Isolation and Characterization of Protein A24, a “Hi&one-like” Non-Histone Chromosomal Protein*

In earlier studies, the nucleolar levels of protein A24 were found to be markedly decreased in the nucleolar hypertrophy induced by thioacetamide or during liver regeneration H. J. Biol. Chem. 250, 5921-5925). To determine the role of protein A24, methods were developed for its isolation in highly purified form. Milligram quantities of highly purified protein A24 were isolated from the 0.4 N H,SO,-soluble proteins of calf thymus chromatin by exclusion chromatography on Sephadex G-100, followed by preparative polyacrylamide gel electrophoresis. Protein A24 was highly purified as shown by its migration as a single spot on two-dimensional polyacrylamide gel electrophoresis, its single NH,-terminal amino acid, methionine, and the production of approximately 50 peptides by tryptic digestion. Like 2A, 3, and was extractable from chromatin with

In earlier studies, the nucleolar levels of protein A24 were found to be markedly decreased in the nucleolar hypertrophy induced by thioacetamide or during liver regeneration (Ballal, N. R., Goldknopf, I. L., Goldberg, D. A., and Busch, H. (1974) Life Sci. 14, 1835-1845; Ballal, N. R., Kang, Y.-J., Olson,and Busch,H. J. Biol. Chem. 250,[5921][5922][5923][5924][5925]. To determine the role of protein A24, methods were developed for its isolation in highly purified form. Milligram quantities of highly purified protein A24 were isolated from the 0.4 N H,SO,-soluble proteins of calf thymus chromatin by exclusion chromatography on Sephadex G-100, followed by preparative polyacrylamide gel electrophoresis. Protein A24 was highly purified as shown by its migration as a single spot on two-dimensional polyacrylamide gel electrophoresis, its single NH,-terminal amino acid, methionine, and the production of approximately 50 peptides by tryptic digestion. Like histones 2A, 2B, 3, and 4, A24 was extractable from chromatin with 0.4 N H,SO, or 3 M NaC1/7 M urea, but unlike most non-histone proteins or histone 1, protein A24 was not extracted with 0.35 M NaCl, 0.5 M HClO,, or 0.6 M NaCl. Protein A24 was present in only 1.9% of the total amount of histones 2A, 2B, 3, and 4; its molecular weight is 27,000.
There is now considerable evidence that some of the nonhistone chromosomal proteins are responsible for the specificity of gene transcription in higher organisms (l-5). Subfractions of these proteins have been purified by a variety of procedures (6-16), some of which have yielded subgroups enriched in proteins that stimulated chromatin transcription in vitro (6,14) or exhibited specificity in binding to DNA (7,15). In addition, several non-histone chromosomal proteins have been purified and characterized to varying extents (10, 17-21).
Protein A24, so designated because of its electrophoretic mobility on two-dimensional polyacrylamide gel electrophoresis, was detected in 0.4 N H,SO,-soluble proteins from nucleoli (22) and nuclei (23-26) but was not found in ribosomes or nucleolar ribonucleoprotein particles (27). The finding that rat liver nucleolar levels of protein A24 were markedly reduced during nucleolar hypertrophy induced by thioacetamide administration (25, 28) or liver regeneration (25,29) suggested that this protein might play a role in gene control (30,31). During liver regeneration, protein A24 was markedly decreased in nucleoli 10 hours prior to the onset of DNA synthesis (29). Since the nucleolus is the site for *These studies were supported by the Cancer Center Grant CA-10893, the United States Public Health Service Grant CA-05154, the Wolff Memorial Foundation, and a generous gift from Mrs. Jack Hutchins.
$ Postdoctoral Trainee of the National Cancer Institutp transcription of ribosomal cistrons in eukaryotes (30) and a protein has been implicated as a repressor of ribosomal gene activity (31), an attempt to purify protein A24 was initiated.
The present results show that A24 is a non-histone chromosomal protein with solubility properties similar to those of histones 2A, 2B, 3, and 4. In this respect, it differs from histone 1 and other non-histone chromosomal proteins. Characterization of protein A24 by amino acid composition, NH,-terminal amino acid, molecular weight, and tryptic digestion shows that it differs from the histones and other purified non-histone chromosomal proteins (10, 17-21).

Preparation
of Rat Liver Nuclei and Chromatin-For studies on the distribution of nuclear proteins in chromatin fractions, rat liver nuclei were prepared (30, 32) and extracted at 4'. To prevent proteolysis, 1 mM phenylmethylsulfonyl fluoride was used throughout (33). Chromatin was prepared (34) by washing the nuclei twice by homogenization at 10 ml/g of nuclei with 0.075 M NaCl. 0.025 M EDTA. pH 8.0, and twice in 0.0: M Tris, pH 8.0. For each wash, centrifuga: tion was at 18,000 x g for 10 min. For electrophoretic analysis of proteins solubilized in these washes, as well as in the subsequent chromatin washes (see "Results"), the supernatants were centrifuged at 100,000 x g for 2 hours to remove sheared deoxyribonucleoprotein, concentrated by ultrafiltration, and dialyzed against electrophoresis sample buffer (0.9 N acetic acid/l0 M urea/l% fl-mercaptoethanol). The proteins soluble in 0.4 N H,SO, were ethanol-precipitated and dried as previously described (17 Fig. 1 (22) were not detected in either the saline/EDTA (0.075 M NaCl/ When the histones and most of the non-histone chromosomal 0.025 M EDTA, pH 8.0) (Fig. lb) or the Tris (Fig. lc) washes proteins were extracted from chromatin with 3 M NaC1/7 M but were found in the 0.4 N H,SO,-soluble proteins of chro-urea, protein A24 was also extracted (Fig. 2~). Protein A24 and matin (Fig. Id). On the other hand, most of the other non-the histones reassociated with the DNA upon one-step or histone proteins were present in reduced amounts in the 0.4 gradient dialysis to low ionic strength. Thus, protein A24 had N H,SO, extract of chromatin (compare Fig. 1, a and d).* tight chromatin binding characteristics similar to those of Protein A24 and the histones were not solubilized when the histones 2A, 2B, 3, and 4, but it was present in much lower amountsthanthehistones chromatin was treated with 0.35 M NaCl (Fig. 2a), which extracts many non-histone chromosomal proteins (12, 13). bility studies, the starting material employed for purification of protein A24 was calf thymus chromatin pre-extracted with 0.35 M NaCl and 0.5 M perchloric acid. As shown in Fig.  36, the 0.4 N H,SO, extract of the starting material was fractionated on Sephadex G-100. The fractions containing protein A24 contaminated with histones 2B and 3 (shaded area, Fig. 3b) were pooled, made to 0.4 N HSO,, and ethanol was added to a concentration of 80% to precipitate the proteins (see "Materials and Methods"). Final purification of protein A24 was accomplished on preparative gel slabs containing 10% polyacrylamide/4.5 M urea/O.9 N acetic acid. Vertical segments of these gels (Fig. 4~) were stained to locate the protein.
The purified protein A24 was eluted by electrophoresis from cut-out horizontal sections of the unstained gel into dialysis bags using 0.9 M acetic acid as electrolyte.
Both one-and two-dimensional polyacrylamide gel electrophoresis showed that the purified protein A24 migrated as a single component in its characteristic position ( Fig. 4b and 3c, respectively).
Analysis of A24-Based on relative electrophoretic mobility in the Na dodecylS0, second dimension of the two-dimensional gels to that of standard proteins (Fig. 3d), the molecular weight of protein A24 is approximately 27,000. Amino acid analyses (Table I) showed that A24 has an acidic/basic amino acid ratio of 0.93; the most plentiful amino acids of A24 are glutamic acid, lysine, and leucine; its content of tyrosine, phenylalanine, and methionine is low (Table I). Protein A24 does not contain tryptophan.
The molecular weight figure was supported by minimum molecular weight calculations (43) based on the finding that lysine and arginine constituted 11.3 and 7.4 mol %, respectively, of the amino acids in protein A24. Thus, there should be 47 residues of these amino acids in one M, = 27,000 molecule of protein A24. Tryptic digestion of protein A24 and two-dimensional separation of the digest (41) resolved approximately 50 peptides (Fig. 5) in agreement with these calculations.
Determination of the NH,-terminal amino acid by the dansyl reaction (38, 39) showed that it is methionine (Table I). No other NO-dansyl amino acids were detected using 100 rg of protein.

DISCUSSION
Protein A24 was isolated from chromatin after pretreatment with 0.35 M NaCl and 0.5 M HClO, to selectively extract most non-histone proteins and histone 1; protein A24 and histones 2A, 2B, 3, and 4 were then extracted from the chromatin residue in 0.4 N H,SO,.
Protein A24 was further purified on Sephadex G-100 and by preparative gel electrophoresis. Its purity was established by one-and two-dimensional polyacrylamide gel electrophoresis, NH,-terminal and amino acid analysis and tryptic digestion. The results obtained show that protein A24 has the unique feature that as a non-histone protein its solubility and its tight binding to chromatin are like those of histones 2A, 2B, 3, and 4. However, unlike these histones which are present in 1:l:l:l molar ratios (44), protein A24 is present in much lower amounts.  Garrard and Bonner (48) reported a "minor protein band" designated X which was found among the rat liver histones obtained from Bio-Rex 70 columns. They also mentioned that it was present in all their histone preparations and was observed by Panyim and Chalkley (49) as well. They reported a high turnover rate for this band during rat liver regeneration. Band X had an electrophoretic mobility similar to protein A243, and in a preliminary report (26) protein A24 was shown to bind and elute from Bio-Rex 70 along with the histones.
Since protein A24 has properties of both histones and nonhistone proteins, it may have unique biological properties. Its localization in nuclear and nucleolar chromatin, and the marked decrease in nucleolar levels of protein A24 during nucleolar hypertrophy (25, 28) suggests it may have a possible role as an rDNA repressor (31). Crippa (31) has reported a protein that inhibits transcription of ribosomal cistrons in amphibian oocytes. The decreased nucleolar content of protein A24 during thioacetamide administration (25, 28) and in liver regeneration (25,29) suggests a possible relationship of protein A24 to the amphibian protein since decreases in protein A24 were correlated with marked increases in rRNA synthesis (30).