Biochemical actions of fibroblast kinin-forming protease.
Purification and further characterization of a kinin-forming acid protease in a mouse fibroblast L929 stationary cell culture line was carried out. Supernatants of dialyzed fibroblast homogenates digested denatured hemoglobin at pH 4.0. The supernatant was fractionated on a G-200 Sephadex column, hydroxylapatite column and finally on a DEAE-A50 Sephadex ion exchange column. A 9.4 fold purification was achieved with a 13.8% yield. The enzyme had a specific activity of 2062 ng kinin per mg protein when measured on a purified rat kininogen using the isolated rat uterus as the bioassay tissue. The protease had a pH optimum of 3.8-4.0. Molecular weights of the enzyme and substrate estimated on a G-200 Sephadex column were 39,000 and 110,000 respectively. Kinin formation was a function of both incubation time and enzyme concentration. Protease activity was localized primarily in the 10,000 g supernatant cell fraction (61.5%) with the 1500 g precipitate cell fraction containing 38.5% of the activity.
D002460:Cell Line; D002850:Chromatography, Gel; D003545:Cysteine / Q000494:pharmacology; D004789:Enzyme Activation; D005347:Fibroblasts / Q000201:enzymology*; D007704:Kininogens / Q000378:metabolism; D007705:Kinins / Q000096:biosynthesis*; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000302:isolation & purification / Q000378:metabolism*
D007704:Kininogens; D007705:Kinins; D010447:Peptide Hydrolases; D003545:Cysteine