Co-ordinate synthesis of ribosomes and elongation factors in the liver of immature chicks following a metabolic shift up

Abstract

Treatment of immature chicks with estradiol results in the induction of egg yolk protein synthesis in the liver ( Heald & McLachlan, 1963, 1965 ). There is a twofold increase in newly synthesized messenger RNA associated with polysomes within the first six hours and an increase in total liver weight, RNA, and protein within 48 hours ( Jost et al. , 1973 ). Conditions have been established for reliable and quantitative determination of elongation factor 1, elongation factor 2 and ribosomal RNA in the livers of immature chicks, in order to study the quantitative relationship between, these components of the protein synthesizing machinery during the “shift-up” situation following estrogen administration. Factor activity of high salt extracts was determined from initial rates of polyphenylalanine synthesis in the presence of excess complementary factor. Direct quantitation of EF-2 § was achieved by incorporation of [ 14 C]ADP-ribose from [ 14 C]NAD in the presence of diphtheria toxin. The reaction conditions were shown to give specific transfer of [ 14 C]ADP-ribose to EF-2 by analysis on gels containing sodium dodecyl sulfate. The fractionated [ 14 C]ADPrEF-2 was used to quantitate EF-2. The amounts of 18 S and 28 S rRNA were used as a measure of the ribosome content of the treated liver. Post-nuclear supernatant fractions were prepared in the presence of diethyl pyrocarbonate and Triton X100 and fractionated on sodium dodecyl sulfate/ sucrose gradients. These conditions were shown to given quantitative determination of 18 S and 28 S rRNA. Analyzed in the above manner, the levels of EF-1, EF-2 and ribosomes were found to increase co-ordinately following estrogen administration, although EF-1 activity was shown to be rate-limiting for poly-phenylalanine synthesis. The stoichiometry of EF-2: ribosome was maintained at unity. However, the ratio of 18 S rRNA: 28 S rRNA was found not to be unity, but 1·5:1.