Kunio Tsurugi

Preferential degradation of newly synthesized ribosomal proteins in rat liver treated with a low dose of actinomycin D.

Abstract The administration of a low dose of actinomycin D to partially hepatectomized rats, which selectively inhibited rRNA synthesis, caused the preferential degradation of newly synthesized ribosomal proteins in regenerating rat liver with an apparent half-life of about 20 to 40 min.

Preferential biosynthesis of ribosomal structural proteins by free and loosely bound polysomes from regenerating rat liver

Homologous cell-free systems were prepared using free, total bound, tightly bound or KCl-sensitive loosely bound (KCl-sensitive) polysomes from regenerating rat liver. [14C]Leucine was incubated with one kind of polysomes and [3H]leucine with another kind. The reaction mixtures were then combined, and ribosomal structural proteins were purified as described previously [4], using two-dimensional polyacrylamide gel electrophoresis as the final step [5]. The 3H to 14C ratios of the purified fractions were estimated to compare the activities of the two kinds of polysomes for biosynthesis of ribosomal structural proteins. The following results were obtained: (1) The activity of free polysomes for biosynthesis of ribosomal structural proteins was about 3.6 or 2.4 times higher than that of total bound polysomes in two experiments in which 14C and 3H labeling was reversed. The radioactivities incorporated by free polysomes into most of the proteins separated on two-dimensional gel were found to be definitely higher than those in the surrounding areas, suggesting that most of the ribosomal structural proteins were synthesized by free polysomes. The activity of free polysomes for biosynthesis of ribosomal structural proteins was about 7 times higher than that of tightly bound polysomes, which were prepared by washing the microsomal membrane fraction with 0.5 M KCl. The radioactivities incorporated by tightly bound polysomes into the proteins separated on two-dimensional gel were only slightly higher than those in the surrounding areas, indicating that these polysomes had very low synthetic activity. (2) Preferential synthesis of histones by free polysomes was also shown using the same procedures. (3) KCl-sensitive polysomes which were released by washing the microsomal membrane fraction with 0.5 M KCl, were shown to have definitely higher activity than tightly bound polysomes for biosynthesis of ribosomal structural proteins. (4) From these results, it is concluded that most of the ribosomal structural proteins are preferentially synthesized by free and KCl-sensitive polysomes in regenerating rat liver.

[42] Analytical methods for synthesis of ribosomal proteins by cell-free systems from rat liver

Publisher Summary This chapter describes the analytical methods for synthesis of ribosomal proteins by two kinds of cell-free systems from regenerating rat liver: postmitochondrial supernatant as well as various kinds of polysomes and cell sap. The former has initiating activity although it is rather low. Ribosomal proteins are purified by acetic acid extraction followed by CM-cellulose column chromatography. These procedures separate ribosomal proteins from the bulk of cell sap proteins. To examine the incorporation into individual ribosomal proteins, fraction I from CM-cellulose chromatography is subjected to two-dimensional acrylamide gel electrophoresis. The radioactivity of stained proteins on two-dimensional gel is then measured. The yield of ribosomal proteins during the purification procedures is variable, it is recommended to use double-labeling techniques to obtain quantitative data on the incorporation into ribosomal proteins or to compare the activities of different kinds of polysomes for biosynthesis of ribosomal proteins. The disadvantage of the chromatographic procedures described in the chapter is that several kinds of ribosomal proteins, including the less basic proteins are lost. To examine the incorporation into these proteins, three-dimensional electrophoresis of acetic acid-soluble protein is available and is described in the chapter.