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Messenger RNA for Human Tissue Plasminogen Activator

OPDENAKKER, Ghislain, WEENING, Hans, COLLEN, Désiré, BILLIAU, Alfons, SOMER, Piet
European journal of biochemistry 1982 v.121 no.2 pp. 269-274
affinity chromatography, bioactive properties, centrifugation, cross reaction, humans, melanoma, messenger RNA, molecular weight, neutralization, oocytes, phenol, plasminogen, polyacrylamide gel electrophoresis, precipitin tests, sodium dodecyl sulfate, sucrose, t-plasminogen activator
A human melanoma cell line (Bowes), which secretes extrinsic (tissue‐type) plasminogen activator, was used as a source for the preparation of mRNA for extrinsic plasminogen activator. The cells were lysed and total RNA was extracted with the phenol method. A poly(A)‐rich RNA fraction was isolated by affinity chromatography on oligo(dT)‐cellulose. This preparation was translated by oocytes into (a) protein(s) that had biological activity in the assay for extrinsic plasminogen activator. On sucrose gradient centrifugation the translatable fraction of the RNA migrated with a sedimentation coefficient of approximately 19 S, a value compatible with the molecular weight of extrinsic plasminogen activator (70000). The translation product was characterized as being similar to or identical with authentic extrinsic plasminogen activator by the following criteria: (a) serological cross‐reactivity with purified extrinsic plasminogen activator in neutralization and immunoprecipitation reactions; (b) plasminogen dependency of fibrinolytic activity and (c) apparent molecular weight of 70000 on sodium dodecyl sulphate/polyacrylamide gel electrophoresis.