Quantification, isolation and structural determination of bradykinin and hydroxyprolyl-bradykinin in tumor ascites.
The presence of kinins in ascitic tumor fluids from rodents and human patients was identified and quantified. In bioassay, kinin content was found to be 1 to 40 ng/ml, and by enzyme immunoassay, 0.6 to 2.5 ng/ml. In particular, a high kinin content, 40 ng/ml, was found in the ascites of a gastric cancer patient by bioassay. Purification of this kinin in the ascites from the gastric cancer patient was performed by ethanol precipitation, gel filtration and reversed-phase high-performance liquid chromatography (HPLC). Two peaks (peak A and peak B) showed kinin activity. Peak A did not correspond to either bradykinin or other known kinins, such as lysyl-bradykinin and T-kinin, whereas peak B corresponded to bradykinin. Peak A contained 8 amino acid residues from bradykinin minus one proline plus an additional hydroxyproline. Sequence analysis of peak A showed that the proline at the third amino acid residue of bradykinin was replaced by hydroxyproline. The retention time of peak A on reversed-phase HPLC was exactly the same as that of synthetic hydroxyprolyl3-bradykinin (Hyp3-bradykinin) but was distinguishable from des-Pro3-bradykinin. Thus, these results demonstrate for the first time the presence of Hyp3-bradykinin in mammalian system.
Matsumura
Y
Y
Department of Microbiology, Kumamoto University Medical School, Japan.
Kimura
M
M
Kato
H
H
Yamamoto
T
T
Maeda
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001202:Ascitic Fluid / Q000032:analysis*; D001920:Bradykinin / Q000302:isolation & purification*; D006801:Humans; D007609:Kallidin / Q000031:analogs & derivatives* / Q000302:isolation & purification; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D009374:Neoplasms, Experimental / Q000503:physiopathology*; D051381:Rats; D011919:Rats, Inbred Strains; D012513:Sarcoma, Experimental / Q000503:physiopathology; D013274:Stomach Neoplasms / Q000503:physiopathology*
C054550:bradykinin, (hydroxy-Pro)(3)-Lys-; D007609:Kallidin; D001920:Bradykinin