi epimastigotes. A highly efficient method to study soluble and particulate enzymes in situ

Abstract

A simple permeabilization procedure has been developed which allows the reliable determination of enzyme activities in situ in epimastigotes of Trypanosoma cruzi. Permeabilization was obtained by treatment with toluene-ethanol during 1 min at room temperature. The efficiency this procedure could be confirmed by the study by glucose 6-phosphate dehydrogenase (G6-PDH) and hexokinase (HK) activities in situ compared to the same data in cell-free extracts. Buffer composition, concentration and parasite concentrations were modified to improve efficiency of the method. Potassium-phosphate buffer was found to be the most effective, particularly at 25 mM. Permeabilization was not affected by buffer pH and did not depend on the concentration of the cell suspension during permeabilization over the range of 50–100 mg wet cells · ml −1. The kinetic properties of glucose 6-phosphate dehydrogenase and hexokinase (a soluble and particulate enzymes, respectively) examined in the permeabilized cells were essentially the same as in cell-free extracts.