Midgut carboxypeptidase from Helicoverpa armigera (Lepidoptera: Noctuidae) larvae: enzyme characterisation, cDNA cloning and expression.
Using synthetic substrates we have characterised carboxypeptidase activity in gut extracts from Helicoverpa armigera larvae. Carboxypeptidase A activity predominates, with only low levels of carboxypeptidase B activity present. Maximum carboxypeptidase A activity occurs over a broad pH range and is inhibited by phenanthroline and potato carboxypeptidase inhibitor. A cDNA clone encoding carboxypeptidase (the first such sequence from a lepidopteran insect) was isolated from a larval gut library. The sequence predicts a secreted polypeptide of Mr 46.6 k with homology to metallocarboxypeptidases from mammalian and invertebrate species. The presence of a serine residue at the active site suggests carboxypeptidase A activity. To further characterise the gene product, the complete cDNA sequence was expressed in insect cells using the baculovirus system. Culture supernatant from these cells contained carboxypeptidase A activity, with no activity against a carboxypeptidase B substrate; the carboxypeptidase B activity in gut extracts must thus be due to a separate enzyme. In agreement with this conclusion, the expressed carboxypeptidase cDNA is a member of a small multigene family. Chronic ingestion of soybean Kunitz trypsin inhibitor by H. armigera larvae results in increased accumulation of carboxypeptidase mRNA in the midgut cells, and an increase in carboxypeptidase A activity detected in gut extract.
Bown
D P
DP
Department of Biological Sciences, University of Durham, UK. d.p.bown@dur.ac.uk
Wilkinson
H S
HS
Gatehouse
J A
JA
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002268:Carboxypeptidases / Q000096:biosynthesis / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004064:Digestive System / Q000187:drug effects / Q000201:enzymology*; D015870:Gene Expression; D007814:Larva / Q000201:enzymology / Q000235:genetics; D007915:Lepidoptera / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera / Q000166:cytology; D014360:Trypsin Inhibitor, Kunitz Soybean / Q000494:pharmacology
D018076:DNA, Complementary; D011994:Recombinant Proteins; D014360:Trypsin Inhibitor, Kunitz Soybean; D002268:Carboxypeptidases