title: Gypsy moth midgut proteinases: purification and characterization of luminal trypsin, elastase and the brush border membrane leucine aminopeptidase.
abstract: The principal digestive proteinases of the gypsy moth, Lymantria dispar, larval midgut were identified, and the subcellular distribution of the enzyme activities was determined. Proteinase activities of fifth-instar larvae were largely attributed to two luminal serine proteinases, a trypsin-like enzyme (TLE) and an elastase 2-like enzyme (ELA). TLE was purified to homegeneity by benzamidine-Sepharose affinity chromatography. With respect to size (M(r) = 25 kDa), substrate specificity, and interaction with trypsin inhibitors, the gypsy moth enzyme resembled mammalian pancreatic trypsin and trypsin-like enzymes from other insects. Gypsy moth elastase (ELA) was purified from the benzamidine-Sepharose flow-through by mono-Q FPLC. ELA exhibited a slightly smaller size (M(r) = 24 kDa) than TLE. The insect enzyme was inhibited by DFP and chymostatin but was unaffected by TPCK. ELA exhibited little esterolytic activity with BTEE. Succinyl-Ala-Ala-Pro-Leu p-nitroanilide was one of the best substrates for ELA, which is characteristic of elastase 2. TLE and ELA constituted c. 6% of the total soluble protein in midgut lumen of actively feeding fifth-instar larvae. Chymotrypsin and carboxypeptidase activities were not detected in any midgut fraction examined. The brush border membrane (BBM) leucine aminopeptidase (LAP) was isolated from CHAPS-solubilized BBM by FPLC. SDS-PAGE results indicated that the aminopeptidase has an apparent molecular size of c. 100 kDa. The aminopeptidase was inhibited by bestatin and was unaffected by serine proteinase inhibitors.
authors.affiliation: United States Department of Agriculture, Forest Service, Delaware, Ohio 43015.
authors.forename: A P
authors.initials: AP
authors.lastname: Valaitis
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004064:Digestive System / Q000166:cytology / Q000201:enzymology*; D007931:Leucyl Aminopeptidase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D008871:Microvilli / Q000201:enzymology; D008969:Molecular Sequence Data; D009036:Moths / Q000201:enzymology*; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D014357:Trypsin / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D007931:Leucyl Aminopeptidase; D010196:Pancreatic Elastase; D014357:Trypsin