BIOCHEMICAL-CHARACTERIZATION AND ULTRASTRUCTURAL-LOCALIZATION OF 2 EXTRACELLULAR TRYPSINS PRODUCED BY METARHIZIUM-ANISOPLIAE IN INFECTED INSECT CUTICLES

Proteinase 2 (Pr2) is a fungal (Metarhizium anisopliae) serine protein ase which has a tryptic specificity for basic residues and which may b e involved in entomopathogenicity, Analytical and preparative isoelect ric focusing methods were used to separate two trypsin components, pro duced during growth on cockroach cuticle, with isoelectric points of 4 .4 (molecular mass, 30 kDa) and 4.9 (27 kDa), The catalytic properties of the proteases were analyzed by their kinetic constants and by a co mbination of two-dimensional gelatin-sodium dodecyl sulfate-polyacryla mide gel electrophoresis and enzyme overlay membranes, Both Pr2 isofor ms preferentially cleave at the carboxyl sides of positively charged a mino acids, preferring arginine; the pI 4.4 Pr2 isoform also possessed significant activity against lysine, Compared with the pathogen's sub tilisin-like enzyme (Prl), the pI 4,4 Pr2 isoform shows low activity a gainst insoluble proteins in a host (Manduca sexta) cuticle, However, it degrades most cuticle proteins when they are solubilized, with high -molecular-weight basic proteins being preferentially hydrolyzed. Poly clonal antibodies raised against each Pr2 isoform were isotype specifi c, This allowed us to use ultrastructural immunocytochemistry to indep endently visualize each isoform during penetration of the host (M, sex ta) cuticle, Both isoforms were secreted by infection structures (appr essoria) on the cuticle surface and by the penetrant hyphae within the cuticle. The extracellular sheath, which is commonly observed around fungal cells, often contained Pr2 molecules, Intracellular labelling w as sparse.