The kinin-forming acid protease system in murine fibroblasts L-929.
Components of an acid protease kinin-forming enzyme system were isolated and purified from the murine fibroblast L-929 cell line grown in stationary cell culture. The enzyme, was purified from the 10,000 g supernatant cell fraction by sequential passage through G-200 Sephadex, hydroxylapatite, DEAE-A50 Sephadex and affinity chromatography columns. The specific activity was determined on a rat plasma kininogen purified on DEAE and G-100 Sephadex columns as assayed for kinin-forming activity on the isolated perfused rat uterus. Both enzyme and substrate showed a single band pattern by disc gel electrophoresis technique. Optimum protease activity was obtained at pH 3.8, and kinin release was both time- and enzyme-dependent at 37 degrees. The molecular weight of the protease and kininogen, estimated on a G-200 Sephadex column, was 39,000 and 115,000 respectively. Two fibroblast kinins were isolated from a mixture of 12,000 g fraction of fibroblast homogenate and rat plasma incubated at 37 degrees, pH 4.0 for 92 hours. The two fibroblast kinins (I, II) were separated and purified on G-25 Sephadex, CM-C50 Sephadex, and Biogel P-4 columns. The purity of the kinins was ascertained by thin layer chromatography. The molecular weight of fibroblast kinin I was estimated to be 1450 with a 14-amino acid composition consisting of AspThrSerProGluGlyAlaVal-LeuTyrPheLysHisArg. Fibroblast kinin II had an estimated molecular weight of 1000 with a 12-amino acid composition that included AspSerProGluGlyAlaLeuTyrLysHisArg. The kinins were relatively rich in arginine and did not contain the bradykinin sequence.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D005347:Fibroblasts / Q000378:metabolism; D007704:Kininogens / Q000097:blood; D007705:Kinins / Q000302:isolation & purification / Q000378:metabolism*; D007739:L Cells / Q000201:enzymology*; D051379:Mice; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000378:metabolism*
D000596:Amino Acids; D007704:Kininogens; D007705:Kinins; D010447:Peptide Hydrolases