title: Properties of purified gut trypsin from Helicoverpa zea, adapted to proteinase inhibitors.
abstract: Pest insects such as Helicoverpa spp. frequently feed on plants expressing protease inhibitors. Apparently, their digestive system can adapt to the presence of protease inhibitors. To study this, a trypsin enzyme was purified from the gut of insects that were raised on an inhibitor-containing diet. The amino-acid sequence of this enzyme was analysed by tandem MS, which allowed assignment of 66% of the mature protein amino acid sequence. This trypsin, called HzTrypsin-S, corresponded to a known cDNA sequence from Helicoverpa. The amino acid sequence is closely related (76% identical) to that of a trypsin, HzTrypsin-C, which was purified and identified in a similar way from insects raised on a diet without additional inhibitor. The digestive properties of HzTrypsin-S and HzTrypsin-C were compared. Both trypsins appeared to be equally efficient in degrading protein. Four typical plant inhibitors were tested in enzymatic measurements. HzTrypsin-S could not be inhibited by > 1000-fold molar excess of any of these. The same inhibitors inhibited HzTrypsin-C with apparent equilibrium dissociation constants ranging from 1 nm to 30 nm. Thus, HzTrypsin-S seems to allow the insect to overcome different defensive proteinase inhibitors in plants.
authors.affiliation: Dipartimento di Biochimica e Biologia Molecolare, Università di Bari, Italy.
authors.forename: Mariateresa
authors.initials: M
authors.lastname: Volpicella
authors.forename: Luigi R
authors.initials: LR
authors.lastname: Ceci
authors.forename: Jan
authors.initials: J
authors.lastname: Cordewener
authors.forename: Twan
authors.initials: T
authors.lastname: America
authors.forename: Raffaele
authors.initials: R
authors.lastname: Gallerani
authors.forename: Wolfram
authors.initials: W
authors.lastname: Bode
authors.forename: Maarten A
authors.initials: MA
authors.lastname: Jongsma
authors.forename: Jules
authors.initials: J
authors.lastname: Beekwilder
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity; D004032:Diet; D004064:Digestive System / Q000201:enzymology*; D019476:Insect Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D007814:Larva / Q000187:drug effects / Q000502:physiology; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009036:Moths / Q000502:physiology*; D010940:Plant Proteins / Q000494:pharmacology*; D011480:Protease Inhibitors / Q000494:pharmacology*; D017386:Sequence Homology, Amino Acid; D014357:Trypsin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D014360:Trypsin Inhibitor, Kunitz Soybean / Q000494:pharmacology; D014361:Trypsin Inhibitors / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D019476:Insect Proteins; D010940:Plant Proteins; D011480:Protease Inhibitors; D014361:Trypsin Inhibitors; D014360:Trypsin Inhibitor, Kunitz Soybean; D014357:Trypsin