Immunological quantitation and immunoadsorption of urokinase-like plasminogen activators secreted by human cells.

Abstract

A highly purified preparation of human urinary plasminogen activator (urokinase) was used to produce specific rabbit urokinase antiserum.The urokinase used for rabbit immunizations contained both M, = 55,000 and 60,000 protein components.The previously unreported M, = 60,000 component is apparently another high molecular weight form of urokinase, since it can activate plasminogen and is indistinguishable in peptide composition from M, = 55,000 urokinase.The urokinase antiserum and 12'I-labeled urokinase have been used to develop a urokinase competition radioimmunoassay which quantitatively determines urokinase in the 3 to 20 ng/sample range.The urokinase competition radioimmunoassay has been used to quantitate urokinase-like plasminogen activator present in conditioned media from several different human cells.A specific antiurokinase immunoglobulin (1g)-Sepharose column was prepared which selectively binds two 50,000 to 60,000 molecular weight proteins produced by Detroit 562 (human pharyngeal carcinoma) and IMR-90 (human embryonic lung) cells in uitro.These two proteins appeared identical with high molecular weight urokinase on the basis of molecular weight, ability to activate plasminogen, and immunological reactivity.Specific antiurokinase Ig-Sepharose did not bind any M, = 73,000 protein which would correspond to the M, = 73,000 plasminogen activator produced by Detroit 652 and IMR-40 cells.In addition, M, = 73,000 plasminogen activator activity produced by other human cells did not react in the urokinase competition radioimmunoassay.This indicates that M, = 73,000 plasminogen activator is not specifically bound by urokinase antibodies and thus does not share common antigenic determinants with urokinase.Urokinase (EC 3.4.99.26), a plasminogen activator found in human urine, has been isolated in both M , = 54,OOO (1,2) and 33,000 (2) forms.The M, = 33,000 form can be produced from the larger form by limited in vitro proteolysis (3), suggesting that a similar mechanism may be responsible for producing M , = 33,000 enzyme in vivo.Both urokinase forms are active site serine-dependent proteases (4) which convert the inactive zymogen plasminogen to the potent, broad spectrum protease Human ovarian carcinoma cells have been shown to pro-