Purification of glycosylphosphatidylinositol-anchoring aminopeptidase in from the plasma membrane of larval midgut epithelial cells of the silkworm,Bombyx mori |
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| Institution: | 1. State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716, China;2. School of Life Sciences, University of Bradford, Bradford, BD7 1DP, UK;1. Guangzhou Key Laboratory of Insect Development Regulation and Application Research, School of Life Sciences, South China Normal University, Guangzhou, 510631, China;2. State Key Laboratory of Silkworm Genome Biology, Southwest University, 400716, China;1. Jiangsu Key Laboratory of Sericutural Biology and Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, Jiangsu 212018, China |
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| 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.
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