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.