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Isolation of different-sized nucleoli from normal and regenerating rat liver
Isolation
213
of nucleoli from rut liver REFERENCES
1. IJIGGERS, J. D., GWATKIX, R. B. L. and HEYNER, S., Exptt Cell Res. 25, 41 (1961). 2. FELL. H. B. and WEISS. L.. J. Exptl Med. 121, 551 (1965). 3. GLAS~TOSE,
S., Proc.
Rby. Sot.
B’126,
315
(1938).
HAY, M. F., Arch. Oral. Viol. 3, 86 (1961). 5. IRVING, J. T., J. Dent. Res. 31, 639 (1952). 4.
6. 7.
KOCH.
\I’.
KOCH;
\I’.
8. 9.
MARSLASD. REYNOLDS,
E.. Anat. Rec. 152, 513 (1965). R., J. Exptt Zoot. 165, 155 (1967). Dent. J. 92, 109 (1952). E. A., Brit. .J. .J., Exptt Cell Res. 47, 42 (1967).
ISOLATION
OF DIFFERENT-SIZED
NORMAL
AND REGENERATING
ELISHEVA Israel
NUCLEOLI
Institute
KAUFMANN,
for
Biological
Received
RAT
A. TRAUB Research, October
FROM
LIVER
and YAEL TEITZ Ness-Ziona,
Israel
9, 1967
rat liver 17 h after two thirds hepatectomy differ from N UCLEOLI from regenerating normal ones in their RNA content [ll], RNA polymerase activity [6] and size [3]. Present methods employed for the isolation of nucleoli from mammalian liver are based on sonication of nuclei and subsequent centrifugation of the sonicate through a dense sucrose solution, which leaves the chromatin and nuclear debris at the top, the nucleoli settling to the bottom of the tube [7]. Such nucleolar preparations, when examined microscopically, contain particles of different sizes. This results, probably, from fragmentation caused by sonication and from the existence of such different sizes in situ. The present communication describes the isolation of different-sized nucleolar preparations from normal and regenerating rat liver. The chemical composition and the activities of NAD pyrophosphorylase (EC 2.7.7.1) and RNA polymerase (EC 2.7.7.6) of the nucleoli were determined. The details of the isolation procedure are as follows: All operations are carried out at 4”. Rat liver nuclei are prepared essentially by the Chauveau method [2] with some modifications [lo]. Freshly isolated nuclei are suspended at a concentration of 100 x 106/ml in batches of 20-40 ml of 0.25 1M sucrose (without Mg2+). This suspension is sonicated in a Raytheon oscillator. The disruption of the nuclei is followed by microscopic observation and is continued until maximally 5 x105/ml of whole nuclei remain. Aliquots of 5 ml sonicate are layered over a linear gradient of 30-70 per cent sucrose and centrifuged at 2000 rpm for 35 min in the swinging bucket rotor No. 269 of the International centrifuge. The sucrose gradient is prepared in a 50 ml lustroid centrifuge tube by allowing layers of 7 ml each of 70, 60, 50, 40 and 30 per cent sucrose solutions to diffuse for 24 h in the cold. At the end of the centrifugation, several opaque bands are observed; the lowest one does not reach the bottom of the Experimental
Cell Research
49
Elisheua Kaufmann,
216
A. Trauh and I’rrel Teitz
1. Size and composition of nucleolar fractions from normal and regenerating liver.
TABLE
Five ml sonicate, prepared from 500 Y 106 nuclei, were sedimented through a 30 i0 “/,, sucrose gradient. Fractions of 2.5 ml were collected and analyzed. After staining with 0.05 96 trgpan blue the nucleoli were counted in a Petroff-Hausser bacteria counter through a phase microscope, and the diameters measured with a calibrated microstage. Protein was determined according to Lowry et al. 141; DNA according to Burton [l]; RNA according to Rlunro and Fleck [5]. The values are the average of 3 experiments. Normal
-A
r-
pg/lO6
Nuclei Nucleoli
Fr.
numher diam. 10” ,u
Protein
IlNA
5 6 7 8 9 10
10 45 90 110 130 40
80 16 8 4 4 2 6
16.0 1.8 2.5 1 .3 0.9 0.5 1.4
TABLE
2. NAD
Regenerating
r---L
2.6 2.2 1,s 1.6 1.3 1.0
--
--
RNA
DNA / RNA
3.2 0.9 1.2 0.7 04 0.2 0.4
5.0 2.0 2.1 1.9 2.1 2.5 3.5
Fr.
numbcr 1v
tliam. p
-Protcin
3 4 5 6 7 8
20 70 105 110 120 75
4.4 3.8 3.1 2.6 2.2 1.6
100 16 9 6 6 5 4
/4106 1)X;\
RN.1
DNA/ RX,\
18.0 1.5 2.1 1 .o 0.7 0.6 0.5
59 1.6 3.1 1.6 1.0 0.8 0.6
3.0 0.9 0.7 0.6 0.G 0.8 0.8
pyrophosphorylase and RNA polymerase in nucleolar normal and regenerating liver.
fractions from
NAD pyrophosphorylase was assayed by suspending 10 Y lUG nuclei or 30 x loo nucleoli in 1 ml of a reaction mixture containing 4pmoles NMN, 5;rmoles ATP, lpmole MgCI, and 3 pmoles phosphate buffer (PH. 7.0). The mixture was incubated for 20 min at 37” and the reaction stopped by the addition of 0.03 ml of 75 % trichloroacetic acid. The resulting NAD was determined according to Racker [8]. RNA polymerase was assayed essentially according to Weiss and Gladstone 112) by suspending 50 x lo6 nuclei or 200 < 10” nucleoli in the reaction mixture of Widnell and Tata [13]. using (I*-C)-GTP (3 x 106 cpm/jdmole). The values are the average of 3 experimenis. Normal
Regenerating 7
RNApolymerese ppmoles GWIP/h
Nuclei Sonicate Nucleoli
Experimenfal
Fr.
per 10”
5 6 7 8 9 10
16 13 7 4 5 4 1 3 Cell
NADpgrophosphorylase m{&moles NADjh
per w2 protein
per 100
Per /Jg protein
0.20 0.15 0.4 0.5 1.3 1.0 0.5 0.5
60 60 8 8 4 3 1 3
0.8 0.8 0.5 1.0 1.0 0.7 0.5 0.5
Kesearch
49
,-RNApolymerase ppmoles GMP/h
NADpyrophosphorylasc mpmoles NAD/h
Fr.
per lo6
per ~g protein
per lo6
per ~2 protein
3 4 5 6 7 8
62 37 17 19 15 13 6 3
0.5 0.3 1.1 2.1 2.5 2.1 1.2 1.0
60 55 7 9 6 5 4 2
0.G 0.6 0.5 1.0 1 .o 0.8 0.8 0.5
Isolation
Fig. 1. nurnhcrs
of nucleoli from rat liver
Suclcolar fractions from normal and regenerating from gradient. (.4) Normal liver; (B) regenerating
liver. liver.
Numbers correspond to fraction Magnification >: 500.
Fractions of 2.5 ml each are collected from the bottom, using a finger pump. The collected fractions are diluted with an equal volume of distilled water and centrifuged at 10,000 g for 20 min. The sedimented material is suspended in 2 ml of 0.88 M sucrose-12 mM MgCl, and an aliquot is examined microscopically. Nucleolar diameters are measured after staining with 0.05 per cent trypan blue with the aid of a calibrated microstage (Graticules Ltd., London). Nucleoli from normal liver were distributed in fractions No. 5-10, while the remaining tubes contained mainly chromatin. Fig. 1 A shows the appearance and Table 1 the size of each fraction (diameter range 2.6-1.0 p); Fig. 1 R shows the corresponding pictures for regenerating liver: the nucleoli were heavier than the normal ones, appearing on the sucrose gradient in fractions No. 3-S with a size range of 4.4-1.6 /I diameter. Each fraction contained nucleoli of relatively homogeneous size. Nucleolar fractions were analyzed for their protein, RNA and DNA content. Table 1 summarizes the results. The chemical composition of the respective nuclei is included. DNA/RNA ratios in all the fractions were much lower for the regenerating tissue than for the normal one. The different fractions were assayed for NAD pyrophosphorylase and RNA polymerase. The results are given in Table 2. Values for nuclei and sonicate are included. tube.
15.-
681803
Experimental
Cell Research
49
218
Elisheva
Kaufmann,
A. Traub and Yael Teitz
Interestingly, in the case of both enzymes, the specific activity was not the same in all fractions. Thus, there was a threefold variation in different fractions in the case of RNA polymerase and a twofold variation with respect to NAD pyrophosphorylase. RNA polymerase was found to be localized to a very high degree in the nucleolus [9J and NAD pyrophosphorylase to the extent of 30-40 per cent. The method described allows the isolation of nucleoli according to their mass. This is reflected in diameter, chemical content and enzyme activity per nucleolus. Specific activities for the enzymes examined vary with different nucleolar fractions. The skillful Schwammbaum
technical assistance of Miss S. Perl, is gratefully acknowledged.
Miss
E. Burstein
and Miss H.
REFERENCES 1. BURTON, K., Biochem. J. 62, 315 (1956). 2. CHAUVEAU, J., MOULB, Y. and ROUILLEK, CH., Ezptl Cell Kes. 11, 317 (1956). 3. HARKNESS, R. D., Brit. Med. Bull. 13, 87 (1957). 4. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. I,. and RANDELL, R. J., J. Viol. Chem. 193, 265 (1951). 5. MUNRO, H. N. and FLECK, A., Analyst 91, 78 (1966). 6. MURAMATSU, M. and BUSCH, H., J. Bio2. Chem. 240, 3960 (1965). 7. MURAMATSU, M., SMETANA, 1~. and BUSCH, H., Cancer Res. 23, 510 (1963). 8. RACKER, E., J. Biol. Chem. 184, 313 (1950). 9. SIEBEHT, G., VILLALOBOS JR., J., Ro, T. S., STEELE, W. J., LINDENMAYER, G., ADA~NS, H. and BUSCH, H., J. Bio/. Chem. 241, 71 (1966). 10. TRAUB, A., KAUFMANN, E. and GINZBURG-TEITZ, Y., Exptl 011 Res. 34 371 (1964). 11. TSUKADA, K. and LIEBERMAN, I., J. Biol. Chem. 239, 1564 (1964). 12. WEISS, S. 1% and GLADSTONE, L., J. Am. Chem. Sot. 81, 4118 (1959). 13. WIDNELL, C. C. and TATA, J. R., Biochem. J. 92, 313 (1964).
Experimental
Cell Research
49
213
of nucleoli from rut liver REFERENCES
1. IJIGGERS, J. D., GWATKIX, R. B. L. and HEYNER, S., Exptt Cell Res. 25, 41 (1961). 2. FELL. H. B. and WEISS. L.. J. Exptl Med. 121, 551 (1965). 3. GLAS~TOSE,
S., Proc.
Rby. Sot.
B’126,
315
(1938).
HAY, M. F., Arch. Oral. Viol. 3, 86 (1961). 5. IRVING, J. T., J. Dent. Res. 31, 639 (1952). 4.
6. 7.
KOCH.
\I’.
KOCH;
\I’.
8. 9.
MARSLASD. REYNOLDS,
E.. Anat. Rec. 152, 513 (1965). R., J. Exptt Zoot. 165, 155 (1967). Dent. J. 92, 109 (1952). E. A., Brit. .J. .J., Exptt Cell Res. 47, 42 (1967).
ISOLATION
OF DIFFERENT-SIZED
NORMAL
AND REGENERATING
ELISHEVA Israel
NUCLEOLI
Institute
KAUFMANN,
for
Biological
Received
RAT
A. TRAUB Research, October
FROM
LIVER
and YAEL TEITZ Ness-Ziona,
Israel
9, 1967
rat liver 17 h after two thirds hepatectomy differ from N UCLEOLI from regenerating normal ones in their RNA content [ll], RNA polymerase activity [6] and size [3]. Present methods employed for the isolation of nucleoli from mammalian liver are based on sonication of nuclei and subsequent centrifugation of the sonicate through a dense sucrose solution, which leaves the chromatin and nuclear debris at the top, the nucleoli settling to the bottom of the tube [7]. Such nucleolar preparations, when examined microscopically, contain particles of different sizes. This results, probably, from fragmentation caused by sonication and from the existence of such different sizes in situ. The present communication describes the isolation of different-sized nucleolar preparations from normal and regenerating rat liver. The chemical composition and the activities of NAD pyrophosphorylase (EC 2.7.7.1) and RNA polymerase (EC 2.7.7.6) of the nucleoli were determined. The details of the isolation procedure are as follows: All operations are carried out at 4”. Rat liver nuclei are prepared essentially by the Chauveau method [2] with some modifications [lo]. Freshly isolated nuclei are suspended at a concentration of 100 x 106/ml in batches of 20-40 ml of 0.25 1M sucrose (without Mg2+). This suspension is sonicated in a Raytheon oscillator. The disruption of the nuclei is followed by microscopic observation and is continued until maximally 5 x105/ml of whole nuclei remain. Aliquots of 5 ml sonicate are layered over a linear gradient of 30-70 per cent sucrose and centrifuged at 2000 rpm for 35 min in the swinging bucket rotor No. 269 of the International centrifuge. The sucrose gradient is prepared in a 50 ml lustroid centrifuge tube by allowing layers of 7 ml each of 70, 60, 50, 40 and 30 per cent sucrose solutions to diffuse for 24 h in the cold. At the end of the centrifugation, several opaque bands are observed; the lowest one does not reach the bottom of the Experimental
Cell Research
49
Elisheua Kaufmann,
216
A. Trauh and I’rrel Teitz
1. Size and composition of nucleolar fractions from normal and regenerating liver.
TABLE
Five ml sonicate, prepared from 500 Y 106 nuclei, were sedimented through a 30 i0 “/,, sucrose gradient. Fractions of 2.5 ml were collected and analyzed. After staining with 0.05 96 trgpan blue the nucleoli were counted in a Petroff-Hausser bacteria counter through a phase microscope, and the diameters measured with a calibrated microstage. Protein was determined according to Lowry et al. 141; DNA according to Burton [l]; RNA according to Rlunro and Fleck [5]. The values are the average of 3 experiments. Normal
-A
r-
pg/lO6
Nuclei Nucleoli
Fr.
numher diam. 10” ,u
Protein
IlNA
5 6 7 8 9 10
10 45 90 110 130 40
80 16 8 4 4 2 6
16.0 1.8 2.5 1 .3 0.9 0.5 1.4
TABLE
2. NAD
Regenerating
r---L
2.6 2.2 1,s 1.6 1.3 1.0
--
--
RNA
DNA / RNA
3.2 0.9 1.2 0.7 04 0.2 0.4
5.0 2.0 2.1 1.9 2.1 2.5 3.5
Fr.
numbcr 1v
tliam. p
-Protcin
3 4 5 6 7 8
20 70 105 110 120 75
4.4 3.8 3.1 2.6 2.2 1.6
100 16 9 6 6 5 4
/4106 1)X;\
RN.1
DNA/ RX,\
18.0 1.5 2.1 1 .o 0.7 0.6 0.5
59 1.6 3.1 1.6 1.0 0.8 0.6
3.0 0.9 0.7 0.6 0.G 0.8 0.8
pyrophosphorylase and RNA polymerase in nucleolar normal and regenerating liver.
fractions from
NAD pyrophosphorylase was assayed by suspending 10 Y lUG nuclei or 30 x loo nucleoli in 1 ml of a reaction mixture containing 4pmoles NMN, 5;rmoles ATP, lpmole MgCI, and 3 pmoles phosphate buffer (PH. 7.0). The mixture was incubated for 20 min at 37” and the reaction stopped by the addition of 0.03 ml of 75 % trichloroacetic acid. The resulting NAD was determined according to Racker [8]. RNA polymerase was assayed essentially according to Weiss and Gladstone 112) by suspending 50 x lo6 nuclei or 200 < 10” nucleoli in the reaction mixture of Widnell and Tata [13]. using (I*-C)-GTP (3 x 106 cpm/jdmole). The values are the average of 3 experimenis. Normal
Regenerating 7
RNApolymerese ppmoles GWIP/h
Nuclei Sonicate Nucleoli
Experimenfal
Fr.
per 10”
5 6 7 8 9 10
16 13 7 4 5 4 1 3 Cell
NADpgrophosphorylase m{&moles NADjh
per w2 protein
per 100
Per /Jg protein
0.20 0.15 0.4 0.5 1.3 1.0 0.5 0.5
60 60 8 8 4 3 1 3
0.8 0.8 0.5 1.0 1.0 0.7 0.5 0.5
Kesearch
49
,-RNApolymerase ppmoles GMP/h
NADpyrophosphorylasc mpmoles NAD/h
Fr.
per lo6
per ~g protein
per lo6
per ~2 protein
3 4 5 6 7 8
62 37 17 19 15 13 6 3
0.5 0.3 1.1 2.1 2.5 2.1 1.2 1.0
60 55 7 9 6 5 4 2
0.G 0.6 0.5 1.0 1 .o 0.8 0.8 0.5
Isolation
Fig. 1. nurnhcrs
of nucleoli from rat liver
Suclcolar fractions from normal and regenerating from gradient. (.4) Normal liver; (B) regenerating
liver. liver.
Numbers correspond to fraction Magnification >: 500.
Fractions of 2.5 ml each are collected from the bottom, using a finger pump. The collected fractions are diluted with an equal volume of distilled water and centrifuged at 10,000 g for 20 min. The sedimented material is suspended in 2 ml of 0.88 M sucrose-12 mM MgCl, and an aliquot is examined microscopically. Nucleolar diameters are measured after staining with 0.05 per cent trypan blue with the aid of a calibrated microstage (Graticules Ltd., London). Nucleoli from normal liver were distributed in fractions No. 5-10, while the remaining tubes contained mainly chromatin. Fig. 1 A shows the appearance and Table 1 the size of each fraction (diameter range 2.6-1.0 p); Fig. 1 R shows the corresponding pictures for regenerating liver: the nucleoli were heavier than the normal ones, appearing on the sucrose gradient in fractions No. 3-S with a size range of 4.4-1.6 /I diameter. Each fraction contained nucleoli of relatively homogeneous size. Nucleolar fractions were analyzed for their protein, RNA and DNA content. Table 1 summarizes the results. The chemical composition of the respective nuclei is included. DNA/RNA ratios in all the fractions were much lower for the regenerating tissue than for the normal one. The different fractions were assayed for NAD pyrophosphorylase and RNA polymerase. The results are given in Table 2. Values for nuclei and sonicate are included. tube.
15.-
681803
Experimental
Cell Research
49
218
Elisheva
Kaufmann,
A. Traub and Yael Teitz
Interestingly, in the case of both enzymes, the specific activity was not the same in all fractions. Thus, there was a threefold variation in different fractions in the case of RNA polymerase and a twofold variation with respect to NAD pyrophosphorylase. RNA polymerase was found to be localized to a very high degree in the nucleolus [9J and NAD pyrophosphorylase to the extent of 30-40 per cent. The method described allows the isolation of nucleoli according to their mass. This is reflected in diameter, chemical content and enzyme activity per nucleolus. Specific activities for the enzymes examined vary with different nucleolar fractions. The skillful Schwammbaum
technical assistance of Miss S. Perl, is gratefully acknowledged.
Miss
E. Burstein
and Miss H.
REFERENCES 1. BURTON, K., Biochem. J. 62, 315 (1956). 2. CHAUVEAU, J., MOULB, Y. and ROUILLEK, CH., Ezptl Cell Kes. 11, 317 (1956). 3. HARKNESS, R. D., Brit. Med. Bull. 13, 87 (1957). 4. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. I,. and RANDELL, R. J., J. Viol. Chem. 193, 265 (1951). 5. MUNRO, H. N. and FLECK, A., Analyst 91, 78 (1966). 6. MURAMATSU, M. and BUSCH, H., J. Bio2. Chem. 240, 3960 (1965). 7. MURAMATSU, M., SMETANA, 1~. and BUSCH, H., Cancer Res. 23, 510 (1963). 8. RACKER, E., J. Biol. Chem. 184, 313 (1950). 9. SIEBEHT, G., VILLALOBOS JR., J., Ro, T. S., STEELE, W. J., LINDENMAYER, G., ADA~NS, H. and BUSCH, H., J. Bio/. Chem. 241, 71 (1966). 10. TRAUB, A., KAUFMANN, E. and GINZBURG-TEITZ, Y., Exptl 011 Res. 34 371 (1964). 11. TSUKADA, K. and LIEBERMAN, I., J. Biol. Chem. 239, 1564 (1964). 12. WEISS, S. 1% and GLADSTONE, L., J. Am. Chem. Sot. 81, 4118 (1959). 13. WIDNELL, C. C. and TATA, J. R., Biochem. J. 92, 313 (1964).
Experimental
Cell Research
49