Abstract
1. Using various purified kallikreins [EC 3. 4. 21. 8] and kininogenases, the process of fragmentation of bovine plasma high-molecular-weight (HMW) kininogen was examined. Bovine plasma kallikrein liberated the previously characterized contact activation inhibitors, fragment 1•2 and fragment X (carbohydrate-free fragment 1•2), in addition to bradykinin, almost simultaneously from the kininogen. Porcine pancreatic and human urinary kallikreins, and snake venom kininogenase released these fragments only slightly, if at all, although all the enzymes produced the vasopeptide kinin very rapidly. On the other hand, the release of kinin from the kininogen by bovine plasmin [EC 3. 4. 21. 7] was insignificant and no release of fragments such as fragment 1•2 and fragment X was observed. Plasmin appeared to destroy rapidly the large polypeptide region, which comprises fragment 1•2, fragment X, and the COOH-terminal part of HMW kininogen. Thus, the release of fragment 1•2 from the kininogen was due to a specific action of plasma kallikrein.
2. Human urinary kallikrein was found to be useful to elucidate a linear polypeptide sequence of the kinin moiety and fragment 1•2 in the kininogen molecule, because the enzyme only releases kinin under certain conditions. After reduction and S-carboxymethylation of the disulfide bonds in urinary kallikrein (URK)-modified HMW kininogen, two polypeptide fragments, named HMW-heavy chain-URK and HMW-light chain-URK, were isolated. These fragments were found to be derived, respectively, from the NH2- and COOH-terminal portions of the parent molecule. Direct Edman degradation up to the 3rd step of HMW-light chain-URK established the sequence Ser-Val-Gin, which is identical with the NH2-terminal sequence of fragment 1•2. Furthermore, on incubation of HMW-light chain-URK with plasma kallikrein, it yielded initially fragment 1•2, fragment X, and the previously identified HMW-light chain. These results indicate that fragment 1•2 and fragment X could be positioned before the HMW-light chain, located in the COOH-terminal portion of whole HMW kininogen.
