Detection of an alkaline peptidase in Leishmania amastigotes and promastigotes.

We have previously characterized a peptidase in Tiypanosomu crirzi that cleaves peptide substrates on the carboxyl side of arginine and lysinc residues at alkaline pH [ I ] . This alkaline peptidase occurs in all stages of the life cycle of T. criizi, and evidence was presented that a similar or identical enzyme was expressed by 15 other species of trypanosomatid, but not by any non-trypanosomatid protozoa or mammalian cells that we tested. The alkaline peptidase of T. criizi is the major enzyme that cleaves Bz-Arg-pNA at pH 8.0. Extracts of six species of Leishmania were found to contain a major alkaline peptidase activity that also cleaves Bz-ArgpNA readily at pH 8.0 [ I ] . This enzyme activity had the same inhibitor and substrate specificity as the 7: cnizi alkaline peptidase in a preliminary investigation. We have now examined the Leishmariiu donovani peptidase that cleaves Bz-Arg-pNA in greater detail. and our findings corroborate our previous data, suggesting that L. donovani does indeed contain an alkaline peptidase very similar t o the T. c r w i enzyme. Detergent extracts of L. donovuni promastigotes were compared with those of amastigotes (prepared from hamster spleens [ 2 ] ) with respect to their ability to cleave a number of peptidyl-p-nitroanilides at pH 8.0, the pH optimum for cleavage of Bz-Arg-pNA. Extracts of both stages of the parasite’s life cycle showed a preference for cleavage of TosGly-Pro-Arg-pNA over Tos-Gly-Pro-Lys-pNA and both generally cleaved longer substrates better than short ones (Table 1 ). Whereas Bz-Arg-pNA was readily cleaved, removal of the N-terminal blocking group, as in Arg-pNA, resulted in considerable reduction in cleavability, although extracts were readily ablc t o cleave Gly-Arg-pNA. The relative rates o f cleavage of different substrates were similar for amastigotes and promastigotes, suggesting that a similar enzyme or enzymes are responsible for hydrolysis of the substrates. The inhibitor profiles for the amastigote and promastigote Bz-Arg-pNA hydrolases were also similar to each other and to the purified T. criizi and C’rithidia jusciciilutu alkaline peptidases (I I I; F. Ashall, D. Harris, H. Roberts. N. Healy & E. Shaw, unpublished work). Thus, the L. doriovarii activity was inhibited by diisopropylfluorophosphate (DFP), TosLys-CH2CI and leupeptin, but was unaffected by phenylmethanesulphonyl fluoride, E64, iodoacetic acid, pepstatin A or 0-phenanthroline. In agreement with a preference for Arg or Lys at PI of substrates, Cbz-Leu-Lys-diazomethane, but not Cbz-Leu-Met-diazomethane, strongly inhibited the enzyme. Since diazomethanes are generally considered to be diagnostic o f cysteine peptidases [ 31 and DFP of serine peptidases 141, the class of peptidase t o which the enzyme belongs remains to be unequivocally determined. The enzyme may be a serine peptidase sensitive to diazomethanes or it may be a cysteine peptidase sensitive to DFP (however, E64 and iodoacetic acid do not inhibit); or it may be a novel class of peptidase.