Minami A, Fukuda H
Biological Institute, Faculty of Science, Tohoku University, Sendai, Japan.
Plant Cell Physiol. 1995 Dec;36(8):1599-606.
Endopeptidase activities during the differentiation of Zinnia cells into tracheary elements (TEs) were examined with several peptidyl 4-methyl-7-coumarylamido (MCA) as substrates. The activity that hydrolysed carbobenzoxy-Phe-Arg-MCA (Z-Phe-Arg-MCA) at pH 5 increased in a differentiation-related manner: this activity, which was not observed in freshly isolated mesophyll cells was induced by the combination of alpha-naphthaleneacetic acid (NAA) and 6-benzyladenine (BA) that is necessary for differentiation of TEs, but not by NAA or BA alone. The activity in cells cultured in TE-inductive medium that contained both NAA and BA increased very rapidly between the 48th and 60th hours of culture, when the number of TE increased rapidly. A protease responsible for this activity with a molecular mass of 30 kDa was partially purified from cells which had been cultured in the TE-inductive medium and included many immature TEs. Strong inhibition by [L-3-trans-carboxyoxiran-2-carbonyl]-L-Leu- -agmatin (E-64), and activation by dithiothreitol (DTT) indicated that this protease belongs to a family of cysteine endopeptidases (EC 3.4.22).
以几种肽基4-甲基-7-香豆素酰胺（MCA）为底物，检测了百日草细胞分化为管状分子（TEs）过程中的内肽酶活性。在pH 5时水解苄氧羰基-Phe-Arg-MCA（Z-Phe-Arg-MCA）的活性以与分化相关的方式增加：这种活性在新鲜分离的叶肉细胞中未观察到，而是由TEs分化所必需的α-萘乙酸（NAA）和6-苄基腺嘌呤（BA）组合诱导产生，单独使用NAA或BA则不能诱导。在含有NAA和BA的TE诱导培养基中培养的细胞，其活性在培养的第48至60小时之间迅速增加，此时TE的数量迅速增加。从在TE诱导培养基中培养且含有许多未成熟TE的细胞中部分纯化出一种分子量为30 kDa的负责该活性的蛋白酶。[L-3-反式-羧基环氧乙烷-2-羰基]-L-亮氨酸-胍丁胺（E-64）的强烈抑制作用以及二硫苏糖醇（DTT）的激活作用表明，该蛋白酶属于半胱氨酸内肽酶家族（EC 3.4.22）。
