Endo S, Yokosawa H, Ishii S
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Hokkaido University.
J Biochem. 1988 Dec;104(6):999-1006. doi: 10.1093/oxfordjournals.jbchem.a122599.
A novel substance P-degrading endopeptidase has been solubilized with Brij 35 from a membrane fraction of rat brain and purified by a procedure involving DEAE-cellulose chromatography, hydroxyapatite chromatography, Sephadex G-100 gel filtration, and Mono-Q HPLC. The activity of the degrading enzyme was monitored by measuring the disappearance of substance P by means of a bioassay and HPLC. SDS-polyacrylamide gel electrophoresis under reducing conditions of the enzyme gave a single band corresponding to a molecular weight of 58,000. The molecular weight of the enzyme was estimated to be 55,000 by gel filtration and the optimum pH for its activity was 7.5.. The purified enzyme cleaved substance P at three bonds, Pro4-Gln5, Gln5-Gln6, and Gln6-Phe7, in the ratio of 2:2:3. EDTA, o-phenanthroline, and p-chloromercuribenzenesulfonic acid strongly inhibited the enzyme, while diisopropyl fluorophosphate, E-64, Z-Gly-ProCH2Cl, phosphoramidon, and captopril had little or no inhibitory effect on it. The cleavage of substance P by the rat brain synaptic membrane was also analyzed under the conditions with or without these inhibitors. The inhibitor-susceptibility of the cleavage sites suggests that the present enzyme, together with endopeptidase-24.11, is involved in the degradation of substance P in the synaptic region.
一种新型的P物质降解内肽酶已用Brij 35从大鼠脑的膜部分中溶解出来，并通过包括DEAE - 纤维素色谱、羟基磷灰石色谱、Sephadex G - 100凝胶过滤和Mono - Q高效液相色谱的程序进行纯化。通过生物测定法和高效液相色谱法测量P物质的消失来监测降解酶的活性。在还原条件下对该酶进行SDS - 聚丙烯酰胺凝胶电泳，得到一条对应分子量为58,000的条带。通过凝胶过滤估计该酶的分子量为55,000，其活性的最适pH值为7.5。纯化后的酶在三个位点Pro4 - Gln5、Gln5 - Gln6和Gln6 - Phe7处切割P物质，比例为2:2:3。EDTA、邻菲罗啉和对氯汞苯磺酸强烈抑制该酶，而二异丙基氟磷酸酯、E - 64、Z - Gly - ProCH2Cl、磷酰胺脒和卡托普利对其几乎没有抑制作用。还在有或没有这些抑制剂的条件下分析了大鼠脑突触膜对P物质的切割情况。切割位点对抑制剂的敏感性表明，本酶与内肽酶 - 24.11一起参与突触区域中P物质的降解。
