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Abstract
A human hepatoma cell line, Hep G2, which synthesizes and secretes several components
of the fibrinolytic system, was examined for the capacity to produce plasminogen activator
(PA). PA activity was found to accumulate in medium conditioned by the cells but was
not detected in cell extracts. Analysis of Hep G2 conditioned medium by SDS-polyacrylamide
gel electrophoresis revealed multiple PAs of Mr 100,000, 60,000, and 52,000 daltons. In addition, a heterogeneous band of activity
was present between the 100,000 and 60,000 dalton PAs. All detectable PA activity
in conditioned medium fractionated at a single position after isoelectric focusing.
The isoelectric point of this material (pl 8.6) was identical to that of purified
urokinase. Anti-urokinase IgG, but not anti-tissue activator IgG, neutralized the
activity of these PAs. Treatment of conditioned medium with 25 mM diisopropylfluorophosphate
for 5 hr at 37 °C inactivated the 52,000 and 60,000 dalton PAs but had no effect on
the 100,000 dalton PA or the heterogeneous zone of activity. The possibility that
the absence of detectable PA activity in the cell extracts resulted from the presence
of fibrinolytic inhibitors was considered. Addition of cell extract to purified urokinase
resulted in a concentration-dependent reduction of urokinase-mediated fibrinolytic
activity, consistent with the presence of such inhibitors. Acidification of the cell
extracts to inactivate the inhibitor(s) revealed the presence of a latent, cell-associated
PA activity. Thus, in addition to the other major components of the fibrinolytic system,
Hep G2 cells produce multiple forms of a urokinase-like PA. Detection of these PAs
may be obscured by the presence of cellular inhibitors.
Abbreviations:
plasminogen activator ((PA)), tissue PA ((t-PA)), minimal essential medium ((MEM)), phosphate-buffered saline ((PBS)), conditioned medium ((CM)), diisopropylfluorophosphate ((DFP)), sodium dodecyl sulfate-polyacrylamide gel electrophoresis ((SDS-PAGE)), molecular weight or relative molecular mass ((Mr)), isoelectric point ((pI))To read this article in full you will need to make a payment
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Article info
Publication history
Accepted:
May 31,
1983
Received:
February 14,
1983
Footnotes
☆Supported by NIH Research Grant 16411.
☆☆Publication No. 2864 from the Immunology Department of the Research Institute of Scripps Clinic.
Identification
Copyright
© 1983 Published by Elsevier Inc.
