Maret G E, Fauchère J L
Department of Biotechnology, Swiss Federal Institute of Technology, ETH, Hönggerberg HPT, Zurich, Switzerland.
Anal Biochem. 1988 Jul;172(1):248-58. doi: 10.1016/0003-2697(88)90439-3.
An endopeptidase was isolated from bovine adrenomedullary granules by fast protein liquid chromatography, including two ion exchange, one hydrophobic interaction, and one gel filtration step. The enzyme assay was based on the HPLC detection of the degradation of the dodecapeptide BAM 12P from the sequence of proenkephalin. After a 1200-fold purification, the enzyme fraction was homogeneous on polyacrylamide gel electrophoresis. The apparent molecular weight of the monomeric protein was 68,000 and its pH optimum was 5.6, in agreement with the internal pH of the granules. The specificity of the protease was determined initially by analysis of the degradation fragments of BAM 12P which showed that cleavage occurred at the double but not at the single Arg site of BAM 12P. The cleavage pattern of other substrates showed that the enzyme also recognized other pairs of basic amino acids. The behavior of the enzyme toward specific inhibitors showed that it was an acid thiol protease different from serine proteases and from lysosomal cathepsin B. The endopeptidase may act as a maturation enzyme in vivo.
通过快速蛋白质液相色谱法从牛肾上腺髓质颗粒中分离出一种内肽酶，该方法包括两个离子交换步骤、一个疏水相互作用步骤和一个凝胶过滤步骤。酶活性测定基于高效液相色谱法检测源自前脑啡肽序列的十二肽BAM 12P的降解情况。经过1200倍的纯化后，酶组分在聚丙烯酰胺凝胶电泳上呈现均一性。单体蛋白的表观分子量为68,000，其最适pH为5.6，与颗粒的内部pH一致。蛋白酶的特异性最初通过分析BAM 12P的降解片段来确定，结果表明切割发生在BAM 12P的双精氨酸位点而非单精氨酸位点。其他底物的切割模式表明该酶也能识别其他碱性氨基酸对。该酶对特定抑制剂的反应表明它是一种酸性巯基蛋白酶，不同于丝氨酸蛋白酶和溶酶体组织蛋白酶B。这种内肽酶可能在体内充当成熟酶。
