Green G D, Shaw E
Arch Biochem Biophys. 1983 Aug;225(1):331-7. doi: 10.1016/0003-9861(83)90037-1.
The presence of a prolyl endopeptidase in the soluble fraction of murine peritoneal macrophages is reported. The prolyl endopeptidase is apparently highly specific for cleaving peptides after proline residues. A sensitive new fluorogenic assay substrate matching this specificity, benzyloxycarbonyl-Ala-Ala-Pro beta-methoxynaphthylamide, is described. The enzyme is rapidly inactivated by benzyloxycarbonyl-Ala-Ala-Pro diazomethyl ketone, one of a class of reagents specific for cysteine proteinases, and by diisopropyl fluorophosphate, an inhibitor of serine proteinases. Culture of macrophages with the addition of low levels of benzyloxycarbonyl-Ala-Ala-Pro diazomethyl ketone to the media allows the selective inhibition of the cytoplasmic enzyme as measured in lysates at the termination of culture. After exposure to inhibitor, macrophages resynthesize the enzyme over a period of days, a process which is inhibited by cycloheximide. Similar amounts of activity were found in both normal peritoneal macrophages and those elicited by prior injection of thioglycollate media. The enzyme from murine macrophages appears similar to that reported in bronchopulmonary lavage fluid and lung tissue and to those isolated from brain and pituitary tissues.
据报道，在小鼠腹膜巨噬细胞的可溶部分存在脯氨酰内肽酶。该脯氨酰内肽酶显然对切割脯氨酸残基后的肽具有高度特异性。描述了一种与这种特异性相匹配的灵敏的新型荧光测定底物，苄氧羰基 - 丙氨酸 - 丙氨酸 - 脯氨酸β - 甲氧基萘酰胺。该酶可被苄氧羰基 - 丙氨酸 - 丙氨酸 - 脯氨酸重氮甲基酮迅速灭活，苄氧羰基 - 丙氨酸 - 丙氨酸 - 脯氨酸重氮甲基酮是一类对半胱氨酸蛋白酶具有特异性的试剂之一，同时也可被二异丙基氟磷酸酯（一种丝氨酸蛋白酶抑制剂）迅速灭活。在培养基中添加低水平的苄氧羰基 - 丙氨酸 - 丙氨酸 - 脯氨酸重氮甲基酮培养巨噬细胞，可在培养结束时在裂解物中检测到对细胞质酶的选择性抑制。暴露于抑制剂后，巨噬细胞会在数天内重新合成该酶，这一过程会被环己酰亚胺抑制。在正常腹膜巨噬细胞和先前注射巯基乙酸盐培养基诱导的巨噬细胞中发现了相似量的活性。小鼠巨噬细胞中的这种酶似乎与支气管肺泡灌洗液和肺组织中报道的酶以及从脑和垂体组织中分离出的酶相似。
