Characterisation of midgut digestive proteases from the maize stem borer Busseola fusca.
BACKGROUND
Insect damage is a major constraint on maize production. Control of Busseola fusca (Fuller) in sub-Saharan Africa is relatively ineffective; the major larval digestive enzymes were characterised with a view to developing future control strategies.


RESULTS
Using BODIPY-FL Casein, maximal activity was at pH 9.5, with six protease forms visualised by gelatin-PAGE. Synthetic substrates and diagnostic inhibitors demonstrated the presence of serine proteases. Chymostatin was a potent inhibitor of general proteolysis (90%), providing strong evidence for the presence of chymotrypsin; it also caused significant inhibition (>95%) with SA(2)PFpNA as substrate. The I(50) values for chymostatin with casein and SA(2)PFpNA were 0.0075 microM and 0.06 microM respectively. Z-Phe-Arg-pNA activity was inhibited by chymostatin and TLCK (50 and 30% respectively), suggesting the presence of trypsin-like activity. BApNA hydrolysis was also strongly inhibited by chymostatin and TLCK (92 and 75%), suggesting trypsin activity, while SBBI, PMSF, pepstatin and E-64 had no significant effect. Interestingly, SBBI (I(50) = 0.39 microM) and SBTI both inhibited general proteolysis by approximately 70%, suggesting that SBBI's dual inhibitory role makes this inhibitor a potentially useful candidate for expression in maize for control of B. fusca.


CONCLUSIONS
These results provide a basis for the rational design of insect-resistant transgenic maize expressing protease inhibitors.
School of Biology, Institute for Research on Environment and Sustainability, Devonshire Building, University of Newcastle upon Tyne, Newcastle NE1 7RU, UK.
Derick
D
George
Natalie
N
Ferry
Emma J
EJ
Back
Angharad Mr
AM
Gatehouse
D000818:Animals; D041981:Gastrointestinal Tract / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007814:Larva / Q000201:enzymology; D009036:Moths / Q000201:enzymology*; D012697:Serine Endopeptidases / Q000378:metabolism*; D003313:Zea mays / Q000469:parasitology
D012697:Serine Endopeptidases