Bollen A M, Makinen K K
Department of Biologic and Material Sciences, School of Dentistry, University of Michigan, Ann Arbor 48109.
Calcif Tissue Int. 1991 Feb;48(2):111-9. doi: 10.1007/BF02555875.
A soluble endopeptidase was purified from rat bone using ammonium sulphate precipitation followed by Fast Protein Liquid Chromatography on gel, anion exchange, and chromatofocusing columns. The enzyme was silver stain-pure (SDS-PAGE) and its apparent molecular weight was 60,000. In general, the enzyme favored the hydrolysis of the X-Y bond in substrates with the sequence of -Pro-X-Y-Pro-, where either X or Y (or both) were hydrophobic residues. The presence of imino acid residues near the scissile bond favored hydrolysis. The enzyme was strongly inactivated by metal chelating agents and p-hydroxymercuribenzoic acid, indicating that SH-groups may be necessary for full activity of the enzyme and that the enzyme may be a metallopeptidase.
利用硫酸铵沉淀法，随后在凝胶、阴离子交换和色谱聚焦柱上进行快速蛋白质液相色谱，从大鼠骨骼中纯化出一种可溶性内肽酶。该酶经银染后呈纯态（十二烷基硫酸钠-聚丙烯酰胺凝胶电泳），其表观分子量为60,000。一般来说，该酶倾向于水解序列为-Pro-X-Y-Pro-的底物中的X-Y键，其中X或Y（或两者）为疏水残基。在可裂解键附近存在亚氨基酸残基有利于水解。该酶被金属螯合剂和对羟基汞苯甲酸强烈灭活，表明巯基可能是该酶充分发挥活性所必需的，且该酶可能是一种金属肽酶。
