/ . Biochem. 84, 395-402 (1978)
Ribonuclease H from Rat Liver II.
Partial Purification and Characterization of Cytosol Ribonuclease H 1 Fumio TASHIRO 1 and Yoshio UENO Department of Microbial Chemistry, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Ichigaya, Shinjuku-ku, Tokyo 162 Received for publication, March 11, 1978
We have detected in rat liver cytosol three enzymes (termed C-l, C-2, and C-3) which cleaved the RNA moiety of RNA-DNA hybrid. These enzymes were separated from each other by DEAE-Sephadex and Sephadex G-200 chromatography. C-l and C-2 specifically act on the RNA moiety of RNA-DNA hybrid, while C-3 degrades single-stranded RNA as well as the RNA of the hybrid. The molecular weights of C-l, C-2, and C-3 are about 110,000, 35,000 and 110,000 daltons, respectively, and their activities are absolutely dependent on divalent cations such as Mg 1+ and Mn 1+ . Cleavage by C-l and C-2 is endonucleolytic, producing mostly oligonucleotides and a small amount of mononucleotides which possess 3'-hydroxyl termini. It seems likely that C-2 is originally present in the nucleus and is released into cytosol because of its loose binding to the nuclear components. As for biochemical properties, C-l is very similar to the cytosol ribonuclease H initially reported by Roewekamp and Sekeris, and C-2 is very similar to the nuclear ribonuclease H reported by us in the preceding paper.
Ribonuclease H of rat liver have been isolated by Roewekamp and Sekeris (J) from cytosol, and by us from nuclei as described in the preceding paper (2). However, the relationship between the cytosol and nuclear enzymes and their possible roles in cells are still not clear. In this communication, it is demonstrated that at least three different enzymes degrading the RNA
region of RNA-DNA hybrid occur in rat liver cytosol. The possible relationship between the cytosol and the nuclear ribonuclease H is also discussed. MATERIALS AND METHODS
1
This work was supported in part by grants from the Ministry of Education, Science and Culture (1977) and the Ministry of Public Health and Welfare (1977), Japan. • To whom request for reprint should be addressed. Enzymes: Ribonuclease H or RNA-DNA-hybrid ribonucleotidohydrolase [EC 3.1.4.34], DNA-dependent RNA polymerase or nucleosidetriphosphate: RNA nucleotidyltransferase [EC 2.7.7.6], Snake venom phosphodiesterase or oligonucleate 5'-nucleotidohydrolase [EC 3.1.4.1]. Vol. 84, No. 2, 1978
395
Buffers—The composition of buffers A and B is described in the accompanying paper (2). Buffer C: 250 mM sucrose, 25 mM KCI, 10 mM MgCl,, and 50 mM Tris-HCI (pH 7.5). Buffer D : 10 mM Tris-HCl (pH 7.9), 1 mM MgCl,, 0.25 mM EDTA, 10% glycerol, and 0.5 mM dithiothreitol. Chemicals—All reagents were as described in the accompanying paper (2). Substrates—[uqRNA-DNA hybrid, [ » Q RNA-fHJDNA hybrid, 'H-labelled T7 DNA, [»H]poly(rU>poly(rA) and [»H]poly(rU>poly(dA)
F. TASHIRO and Y. UENO
396
were prepared as described in the preceding paper (.2). Standard Assay Conditions—[UC]RNA-DNA hybrid (2,000-2,500 cpm) was incubated with enzyme in 300 fx\ of assay mixture containing 50 mM Tris-HCI (pH 7.9) and 0.5 mM dithiothreitol; the assay mixtures were adjusted to contain 10 mM (NH4)SSO4 and 17mM MgCl, for C-2 and 10 mM (NH4),SO4 and 3 mM MgCl, for C-3. After incubation at 37°C for 30 min, acid-soluble material was determined as described in the preceding paper (2). Enzyme activity is defined in the preceding paper (2), and was determined under the standard assay conditions described above. Preparation of Cytosol—Cytosol was usually prepared from three Wistar male rats by the method of Roewekamp and Sekeris (/) with slight modifications. The perfused livers were weighed, minced and homogenized in 3 vol. of buffer C in a PotterElvehjem homogenizer. Nuclei and mitochodna-
lysosome fractions were removed by low-speed centrifugation. Cytosol was then prepared by ultracentrifugation at 105,000 xg for 1.5 h. The clear supernatant was stored at —80°C until use. RESULTS
Purification of Ribonuclease H—The enzyme activity was precipitated from the cytosol fraction with solid (NH4),SO4 at a saturation of 30% to 60%. The precipitate was dissolved in buffer D and dialyzed against the same buffer. The dialysate was centrifuged at 31,000xg for 1 h, the supernatant was applied to a DEAE-Sephadex column, and the enzymes were eluted with a linear gradient from 0 M to 0.5 M NH4C1 in buffer D. The ribonuclease H activity was resolved into two peaks (A and B) as indicated in Fig. 1. The fraction corresponding to peak A (fraction number 5-13) and peak B (fraction number 4057) were each combined, and solid (NH4),SO4
2.0 Q.
3 {0 HI
I 1.0* a o
2T1000
.8
s. 20 30 40 Fraction number
60
Fig. 1. DEAE-Sephadex chromatography of nbonuclease H. The crude extract obtained by ammonium sulphate fractionation (30-60% saturation) was dissolved in 20 ml of buffer D and dialyzed against the same buffer. The dialysate was centnfuged at 31,000X0 for 1 h. The supernatant was applied to DEAESephadex column previously equilibrated with buffer D (bed volume 51 ml, column size 1.8x20 cm, flow rate 24 ml/h). The column was washed with buffer D, then with a linear gradient from 0 to 0.5 M NH4CI in buffer D (100 ml + 100 ml). Fractions of 4 5 ml were collected and assayed for ribonuclease H and nbonuclease activity. Ribonuclease activity was assayed with heat-denatured P^dRNA-DNA hybrid as substrate by determining the acid-soluble radioactivity. , Absorbance at 280nm, , NH,CI concentration; C , ribonuclease H activity; • , ribonuclease activity.
J. Biochem.
CYTOSOL RIBONUCLEASE H FROM RAT LIVER
(0.42 g/ml) was added to afford precipitates, which were collected by centrifugation at 100,000 xg for 1 h. The precipitates were chromatographed on Sephadex G-200 column as described in the preceding paper (2). Figure 2-A shows the elution profile of peak B ribonuclease H activity on Sephadex G-200 chromatography. The enzyme activity was separated into the two peaks, termed C-1 and C-2. The molecular weights of C-1 and C-2 were estimated to be about 110,000 and 35,000, respectively. When heat-denatured [UC]RNA-DNA hybrid (100°C, 5 min) was used as substrate, no acidsoluble radioactivity was detected, indicating that enzyme C-1 and C-2 act exclusively on the RNA region of RNA-DNA hybrid. The ribonuclease activity contained in peak B fraction on DEAESephadex chromatography was not detected on Sephadex G-200 chromatography. This is because the ribonuclease probably has a very small molecular weight or is inactivated during the purification. Figure 2-B shows the elution profile of peak A on Sephadex G-200 chromatography. The enzyme was resolved into four peaks of ribonuclease 2000r
397 TABLE I. Summary of the purification procedure for cytosol nbonuclease H. The reaction mixture for the ribonuclease H is described in " MATERIALS A N D METHODS." The amount of protein was determined by the method of Lowry et al. (5). Total protein (mg)
Specific activity (uruts/^g)
Total activity (units)
Supernatant of 105,000 x p
3, 526.4
0.20
687,644
30-60% (NH 4 ),SO 4
2,303.9
0 23
532,200
1,198.4 198.5
0.07 0 21
83,888 42,089
12.4 0.55 0.84 10.7 309.3 ' 0.05
6,833 8,986 15,154
Steps
DEAE-Sephadex Peak A (C-1 and C-2) Peak B (C-3) Sephadex G-200 C-1 C-2 C-3
DEAE-Sephadex rechromatography C-1 Phosphocellulose C-2
2.8
1.31
3,628
1.8
4 18
7,365
•1000
B a.
SiOOO
60
80 0 20 60 80 Fraction number Fig. 2. Sephadex G-200 chromatography of nbonuclease H. The fractions corresponding to peaks A and B nbonuclease H activity were each combined. Each enzyme was precipitated by the addition of solid (NH 4 ),SO, (0.42 g/ml). The precipitate was collected by centrifugation at 100,000 x g for 1 h, dissolved in 3 ml of buffer A containing 0.5 M NH«C1, and dialyzed against the same buffer for 4 h. The dialysate was applied to a Sephadex G-200 column previously equilibrated with buffer A containing 0.5 M NH4C1 (bed volume 182 ml, column size 2 x 5 8 cm, flow rate 9 ml/h, fraction volume 2.8 ml). (A) and (B) show the elution profiles of peaks B and A ribonuclease H activity of Fig. 1, respectively. The letters a, b, c, d, e, and f represent the elution positions of blue dextran, ^-globulin, bovine serum albumin, ovalbumin, chymotrypsinogen and myoglobin, respectively. , Absorbance at 280 nm; O, ribonuclease H activity; • , ribonuclease activity.
Vol. 84, No. 2, 1978
F. TASHIRO and Y. UENO
398
Fig. 3-A, the enzyme was eluted at approximately 0.05 M NH^Cl as a sharp peak. The fractions having C-2 activity were subjected to phosphocellulose chromatography as shown in Fig. 3-B. A summary of the purification procedure is presented in Table I. Each enzyme from the final purification step was stored at — 20°C in 50% glycerol.
H activity. The main peak enzyme, termed C-3, has a molecular weight of about 110,000. This enzyme acts on heat-denatured [14C]RNA-DNA hybrid (100°C, 5 min) as well as ["QRNA-DNA hybrid as shown in Fig. 7. The three minor peaks were not studied further. The fractions having C-1 activity were rechromatographed on DEAE-Sephadex. As shown in
1000
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Ribonuclease H from Rat Liver II.
Partial Purification and Characterization of Cytosol Ribonuclease H 1 Fumio TASHIRO 1 and Yoshio UENO Department of Microbial Chemistry, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Ichigaya, Shinjuku-ku, Tokyo 162 Received for publication, March 11, 1978
We have detected in rat liver cytosol three enzymes (termed C-l, C-2, and C-3) which cleaved the RNA moiety of RNA-DNA hybrid. These enzymes were separated from each other by DEAE-Sephadex and Sephadex G-200 chromatography. C-l and C-2 specifically act on the RNA moiety of RNA-DNA hybrid, while C-3 degrades single-stranded RNA as well as the RNA of the hybrid. The molecular weights of C-l, C-2, and C-3 are about 110,000, 35,000 and 110,000 daltons, respectively, and their activities are absolutely dependent on divalent cations such as Mg 1+ and Mn 1+ . Cleavage by C-l and C-2 is endonucleolytic, producing mostly oligonucleotides and a small amount of mononucleotides which possess 3'-hydroxyl termini. It seems likely that C-2 is originally present in the nucleus and is released into cytosol because of its loose binding to the nuclear components. As for biochemical properties, C-l is very similar to the cytosol ribonuclease H initially reported by Roewekamp and Sekeris, and C-2 is very similar to the nuclear ribonuclease H reported by us in the preceding paper.
Ribonuclease H of rat liver have been isolated by Roewekamp and Sekeris (J) from cytosol, and by us from nuclei as described in the preceding paper (2). However, the relationship between the cytosol and nuclear enzymes and their possible roles in cells are still not clear. In this communication, it is demonstrated that at least three different enzymes degrading the RNA
region of RNA-DNA hybrid occur in rat liver cytosol. The possible relationship between the cytosol and the nuclear ribonuclease H is also discussed. MATERIALS AND METHODS
1
This work was supported in part by grants from the Ministry of Education, Science and Culture (1977) and the Ministry of Public Health and Welfare (1977), Japan. • To whom request for reprint should be addressed. Enzymes: Ribonuclease H or RNA-DNA-hybrid ribonucleotidohydrolase [EC 3.1.4.34], DNA-dependent RNA polymerase or nucleosidetriphosphate: RNA nucleotidyltransferase [EC 2.7.7.6], Snake venom phosphodiesterase or oligonucleate 5'-nucleotidohydrolase [EC 3.1.4.1]. Vol. 84, No. 2, 1978
395
Buffers—The composition of buffers A and B is described in the accompanying paper (2). Buffer C: 250 mM sucrose, 25 mM KCI, 10 mM MgCl,, and 50 mM Tris-HCI (pH 7.5). Buffer D : 10 mM Tris-HCl (pH 7.9), 1 mM MgCl,, 0.25 mM EDTA, 10% glycerol, and 0.5 mM dithiothreitol. Chemicals—All reagents were as described in the accompanying paper (2). Substrates—[uqRNA-DNA hybrid, [ » Q RNA-fHJDNA hybrid, 'H-labelled T7 DNA, [»H]poly(rU>poly(rA) and [»H]poly(rU>poly(dA)
F. TASHIRO and Y. UENO
396
were prepared as described in the preceding paper (.2). Standard Assay Conditions—[UC]RNA-DNA hybrid (2,000-2,500 cpm) was incubated with enzyme in 300 fx\ of assay mixture containing 50 mM Tris-HCI (pH 7.9) and 0.5 mM dithiothreitol; the assay mixtures were adjusted to contain 10 mM (NH4)SSO4 and 17mM MgCl, for C-2 and 10 mM (NH4),SO4 and 3 mM MgCl, for C-3. After incubation at 37°C for 30 min, acid-soluble material was determined as described in the preceding paper (2). Enzyme activity is defined in the preceding paper (2), and was determined under the standard assay conditions described above. Preparation of Cytosol—Cytosol was usually prepared from three Wistar male rats by the method of Roewekamp and Sekeris (/) with slight modifications. The perfused livers were weighed, minced and homogenized in 3 vol. of buffer C in a PotterElvehjem homogenizer. Nuclei and mitochodna-
lysosome fractions were removed by low-speed centrifugation. Cytosol was then prepared by ultracentrifugation at 105,000 xg for 1.5 h. The clear supernatant was stored at —80°C until use. RESULTS
Purification of Ribonuclease H—The enzyme activity was precipitated from the cytosol fraction with solid (NH4),SO4 at a saturation of 30% to 60%. The precipitate was dissolved in buffer D and dialyzed against the same buffer. The dialysate was centrifuged at 31,000xg for 1 h, the supernatant was applied to a DEAE-Sephadex column, and the enzymes were eluted with a linear gradient from 0 M to 0.5 M NH4C1 in buffer D. The ribonuclease H activity was resolved into two peaks (A and B) as indicated in Fig. 1. The fraction corresponding to peak A (fraction number 5-13) and peak B (fraction number 4057) were each combined, and solid (NH4),SO4
2.0 Q.
3 {0 HI
I 1.0* a o
2T1000
.8
s. 20 30 40 Fraction number
60
Fig. 1. DEAE-Sephadex chromatography of nbonuclease H. The crude extract obtained by ammonium sulphate fractionation (30-60% saturation) was dissolved in 20 ml of buffer D and dialyzed against the same buffer. The dialysate was centnfuged at 31,000X0 for 1 h. The supernatant was applied to DEAESephadex column previously equilibrated with buffer D (bed volume 51 ml, column size 1.8x20 cm, flow rate 24 ml/h). The column was washed with buffer D, then with a linear gradient from 0 to 0.5 M NH4CI in buffer D (100 ml + 100 ml). Fractions of 4 5 ml were collected and assayed for ribonuclease H and nbonuclease activity. Ribonuclease activity was assayed with heat-denatured P^dRNA-DNA hybrid as substrate by determining the acid-soluble radioactivity. , Absorbance at 280nm, , NH,CI concentration; C , ribonuclease H activity; • , ribonuclease activity.
J. Biochem.
CYTOSOL RIBONUCLEASE H FROM RAT LIVER
(0.42 g/ml) was added to afford precipitates, which were collected by centrifugation at 100,000 xg for 1 h. The precipitates were chromatographed on Sephadex G-200 column as described in the preceding paper (2). Figure 2-A shows the elution profile of peak B ribonuclease H activity on Sephadex G-200 chromatography. The enzyme activity was separated into the two peaks, termed C-1 and C-2. The molecular weights of C-1 and C-2 were estimated to be about 110,000 and 35,000, respectively. When heat-denatured [UC]RNA-DNA hybrid (100°C, 5 min) was used as substrate, no acidsoluble radioactivity was detected, indicating that enzyme C-1 and C-2 act exclusively on the RNA region of RNA-DNA hybrid. The ribonuclease activity contained in peak B fraction on DEAESephadex chromatography was not detected on Sephadex G-200 chromatography. This is because the ribonuclease probably has a very small molecular weight or is inactivated during the purification. Figure 2-B shows the elution profile of peak A on Sephadex G-200 chromatography. The enzyme was resolved into four peaks of ribonuclease 2000r
397 TABLE I. Summary of the purification procedure for cytosol nbonuclease H. The reaction mixture for the ribonuclease H is described in " MATERIALS A N D METHODS." The amount of protein was determined by the method of Lowry et al. (5). Total protein (mg)
Specific activity (uruts/^g)
Total activity (units)
Supernatant of 105,000 x p
3, 526.4
0.20
687,644
30-60% (NH 4 ),SO 4
2,303.9
0 23
532,200
1,198.4 198.5
0.07 0 21
83,888 42,089
12.4 0.55 0.84 10.7 309.3 ' 0.05
6,833 8,986 15,154
Steps
DEAE-Sephadex Peak A (C-1 and C-2) Peak B (C-3) Sephadex G-200 C-1 C-2 C-3
DEAE-Sephadex rechromatography C-1 Phosphocellulose C-2
2.8
1.31
3,628
1.8
4 18
7,365
•1000
B a.
SiOOO
60
80 0 20 60 80 Fraction number Fig. 2. Sephadex G-200 chromatography of nbonuclease H. The fractions corresponding to peaks A and B nbonuclease H activity were each combined. Each enzyme was precipitated by the addition of solid (NH 4 ),SO, (0.42 g/ml). The precipitate was collected by centrifugation at 100,000 x g for 1 h, dissolved in 3 ml of buffer A containing 0.5 M NH«C1, and dialyzed against the same buffer for 4 h. The dialysate was applied to a Sephadex G-200 column previously equilibrated with buffer A containing 0.5 M NH4C1 (bed volume 182 ml, column size 2 x 5 8 cm, flow rate 9 ml/h, fraction volume 2.8 ml). (A) and (B) show the elution profiles of peaks B and A ribonuclease H activity of Fig. 1, respectively. The letters a, b, c, d, e, and f represent the elution positions of blue dextran, ^-globulin, bovine serum albumin, ovalbumin, chymotrypsinogen and myoglobin, respectively. , Absorbance at 280 nm; O, ribonuclease H activity; • , ribonuclease activity.
Vol. 84, No. 2, 1978
F. TASHIRO and Y. UENO
398
Fig. 3-A, the enzyme was eluted at approximately 0.05 M NH^Cl as a sharp peak. The fractions having C-2 activity were subjected to phosphocellulose chromatography as shown in Fig. 3-B. A summary of the purification procedure is presented in Table I. Each enzyme from the final purification step was stored at — 20°C in 50% glycerol.
H activity. The main peak enzyme, termed C-3, has a molecular weight of about 110,000. This enzyme acts on heat-denatured [14C]RNA-DNA hybrid (100°C, 5 min) as well as ["QRNA-DNA hybrid as shown in Fig. 7. The three minor peaks were not studied further. The fractions having C-1 activity were rechromatographed on DEAE-Sephadex. As shown in
1000
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