Inactive proenzyme to tissue-type plasminogen activator from human melanoma cells, identified after affinity purification with a monoclonal antibody.
The human 66 000 mol. wt. plasminogen activator (HPA66; tissue-type plasminogen activator) has been purified from melanoma cells by a one-step affinity method with a monoclonal antibody. HPA66 purified in this way consists mainly of a one-polypeptide chain form with small amounts (15%) of a form containing two polypeptide chains held together by one or more disulphide bridges. The one-chain form was converted to the two-chain form by catalytic amounts of plasmin. During the conversion, the enzyme activity of HPA66, as measured by an [125I]plasminogen conversion assay and with a chromogenic substrate, increased linearly with the percentage of the two-chain form. A linear regression analysis showed that all enzyme activity could be accounted for by the two-chain form, while the one-chain form had no measurable enzyme activity (detection limit approximately 5% of the activity of the two-chain form). Together with previous findings of inactive proenzymes to murine and human approximately 50 000 mol. wt. (urokinase-type) plasminogen activators, these findings indicate that plasminogen activators are generally formed from inactive one-chain proenzymes which are converted to active two-chain enzymes by limited proteolysis, thus demonstrating a third step in a cascade reaction leading to extracellular proteolysis.
D000911:Antibodies, Monoclonal; D002460:Cell Line; D002846:Chromatography, Affinity; D004792:Enzyme Precursors / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D008545:Melanoma / Q000378:metabolism*; D008970:Molecular Weight; D010958:Plasminogen / Q000378:metabolism; D010960:Plasminogen Activators / Q000302:isolation & purification / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D004792:Enzyme Precursors; D010958:Plasminogen; D010960:Plasminogen Activators