Purification and properties of a thiol protease from rat liver nuclei

Abstract

A thiol protease was purified about 800-fold from the chromatin fraction of rat liver by employing Sepharose 6B gel filtration, chromatofocusing and Sephadex G-100 gel filtration. It was nearly homogeneous on sodium dodecyl sulfate/polyacrylamide gel electrophoresis and its molecular weight was about 29 000. The isoelectric point of the enzyme was 7.1. The pH optimum for degradation of 3H-labelled ribosomal proteins was 4.5. It is noticeable that the maximal activity was shifted to pH 5.5 by DNA, and that 30 -40 % of the maximal activity was observed at neutral pH in the presence of DNA. The activity was increased about twice by 2-4 mM dithiothreitol. The protease may be specific for the nuclei because it is different from all lysosomal thiol proteases ever known.When the synthesis of rRNA in regenerating rat liver was selectively inhibited by the administration of a low dose of actinomycin D in vivo, the synthesis of ribosomal proteins remained almost normal 1 h after the actinomycin D treatment. These newly synthesized and unbound ribosomal proteins were found to be degraded rapidly with a half-life of 20 -40 min [I]. Furthermore, we found that the ribosomal proteins and histones, synthesized in actinomycin-D-treated rat liver under the conditions as described above, accumulated in liver nuclei after injection of E-64, a thiol protease inhibitor [2]. 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. It was extracted with 0.7 M NaCl from the chromatin fraction and partially purified with Sepharose 6B. The acidic protease was endopeptidase of a thiol type [3]. During the course of these experiments the presence of the same kind of acidic protease was shown in normal rat liver. Partially purified enzyme from normal rat liver was also sensitive to -SH reagents and had almost the same molecular weight as the enzyme from regenerating rat liver. In the present report the purification of the thiol protease to near homogeneity from normal rat liver chromatin, and some of its characteristics are described. MATERIALS AND METHODS MaterialsWistar strain rats weighing 200 -300 g were used. Iodo-[l-14C]acetamide (23 Ci/mol) was obtained from New England Nuclear Co. Sepharose 6B, Sephadex G-100, PBE94 and Chromatin preparationThe chromatin fraction was prepared from rat liver nuclei as described previously [3]. Briefly, rat liver homogenate in medium A (0.25 M sucrose, 5 mM MgCl,, 25 mM KCl and 50 mM Tris/HCl buffer, pH 7.6) was centrifuged at 10000 x g for 10 min and the precipitate was suspended in 10 volumes of 2.3 M sucrose containing 5 m M MgC...

DNA cleavage in rat liver nuclei activated by Mg2+ or Ca2+ + Mg2+ is inhibited by a variety of structurally unrelated inhibitors

“…There is some confusion and contradiction as to the precise effects of protease inhibitors and it is likely that more than one protease is involved. It is of interest that Motizuki et al (1984) have reported the purification of a thiol protease from rat liver nuclei which was activated by DTT and inhibited by iodacetamide. The results of our study provide evidence that DCI and NEM may be targeting a critical thiol group which is essential for endonuclease activity and suggests that these compounds will be useful probes for further investigating this important element of the apoptotic process.…”

Treatment of Starfish Sperm with Egg Jelly Induces the Degradation of Histones

“…Proteases which preferentially hydrolyze histones or other nuclear basic proteins in vitro have been isolated from the chromatin of various types of cells including spermatozoa. These enzymes may be involved in the metabolism of nuclear proteins (17,18), the modulation of higher order chromatin structures (7), and the control of gene expression. However, the capacity of an enzyme to hydrolyze histones in vifro is not enough to corroborate that the enzyme does hydrolyze histones in situ.…”

“…These enzymes may be involved in the metabolism of nuclear proteins (17,18), the modulation of higher order chromatin structures (7), and the control of gene expression. However, the capacity of an enzyme to hydrolyze histones in vifro is not enough to corroborate that the enzyme does hydrolyze histones in situ.…”

Multi‐step DNA cleavage in rat liver nuclei is inhibited by thiol reactive agents

“…There is some confusion and contradiction as to the precise effects of protease inhibitors and it is likely that more than one protease is involved. In this respect it is of interest that Motizuki et al [32] have reported the purification of a thiol protease from rat liver nuclei which was activated by dithiothreitol and inhibited by iodacetamide.…”