Gypsy moth midgut proteinases: purification and characterization of luminal trypsin, elastase and the brush border membrane leucine aminopeptidase.
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.
Valaitis
A P
AP
United States Department of Agriculture, Forest Service, Delaware, Ohio 43015.
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*
D007931:Leucyl Aminopeptidase; D010196:Pancreatic Elastase; D014357:Trypsin