PMID: 6539333Jun 10, 1984Paper

Isolation and characterization of a urokinase-type plasminogen activator (Mr = 54,000) from cultured human endothelial cells indistinguishable from urinary urokinase.

The Journal of Biological Chemistry
F M BooyseJ Scheinbuks

Abstract

A urokinase-type plasminogen activator secreted by subcultured normal human umbilical vein endothelial cells was purified and compared to urinary urokinase (Mr = 54,000). The enzyme was isolated from serum-free conditioned medium in the presence of 0.1% (v/v) Triton X-100 by p-aminobenzamidine-agarose affinity chromatography, followed by Sephacryl S-200 gel filtration, followed by immunoadsorption chromatography on affinity purified specific anti-urokinase IgG-Sepharose CL-4B. This plasminogen activator form was obtained from the culture medium with a yield of about 47% and specific activity of about 93,000 IU/mg of protein, and represented approximately 18% of the total multiple molecular plasminogen activator activity forms present in endothelial cell conditioned medium. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single band of plasminogen activator activity with an estimated molecular weight of about 54,000 that was completely inhibited by diisopropyl fluorophosphate (DFP) as well as a single band of radioactivity with similar molecular weight for both the isolated L-[4,5-3H]leucine and [3H]DFP-labeled enzyme. The radiolabeled protein focused as a single major band with a pI val...Continue Reading

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