Heterogeneity of cysteine proteinases in Leishmania braziliensis and Leishmania major

ABSTRACT

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with acrylamide copolymerized with gelatin (substrate-SDS-PAGE) was used to compare promastigote proteinases from two Leishmania species (L. braziliensis and L. major-like). Substrate-SDS-PAGE resolved at least 6 distinct proteinase activities with relative molecular masses between 20 and 65 kDa. The profile of proteinase activity was the same for both species, although qualitative differences were observed. L. major-like expressed a low molecular weight (20 kDa) proteinase with maximum activity at pH 7.0, which was demonstrable from pH 5.0 to pH 8.0. The 20-kDa protease was recovered in the detergent phase of TX-114 and was inhibited by the sulfhydryl group-blocking reagent E-64 (2.5 mM) and the non-specific inhibitor iodoacetic acid (1 mM). Pepstatin (1 microM) and PMSF (2.5 mM) did not inhibit the 20-kDa enzyme. The present study suggests that both Leishmania species studied express hydrophobic cysteine proteases of different molecular weights, since about 2 x 10(7) parasites were analyzed in each lane and the proteolytic activity developed at 37 degrees C for 16 h