Cell‐free synthesis of rat α2‐macroglobulin and induction of its mRNA during experimental inflammation

Abstract

Poly(A)-rich RNA was isolated from the livers of acutely inflamed rats by extraction with guanidinium HCl and oligo(dT)-cellulose chromatography. After translation in a recticulocyte lysate and immunoprecipitation with a specific antiserum to .alpha.2-macroglobulin, a polypeptide with an apparent MW of 162,000 could be detected. The cell-free synthesis of .alpha.2-macroglobulin was stimulated 8-fold by the addition of RNase inhibitor. Full-length of .alpha.2-macroglobulin polypeptide chains appeared after 35 min in the presence of 1.85 mM Mg2+ and 100 mM K+. A nucleotide number of about 5100 was estimated for .alpha.2-macroglobulin by means of sucrose gradient centrifugation of poly(A)-rich RNA followed by translation in vitro and immunoprecipitation of .alpha.2-macroglobulin. In normal liver .alpha.2-macroglobulin mRNA represented about 0.0007% of total translatable RNA. Acute inflammation generated by intramuscular injection of turpentine led to a 66-fold increase in translatable .alpha.2-macroglobulin mRNA after 18 h, followed by a rapid decrease. In accordance to the induction of .alpha.2-macroglobulin mRNA, serum concentrations of .alpha.2-macroglobulin increased to about 2 mg/ml. Unlike .alpha.2-macroglobulin mRNA, serum .alpha.2-macroglobulin levels remained unchanged up to 60 h.