Yoshimoto T, Sattar A K, Hirose W, Tsuru D
School of Pharmaceutical Sciences, Nagasaki University.
J Biochem. 1988 Oct;104(4):622-7. doi: 10.1093/oxfordjournals.jbchem.a122522.
High prolyl endopeptidase (post-proline cleaving enzyme) [EC 3.4.21.26] activity was detected in fruit bodies of shakashimeji (Lyophyllum cinerascens), tsukuritake (mushroom: Agaricus bisporus), hirohachichitake (Lactarius hygrophoroides), and yaburebenitake (Russula lepida) which belong to the genus Basidiomycetes. Cell-free extract of shakashimeji showed high activities of proline iminopeptidase and arylamidase as well as prolyl endopeptidase. The prolyl endopeptidase was purified from the extract of shakashimeji by sequential chromatographies on DEAE-Toyopearl, DEAE-Sephadex and hydroxyapatite, and high-performance liquid chromatography with a DEAE-5PW column. The purified enzyme was homogeneous as judged by disc gel electrophoresis. The enzyme was most active at pH 6.8 as checked with Z-Gly-Pro-beta-naphthylamide as a substrate and was stable in the range of pH 5.8-7.4. The isoelectric point of the enzyme was 5.2 and the molecular weight was estimated to be 76,000 by gel filtration on Sephadex G-150 and by sodium dodecyl sulfate (SDS) gel electrophoresis, suggesting that the enzyme was a monomer. The enzyme was completely inhibited by diisopropyl fluorophosphate (DFP), Z-Gly-Pro-CH2Cl, and Z-Pro-prolinal, while it was not inhibited by p-chloromercuribenzoate (PCMB), phenylmethylsulfonyl fluoride (PMSF), or metal chelators. It was estimated that at least five subsites were concerned with the enzyme-substrate binding. Among them, the S1, S2, and S1' sites showed high stereospecificity, as in mammalian, microbial, and plant enzymes. The enzyme hydrolyzed TRH at the carboxyl side of the proline residue. The mushroom enzyme, that was sensitive to DFP, Z-Pro-prolinal, and Z-Gly-Pro-CH2Cl, but not to PCMB, were quite similar in characteristics to the Flavobacterium enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
在属于担子菌纲的白盖环柄菇（灰环粘盖牛肝菌）、双孢蘑菇、湿色乳菇和红汁乳菇的子实体中检测到了高脯氨酰内肽酶（脯氨酸后切割酶）[EC 3.4.21.26]活性。白盖环柄菇的无细胞提取物显示出脯氨酸亚氨肽酶、芳基酰胺酶以及脯氨酰内肽酶的高活性。通过在DEAE - Toyopearl、DEAE - Sephadex和羟基磷灰石上的连续色谱法以及使用DEAE - 5PW柱的高效液相色谱法，从白盖环柄菇的提取物中纯化出了脯氨酰内肽酶。通过圆盘凝胶电泳判断，纯化后的酶是均一的。以Z - Gly - Pro - β - 萘酰胺为底物检测时，该酶在pH 6.8时活性最高，并且在pH 5.8 - 7.4范围内稳定。该酶的等电点为5.2，通过在Sephadex G - 150上的凝胶过滤和十二烷基硫酸钠（SDS）凝胶电泳估计其分子量为76,000，表明该酶是单体。该酶被二异丙基氟磷酸（DFP）（译者注：原文中此处DFP英文表述有误，正确为diisopropyl fluorophosphate）、Z - Gly - Pro - CH₂Cl和Z - Pro - 脯氨醛完全抑制，而不被对氯汞苯甲酸（PCMB）、苯甲基磺酰氟（PMSF）或金属螯合剂抑制。据估计，至少有五个亚位点与酶 - 底物结合有关。其中，S1、S2和S1'位点表现出高立体特异性，与哺乳动物、微生物和植物的酶类似。该酶在脯氨酸残基的羧基侧水解促甲状腺激素释放激素（TRH）。这种对DFP、Z - Pro - 脯氨醛和Z - Gly - Pro - CH₂Cl敏感但对PCMB不敏感的蘑菇酶在特性上与黄杆菌属的酶非常相似。（摘要截断于250字）
