J. Biochem, 1982, Vol. 92, No. 3 679-688
© 1982 Japanese Biochemical Society
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A Method for Preparation of a Single Chain High-Molecular-Weight (HMW) Kininogen from Bovine Plasma1,2
Department of Biology, Faculty of Science, Kyushu University 33 Higashi-ku, Fukuoka, Fukuoka 812
Although a method for purification of high-molecular-weight (HMW) kininogen from bovine plasma has been reported (Komiya et al. (1974) J. Biochem. 76, 811–822), the preparation of HMW kininogen consisted of a mixture of two molecular species, a single chain kininogen and a two chain kininogen, the latter of which suffered limited proteolysis of the polypeptide chain. We succeeded here in establishing a procedure for preparation of a single chain HMW kininogen. The method consisted of three steps, chromatographies on columns of DEAE-Sephadex A-50 and CM-Sephadex C-50 and gel-filtration on a column of Sephadex G-150 in 0.02 M Tris-HCl buffer, pH 8.0, containing 1 m NaCl. Although HMW kininogen existed as a molecular complex with prekallikrein at a low salt concentration, the complex was dissociated and the components separated from each other by the final step with molecular sieving at a high salt concentration. The kininogen prepared by the present method showed a single band on SDS-polyacrylamide gel electrophoresis in the presence and absence of 2-mercaptoethanol and on disc polyacrylamide gel electrophoresis at pH 7.2, indicating that it consists of a single chain polypeptide. The single chain kininogen was indistinguishable from the previous preparation, with regard to the amino acid composition and immunological properties. Moreover, no significant difference was observed in their accelerating activities on the kaolin-mediated activation of Factor XII in the presence of prekallikrein. The single chain kininogen was also demonstrated to be free from kallikrein, prekallikrein, Factor XIIa and Factor XII based on the following results; (1) it was not degraded into the two chain protein upon incubation at 37°C for 24 h, (2) it did not hydrolyze carbobenzoxy-phenylalanyl-arginine-4-methylcoumaryl-7-amide, which is a very sensitive substrate for plasma kallikrein, even in the presence of each of kaolin, Factor XII or XIIa and prekallikrein, and (3) it did not correct the prolonged clotting time of Factor XII deficient plasma.
1 This study was supported in part by Grants-in-Aid (544081 and 558064) for Scientific Research from the Ministry of Education, Science and Culture of Japan, and by a Yamada Science Foundation Grant for 1980.
2 Reported at the 53rd annual meeting of the Japanese Biochemical Society held in Tokyo (1980). Seikagaku (in Japanese) 52, 734.
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