The role of leucine aminopeptidase in the moulting of nematode parasites

Abstract

1. 1. A leucine aminopeptidase in the exsheathing fluid of infective juveniles of Haemonchus contortus may be regarded, on the following evidence, as the enzyme which attacks the sheath and so completes the second moult: (a) the enzyme was secreted by juveniles which had been stimulated to exsheathe; (b) the action of the enzyme of the sheath and on other substrates required Mg 2+ or Mn 2+ as a cofactor; (c) diaminoethanetetra-acetic acid was an effective inhibitor of the enzyme and di-isopropylfluorophosphate had no effect; (d) fractionation of the exsheathing fluid by electrophoresis on acrylamide columns showed that maximum activity of leucine aminopeptidase, obtained from less than 1 per cent of the total protein subjected to electrophoresis, was associated with maximum biological activity; fractions containing no leucine aminopeptidase did not attack isolated sheaths. 2. 2. The enzymes in exsheathing fluids of H. contortus and Trichostrongylus colubriformis were true leucine aminopeptidases. With Mg 2+ or Mn 2+ as a co-factor they hydrolysed l-leucinamide (and l-leucyl-β-naphthylamide). They were strongly inhibited by diaminoethanetetra-acetic acid, but not by N-methylmaleimide, p-chloromercuribenzoate or di-isopropylfluorophosphate. The optimum pH was about 9·5. In the absence of Mg 2+ or Mn 2+ and below pH 7, the enzymes were highly unstable; concentration by freeze-drying and ultrafiltration at 5°C led to considerable losses in activity. 3. 3. The leucine aminopeptidases in the exsheathing fluids of H. contortus and T. colubriformis differed from each other and from mammalian leucine aminopeptidase in the range of substrates they attacked. Thus the enzyme from H. contortus attacked isolated sheaths of its own species but it did not attack isolated sheaths from T. colubriformis, and vice versa. The enzyme from pigs' kidneys did not attack isolated sheaths from H. contortus and had only a slight effect on isolated sheaths of T. colubriformis. The three enzymes also differed in their relative activity against l-leucinamide and l-leucyl-β-naphthylamide; the enzymes in exsheathing fluid were relatively more active against l-leucyl-β-naphthylamide.