Biosynthesis Of Human Fibrinogen In A Cell Free System

In order to investigate the early events involved in the biosynthesis of human fibrinogen mRNA was isolated from human liver and translated in a rabbit reticulocyte system. The translation was carried out in the presence of ³⁵S-Methionine. Neosynthesized fibrinogen was isolated by immuno- adsorption on microcolumns of anti-fibrinogen IgG coupled to ultrogel and analyzed by vertical slab polyacrylamide gel electrophoresis followed by autoradiography of the gel. Additional identification was achieved by immunological competition with non labeled plasmatic fibrinogen. The results indicated that each polypeptide chain was synthesized separately. The neosynthesized Aα chain exhibited a higher molecular weight that the plasmatic Aα chain. Based on electrophoretic mobility a difference in molecular weight of 2.000 dalton was estimated suggesting the existence of a signal sequence on the nascent Aα chain. The neosynthesized Bβ and γ chains showed lower molecular weight, when compared to the corresponding plasmatic Be and y chains. Since the Bβ and the γ chains are normally glycosylated, the presence of a signal peptide on the nascent chains was not definitely established from electrophoretic mobility alone. In addition total human mRNA were partially fractionated on a sucrose gradient and the different fractions were translated. The precursor of the Aα, Bβ and γ chains were synthesized by mRNA present in the 18 S fraction. These observations established that human fibrinogen was not synthesized as a single chain large precursor and that separate mRNA exist for each polypeptidic chain.