Differential in vitro translation and independent in vivo regulation of mRNA's for subunits of ligandin

Abstract

Synthesis of both subunits (Y a and Y b) of ligandin in equal amounts was observed when poly(A) + mRNA isolated from the post-mitochondrial fraction was translated in an in vitro wheat-germ system and the products were immunoprecipitated by monospecific antibody to ligandin and analyzed by SDS-polyacrylamide gel electrophoresis and fluorography. When the Mg 2+ or K + concentrations were increased in the in vitro wheat-germ system the ratio of synthesis of Y b Y a subunits was 3. With a mRNA-dependent reticulocyte lysate, the synthesis of Y a subunits was 20–30% higher than Y b subunits. At a fixed K + and Mg 2+ concentration, the ratio of incorporation of [ 35S]methionine into Y b Y a subunits remained 1 and 0.7 in wheat-germ and reticulocyte lysate systems, respectively, up to 60 min. When poly(A) + mRNA was fractionated on a 5–20% sucrose gradient, ligandin mRNA was present in fractions having a peak sedimentation value of 11 S. When poly(A) + mRNA was fractionated by gel electrophoresis, fractions enriched in mRNA for each subunit were obtained. By administration of [ 3H]leucine followed by determination of radioactivity in ligandin and total proteins by immunoprecipitation and trichloroacetic acid precipitation, respectively, synthesis of the Y a subunits was selectively stimulated by phenobarbital administration. When poly(A) + mRNA from liver of rats administered phenobarbital was translated in vitro a selective increase in the mRNA content of Y a subunits was observed. When poly(A) + RNA from testes was translated in the wheat-germ system and products analyzed, Y b subunits were the predominant subunit (> 90%) synthesized, reflecting the subunit composition of testicular ligandin. These results suggest that in spite of the close sequence homology between the two subunits of ligandin, there are separate mRNA's for each subunit which are independently regulated.