PURIFICATION AND CHARACTERIZATION OF THE PLASMINOGEN-ACTIVATOR INHIBITORS PAI-1, PAI-2, AND PN-1 FROM THE HUMAN GLIOBLASTOMA U138

This investigation presents data which indicate that the plasminogen a ctivator inhibitor (PAI) activity secreted from U138 cells is composed of three separate PAIs: PAI-1, PAI-2, and PN-1. It was demonstrated t hat the U138 PAI-1-like protein had an apparent molecular mass of 50 k ilodaltons (kDa) and was purified to apparent homogeneity by elution f rom an anti-PAI-1 immunoaffinity column. These fractions were also rea ctive with a second anti-PAI-1 monoclonal antibody using immunoblottin g techniques. Northern blot analysis of RNA isolated from unstimulated U138 cells demonstrated positive hybridization with the cDNA specific for human PAI-1. The U138 PAI-2-like protein was adherent to an anti- PAI-2 immunoaffinity column and was demonstrated to be nonadherent to concanavalin A - agarose, heparin-Sepharose, and the anti-PAI-1 immuno affinity column. The eluted U138 PAI-2-like protein was demonstrated t o have an apparent molecular num of 60 kDa and was also reactive with a second anti-PAI-2 monoclonal antibody using immunoblotting technique s. Further, the cDNA specific for PAI-2 was demonstrated to hybridize to a 2.5-kilobase message from RNA isolated from U138 cells. A third P AI was detected that was nonadherent to concanavalin A - agarose and b oth of the anti-PAI columns. This 50-kDa PAI was adherent to heparin-S epharose and thrombin-agarose columns, and was not reactive with any a ntibodies for either PAI-1 or PAI-2. Northern blot analysis of U138 RN A demonstrated positive hybridization with an oligodeoxynucleotide spe cific for PN-1. This investigation demonstrates with biochemical, immu nological, and molecular data that the U138 glioblastoma constitutivel y produces three PAIs.