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

Abstract

The purification of a plasminogen-activating serine protease with a MW of .apprx. 48,000 from sarcoma-virus-transformed murine cells was previously reported. 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 dodecyl sulfate [SDS-PAGE]. Proenzyme was quantitatively converted to active enzyme by incubation with catalytic amounts of plasmin. Analysis by SDS-PAGE under reducing and non-reducing conditions showed that the inactive form consisted of a single polypeptide chain with a MW of .apprx. 48,000, while the active form consisted of 2 chains with MW values of .apprx. 18,000 and 29,000, held together by > 1 disulfide bridges. The active-site reagent DFP in radiolabeled form was incorporated into the 29,000 MW chain of the 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 extracellular proteolysis. Regulatory and methodological implications of this finding are discussed.