A
urokinase-type plasminogen activator was purified from
conditioned media of several human cell cultures, but preferably from the human
lung adenocarcinoma line CALU-3 (ATCC, HTB-55), using a combination of chromatography on
zinc chelate-Sepharose, SP-
Sephadex C-50, and
Sephadex G-100. Final yields of 65-100 micrograms/liter of starting material were obtained with a 290-fold purification factor and a recovery of 30%. The purified
plasminogen activator consists of a single
polypeptide chain with Mr 54,000 as determined by
sodium dodecyl sulfate-
polyacrylamide gel electrophoresis and is very similar or identical to
single-chain urokinase-type plasminogen activator on the basis of immunodiffusion,
amino acid composition, and the lack of specific binding to
fibrin. It has very low amidolytic activity on Pyroglu-Gly-Arg-rho-nitroanilide and is converted to two-chain
urokinase by limited exposure to
plasmin. It has a specific activity of 60,000 IU/mg on
fibrin plates and directly activates
plasminogen following Michaelis-Menten kinetics with Km = 1.1 microM and kappa cat = 0.0026 S-1. It is concluded that the
plasminogen activator purified from CALU-3-conditioned media is physically and kinetically identical to
single-chain urokinase-type plasminogen activator. With the present straightforward purification method and a readily available source, sufficient amounts of
single-chain urokinase-type plasminogen activator can be obtained for more detailed investigations of its biochemical,
biological, and thrombolytic properties.