Abstract
High-molecular-weight kininogen has been isolated from rat plasma in three steps in a relatively high yield. The purified preparation gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence and presence of 2-mercaptoethanol, and the apparent Mr was estimated as 100,000. On incubation with rat plasma kallikrein, rat high Mr kininogen yielded a kinin-free protein consisting of a heavy chain (Mr = 64,000) and a light chain (Mr = 46,000), liberating bradykinin. The kinin-free protein was S-alkylated, and its heavy and light chains were separated by a zinc-chelating Sepharose 6B column. The amino acid compositions of rat high Mr kininogen and its heavy and light chains were very similar to those of bovine high Mr kininogen and its heavy and fragment 1.2-light chains, respectively. A high histidine content in the light chain of rat high Mr kininogen indicated the presence of a histidine-rich region in this protein as in bovine high Mr kininogen, although this region was not cleaved by rat plasma kallikrein. Rat high Mr kininogen corrected to normal values the prolonged activated partial thromboplastin time of Brown-Norway Katholiek rat plasma known to be deficient in high Mr kininogen and of Fitzgerald trait plasma. The kinin-free protein had the same correcting activity as intact high Mr kininogen. Rat high Mr kininogen also accelerated approximately 10-fold the surface-dependent activation of rat factor XII and prekallikrein, which was mediated with kaolin, amylose sulfate, and sulfatide. These results indicate that rat high Mr kininogen is quite similar to human and bovine high Mr kininogens in terms of biochemical and functional properties.
Highlights
High-molecular-weight kininogen has been isolated krein [10, 11]
- ating thepreviously known fragment 1.2 [25] and thecarbohydrate-free fragment 1 2 [34]very rapidly from bovine high MI kininogen. These results indicate that rat plasma kallikrein cleaves rat high M,kininogen into a protein with two chains bridged by disulfidebonds, liberating kinin, and that it does not liberate any fragments from the kininogen, which is different from bovine highMIkininogen
Kinin released from rat high M,kininogen by rat plasma kallikrein was identified as bradykinin
Summary
Region was not cleaved by rat plasma kallikrein. Rat high M, kininogen corrected to normal values. Protein was eluted with a linear salt gradient formed from liters each of the equilibration buffer and the buffer containing 0.3 M NaCl. As shown. Rat Plasma High-molKeciunlianro-gweenight was applied to a column (3.2 X 25 cm) of zinc-chelating Sepharose 6B,equilibrated with 0.02 M Tris-HC1, pH 8.0, containing 0.05 M NaCl. The column was washedwith 500 ml of 0.02 M Tris-HC1,pH 8.0, containing 1M NaCl and subsequently eluted first with a linear gradient formed from 1liter each of the washing buffer and thesame buffer containing 30. 2), 5.3pgof prekallikrein (lanes 4 and 5), 3.6 pgof plasma Elution was performed a t a flow rate of 180 ml/h with a linear kallikrein (lanes and 7), and standardmarker proteins
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