Characterization of Helicoverpa armigera gut proteinases and their interaction with proteinase inhibitors using gel X-ray film contact print technique.
Since Helicoverpa armigera is a devastating pest, an attempt was made to separate its gut proteinases and assess their diversity. Gelatin coating present on the X-ray film was used as a substrate to detect electrophoretically separated proteinases of H. armigera gut extract on native polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate (SDS)-PAGE and isoelectric focusing gels. The method involves electrophoresis, followed by washing the gel with Triton X-100 in case of SDS-PAGE, equilibration of the gel in proteinase assay buffers, overlaying the gel on X-ray film followed by washing the film with hot water to remove hydrolyzed gelatin revealing bands of proteinase activity. Using this protocol, at least six different proteinase isoforms were detected in H. armigera gut contents while three isoproteinases were identified in a commercial bacterial proteinase preparation. Adoption of the technique facilitated characterization of the H. armigera gut proteinases (HGP) and provided an easy tool to study the properties of the individual proteinases without purification. The approximate molecular masses of HGP as determined by SDS-PAGE were: 172.9, 59.3, 54.9, 47.6, 44.1 and 41.6 kDa, and of bacterial proteinases: 180.7, 127.3 and 95.3 kDa. The isoelectric point (pI) values of HGP and bacterial proteinase were in the range of 5.1-7.1 and 3.5-7.7, respectively. Some of the HGP isoforms were found to be highly pH-sensitive and showed activity only at pH 10.0. The major HGPs were inhibited by phenylmethylsulfonyl fluoride but not by (4-amidinophenyl)-methanesulfonyl fluoride. Incubation of HGP-resolved electrophoretic gel strips in chickpea or winged bean proteinase inhibitor solution permitted identification of specific inhibitors of individual proteinases and revealed that the major HGPs were insensitive to chickpea inhibitors whereas winged bean inhibitors effectively inhibited all the HGPs. Our results suggest that considerable variability exists among the isoproteinases of H. armigera gut with respect to their pH optima and sensitivity towards chemical and plant proteinase inhibitors. Such diversity is of immense biological significance as it explains the polyphagous nature of the insect which imparts unique adaptability to it against the defensive proteinase inhibitors of its wide range of host plants.
Harsulkar
A M
AM
Plant Molecular Biology Unit, Division of Biochemical Sciences, National Chemical Laboratory, Pune, India.
Giri
A P
AP
Gupta
V S
VS
Sainani
M N
MN
Deshpande
V V
VV
Patankar
A G
AG
Ranjekar
P K
PK
D000180:Acrylic Resins; D000818:Animals; D004064:Digestive System / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D010450:Endopeptidases / Q000378:metabolism*; D019476:Insect Proteins / Q000378:metabolism*; D007525:Isoelectric Focusing; D009036:Moths / Q000201:enzymology; D010664:Phenylmethylsulfonyl Fluoride / Q000031:analogs & derivatives* / Q000378:metabolism*; D011480:Protease Inhibitors / Q000378:metabolism*; D012967:Sodium Dodecyl Sulfate; D014962:X-Ray Film
D000180:Acrylic Resins; D019476:Insect Proteins; D011480:Protease Inhibitors; C016680:polyacrylamide gels; D012967:Sodium Dodecyl Sulfate; D010664:Phenylmethylsulfonyl Fluoride; C027226:(4-amidinophenyl)methanesulfonyl fluoride; D010450:Endopeptidases