Satoh M, Yokosawa H, Ishii S
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Hokkaido University.
J Biochem. 1988 Mar;103(3):493-8. doi: 10.1093/oxfordjournals.jbchem.a122298.
A trypsin-like enzyme has been purified to apparent homogeneity from neuroblastoma cell membranes by a procedure including extraction with Triton X-100, soybean trypsin inhibitor-immobilized Sepharose 4B affinity chromatography, and gel filtration. SDS-polyacrylamide gel electrophoresis under reducing conditions of the purified enzyme gave a single band corresponding to a molecular weight of 28,000. The molecular weight of the enzyme was also estimated to be 32,000 by gel filtration. The pH optimum of the activity was 8.5-9.0. The purified enzyme was inhibited by diisopropylphosphorofluoridate, p-aminobenzamidine, and leupeptin, and moderately by chymostatin, but not, or only scarcely, by bestatin, phosphoramidon, p-chloromercuribenzoate, and N-ethylmaleimide. The substrate subsite specificity of the purified enzyme was broad toward various peptidyl-arginine (or lysine) 4-methylcoumaryl-7-amides, but it cleaved dynorphin(1-17) only at two sites, i.e., between the Arg6-Arg7 and Lys11-Leu12 bonds, both of which correspond to the initial cleavage sites of dynorphin with a membrane preparation of neuroblastoma cells. A trypsin-like enzyme was also purified from a synaptic membrane preparation of rat brain, which shows almost the same properties as those of the enzyme from the neuroblastoma cell membrane. Thus, the trypsin-like enzyme present in the synaptic membrane would participate in the degradation of dynorphin.
通过包括用 Triton X - 100 提取、大豆胰蛋白酶抑制剂固定化的 Sepharose 4B 亲和层析和凝胶过滤在内的一系列步骤，已从神经母细胞瘤细胞膜中纯化出一种类似胰蛋白酶的酶，其纯度达到表观均一。在还原条件下对纯化后的酶进行 SDS - 聚丙烯酰胺凝胶电泳，得到一条对应分子量为 28,000 的单带。通过凝胶过滤法估计该酶的分子量也为 32,000。该酶活性的最适 pH 为 8.5 - 9.0。纯化后的酶被二异丙基氟磷酸酯、对氨基苯甲脒和亮肽素抑制，被抑肽酶中度抑制，但不被贝司他汀、磷酰胺素、对氯汞苯甲酸和 N - 乙基马来酰亚胺抑制或仅受到微弱抑制。纯化后的酶对各种肽基 - 精氨酸（或赖氨酸）4 - 甲基香豆素 - 7 - 酰胺的底物亚位点特异性较宽，但它仅在两个位点切割强啡肽(1 - 17)，即 Arg6 - Arg7 和 Lys11 - Leu12 键之间，这两个位点都对应于神经母细胞瘤细胞膜制剂切割强啡肽的初始切割位点。还从大鼠脑的突触膜制剂中纯化出一种类似胰蛋白酶的酶，它表现出与神经母细胞瘤细胞膜中的酶几乎相同的性质。因此，存在于突触膜中的类似胰蛋白酶的酶可能参与强啡肽的降解。
