Hattori Maki, Isomura Shigeki, Yokoyama Eiji, Ujita Minoru, Hara Akira
Department of Applied Biological Chemistry, Faculty of Agriculture, Meijo University, Tempaku-ku, Nagoya, Japan.
J Biosci Bioeng. 2005 Dec;100(6):631-6. doi: 10.1263/jbb.100.631.
A trypsin-like protease, P-1-1, was purified from the culture supernatant of the fungus Cordyceps militaris by (NH(4))(2)SO(4) precipitation, chromatography on DEAE Bio-Gel Agarose, TSKgel CM-5PW, and gel-filtration with HiLoad 26/60 Superdex 75 pg, and its properties were examined. Purified P-1-1 showed a single band by SDS-PAGE and was estimated to have a molecular mass of 23,405 by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The optimum pH of the enzyme was between 8.5 and 12.0. It was inhibited strongly by leupeptin and diisopropyl fluorophosphate (DFP), and definitely did by N(alpha)-tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK), phenylmethanesulfonyl fluoride (PMSF) and chymostatin. The carbonyl group sides of Arg and Lys were confirmed as the sites of cleavage by the enzyme toward cecropin B. These results indicate that P-1-1 is a trypsin-type serine protease. The N-terminal amino acid sequence of P-1-1 showed a high homology with those of trypsins or chymotrypsin derived from Diptera insects.
从蛹虫草真菌的培养上清液中通过硫酸铵沉淀、DEAE琼脂糖凝胶柱层析、TSKgel CM - 5PW柱层析以及HiLoad 26/60 Superdex 75 pg凝胶过滤法纯化出一种类胰蛋白酶P - 1 - 1，并对其性质进行了研究。纯化后的P - 1 - 1在SDS - PAGE上显示为单一条带，通过基质辅助激光解吸/电离质谱（MALDI - MS）估计其分子量为23405。该酶的最适pH在8.5至12.0之间。它受到亮抑酶肽和二异丙基氟磷酸（DFP）的强烈抑制，并且肯定受到N - α - 甲苯磺酰 - L - 赖氨酸氯甲基酮盐酸盐（TLCK）、苯甲基磺酰氟（PMSF）和抑糜酶素的抑制。该酶对天蚕素B的切割位点确定为精氨酸（Arg）和赖氨酸（Lys）的羰基侧。这些结果表明P - 1 - 1是一种胰蛋白酶型丝氨酸蛋白酶。P - 1 - 1 的N端氨基酸序列与双翅目昆虫的胰蛋白酶或胰凝乳蛋白酶的N端氨基酸序列具有高度同源性。
