Purification and Characterization of the Ca2+ Plus Mg2+-Dependent
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J. Biochem. 89, 143-152 (1981) Purification and Characterization of the Ca2+ Plus Mg2+-Dependent Endodeoxyribonuclease from Calf Thymus Chromatin1 Michitoshi NAKAMURA, Yoshiyuki SAKAKI , Nobuko WATANABE, and Yasuyuki TAKAGI Department of Biochemistry, Kyushu University School of Medicine , Hi gashi-ku, Fukuoka, Fukuoka 812 Received for publication, June 23, 1980 Ca2+ plus Mg2+-dependent endodeoxyribonuclease was extracted from calf thymus chromatin and purified to a state free from contamination by other DNases. This DNase required both Ca2+ and Mg2+, or Mn2+ alone for its activity and the opti mum pH for activity was at 6.5-7.5. No specificity for the 5•Œ-base was observed . The molecular weight of the DNase was estimated to be about 25,000-30,000 by glycerol gradient centrifugation. Actin and antibody for pancreatic DNase (DNase I) did not inhibit the enzyme, whereas both strongly inhibited DNase I, suggesting that these two DNases are different enzymes. The existence of Ca2+ plus Mg2+-dependent endo However, when the enzyme was partially deoxyribonuclease ("Ca2+-Mg2+ DNase") in mam purified from rat liver chromatin and characterized malian cell nuclei was predicted by Burgoyne to some extent (3, 5, 6), it was found, as pointed et al. (1) and Hewish and Burgoyne (2), who out by Sierakowska and Shugar (7), that the showed that incubation of isolated nuclei in the properties of this DNase (such as ion requirements, presence of Ca2+ plus Mg2+ causes a remarkable optimum pH and mode of action) were very similar fragmentation of nuclear DNA. This "Ca2+- to those of DNase I, which also requires Ca2+ and Mg2+ DNase" activity was found in chromatin of Mg2+ for activity in the presence of SH reagents a variety of cells, and particularly of rapidly such as 2-mercaptoethanol (8). Thus the question dividing cells such as those in regenerating liver arose as to whether or not so-called "Ca2+-Mg 2+ (3). Furthermore, some evidence suggesting that DNase" is really a different enzyme from DNase the activity is connected with the poly-ADP ribo I. This problem was highlighted by the recent sylation reaction in mammalian nuclei has been work from our laboratory which showed that reported (4). Thus, this DNase appears to be mammalian cell nuclei contain DNase I-type involved in some essential process of cell prolifera enzyme (9). Therefore, it became necessary to tion, but its real function remains to be determined. obtain a highly purified preparation of the "Ca2+- Mg2+ DNase" free from other contaminating 1 This work was supported by a Grant-in-Aid for Cancer nucleases and to investigate its biochemical prop Research from the Ministry of Education, Science and erties in detail, in order to compare the enzyme Culture of Japan. with DNase I. Vol. 89, No. 1, 1981 143 144 M. NAKAMURA, Y. SAKAKI, N. WATANABE, and Y. TAKAGI In this paper, a procedure for the purification (1.95 M sucrose, 50 mm Tris-HCl, pH 7.5, 3 mm of this DNase from calf thymus chromatin is CaC12, and 1 mm EDTA) then centrifuged for 30 described. It has been shown that this DNase min at 7,500•~g. The pellet was washed again is a different enzyme from DNase I, though its with 0.25 M sucrose-buffer and used as nuclei. properties are very similar to those of the latter. Assay of DNase Activity-(I) DNA gel method: Polyacrylamide gel containing 32P-labeled MATERIALS AND METHODS DNA was prepared as described by Melgar and Goldthwait (16). Standard reaction mixture (200 Chemicals-CNBr-activated Sepharose 4B was ƒÊl) consisted of 100 pl of the suspension of [32P]- obtained from Pharmacia. Heparin (Sodium salt, DNA gel (2.6-5.3 nmol DNA) in 50 mm Tris-HC1, 155 I.U. per mg) was a product of Nakarai Chem pH 7.5, 75 ƒÊl of a solution containing 5 mm Ca2+ ical Co., and heparin-agarose gel was prepared by 5 mm Mn2+, and 10 mm 2-mercaptoethanol and the method of Bickle et al. (10). Pancreatic DNase 25 ƒÊl of enzyme in buffer A (20 % glycerol, 50 mm (DNase I) was purchased from Worthington Bio- Tris-HCl, pH 7.5, 0.1 M NaCl, 1 mm 2-mercapto chemical Co. and further purified by isoelectric ethanol, and 2 mm EDTA). The mixture was focusing (11). Frozen thymus of newborn calf incubated at 37•Ž for 60 min, then the reaction was kindly supplied by the Mitsubishi-Kasei was stopped by the addition of 200 ƒÊl of ice-cold Institute of Life Sciences, Machida, Tokyo. Anti- 20 mm EDTA. The radioactivity in supernatant body for DNase I partially purified from rabbit obtained after centrifugation at 8,000 rpm for 2 serum through ammonium sulfate fractionation min was measured by means of a gas flow counter. and DEAE cellulose chromatography was a gift One unit of enzyme is defined as that amount from Dr. A. Funakoshi, Department of Internal catalyzing the release of 0.1 nmol of DNA from Medicine, Kyushu University School of Medicine the gel into the supernatant under the conditions (12). Actin was prepared from rabbit dry muscle described above. according to the method described by Spudich (2) Acid-soluble fraction method: The reac and Watt (13). 32P-Labeled DNA was obtained tion was conducted as mentioned above, except from Escherichia coli as described by Anai et al. that [32P]DNA solution was used instead of [32P]- (14), and heat-denatured DNA was prepared by DNA gel, and stopped by the addition of 100 ƒÊl heating the DNA at 100•Ž for 10 min in 10 mm of 7 % perchloric acid and 100 ƒÊl of ovalbumin Tris-HCl (pH 7.5) and 1 mm EDTA, then cooling (0.5 mg/ml). After 10 min at 0•Ž, the precipitate it rapidly in ice. Supercoiled pBR322 DNA and was removed by centrifugation at 8,000 rpm for linear , -phage DNA were gifts from Dr. H. 2 min and the radioactivity of the supernatant Nomiyama, this department. The modulator pro was measured (14). tein was kindly provided by Dr. S. Kakiuchi, Assay of RNase Activity-32P-Labeled RNA Osaka University Medical School. was prepared from E. coli ribosomes. RNase Isolation of Nuclei-Nuclei were isolated from activity was measured in terms of the release of frozen calf thymus by the method of Ueda (15). 32P from the RNA into the acid-soluble fraction Three hundred g of thymus was minced and mixed under the same conditions as for DNase assay, with 3,000 ml of 0.25 M sucrose-buffer (0.25 M su except that [32P]RNA was used as a substrate. crose, 50 mm Tris-HCl, pH 7.5, 3 mm CaCl2, and I Assay of Phosphatase Activity-Phosphatase mm EDTA). After being homogenized in a Waring activity was monitored in terms of the conversion blender for 3 min, the homogenate was centrifuged of ƒÏ-nitrophenyl phosphate to nitrophenol and at 900 x g for 10 min, and the precipitate was inorganic phosphate. washed twice with the same buffer. The pellet 5•Œ-Terminal Base Analysis of the Reaction obtained was suspended in the same buffer and Products-2-Phage DNA was extensively digested the volume was adjusted to 800 ml. This suspen by the enzyme. The products were dephospho sion containing nuclei was mixed with 1,600 ml rylated with alkaline phosphatase and rephos of 2.3 M sucrose-buffer (2.3 M sucrose, 50 mm phorylated by means of T4-polynucleotide kinase Tris-HCl, pH 7.5, 3 mm CaCl2, and 1 mm EDTA), with ƒÁ-[32P]ATP added as a substrate, as described and layered over 1.25 vol. of 1.95 M sucrose-buffer by Maxam and Gilbert (17). 5•Œ-Labeled products J. Biochem. PURIFICATION OF Ca"-Mg2+ DNase 145 were further digested into mononucleotides by TABLE 1. DNase activity at various steps of washing. treatment with DNase I and exonuclease ‡V, and 32P-labeled nucleotides were separated by paper chromatography on Whatman No. 1 filter paper. The solution for development contained 60 ammonium sulfate, 2% n-propanol, and 0.1 M sodium phosphate buffer, pH 6.8. Spots were detected by autoradiography, and identified by comparing their migration with that of authentic nucleotides. Each spot was cut out and the radioactivity was measured with a gas flow counter. RESULTS Purification of "Call -Mg2+ DNase"-Extrac a The total volume of each fraction was the same (400 tion of "Ca2+-Mg2+ DNase": All operations were nil). An aliquot of 25 ƒÊl of each fraction was taken, carried out at 0-4•Ž and centrifugation were and its DNase activity was measured by the DNA gel method, with the divalent cations indicated in the table. performed at 900 x g for 10 min, unless otherwise Numbers are in activity units (defined in the text). noted. Nuclei were prepared from 300g of thymus as described in " MATERIALS AND METHODS." To disrupt the nuclear membrane and to remove nucleoplasm and proteins loosely to remove DNases, most of which do not require bound to chromatin, nuclei were repeatedly Call for activity, and DNasc activated by Call washed with ice-cold buffers as follows. First, plus Mg2+ was extracted from crude chromatin the nuclei were suspended in 400 ml of Tris-EDTA with 0.4 M NaCl. The same activity was found in buffer (25 mM EDTA, 50 mm Tris-HCl, pH 7.5, extracts of purified chromatin, and no significant 5 % glycerol, and 1 mm 2-mercaptoethanol) and difference in further purification was observed centrifuged (1st Tris-EDTA washing). The pellet between these two extracts.