Purification and Characterization of the Plasminogen Activator Secreted by a Rat Brain Tumor Cell Line in Culture

Summary

The plasminogen activator secreted by a cultured rat brain tumor cell line (RT4-71-1) (Imada M. and Sueoka N., Develop.Biol. 69, 97-107, 1978) was purified by chromatography on zinc chelate-agarose, concanavalin A-agarose and Sephadex G-150 in the presence of 0.01% (vol/vol) Tween 80. Aprotinin was added to the culture medium to a concentration of 20 KIU per ml and to the buffers in the first two chromatographic steps to a concentration of 10 KIU per ml. Approximately 90 μg purified material was obtained from 1 1 of culture medium with a yield of 39% and a purification factor of 200. Sodium dodecylsulfate-polyacryl amide gel electrophoresis in the presence of reducing agents showed one main band with M _r of about 60,000, and a minor band with M _r about 30,000. Fibrinolytic activity was associated with the main band. The rat brain tumor plasminogen activator bound to a fibrin clot to a similar extent as human tissue plasminogen activator, whereas urokinase did not bind. In quenching experiments of the fibrinolytic activities the purified rat brain tumor plasminogen activator appeared to be immunologically related to the human tissue plasminogen activator but unrelated to urokinase.

Keywords

Rat brain tumor plasminogen activator - Tissue plasminogen activator - Cell culture - Thrombolysis

• References

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