Yoshida N, Tsuruyama S, Nagata K, Hirayama K, Noda K, Makisumi S
Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka.
J Biochem. 1988 Sep;104(3):451-6. doi: 10.1093/oxfordjournals.jbchem.a122488.
A protease was purified 163-fold from Pronase, a commercial product from culture filtrate of Streptomyces griseus, by a series of column chromatographies on CM-Toyopearl (Fractogel), Sephadex G-50, hydroxyapatite, and Z-Gly-D-Phe-AH-Sepharose 4B using Boc-Ala-Ala-Pro-Glu-pNA as a substrate. The final preparation was homogeneous by polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and gel isoelectric focusing. Studies on the substrate specificity with peptide p-nitroanilides revealed that this protease preferentially hydrolyzed peptide bonds on the carbonyl-terminal side of either glutamic acid or aspartic acid. It was most active at pH 8.8 for the hydrolysis of Boc-Ala-Ala-Pro-Glu-pNA. The molecular weight of the protease was estimated to be 20,000 by gel filtration on Sepharose 6B using 6 M guanidine hydrochloride as an eluent, and 22,000 by SDS-PAGE in the presence of 2-mercaptoethanol. The isoelectric point of the enzyme was 8.4. The enzyme was inactivated by diisopropyl phosphofluoridate (DFP) but not by p-chloromercuribenzoate (PCMB) or EDTA.
以Boc-Ala-Ala-Pro-Glu-pNA为底物，通过在CM- Toyopearl（Fractogel）、Sephadex G-50、羟基磷灰石和Z-Gly-D-Phe-AH-Sepharose 4B上进行一系列柱色谱，从灰色链霉菌培养滤液的商业产品链霉蛋白酶中纯化出一种蛋白酶，纯化倍数为163倍。通过聚丙烯酰胺凝胶电泳（PAGE）、十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）和凝胶等电聚焦分析，最终制备的蛋白酶呈均一状态。对肽对硝基苯胺底物特异性的研究表明，该蛋白酶优先水解谷氨酸或天冬氨酸羰基末端侧的肽键。它在pH 8.8时对Boc-Ala-Ala-Pro-Glu-pNA的水解活性最高。使用6 M盐酸胍作为洗脱剂，通过Sepharose 6B凝胶过滤法估计该蛋白酶的分子量为20,000，在2-巯基乙醇存在下通过SDS-PAGE测定其分子量为22,000。该酶的等电点为8.4。该酶被二异丙基氟磷酸酯（DFP）灭活，但不被对氯汞苯甲酸（PCMB）或乙二胺四乙酸（EDTA）灭活。
