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

International Journal of Biochemistry Pub Date : 1994-08-01 DOI:10.1016/0020-711X(94)90068-X

Masahiro Tomita , Hiroshi Obara , Yoshiki Takesue , Hiro-Omi Tamura , Shigetoshi Miyajima , Ryo Taguchi , Hiroh Ikezawa

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引用次数: 14

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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家蚕幼虫中肠上皮细胞质膜中糖基磷脂酰肌醇锚定氨基肽酶的纯化

1.1. 利用磷脂酰肌醇特异性磷脂酶C从家蚕幼虫中肠中选择性释放氨肽酶N，并经离子交换、凝胶过滤纯化为均相状态。cona - sepharose和4-氨基苯膦酸-琼脂糖柱层析。纯化后的氨基肽酶N比活性为190.8 U/mg。经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测定其分子量约为100kda。纯化的氨基肽酶N分子优先水解Leu-、Ala-和met -对硝基苯胺作为底物。其中，亮氨酸-对硝基苯胺是家蚕幼虫中肠中氨基肽酶N的最佳底物。用磷脂酰肌醇特异性磷脂酶C和碱性磷酸酶和碱性磷酸二酯酶I对家蚕中肠进行了水解。

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International Journal of Biochemistry

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