Plasminogen activator released as inactive proenzyme from murine cells transformed by sarcoma virus.
We have previously reported the purification of a plasminogen-activating serine protease with an approximate Mr of 48 000 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 polyacrylamide gel electrophoresis with sodium dodecylsulphate under reducing and non-reducing conditions showed that the inactive form consisted of a single polypeptide chain with an Mr of approximately 48 000, while the active form consisted of two chains with Mr values of approximately 18 000 and 29 000 , held together by one or more disulphide bridges. The active-site reagent diisopropylfluorophosphate in radiolabelled form was incorporated into the 29 000-Mr 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 and additional step in a cascade-like reaction leading to extracellular proteolysis. Regulatory as well as methodological implications of this findings are discussed.
D000818:Animals; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D004792:Enzyme Precursors / Q000235:genetics* / Q000302:isolation & purification; D005341:Fibrinolysin / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008970:Molecular Weight; D010960:Plasminogen Activators / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D009053:Sarcoma Viruses, Murine / Q000235:genetics*
D004792:Enzyme Precursors; D010960:Plasminogen Activators; D005341:Fibrinolysin