Biochemical and physiological studies on two T-kininogen species using monoclonal antibodies.
Rat T-kininogens were characterized in plasma, urine and liver slice incubation medium in different physiological conditions using monoclonal antibodies that allow to distinguish between the two forms of T-kininogen. T-kininogen purified from the plasma of both normal and inflamed Wistar and Sprague-Dawley rats was found to contain the two forms of T-kininogen, TI and TII, separated by non-denaturing polyacrylamide gel electrophoresis. The two forms were also found in the plasma of several strains of normal and inflamed rats, except in the plasma of the Buffalo rat which contained only TII-kininogen. The two forms of T-kininogen were also found in the media in which liver slices from inflamed and non-inflamed wistar rats had been incubated. The urine T-kininogen of normal rats was chiefly TI-kininogen, but both forms were found in the urine of inflamed rats. T-kininogen in the plasma of normal and inflamed rats was further characterized by chromatography on Con A-Ultrogel. In normal plasma, we observed a ratio of non-retained to retained T-kininogen of 0.41. The retained T-kininogen was eluted as two peaks, one eluted with 45 mM and the other with 120 mM alpha-methyl-D-glucoside. The ratio of non-adsorbed to adsorbed T-kininogen in inflamed Wistar rat plasma was 1.40 and the retained material was almost exclusively in a single peak, which eluted with 50 mM alpha-methyl-D-glucoside. The non-adsorbed and adsorbed fractions contained both forms of T-kininogen, but the protein bands in the non-retained fraction had greater mobilities on non-denaturing PAGE. SDS-PAGE analysis of T-kininogen deglycosylated by N-glycosidase F showed a major band with a molecular mass of 50 kDa, whereas the molecular mass of the native form was 66 kDa. We concluded that both forms of T-kininogen exist in the non-inflamed and the inflamed rat plasma, except for the Buffalo rat, and that T-kininogen displays a different chromatographic pattern on Con A-Ultrogel after inflammation suggesting altered glycosylation.
Bouhnik
J
J
INSERM U 367, Paris, France.
Baussant
T
T
Savoie
F
F
Lesage
S
S
D000581:Amidohydrolases; D000818:Animals; D000911:Antibodies, Monoclonal; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006031:Glycosylation; D007249:Inflammation / Q000097:blood / Q000652:urine; D007704:Kininogens / Q000032:analysis* / Q000097:blood / Q000302:isolation & purification; D008099:Liver / Q000201:enzymology; D008297:Male; D008757:Methylglucosides; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D051381:Rats; D011919:Rats, Inbred Strains
D000911:Antibodies, Monoclonal; D007704:Kininogens; D008757:Methylglucosides; C027020:methylglucoside; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase