Inactive proenzyme to tissue-type plasminogen activator from human melanoma cells, identified after affinity purification with a monoclonal antibody.

Abstract

The human 66,000 MW plasminogen activator (HPA66; tissue-type plasminogen activator) was purified from melanoma cells by a 1-step affinity method with a monoclonal antibody. HPA66 purified in this way consists mainly of a 1-polypeptide chain form with small amounts (15%) of a form containing 2 polypeptide chains held together by 1 or more s-s bridges. The 1-chain form was converted to the 2-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 2-chain form. A linear regression analysis showed that all enzyme activity could be accounted for by the 2-chain form, while the 1-chain form had no measurable enzyme activity (detection limit .apprx. 5% of the activity of the 2-chain form). Together with previous findings of inactive proenzymes to murine and human .apprx. 50,000 MW (urokinase-type) plasminogen activators, these findings indicate that plasminogen activators are generally formed from inactive 1 chain proenzymes which are converted to active 2-chain enzymes by limited proteolysis, demonstrating a 3rd step in a cascade reaction leading to extracellular proteolysis.