Purification of glycosylphosphatidylinositol-anchoring aminopeptidase in from the plasma membrane of larval midgut epithelial cells of the silkworm, Bombyx mori

Abstract

1. 1. Aminopeptidase N was selectively released from larval midgut of silkworm, Bombyx mori, by phosphatidylinositol-specific phospholipase C, and purified to a homogeneous state by ion exchange, gel filtration. Con A-Sepharose and 4-aminobenzyl phosphonic acid-agarose column chromatographies. 2. 2. The purified aminopeptidase N preparation showed 190.8 U/mg of specific activity. Its molecular weight was estimated to be around 100 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 3. 3. Purified aminopeptidase N molecule preferentially hydrolyzed Leu-, Ala- and Met- p-nitroanilide as substrates. Especially, Leu- p-nitroanilide proved to be the best substrate for aminopeptidase N from larval midgut of silkworm. 4. 4. By treatment with phosphatidylinositol-specific phospholipase C, two other hydrolases, alkaline phosphatase and alkaline phosphodiesterase I, were also solubilized from silkworm midgut.