Study of proteolytic activities released by incubation of trypanosomes (Trypanosoma brucei brucei) in pH 5.5 and pH 7.0 phosphate/glucose buffers.
Proteolytic activities released by overnight incubation of Antwerpeen Trypanozoon antigenic type (AnTat) 1.1 trypanosomes at 4 degrees 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-alpha-benzyloxycarbonyl-L-phenyl-alanyl-L-arginine-7-amido-4- methylcoumarin (Z-Phe-Arg-NMec) and N-alpha-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 microM L-trans-epoxysuccinyllencylamido(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 microM E-64. 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 microM 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).
Unité Inserm N. 16, Lille, France.
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Boutignon
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Huet-Duvillier
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Demeyer
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Richet
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Degand
D000818:Animals; D002021:Buffers; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004151:Dipeptides / Q000378:metabolism; D010450:Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013379:Substrate Specificity; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
D002021:Buffers; D004151:Dipeptides; D005456:Fluorescent Dyes; D010450:Endopeptidases