Purification and characterization of rat low molecular weight kininogen.
Low molecular weight (LMW) kininogen was isolated from pooled rat plasma by chromatography on DEAE-Sephadex A-50, CM-Sephadex C-50, Blue-Sepharose CL-6B, and Sephadex G-100. It was shown to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoelectrophoresis. The molecular weight of rat LMW kininogen was determined to be 72,000 by SDS-PAGE. The LMW kininogen contained 83.5% protein, 4.0% hexose, 5.5% hexosamine, and 2.7% sialic acid. Kinin liberated from LMW kininogen by trypsin treatment was identified as an Ile-Ser-bradykinin(T-kinin) by analysis involving ion exchange column chromatography on CM-Sephadex C-25 and high performance liquid chromatography on a reverse-phase column (ODS-120T). LMW kininogen formed kinin with rat submaxillary gland kallikrein, but the kinin liberated was only 14% of the total kinin content, that is, that released by trypsin. In order to determine the immunochemical properties of LMW kininogen, specific antiserum was prepared in rabbits. The antiserum cross-reacted with high molecular weight (HMW) kininogen, but spur formation was observed between the LMW and HMW kininogens. The kininogen level in rat plasma was estimated to be 433 microgram/ml by a quantitative single radial immunodiffusion test.
D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D002852:Chromatography, Ion Exchange; D005341:Fibrinolysin / Q000378:metabolism; D006801:Humans; D005779:Immunodiffusion; D007610:Kallikreins / Q000378:metabolism; D007704:Kininogens / Q000097:blood* / Q000302:isolation & purification; D007705:Kinins / Q000378:metabolism; D008297:Male; D008832:Microchemistry; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains; D014357:Trypsin / Q000378:metabolism
D007704:Kininogens; D007705:Kinins; D007610:Kallikreins; D014357:Trypsin; D005341:Fibrinolysin