Eguchi M, Kuriyama K
J Biochem. 1985 May;97(5):1437-45. doi: 10.1093/oxfordjournals.jbchem.a135198.
Membrane-bound alkaline proteases from the midgut epithelia of the silkworm, Bombyx mori, were solubilized with 1% Lubrol-WX, at pH 11.2. They were purified by gel filtration on Sepharose 6B and Ultrogel AcA-202 columns and a preparative polyacrylamide gel electrophoresis. Two proteases, caseinolytic (6B3-Tc) and benzoyl-arginine-p-nitroanilide-lytic (6B3-Tb) were obtained. Both enzymes were homogeneous as judged by polyacrylamide electrophoresis. These enzymes showed high pH optima, 11.2, and pI values, above 11, and were extremely stable over a wide range of pH. The Km values for 6B3-Tb and Tc were 0.476 mM and 2.5 mg/ml respectively. Hammarsten casein and mulberry leaf protein were rapidly hydrolyzed by Tc, whereas the hydrolytic activity of Tb for Azocoll was higher than that of Tc. The protease Tb was strongly inhibited by diisopropylfluorophosphate, p-chloromercuribenzoate, benzamidine, leupeptin, and soybean trypsin inhibitor; Tc was inhibited by diisopropylfluorophosphate, tosyl phenylalanine chloromethylketone and chymostatin, but not by tosyl lysine chloromethylketone, p-chloromercuribenzoate, or iodoacetamide. The molecular weights of the proteases were estimated to be 12,800 (Tb) and 13,300 (Tc) by Sephacryl S-300 gel filtration. The amino acid analyses showed that both proteases contain a large number of acidic amino acids but a relatively small number of basic amino acids.
家蚕中肠上皮细胞的膜结合碱性蛋白酶在pH 11.2条件下用1% Lubrol-WX溶解。通过在Sepharose 6B和Ultrogel AcA - 202柱上进行凝胶过滤以及制备性聚丙烯酰胺凝胶电泳对其进行纯化。得到了两种蛋白酶，酪蛋白水解酶（6B3 - Tc）和苯甲酰精氨酸对硝基苯胺水解酶（6B3 - Tb）。通过聚丙烯酰胺电泳判断，这两种酶均为纯品。这些酶表现出较高的最适pH值（11.2）和pI值（高于11），并且在很宽的pH范围内都极其稳定。6B3 - Tb和Tc的Km值分别为0.476 mM和2.5 mg/ml。哈马斯坦酪蛋白和桑叶蛋白能被Tc快速水解，而Tb对偶氮酪蛋白的水解活性高于Tc。蛋白酶Tb受到二异丙基氟磷酸酯、对氯汞苯甲酸、苯甲脒、亮抑酶肽和大豆胰蛋白酶抑制剂的强烈抑制；Tc受到二异丙基氟磷酸酯、甲苯磺酰苯丙氨酸氯甲基酮和糜蛋白酶抑制剂的抑制，但不受甲苯磺酰赖氨酸氯甲基酮、对氯汞苯甲酸或碘乙酰胺的抑制。通过Sephacryl S - 300凝胶过滤估计，这两种蛋白酶的分子量分别为12,800（Tb）和13,300（Tc）。氨基酸分析表明，这两种蛋白酶都含有大量酸性氨基酸，但碱性氨基酸数量相对较少。
