Cawley N X, Olsen V, Zhang C F, Chen H C, Tan M, Loh Y P
Section on Cellular Neurobiology, Laboratory of Developmental Neurobiology, NICHD, National Institutes of Health, Bethesda, Maryland 20892,
J Biol Chem. 1998 Jan 2;273(1):584-91. doi: 10.1074/jbc.273.1.584.
A C-terminally truncated form of yapsin 1 (yeast aspartic protease 3), the first member of the novel sub-class of aspartic proteases with specificity for basic residues (designated the Yapsins), was overexpressed and purified to apparent homogeneity, yielding approximately 1 microg of yapsin 1/g of wet yeast. N-terminal amino acid analysis of the purified protein confirmed that the propeptide was absent and that the mature enzyme began at Ala68. The mature enzyme was shown to be composed of approximately equimolar amounts of two subunits, designated alpha and beta, that were associated to each other by a disulfide bond. C-terminally truncated proyapsin 1 was also expressed in the baculovirus/Sf9 insect cell expression system and secreted as a zymogen that could be activated upon incubation at an acidic pH with an optimum at approximately 4.0. When expressed without its pro-region, it was localized intracellularly and lacked activity, indicating that the pro-region was required for the correct folding of the enzyme. The activation of proyapsin 1 in vitro exhibited linear kinetics and generated an intermediate form of yapsin 1 or pseudo-yapsin 1.
天冬氨酸蛋白酶3（酵母天冬氨酸蛋白酶3）的C末端截短形式是一种对碱性残基具有特异性的新型天冬氨酸蛋白酶亚类（命名为Yapsins）的首个成员，其被过表达并纯化至表观均一性，每克湿酵母可产生约1微克天冬氨酸蛋白酶3。对纯化蛋白的N末端氨基酸分析证实前肽缺失，成熟酶从Ala68开始。成熟酶显示由大约等摩尔量的两个亚基组成，分别命名为α和β，它们通过二硫键相互关联。C末端截短的前天冬氨酸蛋白酶3也在杆状病毒/Sf9昆虫细胞表达系统中表达，并作为一种酶原分泌，在酸性pH下孵育时可被激活，最适pH约为4.0。当在没有其前体区域的情况下表达时，它定位于细胞内且缺乏活性，这表明前体区域是酶正确折叠所必需的。前天冬氨酸蛋白酶3在体外的激活表现出线性动力学，并产生天冬氨酸蛋白酶3的中间形式或假天冬氨酸蛋白酶3。
