Plasminogen Activator Released as Inactive Proenzyme from Murine Cells Transformed by Sarcoma Virus

We have previously reported the purification of a plasminogen‐activating serine pro1 ease with an approximate M r of 48000 from sarcoma‐virus‐transformed murine cells. We now report that under serum‐free conditions the enzyme is released from the cells in an inactive form. After affinity chromatography with 4‐aminobenzamidine‐cellulose, ion‐exchange chromatography and gel filtration, the proenzyme could be obtained from culture fluid as a pure, homogeneous protein as evaluated by polyacrylamide gel electrophoresis with sodium dodecylsulphate. Proenzyme was quantitatively converted to active enzyme by incubation with catalytic amounts of plasmin. Analysis by polyacrylainide gel electrophoresis with sodium dodecylsulphate under reducing and non‐reducing conditions showed that the inactive form consisted of a single polypeptide chain with an M r of approximately 48000, while the active form consisted of two chains with M r values of approximately 18000 and 29000, held together by one or more disulphide bridges. The active‐site reagent diisopropylfluorophosphate in radiolabelled form was incorporated into the 29000‐ M r chain of thc active enzyme, but not into the inactive form. These findings provide conclusive evidence for the existence of an inactive proenzyme to this. plasminogen activator and thus demonstrate an additional step in a cascade‐like reaction leading to extrac ellular proteolysis. Regulatory as well as methodological implications of this finding are discussed.