Aminopeptidase-Like Activity in Hemolymph Plasma from Larvae of the Gypsy Moth, Lymantria dispar (Lepidoptera: Lymantriidae)

Aminopeptidase was isolated from the plasma fraction of hemolymph from last instar larvae of the gypsy moth Lymantria dispar. Activity was detected using the synthetic substrate L-alanine-4-nitroanilide. Total aminopeptidase activity per microliter of plasma varied with developmental stage. Activity was detected throughout the last (fifth) larval instar and increased throughout the pupal stage. The relatively large volumes of hemolymph that could be collected from day-1, fifth instar larvae made this the stage of choice for plasma preparation and enzyme purification. Hemolymph plasma was fractionated by TSK-DEAE-5PW ion exchange chromatography. Seventy percent of all aminopeptidase-like activity was detected in column fractions eluting near 125 mM NaCl. The enzyme was further purified using a combination of ion exchange and size exclusion (TSK-G2000SWXL ) methods. Purity was assessed by SDS-polyacrylamide gel electrophoresis. Native and denaturing electrophoresis results suggest that the enzyme, termed AP-125, is a hexamer of approximately 420,000 MW. The enzyme is inhibited competitively by amastatin (2 μM) and noncompetitively by leuhistin (1 μM). AP-125 is a new insect aminopeptidase, differing from those reported thus far in molecular weight, subunit composition and inhibitor response. Copyright  1997 Elsevier Science Inc. comp biochem physiol 116B;1:11–18, 1997.