Actions of Urinary Kallikrein, Plasmin, and Other Kininogenases on Bovine Plasma High-Molecular-Weight Kininogen1

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Abstract
1.Using various purified kallikreins [EC 3.4.21.8] and kininogenases, the process of frag inentation of bovine plasma high-moleculare-weight (HMV) kininogen was examined. Bovine plasma kallikrein liberated the previously characterized contact activation inhibitors, fragment 1.2 and fragment X (carbohydrate-free fragement 1.2), in addition to bradykinin, almost simultaneously from the kininogen. Porcine parncreatic and human urinary kallikreins, and snake venom kininogenease released these fragements only slightly, if at all, although all the enzymes produced thne vasopeptide kinin very rapidly. On the other band, the release of kiniu from the kininogen by bovine plasmin [EC 3.4.21.7.] was insignificant and no release of fragments the large polypeptide region, which comprises fragement 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 specific action of plasma kallikrein. 2.Human urinary kallikrein was found to be useful to clucidate a linear polypeptide sequence of the kinin moiety and fragement 1.2 in the kiniogen molecule, because the enzyme only releases kinin under certain conditions. After reduction and S-carboxymethylaton of the disulfide bonds in urinary kallikrein (URK) modifies HMW kininogen, two polypeptide fragments, named HMW-heavy chain-URK and HMV-light chain-URK, were isolated. These fragments were found to be derived, respectively, from the NH2-and COOR-terminal portions of the parent molecule. Direct Edman degradation up the the 3rd step of HMW-light chain URK established the sequence Ser-Val-Gln, which is identical with the Nh2 terminal sequence of fagment 1.2. Furthemore, on incubation of HMW-light chain-URK with plasma kallikrein, it yiedede initially fragment 1.2, fragment X, and the previously identified HMW light chain. These results indicate that fragement 1.2 and fragment X could be positioned before the HMW-light chain, located in the COOH-terminal protionh of whole HMW kiniogen.