Characterization and cDNA cloning of midgut carboxypeptidases from Trichoplusia ni.
Carboxypeptidase A and carboxypeptidase B activities from the midgut of Trichoplusia ni larvae were characterized. In the T. ni larval midgut, the primary digestive carboxypeptidase activity was attributed to carboxypeptidase A, which was eight times more active than carboxypeptidase B. Both the midgut carboxypeptidase A and carboxypeptidase B exhibited maximal activities at pH 8.0-8.5 and were similarly susceptible to inhibition by potato carboxypeptidase inhibitor and phenanthroline. The midgut carboxypeptidase activities were analyzed in T. ni larvae fed on various diet sources and the results indicated that midgut carboxypeptidase activities per milligram of gut were similar regardless of the amount of dietary proteins or amino acids. However, midgut carboxypeptidase A activity was significantly higher in larvae exposed to soybean trypsin inhibitor and was significantly lower in larvae fed on broccoli foliage. From the T. ni larval midgut, five putative carboxypeptidase cDNAs were cloned, demonstrating that midgut carboxypeptidase activities are composed of multiple carboxypeptidase types. Sequence analysis indicated that the midgut carboxypeptidases were produced as secreted proenzymes which could be activated after removal of an N-terminal activation fragment by a trypsin. Two cloned cDNAs are predicted to code for carboxypeptidase A and one cDNA is predicted to code for a putative carboxypeptidase B. The other two cDNAs are highly similar to carboxypeptidase A and carboxypeptidase B in sequences, but their activity was not predictable.
Wang
Ping
P
Department of Entomology, Cornell University, New York State Agricultural Experiment Station, Geneva 14456, USA. pw15@cornell.edu
Li
Guoxun
G
Kain
Wendy
W
D000595:Amino Acid Sequence; D000818:Animals; D043424:Carboxypeptidase B / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D002268:Carboxypeptidases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D043422:Carboxypeptidases A / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D004032:Diet; D004064:Digestive System / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007814:Larva / Q000187:drug effects / Q000201:enzymology / Q000254:growth & development; D007915:Lepidoptera / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D008969:Molecular Sequence Data; D010802:Phylogeny; D011480:Protease Inhibitors / Q000494:pharmacology; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D018076:DNA, Complementary; D011480:Protease Inhibitors; D002268:Carboxypeptidases; D043422:Carboxypeptidases A; D043424:Carboxypeptidase B