STUDY OF PROTEOLYTIC ACTIVITIES RELEASED BY INCUBATION OF TRYPANOSOMES (TRYPANOSOMA-BRUCEI-BRUCEI) IN PH5.5 AND PH7.0 PHOSPHATE GLUCOSE BUFFERS

Proteolytic activities released by overnight incubation of Antwerpeen Trypanozoon antigenic type (AnTat) 1.1 trypanosomes at 4°C in pH 5.5 and pH 7.0 phosphate/glucose buffers were analyzed in the supernatants obtained after centrifugation of the parasite suspensions. The assays used the fluorogenic substrates N-α-benzyloxycarbonyl-L-phenylalanyl-L- arginine-7amido-4-methylcoumarin (Z-Phe-Arg-NMec) and N-α-benzyloxycarbonyl-L-arginyl-L-arginine-7-amido-4-methylcoumarin (Z-Arg-Arg-NMec) at two different pHs (6.0 and 8.3). Z-Phe-Arg-NMec hydrolysis was inhibited by 2 μM L-trans-mepoxysuccinyllencylamido(4-guanidino)butane (E-64) to a greater extent in the pH 7.0 supernatant than in the pH 5.5 supernatant. Z-Arg-Arg-NMec hydrolysis by the two supernatants was not significantly inhibited by 2 μM E-664. At pH 8.3 this activity was increased more than 2-fold by the addition of dithiothreitol. The hydrolysis activities were analyzed in collected eluates after fractionation of the supernatants by gel permeation high-performance liquid chromatography. Z-Phe-Arg-NMec hydrolytic activity inhibited by 2 μM E-64 was maximal at a retention time of 33 min (approx. Mr 30 000). In addition, a hydrolytic activity against the substrates Z-Phe-Arg-NMec and Z-Arg-Arg-NMec gave a peak showing a maximum at a retention time of 29 min (approx. Mr 70 000). © 1990.