Degradation of Newly Synthesized Ribosomal Proteins and Histones in Regenerating Rat Liver with and without Treatment with a Low Dose of Actinomycin D

Abstract

The metabolism of newly synthesized ribosomal proteins in regenerating livers of rats pretreated with a low dose of actinomycin D which selectively inhibits rRNA synthesis, has been. investigated. After 15 min of labeling most newly synthesized proteins were found in the cell sap of regenerating livers of rats pretreated for 1 h with actinomycin D and disappeared from this fraction during a subsequent chase in the presence of cycloheximide. However, no proteinase activity for ribosomal proteins was detected in cell sap. After treatment in vivo with E-64, a thiol-proteinase inhibitor which inhibits the ribosomal protein-hydrolysing activities of nuclear and mitochondria1 plus lysosomal fractions of rat liver, a fraction of newly synthesized ribosomal proteins was found to accumulate in nuclei ofactinomycin-D-pretreated regenerating rat liver. These results may indicate that ribosomal proteins synthesized in the absence of rRNA synthesis accumulated in cell sap, and were then transported into nuclei where they were degraded by a proteinase sensitive to E-64. In the absence of actinomycin D pretreatment smaller amounts of ribosomal proteins accumulated in nuclei of regenerating livers of E-64-treated rats. Furthermore, histones (except histone H1) also accumulated in nuclei of regenerating livers of E-64-treated rats with and without actinomycin D pretreatment. Thus, these two kinds of proteins, especially histones, are synthesized in excess of nascent rRNA or DNA, and are degraded preferentially in nuclei in regenerating rat liver.

Presence of a Thiol Protease in Regenerating Rat-Liver Nuclei. Partial Purification and Some Properties

“…In this paper we describe the presence of a thiol protease in nuclei of regenerating rat liver which may participate in the degradation of newly synthesized ribosomal proteins and histones as reported previously [4]. Partial purification of the enzyme and some properties of it are also reported.…”

“…Radioactivity was measured using a toluene-based scintillater containing 30 o/, Triton X-100 as described previously [4] and protein was determined by the method of Lowry et al [16].…”

“…It was found that newly synthesized ribosomal proteins are degraded rapidly with a half-life of 20-40 min [l] in regenerating rat liver treated with actinomycin D. Recently we showed that the newly synthesized ribosomal proteins accumulated in liver nuclei after injection of E-64, a thiol protease inhibitor [2,3], into actinomycin-D-treated rat liver [4]. Newly synthesized histones also accumulated in nuclei after E-64 treatment, regardless of actinomycin D pretreatment [4]. From these results we concluded that these two kinds of proteins which have not associated with the nascent rRNA or DNA are degraded post-translationally by thiol protease(s) in regenerating rat liver nuclei.…”

“…Bonner and colleagues isolated a 200 000-M, protease [5,6] and Carter and Chae reported a protease which is active in 2 M NaCl and 5 M urea [7,8]. Since these proteases are inhibited by diisopropylfluorophosphate (iPrd'-F), a specific inhibitor of serine protease, they are different from the protease which were inhibited by administration of E-64 in vivo as described previously [4].…”

Purification and properties of a thiol protease from rat liver nuclei

“…From the results we have concluded that these two kinds of proteins, which are not associated with the nascent rRNA or DNA, are degraded post-translationally by thiol protease(s) in the nuclei of regenerating rat liver. It must be added that the degradation of newly synthesized and unassociated ribosomal proteins and especially that of histones may occur in the regenerating liver even without actinomycin D treatment [2]. More recently we demonstrated the presence of an acidic protease in the pure nuclei of regenerating rat liver.…”