Rodent kinin-forming enzyme systems--I. Purification and characterization of plasma kininogen.
Low molecular weight (LMW) kininogen was purified 70-fold with a 16% yield from fresh rat plasma by DEAE-Sephadex chromatography, ammonium sulfate precipitation, Sephadex G-200 gel filtration, SP-Sephadex chromatography, CM-cellulose chromatography, and Sephadex G-200 gel filtration. Ferguson plots of polyacrylamide gel electrophoretic patterns revealed four bands with relative molecular weights of 64,000, 123,500, 252,436 and 357,900 (ratio of 1:2:4:6). Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis provided a single protein band with a molecular weight of 72,000, suggesting that the four kininogen bands had been caused by the aggregation of a single oligomeric protein. The purified LMW rat kininogen Fraction B (3.9 micrograms bradykinin/mg) was used to elicit an antiserum in the rabbit. Monospecificity of the antiserum was demonstrated by immunoelectrophoresis (Laurell rocket and Grabar methods) and, thus, the homogeneity of the kininogen was also. The purified kininogen (both Fractions A and B) formed kinin with human urinary kallikrein, rat urinary kallikrein and hog pancreatic kallikrein. Murphy-Sturm lymphosarcoma acid protease also formed kinin when incubated with the kininogen at pH 3.0. The isoelectric point for both fractions was at pH 4.3. Amino acid analyses showed the two kininogen fractions to be rich in acidic amino acids and to have a total carbohydrate content of 8.5% consisting of galactose (1.2 to 1.5%), mannose (1.9 to 2.1%), N-acetylglucosamine (4.3 to 5.1%), N-acetylgalactosamine (0.3%), and sialic acid (0.68%).
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D055598:Chemical Phenomena; D011232:Chemical Precipitation; D002621:Chemistry; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007122:Immunoelectrophoresis; D007525:Isoelectric Focusing; D007704:Kininogens / Q000097:blood* / Q000302:isolation & purification; D007705:Kinins / Q000378:metabolism; D008970:Molecular Weight; D051381:Rats
D000596:Amino Acids; D002241:Carbohydrates; D007704:Kininogens; D007705:Kinins