In vitro biosynthesis of plasminogen in a cell-free system directed by mRNA fractions isolated from monkey liver

Abstract

MRNA was isolated from total RNA of monkey liver by oligo(dT)-cellulose chromatography and was translated in a rabbit reticulocyte cell-free system. Analysis of the translation products immunoprecipitated with specific antibodies to monkey plasma plasminogen revealed a molecule with characteristics similar to those of native plasminogen. The purification of the mRNA by centrifugation on sucrose gradients indicated the presence of plasminogen mRNA in both the 23S and 18S RNA fractions. Both plasminogen mRNA can be further purified by chromatography on Sepharose 4B. Affinity chromatography of the proteins synthesized in vitro by total mRNA from liver, as well as by the purified mRNA, on L-lysine-substituted Sepharose revealed that both major plasma plasminogen forms (1 and 2) are synthesized, as precursors, in the system. The in vitro synthesized plasminogen is similar in its physical and chemical properties to native plasma plasminogen as determined by its ability to bind to L-lysine-substituted Sepharose and its molecular interaction with streptokinase. The purified mRNA were also translated in the presence of dog pancreas microsomal membranes, and the proteins sequestered inside the membranes were released and fractionated on concanavalin A-Sepharose. The 23S mRNA directed the synthesis of a plasminogen molecule similar to the circulating plasma plasminogen form 1, whereas the 18S mRNA directed the synthesis of a molecular similar to the circulating plasma plasminogen form 2. Evidently, the synthesis of the 2 major circulating plasma plasminogen forms is directed in the liver by separate mRNA.