Purification and Characterization of Proteolytic Enzymes of Leishmania mexicana mexicana Amastigotes and Promastigotes

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Abstract
L. m. mexicana amastigote and promastigote soluble proteinases were purified using gel filtration and ion exchange chromatography. For the amastigotes, 2 main proteinase activity peaks were separated with both methods. These accounted for .apprx. 10% and 90% of the total activity. Characterization of 2 activities for substrate specificity and sensitivity to inhibitors indicated that the major peak from both column methods contained enzymes with the characteristics of cysteine proteinases. SDS [sodium dodecylsulfate]-polyacrylamide gel electrophoresis of the enzyme from the major peak purified by gel filtration revealed 1 polypeptide with a MW in the region of 31,000. The activity of the minor peak eluted from the columns was of higher MW (67,000) and was similar to metalloproteinases. Purification of the soluble proteinases in the promastigote of L. m. mexicana produced only 1 activity peak from both column techniques. This activity (MW 67,000) corresponded to the high MW proteinase of the amastigote. The purified proteinases were active on 4-nitroanilide and 7-amino-4-methylcoumarin derivates of various small peptides. The high MW proteinases of amastigotes and promastigotes were similarly active against most of the peptides, suggesting a low specificity of the enzymes. The low MW amastigote proteinases were particularly active against 2 of the substrates, namely BZ-Pro-Phe-Arg-Nan and Z-Phe-Arg-MCA. A highly active, substrate-specific, soluble proteinase, with characteristics of a cysteine proteinase, is produced upon transformation of the L. m. mexicana promastigote to amastigote. The discovery and characterization of this enzyme offers opportunities for the development of new antileishmanial agents.