Gypsy moth midgut proteinases: purification and characterization of luminal trypsin, elastase and the brush border membrane leucine aminopeptidase

Insect Biochem Mol Biol. 1995 Jan;25(1):139-49. doi: 10.1016/0965-1748(94)00033-e.

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.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Digestive System / cytology
  • Digestive System / enzymology*
  • Leucyl Aminopeptidase / antagonists & inhibitors
  • Leucyl Aminopeptidase / isolation & purification*
  • Leucyl Aminopeptidase / metabolism
  • Microvilli / enzymology
  • Molecular Sequence Data
  • Moths / enzymology*
  • Pancreatic Elastase / antagonists & inhibitors
  • Pancreatic Elastase / isolation & purification
  • Pancreatic Elastase / metabolism*
  • Substrate Specificity
  • Trypsin / isolation & purification
  • Trypsin / metabolism*

Substances

  • Leucyl Aminopeptidase
  • Pancreatic Elastase
  • Trypsin