Purification and characterization of kinin-forming acid protease from mouse fibroblasts L-929

Purification and further characterization was carried out on a kinin-forming acid protease isolated from a rodent fibroblast cell line L-929 grown in stationary cell culture (N. Back and R. Steger,[7]). The cells, cultured in minimal essential medium containing 10% fetal calf serum and 0.4% lactalbumin, were homogenized, the homogenate dialyzed for 18 hr against 0.01 M phosphate buffer at pH 6.8 in 0.1 M NaCl and 1.0 mM EDTA, and centrifuged at 10000 rpm for 45 min. The supernatant, which digested denatured hemoglobin at pH 4.0, was fractionated first on a G-200 Sephadex column. Kinin-forming activity, compared with that of the supernatant on an isolated perfused rat uterus preparation, was identified in fractions 25–40 when incubated for 24 hr at pH 4.0 with rat plasma kininogen substrate. The active fractions were pooled and purified further on a hydroxy-patite column. Treatment of the active fractions with 5 mM cysteine increased the activity 2-fold. Final purification was carried out on a DEAE-A50 Sephadex ion exchange column. The purification factor, compared to the initial supernatant, was 9.4 with a 13.8% yield and a specific activity of 2062.5 ng kinin per mg protein. Dialyzed and centrifuged rat plasma fractionated on a DEAE-A50 Sephadex column initially yielded two apparent kininogen species which resolved into a single major molecular species following passage through a G-100 Sephadex column. The purified enzyme and substrate preparations were used to establish the optimum kinin-forming activity at pH 3.8–4.0. The molecular weights of the enzyme and kininogen were estimated on a G-200 Sephadex column to be 38000–39000 and 115000 respectively. The amount of kinin formed was a function of incubation time and enzyme concentration. The acid protease activity was found localized primarily in the 10000 g supernatant cell fraction. The 500 g cell fraction also exhibited activity.