Page J D, Colman R W
Thrombosis Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
J Biol Chem. 1991 May 5;266(13):8143-8.
The predominant autolytic form of human kallikrein, beta-kallikrein, was used to localize the high molecular weight kininogen (HK) binding site on kallikrein as well as the substrate recognition site for activated factor XII on prekallikrein. beta-Kallikrein is formed by autolysis of the kallikrein heavy chain to give two fragments of approximately 18 and 28 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A ligand binding technique established that the HK binding site on kallikrein residues on the 28-kDa fragment of the heavy chain. Limited NH2-terminal sequencing of this portion of beta-kallikrein showed that this fragment of the heavy chain consists of the COOH-terminal 231 amino acids of the heavy chain. A panel of five murine monoclonal antibodies to human prekallikrein (PK) were found to have epitopes on this same fragment of the heavy chain. None of the monoclonal antibodies were able to block binding of HK to PK. Three of the monoclonal antibodies (13G11, 13H11, and 6A6) were able to inhibit the activation of PK to kallikrein in both a plasma system and a purified system. The 28-kDa fragment of the PK heavy chain was purified and was able to compete with HK for binding to PK. The HK binding site and the site of recognition of factor XII are separate and distinct on PK, and both are contained in the COOH-terminal 231 amino acids of the PK heavy chain.
人类激肽释放酶的主要自溶形式——β-激肽释放酶，被用于定位激肽释放酶上高分子量激肽原（HK）的结合位点以及前激肽释放酶上活化因子XII的底物识别位点。β-激肽释放酶是由激肽释放酶重链自溶形成的，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳可得到两条分子量约为18 kDa和28 kDa的片段。一种配体结合技术确定了激肽释放酶上HK的结合位点位于重链的28 kDa片段上。对β-激肽释放酶这一部分进行有限的氨基末端测序表明，重链的这一片段由重链的羧基末端231个氨基酸组成。发现一组针对人类前激肽释放酶（PK）的五种鼠单克隆抗体在重链的同一片段上具有表位。没有一种单克隆抗体能够阻断HK与PK的结合。其中三种单克隆抗体（13G11、13H11和6A6）能够在血浆系统和纯化系统中抑制PK激活为激肽释放酶。PK重链的28 kDa片段被纯化，并且能够与HK竞争结合PK。PK上HK的结合位点和因子XII的识别位点是分开且不同的，两者都包含在PK重链的羧基末端231个氨基酸中。
