Purification of zymogen to plasminogen activator from human glioblastoma cells by affinity chromatography with monoclonal antibody

Abstract

Incorporation of the serine protease active site reagent DFP into a plasminogen activator with an MW of .apprx. 52,000 released from cultured human glioblastoma cells was strongly enhanced by incubation with plasmin. This observation led to the isolation of an inactive form of the enzyme from serum-free conditioned culture fluid by affinity chromatography on a column of a Sepharose-bound monoclonal antibody raised against urokinase. An 831-fold purification was obtained with a yield of 41%. The purified molecule was homogeneous as evaluated by polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS), having 1 stainable band under nonreducing as well as reducing conditions with an MW of .apprx. 52,000. It was unable to activate plasminogen, but catalytic amounts of plasmin converted it into active enzyme. After SDS-polyacrylamide gel electrophoresis, the active enzyme showed 1 band under nonreducing conditions, but after reduction, 2 bands with MW values of .apprx. 20,000 and 32,000 were observed. The active enzyme incorporated [3H]DFP into the .apprx. MW 32,000 band, while no incorporation was observed into the inactive form. The MW 52,000 human plasminogen activator exists in a proenzyme form consisting of a single polypeptide chain that by proteolysis between half-cystine residues is converted into the active enzyme consisting of 2 chains with MW of .apprx. 20,000 and 32,000, the active site being on the latter chain. The results are consistent with the active form of the enzyme being identical with the higher MW form of urokinase, and together with recent observations that a murine plasminogen activator is released from sarcoma virus transformed cells as an inactive proenzyme, they suggest that zymogens to plasminogen activators are of more general occurrence.