Characterization of bovine prothrombin mRNA and its translation product

Abstract

Prothrombin mRNA was enriched 20- to 60-fold by using specific immunoadsorption of bovine liver polysomes. The enriched mRNA was translated in a cell-free protein synthesizing system derived from rabbit reticulocytes, and the radiolabeled translation product was isolated by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The radiolabeled prothrombin synthesized in the cell-free system was then subjected to automated Edman degradation and shown to contain a leader sequence of at least 30 residues that was rich in leucine, phenylalanine and alanine. To fully characterize the leader sequence for prothrombin, a bovine liver c[complementary]DNA library was constructed containing DNA inserts of over 1000 base pairs. Two cDNA clones coding for bovine prothrombin were isolated from the library and their nucleotide sequences determined. A leader sequence of 43 amino acids was predicted from the sequence of the cDNA, and the first 30 residues were in agreement with the partial sequence obtained by the cell-free protein synthesizing system. From the amino acid sequence of the leader sequence, it is proposed that bovine prothrombin is synthesized with a prepro leader sequence starting with a methionine residue at position -43. The amino acid sequence of the mature prothrombin molecule circulating in plasma was also predicted from the cDNA and in good agreement with that determined previously by conventional amino acid sequence analysis.