AN AMINOPEPTIDASE FROM THE MOLTING FLUID OF THE TOBACCO HORNWORM, MANDUCA-SEXTA

A neutral metalloprotease (moulting fluid protease 2; MFP-2) from the moulting fluid of Manduca sexta (Lepidoptera:Sphingidae) pharate adults has been purified and partially characterized. The enzyme has a native molecular mass of 240 kDa as determined by HPLC gel filtration. SDS-PAGE of MFP-2 gave a single band of molecular mass 39.4 kDa indicating that the native enzyme probably exists as a hexamer. MFP-2 degrades a broad range of synthetic amino acid-beta-naphthylamide substrates with a preference for methionine-, leucine- or alanine beta-naphthylamides. Activity is inhibited by amastatin, 1,10-phenanthroline and, to a lesser extent, ethylenediaminetetraacetic acid (EDTA). The inhibitory effects of divalent metal ion chelation are most effectively overcome by the addition of cobalt ions. MFP-2 alone has low cuticle degrading activity but acts with another moulting fluid enzyme, MFP-1, to degrade Manduca pupal cuticle.