大鼠小肠黏膜α,α-海藻糖酶的纯化及性质
Purification and properties of alpha,alpha-trehalase from the mucosa of rat small intestine.
作者信息
Sasajima K, Kawachi T, Sato S, Sugimura T
出版信息
Biochim Biophys Acta. 1975 Sep 22;403(1):139-46. doi: 10.1016/0005-2744(75)90017-0.
ALPHA,ALPHA-Trehalase (EC 3.2.1.28, alpha,alpha-trehalose glucohydrolase) was solubilized from the microvillous membrane of the intestinal mucosa of rats with Triton X-100 and butanol. It was purified 6350-fold by gel filtration on Sephadex G-150 and chromatography on DE-52 and hydroxyapatite. The purified enzyme, with a specific activity of about 127 units per mg of protein, showed almost a single band of protein and activity on polyacrylamide gel electrophoresis. Its molecular weight was estimated to be 96 000 on Sephadex G-150 and 90 000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its pH optimum was 5.5-5.7 and its Km value for trehalose was 5.4 mM. Its activity was inhibited 30 and 100% by 1 mM p-chloromercuribenzoate and 0.1 mM HgCl2, respectively and 30% by 1 mM MgCl2. Moreover, its activity was inhibited completely by 10 mM tris(hydroxymethyl)aminomethane and about 60% by 10 mM sucrose and cellobiose. The enzyme showed a high specificity for trehalose.
α,α-海藻糖酶（EC 3.2.1.28，α,α-海藻糖葡萄糖水解酶）用Triton X-100和丁醇从大鼠肠黏膜微绒毛膜中溶解出来。通过在Sephadex G-150上进行凝胶过滤以及在DE-52和羟基磷灰石上进行色谱分离，该酶被纯化了6350倍。纯化后的酶，每毫克蛋白质的比活性约为127单位，在聚丙烯酰胺凝胶电泳上显示出几乎单一的蛋白质条带和活性。在Sephadex G-150上其分子量估计为96000，在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上为90000。其最适pH为5.5 - 5.7，对海藻糖的Km值为5.4 mM。其活性分别被1 mM对氯汞苯甲酸和0.1 mM HgCl2抑制30%和100%，被1 mM MgCl2抑制30%。此外，其活性被10 mM三（羟甲基）氨基甲烷完全抑制，被10 mM蔗糖和纤维二糖抑制约60%。该酶对海藻糖表现出高度特异性。
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