/ . Biochem., 77, 1007-1014 (1975)
Chromosomal Protein from Calf Thymus Michiteru YOSHIDA, Aiko KIKUCHI, and Kensuke SHIMURA Laboratory of Biochemistry, Department of Agricultural Chemistry, Tohoku University, Sendai, Miyagi 980 Received for publication, October 22, 1974
1) A method is described for the separation and fractionation of nonhistone chromosomal proteins from salt-urea dissociated calf thymus chromatin. After precipitating DNA in the dissociated chromatin solution with LaCl s , the chromosomal proteins in the supernatant were fractionated by SP-Sephadex C-25 column chromatography using a combination of NaCl stepwise and linear gradient elutions. Much care was taken to prevent proteolytic degradation of the chromosomal proteins during the preparation. 2) Among the protein fractions separated by this chromatography, twenty subtractions were found to be homogeneous on SDS-polyacrylamide gel electrophoresis. These purified proteins account for about 18% of the whole chromosomal protein. Eleven subtractions of these purified nonhistone proteins had ratios of acidic to basic amino acids above 1.0 and the nine remaining subtractions had ratios below 1.0, corresponding to nonhistone proteins of basic character. 3) The molecular weights of the purified nonhistone proteins ranged from 7,400 to 19,000.
The importance of nonhistone chromosomal proteins in controlling the specificity of RNA transcription in eukaryotic cells has been -shown by several studies (1—6). Therefore, it is desirable to isolate each component of nonhistone chromosomal proteins and investigate their characteristics to elucidate the roles of the proteins. However, only a little information is available regarding the properties of these proteins due to the absence of appropriate techniques for isolation and preparation of the proteins from nuclei or chromatin of eukaryotic cells {6—16). Recently, we have developed a method of isolating the chromosomal proteins by remov"Vol. 77, No. 5, 1975
ing DNA with LaClj from dissociated chromatin (17). Using this method, we tried to fractionate and purify nonhistone chromosomal proteins from calf thymus chromatin by SPSephadex C-25 column chromatography and to elucidate some characteristics of the purified components. EXPERIMENTAL
Materials — SP-Sephadex C-25 (40-120 ft, 2.3±0.3 meq/g) was purchased from Pharmacia Fine Chemicals; 2-mercaptoethanol from Tokyo Kasei Company; LaCls-7Hi0 from Wako Pure Chemical Industries. Specially prepared re-
1007
Downloaded from https://academic.oup.com/jb/article-abstract/77/5/1007/2185186 by Goteborgs Universitet user on 23 January 2019
Fractionation, Isolation, and Characterization of Nonhistone
1008
To measure autolytic activity, 1.2 ml of each sample obtained above was taken in a small test tube and incubated at 30°. The incubation was stopped by chilling in ice water, followed by the addition of 1.0 ml of 0.44 M trichloroacetic acid (75). After 30 min, the resulting precipitate was centrifuged off at 800 XQ for 10 min and the supernatant was used for determination of solubilized proteins by the procedure of Folin and Ciocalteu {19). Preparation of Chromatin from Calf Thymus—.The preparation of chromatin from calf thymus was carried out by the method described previously (17) with a slight modification. All operations were performed at 4°.
Minced calf thymus was homogenized in 6 volumes of 0.14 M NaCl 0.01 M sodium citrate (pH 7.0)-2.5 mM £-chloromercuribenzoate (PCMB} with a Waring blender and passed through twcv layers of gauze. The filtrate was centrifuged at 2,000 Xfir for 15 min. The sediment was then washed five times with 0.14 M NaCl-0.01 M sodium citrate (pH 7.0)-l mM PCMB in the same way. The precipitate was resuspended in 0.05 M Tris-HCl buffer (pH 7.6) (3 ml/g fresh weight of calf thymus), and centrifuged at 6,000 Xg for 10 min. The washing was repeated once in the same manner. Then, the precipitate was extracted with 2.0 M NaCl-1.0 mM PCMB in a glass homogenizer and centrifuged at 35,000xg for 60 min. The supernatant was used for the isolation of whole chromosomal proteins. Isolation of Whole Chromosomal Protein from the Washed Chromatin—The isolation of the chromosomal protein from the chromatin fraction was carried out by the LaClj method described previously (17) with a slight modification. In order ta dissociate the chromatin into DNA and chromosomal proteins, solid NaCl, 8 M deionized urea and 1M Tris-HCl buffer (pH 7.9) were added to the chromatin solution obtained above to give final concentrations of 2.0 M NaCl, 5M urea, and 0.05 M TrisHCl. The DNA concentration in the solution, was adjusted to 300 fig per ml by adding 2 M NaCl-5M urea-0.05M Tris-HCl buffer (pH 7.9), and the solution was homogenized in a glass homogenizer (10 strokes). To the dissociated chromatin solution, LaCls previously dissolved in a small volume of 2 M NaCl-5 M urea-0.05 M Tris-HCl buffer (pH 7.9) was added to a concentration of 0.0135 M, and the solution was stirred gently. After 3 hr, the resulting gelatinous precipitate of DNA was removed by centrifugation at 35,000xg for 120 min. The supernatant was adjusted to pH 4.0 with acetic acid, and concentrated to 8 mg protein per ml on an Amicon Ultrafiltration Membrane UM-2. The chromosomal protein fraction was thea subjected to gel filtration on a Sephadex G-15 (medium) column previously equilibrated with standard solution I (5 M urea-0.02 M acetic acid3 mM 2-mercaptoethanol) containing 0.25 M NaCl. The whole chromosomal protein, eluted
/ . Biochtm.
Downloaded from https://academic.oup.com/jb/article-abstract/77/5/1007/2185186 by Goteborgs Universitet user on 23 January 2019
agents, acrylamide(monomer), N, N, N', N'tetramethylethylenediamine, N, N'-methylenebis(acrylamide) and Gx>massie brilliant blue, were obtained from Nakarai Chemicals. Acrylamide(monomer) and N, N'-methylenebis(acrylamide) were further purified by recrystallization. Urea was purchased from Wako Pure Chemical Industries, and was deionized with Amberlite MB-3 just before use. All other reagents were of the purest grade commercially available. Assay for Autolytic Activity of Calf Thymus Homogenate—In order to determine autolytic activity in a preparation of calf thymus homogenate, fresh calf thymus obtained immediately after slaughter of the animal and frozen in solid COE was homogenized with 6 volumes of 0.14 M NaCl-0.01 M sodium citrate (pH 7.0) with a Waring blender, and the homogenate was filtered through two layers of gauze. The filtrate (Sample A) was centrifuged at 2,000 XQ for 15 min. The precipitate was washed five times more with NaCl-sodium citrate in the' same way. Half of the resulting precipitate was resuspended in the NaCl-sodium citrate (3 ml/g fresh weight of calf thymus) (Sample B). The other half of the precipitate was resuspended in 0.05 M Tris-HCl (pH 7.6) (3 ml/g fresh weight of calf thymus), and centrifuged at 6,000 x Q for 10 min. The washing was repeated once in the same manner. The precipitate was resuspended in 0.05 M Tris-HCl (pH 7.6) (3 ml/g fresh weight of calf thymus) (Sample C). All these operations were carried out at 0—4°.
M. YOSHIDA, A. KIKUCHI, and K. SHIMURA
FRACTIONATION OF NONHISTONE CHROMOSOMAL PROTEINS
Further Fractionation of Chromosomal Protein Fractions by SP-Sephadex C-25 Column Chromatography {Chromatography II)—Each fraction from chromatography I, each containing 10—20 mg protein, was applied to an SPSephadex C-25 column (1.0x15 cm) previously equilibrated with standard solution II (5 M urea0.02 M sodium acetate buffer (pH 5.5)-3 mM 2mercaptoethanol) containing NaCl of the same concentration as in the starting buffer for the NaCl linear gradient elution. In general, 350 ml of linear NaCl gradient in standard solution II was passed through each column. The initial and final concentrations of NaCl used are indicated in Fig. 4. The flow rates were kept approximately constant at a rate of 3 ml per hr. Electrophoresis of Protein—Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis was performed by a modification of the technique of Weber and Osborn (20). Protein solutions were dialyzed for 72 hr against a large volume of water with a Visking tube preheated to 90°, and lyophilized. The proteins were then incubated at 37° overnight in 0.01 M Na phosphate buffer (pH 7.1)-1% SDS1% 2-mercaptoethanol-5 M urea. The running gels (6x85 mm) were made up from 0.1 M sodium phosphate buffer (pH 7.1), 15% acrylamide, 0.39% N, N'-methylenebis(acrylamide), 0.045% N, N, N', N' - tetramethylethylenedi amine, 0.05% ammonium persulfate and 5 M Vol. 77, No. 5, 1975
urea. The samples were run at a constant current of 8 mA per tube for about 5 hr until bromophenol blue marker reached a distance of 70 mm from the starting gel face, and were then stained with 0.25% Coomassie brilliant blue in 7% acetic acid-45% methanol for 2 hr, and destained by diffusion in 7% acetic acid5% methanol. Amino Acid Analysis—100—200 fig of protein was dialyzed against a large volume of water for 72 hr with a Visking tube preheated at 90°, lyophilized, and then hydrolyzed in 6 N HC1 at 110° for 24 hr in an evacuated tube. The hydrolyzate was evaporated to dryness at 45° with a rotary evaporator, and analyzed with a Hitachi Model KLA-3B Amino Acid Analyzer. Other Methods—Calf thymus whole histone was separated by the procedure described previously (21, 22). Protein concentration was determined by the method of Lowry etal. (23). DNA was determined by the diphenylamine method of Ashwell (24) and also by reading the ultraviolet absorbance at 260 nm, taking 22 as the absorbance corresponding to 1 mg/ml of DNA. RESULTS Activity of Autolysis in Calf Thymus Homogenate—It is widely accepted that nonhistone chromosomal protein contains many kinds of components (6—16). However, it is important to ascertain that this wide heterogeneity does not originate from degradation of the proteins in the process of preparation. Thus, the proteolytic activity was assayed in a preparation of calf thymus homogenate and also in chromatin. When the homogenate in 0.14 M NaCl-O.OlM sodium citrate (Sample A) was incubated at 30°, an apparent autolysis activity was detected, as shown in Fig. l(a). Low but detectable activities were also observed in chromatins washed with 0.14 M NaCl0.01 M sodium citrate (Sample B) and 0.05 M Tris-HCl buffer (pH 7.6) (Sample C). However, the activities in both the homogenate and the chromatins were almost negligible when they were incubated at 4°. Effect of PCMB on the Autolysis — Al-
Downloaded from https://academic.oup.com/jb/article-abstract/77/5/1007/2185186 by Goteborgs Universitet user on 23 January 2019
at the void volume, was subjected to further fractionation. Partial Fractionation of Whole Chromosomal Protein by SP-Sephadex C-25 Column Chromatography (Chromatography I)—Whole chromosomal protein eluted from a SephadexG-15 column was applied to a SP-Sephadex C25 column (3.7x20 cm) equilibrated with 0.25 M NaCl in standard solution I, followed by elution with 500 ml of the same solution. Then, 800 ml each of 0.30, 0.35, 0.40, 0.45, 0.50, and 1.0 M NaCl in standard solution I was passed through the column to elute the proteins. The flow rate was kept approximately constant at a rate of 30 ml per hr throughout the operations. After determination of the protein content, each protein fraction was collected and concentrated on an Amicon Ultrafiltration Membrane UM-2.
1009
1010
M. YOSHIDA, A. KIKUCHI, and K. SHIMURA
60
90
several autolysis inhibitors were examined at 30°. Among them, PCMB was found to be most effective in preventing autolysis, as shown in Fig. l(b). At a concentration of 2.5 mM PCMB, more than 90% inhibition was observed in Sample A.
120
TIME (mln)
100
•E 5 0 -
10-' 10" PCMB (M)
Fig. 1. (a) Autolytic activity in homogenate and washed chromatins. The assay method for autolysis is described in " EXPERIMENTAL." —O—, Sample A at 30°; —•—, Sample B at 30°; —x—, Sample C at 30; - - O - - , Sample A at 4°; - - • - - , Sample B at 4°; --X--, Sample C at 4°. (b) Effect of PCMB on autolysis in the homogenate (Sample A). NaCl (M) «-0.25-» — 0 3 0 — - 0 3 5 — • — 0 4 0 — • - O . 4 5 — » * 0 5 0 - » * 1 0 '
too
C-41
"580
C-51 G42 C-7
z 60 UJ
g40
C-31
a. 20 n
50
MO
1I
1
150 200 250 FRACTION NO.(15ml)
Gfri
ft
(1
300
Fig. 2. Partial fractionation of whole chromosomal protein by SP-Sephadex C-25 column chromatography (Chromatography I). NaCl concentrations used in stepwise elution are given on top of the figure.
FRACTION
Fig. 3. SP-Sephadex C-25 column chromatography (Chromatography II) of the chromosomal protein fractions from Chromatography I. NaCl linear gradients used are shown on the right of the figure. / . Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/77/5/1007/2185186 by Goteborgs Universitet user on 23 January 2019
though no actual autolysis activity of the chromosomal proteins was observed at 4°, as described above, it is desirable to avoid the possibility of proteolytic degradation of the proteins during the course of preparation. Thus,
FRACTIONATION OF NOiNHISTONE CHROMOSOMAL PROTEINS
captoethanol, and various concentrations of NaCl. Under these conditions, the whole chromosomal protein was fractionated into 13 fractions, as shown in Fig. 2. The fractions were named in regular series as C-l-1, C-l-2, C-2-1, and so on. The total recovery of proteins from the column was 80%, and the ratios of protein content of each fraction to the total protein recovered were 3.3% in C-l-1; 2.5% in C-l-2; 4.5% in C-2-1; 3.0% inC-2-2; 7.1% in C-3-1; 5.5% in C-3-2; 18.4% in C-4-1; 23.9% in C-4-2; 16.3% in C-5-1; 4.8% in C-5-2; 5.2% in C-6-1; 2.8% in C-6-2; and 2.8% in C-7, as shown in Fig. 2. Further Fractionation of the Chromosomal Protein Fractions by SP-Sephadex C-25 Column Chromatography {Chromatography II)— The 13 fractions obtained above were further fractionated by SP-Sephadex C-25 chromatography using linear gradient elutions of NaCl. All of the protein fractions were separated into several subfractions, as shown in Fig. 3. The subfractions were named in regular series as C-l-1-1, C-l-1-2, and so on. Amino Acid Compositions and Moleculat Weights of the Chromosomal Protein Subfractions— All protein subfractions obtained by Chromatography II were examined for homogeneity and molecular size by SDS-polyacrylamide gel electrophoresis. Among them, the 22 subfractions shown in Fig. 4 appeared to be electrophoretically pure, although the re-
C-2H C-2K C-2-19 C-2-H0 C-2-2-2 C-226 C-31-3 C-321 0 3 2 3 CAH CAM 0 « 4 M M
CHYi
G5H
0 5 * G « 7 O5?.
Chromosomal Protein from Calf Thymus Michiteru YOSHIDA, Aiko KIKUCHI, and Kensuke SHIMURA Laboratory of Biochemistry, Department of Agricultural Chemistry, Tohoku University, Sendai, Miyagi 980 Received for publication, October 22, 1974
1) A method is described for the separation and fractionation of nonhistone chromosomal proteins from salt-urea dissociated calf thymus chromatin. After precipitating DNA in the dissociated chromatin solution with LaCl s , the chromosomal proteins in the supernatant were fractionated by SP-Sephadex C-25 column chromatography using a combination of NaCl stepwise and linear gradient elutions. Much care was taken to prevent proteolytic degradation of the chromosomal proteins during the preparation. 2) Among the protein fractions separated by this chromatography, twenty subtractions were found to be homogeneous on SDS-polyacrylamide gel electrophoresis. These purified proteins account for about 18% of the whole chromosomal protein. Eleven subtractions of these purified nonhistone proteins had ratios of acidic to basic amino acids above 1.0 and the nine remaining subtractions had ratios below 1.0, corresponding to nonhistone proteins of basic character. 3) The molecular weights of the purified nonhistone proteins ranged from 7,400 to 19,000.
The importance of nonhistone chromosomal proteins in controlling the specificity of RNA transcription in eukaryotic cells has been -shown by several studies (1—6). Therefore, it is desirable to isolate each component of nonhistone chromosomal proteins and investigate their characteristics to elucidate the roles of the proteins. However, only a little information is available regarding the properties of these proteins due to the absence of appropriate techniques for isolation and preparation of the proteins from nuclei or chromatin of eukaryotic cells {6—16). Recently, we have developed a method of isolating the chromosomal proteins by remov"Vol. 77, No. 5, 1975
ing DNA with LaClj from dissociated chromatin (17). Using this method, we tried to fractionate and purify nonhistone chromosomal proteins from calf thymus chromatin by SPSephadex C-25 column chromatography and to elucidate some characteristics of the purified components. EXPERIMENTAL
Materials — SP-Sephadex C-25 (40-120 ft, 2.3±0.3 meq/g) was purchased from Pharmacia Fine Chemicals; 2-mercaptoethanol from Tokyo Kasei Company; LaCls-7Hi0 from Wako Pure Chemical Industries. Specially prepared re-
1007
Downloaded from https://academic.oup.com/jb/article-abstract/77/5/1007/2185186 by Goteborgs Universitet user on 23 January 2019
Fractionation, Isolation, and Characterization of Nonhistone
1008
To measure autolytic activity, 1.2 ml of each sample obtained above was taken in a small test tube and incubated at 30°. The incubation was stopped by chilling in ice water, followed by the addition of 1.0 ml of 0.44 M trichloroacetic acid (75). After 30 min, the resulting precipitate was centrifuged off at 800 XQ for 10 min and the supernatant was used for determination of solubilized proteins by the procedure of Folin and Ciocalteu {19). Preparation of Chromatin from Calf Thymus—.The preparation of chromatin from calf thymus was carried out by the method described previously (17) with a slight modification. All operations were performed at 4°.
Minced calf thymus was homogenized in 6 volumes of 0.14 M NaCl 0.01 M sodium citrate (pH 7.0)-2.5 mM £-chloromercuribenzoate (PCMB} with a Waring blender and passed through twcv layers of gauze. The filtrate was centrifuged at 2,000 Xfir for 15 min. The sediment was then washed five times with 0.14 M NaCl-0.01 M sodium citrate (pH 7.0)-l mM PCMB in the same way. The precipitate was resuspended in 0.05 M Tris-HCl buffer (pH 7.6) (3 ml/g fresh weight of calf thymus), and centrifuged at 6,000 Xg for 10 min. The washing was repeated once in the same manner. Then, the precipitate was extracted with 2.0 M NaCl-1.0 mM PCMB in a glass homogenizer and centrifuged at 35,000xg for 60 min. The supernatant was used for the isolation of whole chromosomal proteins. Isolation of Whole Chromosomal Protein from the Washed Chromatin—The isolation of the chromosomal protein from the chromatin fraction was carried out by the LaClj method described previously (17) with a slight modification. In order ta dissociate the chromatin into DNA and chromosomal proteins, solid NaCl, 8 M deionized urea and 1M Tris-HCl buffer (pH 7.9) were added to the chromatin solution obtained above to give final concentrations of 2.0 M NaCl, 5M urea, and 0.05 M TrisHCl. The DNA concentration in the solution, was adjusted to 300 fig per ml by adding 2 M NaCl-5M urea-0.05M Tris-HCl buffer (pH 7.9), and the solution was homogenized in a glass homogenizer (10 strokes). To the dissociated chromatin solution, LaCls previously dissolved in a small volume of 2 M NaCl-5 M urea-0.05 M Tris-HCl buffer (pH 7.9) was added to a concentration of 0.0135 M, and the solution was stirred gently. After 3 hr, the resulting gelatinous precipitate of DNA was removed by centrifugation at 35,000xg for 120 min. The supernatant was adjusted to pH 4.0 with acetic acid, and concentrated to 8 mg protein per ml on an Amicon Ultrafiltration Membrane UM-2. The chromosomal protein fraction was thea subjected to gel filtration on a Sephadex G-15 (medium) column previously equilibrated with standard solution I (5 M urea-0.02 M acetic acid3 mM 2-mercaptoethanol) containing 0.25 M NaCl. The whole chromosomal protein, eluted
/ . Biochtm.
Downloaded from https://academic.oup.com/jb/article-abstract/77/5/1007/2185186 by Goteborgs Universitet user on 23 January 2019
agents, acrylamide(monomer), N, N, N', N'tetramethylethylenediamine, N, N'-methylenebis(acrylamide) and Gx>massie brilliant blue, were obtained from Nakarai Chemicals. Acrylamide(monomer) and N, N'-methylenebis(acrylamide) were further purified by recrystallization. Urea was purchased from Wako Pure Chemical Industries, and was deionized with Amberlite MB-3 just before use. All other reagents were of the purest grade commercially available. Assay for Autolytic Activity of Calf Thymus Homogenate—In order to determine autolytic activity in a preparation of calf thymus homogenate, fresh calf thymus obtained immediately after slaughter of the animal and frozen in solid COE was homogenized with 6 volumes of 0.14 M NaCl-0.01 M sodium citrate (pH 7.0) with a Waring blender, and the homogenate was filtered through two layers of gauze. The filtrate (Sample A) was centrifuged at 2,000 XQ for 15 min. The precipitate was washed five times more with NaCl-sodium citrate in the' same way. Half of the resulting precipitate was resuspended in the NaCl-sodium citrate (3 ml/g fresh weight of calf thymus) (Sample B). The other half of the precipitate was resuspended in 0.05 M Tris-HCl (pH 7.6) (3 ml/g fresh weight of calf thymus), and centrifuged at 6,000 x Q for 10 min. The washing was repeated once in the same manner. The precipitate was resuspended in 0.05 M Tris-HCl (pH 7.6) (3 ml/g fresh weight of calf thymus) (Sample C). All these operations were carried out at 0—4°.
M. YOSHIDA, A. KIKUCHI, and K. SHIMURA
FRACTIONATION OF NONHISTONE CHROMOSOMAL PROTEINS
Further Fractionation of Chromosomal Protein Fractions by SP-Sephadex C-25 Column Chromatography {Chromatography II)—Each fraction from chromatography I, each containing 10—20 mg protein, was applied to an SPSephadex C-25 column (1.0x15 cm) previously equilibrated with standard solution II (5 M urea0.02 M sodium acetate buffer (pH 5.5)-3 mM 2mercaptoethanol) containing NaCl of the same concentration as in the starting buffer for the NaCl linear gradient elution. In general, 350 ml of linear NaCl gradient in standard solution II was passed through each column. The initial and final concentrations of NaCl used are indicated in Fig. 4. The flow rates were kept approximately constant at a rate of 3 ml per hr. Electrophoresis of Protein—Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis was performed by a modification of the technique of Weber and Osborn (20). Protein solutions were dialyzed for 72 hr against a large volume of water with a Visking tube preheated to 90°, and lyophilized. The proteins were then incubated at 37° overnight in 0.01 M Na phosphate buffer (pH 7.1)-1% SDS1% 2-mercaptoethanol-5 M urea. The running gels (6x85 mm) were made up from 0.1 M sodium phosphate buffer (pH 7.1), 15% acrylamide, 0.39% N, N'-methylenebis(acrylamide), 0.045% N, N, N', N' - tetramethylethylenedi amine, 0.05% ammonium persulfate and 5 M Vol. 77, No. 5, 1975
urea. The samples were run at a constant current of 8 mA per tube for about 5 hr until bromophenol blue marker reached a distance of 70 mm from the starting gel face, and were then stained with 0.25% Coomassie brilliant blue in 7% acetic acid-45% methanol for 2 hr, and destained by diffusion in 7% acetic acid5% methanol. Amino Acid Analysis—100—200 fig of protein was dialyzed against a large volume of water for 72 hr with a Visking tube preheated at 90°, lyophilized, and then hydrolyzed in 6 N HC1 at 110° for 24 hr in an evacuated tube. The hydrolyzate was evaporated to dryness at 45° with a rotary evaporator, and analyzed with a Hitachi Model KLA-3B Amino Acid Analyzer. Other Methods—Calf thymus whole histone was separated by the procedure described previously (21, 22). Protein concentration was determined by the method of Lowry etal. (23). DNA was determined by the diphenylamine method of Ashwell (24) and also by reading the ultraviolet absorbance at 260 nm, taking 22 as the absorbance corresponding to 1 mg/ml of DNA. RESULTS Activity of Autolysis in Calf Thymus Homogenate—It is widely accepted that nonhistone chromosomal protein contains many kinds of components (6—16). However, it is important to ascertain that this wide heterogeneity does not originate from degradation of the proteins in the process of preparation. Thus, the proteolytic activity was assayed in a preparation of calf thymus homogenate and also in chromatin. When the homogenate in 0.14 M NaCl-O.OlM sodium citrate (Sample A) was incubated at 30°, an apparent autolysis activity was detected, as shown in Fig. l(a). Low but detectable activities were also observed in chromatins washed with 0.14 M NaCl0.01 M sodium citrate (Sample B) and 0.05 M Tris-HCl buffer (pH 7.6) (Sample C). However, the activities in both the homogenate and the chromatins were almost negligible when they were incubated at 4°. Effect of PCMB on the Autolysis — Al-
Downloaded from https://academic.oup.com/jb/article-abstract/77/5/1007/2185186 by Goteborgs Universitet user on 23 January 2019
at the void volume, was subjected to further fractionation. Partial Fractionation of Whole Chromosomal Protein by SP-Sephadex C-25 Column Chromatography (Chromatography I)—Whole chromosomal protein eluted from a SephadexG-15 column was applied to a SP-Sephadex C25 column (3.7x20 cm) equilibrated with 0.25 M NaCl in standard solution I, followed by elution with 500 ml of the same solution. Then, 800 ml each of 0.30, 0.35, 0.40, 0.45, 0.50, and 1.0 M NaCl in standard solution I was passed through the column to elute the proteins. The flow rate was kept approximately constant at a rate of 30 ml per hr throughout the operations. After determination of the protein content, each protein fraction was collected and concentrated on an Amicon Ultrafiltration Membrane UM-2.
1009
1010
M. YOSHIDA, A. KIKUCHI, and K. SHIMURA
60
90
several autolysis inhibitors were examined at 30°. Among them, PCMB was found to be most effective in preventing autolysis, as shown in Fig. l(b). At a concentration of 2.5 mM PCMB, more than 90% inhibition was observed in Sample A.
120
TIME (mln)
100
•E 5 0 -
10-' 10" PCMB (M)
Fig. 1. (a) Autolytic activity in homogenate and washed chromatins. The assay method for autolysis is described in " EXPERIMENTAL." —O—, Sample A at 30°; —•—, Sample B at 30°; —x—, Sample C at 30; - - O - - , Sample A at 4°; - - • - - , Sample B at 4°; --X--, Sample C at 4°. (b) Effect of PCMB on autolysis in the homogenate (Sample A). NaCl (M) «-0.25-» — 0 3 0 — - 0 3 5 — • — 0 4 0 — • - O . 4 5 — » * 0 5 0 - » * 1 0 '
too
C-41
"580
C-51 G42 C-7
z 60 UJ
g40
C-31
a. 20 n
50
MO
1I
1
150 200 250 FRACTION NO.(15ml)
Gfri
ft
(1
300
Fig. 2. Partial fractionation of whole chromosomal protein by SP-Sephadex C-25 column chromatography (Chromatography I). NaCl concentrations used in stepwise elution are given on top of the figure.
FRACTION
Fig. 3. SP-Sephadex C-25 column chromatography (Chromatography II) of the chromosomal protein fractions from Chromatography I. NaCl linear gradients used are shown on the right of the figure. / . Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/77/5/1007/2185186 by Goteborgs Universitet user on 23 January 2019
though no actual autolysis activity of the chromosomal proteins was observed at 4°, as described above, it is desirable to avoid the possibility of proteolytic degradation of the proteins during the course of preparation. Thus,
FRACTIONATION OF NOiNHISTONE CHROMOSOMAL PROTEINS
captoethanol, and various concentrations of NaCl. Under these conditions, the whole chromosomal protein was fractionated into 13 fractions, as shown in Fig. 2. The fractions were named in regular series as C-l-1, C-l-2, C-2-1, and so on. The total recovery of proteins from the column was 80%, and the ratios of protein content of each fraction to the total protein recovered were 3.3% in C-l-1; 2.5% in C-l-2; 4.5% in C-2-1; 3.0% inC-2-2; 7.1% in C-3-1; 5.5% in C-3-2; 18.4% in C-4-1; 23.9% in C-4-2; 16.3% in C-5-1; 4.8% in C-5-2; 5.2% in C-6-1; 2.8% in C-6-2; and 2.8% in C-7, as shown in Fig. 2. Further Fractionation of the Chromosomal Protein Fractions by SP-Sephadex C-25 Column Chromatography {Chromatography II)— The 13 fractions obtained above were further fractionated by SP-Sephadex C-25 chromatography using linear gradient elutions of NaCl. All of the protein fractions were separated into several subfractions, as shown in Fig. 3. The subfractions were named in regular series as C-l-1-1, C-l-1-2, and so on. Amino Acid Compositions and Moleculat Weights of the Chromosomal Protein Subfractions— All protein subfractions obtained by Chromatography II were examined for homogeneity and molecular size by SDS-polyacrylamide gel electrophoresis. Among them, the 22 subfractions shown in Fig. 4 appeared to be electrophoretically pure, although the re-
C-2H C-2K C-2-19 C-2-H0 C-2-2-2 C-226 C-31-3 C-321 0 3 2 3 CAH CAM 0 « 4 M M
CHYi
G5H
0 5 * G « 7 O5?.
