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

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