MIDGUT CARBOXYPEPTIDASE FROM HELICOVERPA-ARMIGERA (LEPIDOPTERA, NOCTUIDAE) LARVAE - ENZYME CHARACTERIZATION, CDNA CLONING AND EXPRESSION

Using synthetic substrates we have characterised carboxypeptidase acti vity in gut extracts from Helicoverpa armigera larvae. Carboxypeptidas e A activity predominates, with only low levels of carboxypeptidase B activity present. Maximum carboxypeptidase A activity occurs over a br oad pH range and is inhibited by phenanthroline and potato carboxypept idase inhibitor. A cDNA clone encoding carboxypeptidase (the first suc h sequence from a lepidopteran insect) was isolated from a larval gut library. The sequence predicts a secreted polypeptide of Mr 46.6 k wit h 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 b aculovirus system. Culture supernatant from these cells contained carb oxypeptidase 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 ex pressed 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 t he midgut cells, and an increase in carboxypeptidase A activity detect ed in gut extract. (C) 1998 Elsevier Science Ltd. All rights reserved.