THE JOURNAL OF B~o~oarcar, CHEMISTRY Vol. 249, No. 17, Issue of September 10, pp. 5580-5585, 1974

Printed in U.S.A.

Phosphorylation of Acid-soluble Proteins in Isolated Nucleoli

of Novikoff Hepatoma Ascites Cells

EFFECTS OF DIVALENT CATIONS*

(Received for publication, March 11, 1974)

YEOU-JAN KANG, MARK 0. J. OLSON, AND HARRIS BUSCH

From the Nuclear Protein Laboratory, Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77025

SUMMARY

Isolated nucleoli from rat Novikoff hepatoma ascites cells incorporated 32P of [T-~~P]ATP into acid-soluble nucleolar proteins in vitro in systems containing 0.25 M sucrose, 5 mM MgCL, 12.5 mM NaCl, and 0.05 M Tris-HCl buffer (pH 7.3) at 37”. Although the total uptake of 32P reached a maximum after 15 min of incubation, the uptake into some protein bands increased with longer incubation times. As shown by two- dimensional gel electrophoresis and autoradiography, all the proteins labeled in uitro, including the high molecular weight proteins designated as C23-24, B27, and B13 were shown previously to be labeled in uivo (OLSON, M. 0. J., ORRICK, L. R., JONES, C., AND BUSCH, H. J. Biol. Chem. 249, 2823-2827). Divalent cations were necessary for phos- phorylation in vitro but different profiles of phosphoryiation were observed when Mg2f was replaced with Co2+, Znzi, or Mn2+. The presence of Zn2+ produced the highest 32P up- take associated with a marked increase in phosphorylation in proteins A17-19 and B23-25 compared to Mg2f which produced high labeling in spots B13, and C23-24. These data indicate that the nucleolus contains phosphoprotein kinase(s) which exhibit different specificities in the presence of different divalent cations.

Phosphorylation of nuclear proteins has been suggested to serve a role in the regulation of gene activities (l-5). Recently, selective phosphorylation of nucleolar acid-soluble proteins of Novikoff hepatoma ascites cells was shown to occur in viva (6). Analyses of the phosphorylation of these nucleolar acid-soluble proteins by two-dimensional polyacrylamide gel electrophoresis showed that approximately 40 nucleolar proteins were phos- phorylated (6).

Since isolated nucleoli are simple subcellular organelles which provide a convenient system for the study of biochemical events involved in the control of genetic activities (7), they have been

* This study was supported by National Science Foundation Grant GB-38012 and United States Public Health Service Center Grant CA-10893.

used for a number of enzymatic studies. Several studies have reported the presence of specific DNA-dependent RNA poly merases (8, 9), methylases for transfer (10) and preribosomal RNA (10, 1 l), and systems for the aminoacylation of transfer RNA (12) and incorporation of amino acids into nucleolar proteins (13) in isolated nucleoli.

The present study was designed as an initial approach to the investigation of the significance and factors controlling the

phosphorylation of nucleolar proteins in vitro. It was found that the proteins phosphorylated in vitro by properly supple- mented isolated nucleoli were also labeled in Z&JO following expo- sure to [Vlorthophosphate (6).

MATERIALS AND METHODS

Isolation oj Nucleoli from Novikoff Hepatoma Ascites Cells- Nucleoli were isolated from Novikoff hepatoma ascites cells by the sucrose-calcium procedure employing sonic oscillation as described previously (6, 7, 14-17).

In Vitro Incubation of Nucleoli-Isolated nucleoli containing about 10 mg of protein (determined by the absorbance ratio o‘f 280:260 nm in 6 M guanidine hydrochloride and 0.02 M Tris-HCl buffer, pH 8.0) (18) were incubated in a medium containing 0.25 M sucrose, 5 mM MgC12, 12.5 mM NaCI, and 0.05 M Tris-HCI buffer (pH 7.3) and 0.125 mCi of [Y-~*P]ATP (64 to 120 Ci per mmole, International Chemical and Nuclear Co., Irvine, California) in a total volume of 2.1 ml. The mixtures were placed in disposable test tubes which were gentlv shaken durine incubation at 37” in the thermostatically c&trol”led water bath-of a gyrotory shaker (New Brunswick Scientific Co., N. J.). At appropriate time intervals, the reaction was stopped by cooling the reaction tubes in ice-cold water and the subsequent addition of 0.4 ml of ice-cold 2 N H2S04. The nucleolar acid-soluble proteins were then ex- tracted at 4’ and precipitated with 4 volumes of absolute ethanol 0% 19).

Electrophoresis of Nucleolar Acid-soluble Proteins-Two-dimen- sional polyacrylamide gels were prepared and subjected to elec- trophoresis by the method of Orrick et al. (6, 20, 21). The radio- active protein spots were detected on DuPont Cronex x-ray film as described earlier (6).

Quantitative Determination of Phosphorylation-After precipi- tation from ethanol, 3 to 8 mg of acid-soluble proteins were washed twice with 3-ml portions of ethanol-ether (3:1, v/v) and once with 3 ml of 25% trichloroacetic acid containing 0.2 M sodium pyro- phosphate (5, 22). To remove the nucleic acids, the proteins were suspended in 1 ml of 5yo trichloroacetic acid at 96” for 15 min. The proteins were then precipitated by the addition of 3 ml of 307, trichloroacetic acid. After spinning at 20,000 X g for 15 min, the supernatant fraction usually contained less than 8% of the total radioactivity. The protein pellet was again washed

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I I O 0

I I I I I I I 5 10 15 20 25 30

TIME OF INCUBATION (MIN)

FIG. 1. The time course of the in vi&o phosphorylation of nucleolar acid-soluble proteins during incubation of isolated nucleoli at 37”. The incubation mixture of 2.1 ml contained 0.25 M sucrose, 5 mM MgClz, 12.5 mM NaCl, 0.125 mCi of [Y-~~P]ATP (64 to 120 Ci per mM), 0.05 M Tris-HCI buffer (pH 7.3), and iso- lated nucleoli containing 10 mg of protein.

12

x 6 I a v

4

was rapid for the first 5 min and a plateau was reached by 15 min of incubation.

On one-dimensional 10% polyacrylamide disc gel electro- phoresis, no significant differences were found in the patterns of the stained protein (Fig. 2, bot!om) during different time intervals of incubation of nucleoli in vitro. However, when the azP was determined in the protein bands after slicing the gels, significant differences were found in the radioactivity in the stained protein bands. Tn addition, at different times, there were changes in labeling of specific protein bands. As shown in Fig. 2, a radio- active peak in slices 55 to 57 corresponding to the area of spot B27 on the two-dimensional slab gels (Fig. 3) was maximally phosphorylated within 5 min. However, the uptake of radio- active phosphate was found to increase with longer incubation times into peaks at slices 50 and 68, which correspond to spots B13 and C23-24, respectively, on the slab gels (Fig. 3).

Two-Dimensional Gel Electroplwretic Analysis of Nucleolar Acid-soluble Phosphoproteins-Two-dimensional gel electrophore-

0-0 15 min

10 20 30 40 50 60 70 80 CATHODE ANODE

GEL SLICE NUMBER

twice with 3 ml of 25% trichloroacetic acid and then dissolved in 1.0 ml of 1 N KOH. Aliquots of the solution were analyzed for protein concentration and for radioactivity after dilution with Aquasol (New England Nuclear, Boston, Mass.). The protein concentrations were determined by the method of Lowry et al. (23) with bovine serum albumin (Miles Laboratories, Kankakee, Ill.) as a standard.

Determination of Radioactivity on One- and Two-Dimensional Gels-After destaining, the one-dimensional disc polyacrylamide gels were frozen in acetone and Dry Ice and cut into l-mm slices using a Bio-Rad (Richmond, Calif.) model 190 gel slicer. Each slice was dried overnight at room temperature in a scintillation vial. After addition of 8 ml of Aquasol, the radioactivity was measured in a Beckman (Palo Alto, Calif.) LS-230 liquid scintil- lation counter. With the two-dimensional gels, the spots found to be radioactive by autoradiography were excised with a cork borer. The radioactivity of each spot was determined as above.

RESULTS

Kinetics of in Vitro Phosphorylation of Nucleolar Acid-soluble Proteins-Fig. 1 shows the time course of phosphorylation of

nucleolar acid-soluble proteins in vitro. The uptake of isotope

FIG. 2. Comparison of one-dimen- sional disc gel electrophoretic patterns of nucleolar phosphoproteins labeled with [r-32P]ATP after different time intervals of incubation of nucleoli in vitro.

sis (6, 20) not only demonstrated the heterogeneity of the nucle- olar proteins in the incubated isolated nucleoli (Fig. 3, a and b) but also showed that no major changes in the protein profile occurred for the duration of these experiments. The nomen- clature for the spots in the regions A, B, and C (Fig. 3, a and b) is the same as that described earlier (20, 21). At 24 hours of

exposure, three intense spots were observed in the autoradiograph (Fig. 3c, inset) which correspond to C23-24, B27, and B13 on the gel slab. Further exposure up to 10 days (Fig. 3c) showed incorporation of radioactive phosphate into a number of protein spots including C17-18, C8, C2, B35, B20, B9, A25, A20, A16, and A7.r Since many protein spots contained little or no radioactivity (Fig. 3, a and c), there is clearly selective phos- phorylation of the nucleolar acid-soluble proteins in vilro.

Quantifalive Estimation of Phosphorylation of Protein Spots- The amounts of radioactivity in the protein spots differed in the

1 In the A region, a radioactive spot designated as A8P was observed which did not coincide exactly with spot A8. This spot was also phosphorylated in viva (6).

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b

NOVIKOFF HEPATOMA NUCLEOLAR PROTEINS

B 1 c

FIG. 3. a, two-dimensional polyacrylamide gel electrophoresis of 250 pg of Novikoff hepatoma acid-soluble nucleolar proteins from nucleoli after 15 min of incubation in vitro as described in Fig. 1. Samples were first loaded on disc gels of 10% acrylamide- 4.5 M urea and run in the first dimension for 5.5 hours at 120 volts constant voltage. For the second dimension, a 127c acrylamide- 0.1% sodium dodecyl sulfate slab gel was run for 16 hours at 50 ma constant amperage and then stained with Coomassie brilliant blue R. b, diagrammatic representation of the protein pattern of a showing the numbering system for the protein spots. The spots are in the order of decreasing intensity: closed circles >

TABLE I Effects of divalent cations on incorporation of [+P]ATP into

nucleolar acid-soluble proteins

Isolated nucleoli were incubat.ed in syst.ems containing 0.25 M sucrose, 12.5 mM NaCl, 0.125 mCi of [@2P]ATP, 0.05 M Tris-HCl buffer (pH 7.3), and 5 mM MgC12, MnCL, ZnClt, or CoCll as indi- cated. The incubation was carried out for 15 min at 37”, then stopped by quick cooling of the reaction tubes in ice-cold water and the subsequent addition of 0.4 ml of cold 2 N sulfuric acid. The acid-soluble nucleolar proteins were extracted with 0.4 N HaSOd at 4’ and precipitated with ethanol. The protein residues were washed several times with ethanol-ether and trichloroacetic acid as described under “Experimental Procedure.” Aliquots of the protein residues dissolved in 0.1 N KOH were taken out for the determination of protein concentrations and radioactivity.

Divalent cation

Without divalent cation Ca2+ co2+ Mg2+ Mn2+ ZnZ+

Radioactivity Relative radioactivity

cpm/mg protein relative cpm/mga

2,400 1.6 1,000 0.6

47,000 30.3 47,800 30.8

109,200 70.4 155,100 100

- a The values are expressed as percentages of counts per min per

mg relative to the incorporation observed in the presence of Zn2+.

three regions and varied considerably from spot to spot within each region (Fig. 4 a to c). For example, in the C region spots C23 and (124 were so highly phosphorylated that a separate ordinate was required (Fig. 4c) ; spots C4 and C6 contained less than 50 cpm. In the A region the protein spot which contained the most radioactivity was spot A20 with only 90 cpm. Dif- ferences in labeling were also found in the 13 region. These results provide further evidence for selective phosphorylation of nucleolar protein.

Efects of Divalent Cations on in Vitro Phosphorylation of Nucleolar Acid-soluble Proteins-The presence of divalent cations was necessary for in vitro phosphorylation; in the absence of any divalent cations, only 2400 cpm per mg were incorporated into the nucleolar acid-soluble proteins (Table I). Addition of Mg2+ (8), Mn2+ (8), and Zn*+ increased the labeling of the proteins by factors of 20, 45, and 65, respectively (Table I). In the presence of Co2+, the uptake of isotope was similar to that with Mg2+. However, Ca2+ had inhibitory effects on the phosphoryl- ation since it reduced the phosphorylation to half the endogenous 32P incorporation.

In addition to these quantitative differences in labeling of whole nucleolar proteins, Fig. 5 shows marked differences in the incorporation of 32P into different protein bands on one-dimen- sional polyacrylamide disc gels. By comparison with labeling

cross-hatched circles > open circles. Minor spots are shown as broken circles. c, visualization of nucleolar phosphoproteins from Novikoff hepatoma ascites cells after 15 min of in vitro incubation of isolated nucleoli with [@P]ATP. The system for in vitro incubation was the same as described in Fig. 1. The acid-soluble nucleolar proteins were subjected to electrophoresis as described in a. After electrophoresis, the slab gel was sand- wiched between DuPont Cronex x-ray films in the dark. The film was exposed for 1 day (inset) or 10 days and then developed. The radioactive spots on the x-ray film were matched with the protein spots on b as indicated by the same numbering system. A8P indicates this radioactive spot does not coincide exactly with the stained spot on the slab gel.

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a

ml -

80 -

z 60

0

40

20

L GAR

1

12- -b

10 -

8-

2-

n-l 1’ 3 1

I 1 I 6P

AREGION 16 16

2 3’ 6 I 10 12 14 16

9 11 13 15 1 'OTNUMBER

B REGION

hobE 7 19 21 23

u ‘4

2 5

1 3 5 7 10 12 14 16 18 21 23 25 27 32 34 2 4 6 9 11 13 15 17 20 22 24 26 28 33 35

SPOTNUMBER

C REGION

14 c t

t 12

t

2 4 6 8 12 14 16 18 20 22 24 26 26A SPOTNUMBER

24

20

16 “0 72 I

12 ::

8

4

0

FIG. 4. Quantitative estimation of uptake of 3*P into protein spots in the two-dimensional electrophoretogram of acid-soluble

map of Fig. 3b. a, A region of slab gel map: b, B region of slab

nucleolar proteins from Novikoff hepatoma ascites cells. The gel map; c, C region of slab gel map as shown in Fig. 36. Cross- hatched bars indicate the expanded counts per min scale on the

14 - - MS*’ - Mn+’

10 - “,

x 8-

5 ”

6-

4 4

2 2

0 0

10 20 30 40 50 60 70 80 CATHODE ANODE

GEL SLICE NUMBER

Fro. 5.~ Comparison of one-dumensional disc gel electrophoretic pattern of nucleolar phosphoproteins labeled with [Y-~~P]ATP with 15 min of incubation in vitro in the presence of 5 mM concen- trations of MgClz (O--O), MnClz (O---O), ZnClt (A--A), CoC12 (O---O), or CaCls (A-A).

in the presence of Mg2+, there was a decrease in the isotope content of peaks in slices 48 to 50 and 65 to 68 in the presence of Zn2+. On the other hand, Zn 2+ greatly increased the radio- activity in the peak in slices 55 to 57 which corresponds to pro- tein spots B27 and B23-25 (Figs. 3, b and c, and 6,). An addi- tional peak in slice numbers 31 to 33 corresponding to A17-19 (Figs. 3 and 6, a and c) was also observed when Mg2+ was replaced by either ZrPf or Co2+.

The presence of the various divalent cations produced quali-

tative and quantitative differences in the radioactivity in various spots (Figs. 3c and 6; Table II). For example, spots 1323-25 were increasingly labeled in the presence of Co2+, Mn2+, and Zn2+ and not in the presence of Mg2+ (Fig. 3~). Spot El3 was labeled more in the presence of Mg2+ than with the other cations. Spots AU-19 (lysine-rich histones) mere moderately labeled

only in the presence of ZrP+. On the other hand, spot B27 was moderately labeled only in the presence of Mg*+ and Zn2+ (Table II). There was a high level of phosphorylation of protein spots C23-24 (Figs. 3c and 6, a to c) in the presence of all four divalent cations. The incorporation of Y’ into nucleolar acid-soluble proteins in the absence of divalent cations or the presence of Ca2+ was too low to be detected by autoradiography. By comparison with the divalent ions, cyclic AMF2 and papav- erine produced little change in phosphorylation of nucleolar proteins (Fig. 7).

DISCUSSION

In addition to phosphorylation of other nuclear proteins (4,22,24-27)) phosphorylation of nucleolar acid-soluble proteins in viva has recently been demonstrated (6, 28). Since several nucleolar proteins were found to be highly phosphorylated (Figs. 3c and 4) in nucleoli in vitro, phosphoprotein kinases (28j may be added to the list of nucleolar enzymes which includes

2 The abbreviation used is: cyclic AMP, adenosine 3’:5’-mono- phosphate.

numbers on the abscissa correspond to the spot numbers on the right-hand side. The system of incubation is the same as in Fig. 1.

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TABLE II Major qualitative and quantitative diflerences in incorporation of

32P into nucleolar protein spots in presence of various divalent cations

With lo-day exposures of slab gels on x-ray films, different pro- tein spots contained different amounts of 3*P as detected by auto- radiography (Figs, 3c and 6). Differences in the amounts of radio- activity in the protein spots showed different intensities of spots on x-ray films. These were expressed in the order of decreasing radioactivity as ++++ for greater than 20,000 cpm, +++ for 709 to 20,006 cpm, ++ for 106 to 700 cpm, + for 40 to 100 cpm, and - for less than 40 cpm or absence of radioactivity.

A1-4a A7 A8P A20 A25 Al6 Al7 Al%19 B5 B9 B13 B23-25 B27 C2 C23-24

Relative radioactivity in spot

co2+

- -

++ + - - -

-t + -

+ ++

+ +

++-I-+

Mgz+

-

+ +

+ +

+ -

-

+

++

+++ -

+++ +

++-I-+

MS+

+ -

+

-

-

-

+

4 -

f

+++

-t- ++

++++

- ZIG+

+ + +

- -

+

+++

++ -I-+++

-I-+ +++

-l-+-l-+

FIG. 6. Visualization of nucleolar phosphoproteins from Novi- koff hepatoma ascites cells after a 15-min incubation in vitro of isolated nucleoli with [r-a*P]ATP in the presence of 5 mM of either (a) CoC12, (b) MnClt, or (c) ZnClz. The procedures for the two-dimensional gel electrophoresis and the exposure of the x-ray film were the same as for Fig. 3.

Q This is a radioactive spot which migrates in the center of the region encompassed by stained spots Al, 2, and 4. It was also phosphorylated in vivo (6).

RNA polymerase (8, 9, 15), an RNase (17), NAD pyrophos- phorylase (17)) ATYase A (17)) and RNA methylase (10, 11). Although these results demonstrate the presence of phospho- protein kinase in nucleoli, they do not establish a unique locali- zation for this phosphoprotein kinase activity.

Comparison of the phosphorylation of nucleolar acid-soluble proteins shows that the same protein spots, including spots

I I, I I I I I I I I,

0 5x1o-9 5x1o-7 5x1o-5 5x1o-3

CAMP CONCENTRATION (Ml

FIG. 7. Effects of increasing concentrations of cyclic AMP on the phosphorylation of nucleolar acid-soluble protein in isolated nucleoli from Novikoff hepatoma ascites cells. The in vitro incubation systems were the same as for Fig. 1 except for the presence of various concentrations of cyclic AMP as indicated.

C23-24, B27, and I313 (6) were labeled both in viva and in vitro. In the system containing Mg*+ (Fig. 3c), several protein spots that were phosphorylated in viva were not phosphorylated in vitro, including spots A17-19, 1315, B22, 1323-25, C6, and C25 (6, 20). However, under proper conditions with replace- ment of Mg2f by Co 2+, Mnz+, or Znzf (Table II), the isolated

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nucleoli phosphorylated all the proteins phosphorylated in viva

(6). Quantitative differences in phosphorylation were also ob-

served between in vivo and in vitro systems. This variation could be due to different turnover rates or different divalent cation concentrations in vivo and in vitro.

Zn2f produced the highest level of- phosphorylation of the nucleolar proteins (Table I). In histological studies, a high Zn2+ concentration has been found in nucleoli of prostatic cancer cells (29). Also Znzf and Mrl*f caused higher endogenous phosphorylation of proteins in synaptic membrane fractions from rat brain; Ca2+ was inhibitory (30). Divalent cations may act to change phosphoprotein kinase activities, phospho- protein phosphatase activities (30), or the availability of serine hydroxyl groups for phosphorylation.

Possible relationships have been suggested between the phos phorylation of nuclear nonhistone proteins and gene regulation (4, 31, 32), cell division (26) or protein binding to deoxyribo- nucleic acid (33). Studies are currently underway to isolat’e and purify the nucleolar phosphoprotein kinases to identify which of the proteins phosphorylated are involved in matura- tion of the nucleolar preribosomal RNP particles and are related to phosphorylated polysomal proteins.

Acknowledgments-The authors wish to thauk Mr. Christo-

pher Jones for technical assistance and i\ilrs. Rose Izusch for transplantation of the Novikoff tumor cells.

REFERENCES

1. LANGAN, T. A. (1967) in Regulation of Nucleic Acid and Pro- tein Biosynthesis (KONINGSBERGER, V. V., AND BOSCH, L., eds) pp. 233-242, Elsevier, Amsterdam

2. KLEINSMITH, L. J., AND ALLFREY, V. G. (1969) Biochim. Biophys. Acta 176, 123-135

3. PLATZ, R. D.. KISH, V. M., AND KLEINSMITH, L. J. (1970)

7. BUSCH, H., AND SMETANA, K. (1970) The Nucleolus, Academic Press, New York

8. Ro, T. S., AND BUSCH, H. (1964) Cancer Res. 24, 1630-1633 9. LIAU, M. C., HNILICA. L. S., AND HURLBERT. R. B. (1965)

Proc. Nat. Acad. Sci: U. S.’ A. 63, 626-633 ’ 10. LIAU, M. C.. FLATT. N. C.. AND HURLBERT. R. B. (1970)

B&him. Bkophys. kcta 224, 282-285 ’ ’ ’ 11. CULP, L. A., AND BROWN, G. M. (1968) Arch. Biochem. Bio-

phys. 124. 483492 12. LAMKIN, A. F., SMITH, D. W., AND HURLBERT, R. B. (1973)

Biochemistry 12, 4137-4145. 13. LAMKIN, A. F., AND HURLBERT, R. B. (1972) Biochim. Biophys.

Acta 272, 321-326 14. MURAMATSU, M., SMETANA, K., AND BUSCH, H. (1963) cancer

Res. 23, 510-518 15. Ro, T. S., MURAMITSU, M., AND BUSCH, H. (1964) Biochem.

Biophys. Res. Commun. 14, 149-155 16. BUS&, H. (1967) Methods Enzymol. 12, 448464 17. SIEBERT, G.. VILLALOBOS. J.. JR.. Ro. T. S.. STEELE. W. J..

LINDE~MA~ER, G., ADAMS; H.,’ AN; BUSCH, H. (i966) J: Biol. Chem. 241, 71-78

18. LAYNE, E. (1957) Methods Enzymol. 3, 447-454 19. KNECHT, M., AND BUSCH, H. (1971) Life Sci. 10, 1297-1309 20. ORRICK, L. R., OLSON, %I. 0.. J., AND-BUSCH, H. (1973) Proc.

Nat. Acad. Sci. U. S. A. 70. 1316-1320 21. YEOMAN. L. C., TAYLOR. C. W., AND BUSCH, H. (1973) Bio-

them. Biophys. Res. Cokmun. 61, 956-966 22. JOHNSON. E. M.. AND ALLFREY. V. G. (1972) Arch. Biochem.

Biophyi. 162, $86-794 ’ ’ ’ 23. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RAN-

DALL, R. J. (1951) J. Biol. Chem. 193, 265-275 24. KLEINSMITH, L. J.. HEIDEMA. J.. AND CARROLL. A. (1970)

Nature 226, 1025-iO26 ’ 25. BALHORN. R.. BALHORN. M.. AND CHALKLEY. R. (1972) Dev.

Biol. 29, 196-203 ’ ’ , \ I

26. PLATZ, R. D., STEIN, G. S., AND KLEINSMITH, L. J. (1973) Biochem. Biophys. kes. Commun. 61, 735-740

27. LANGAN. T. A. (1968) Science 162. 579-580 28. GRUMM;, I. (197‘4) Fed. Eur. Biochem. Sot. Lett. 39, 125-128 29. GYBRKEY, F., MIN, K.-W., HUFF, J. A., AND GYBRKEY, P.

(1967) Cancer Res. 27, 1348-1353 Fed.‘Eur. Bkochem: Sot. L&t. 12, 38-40 30. U~DA, T., MAENO, H., AND GREENGARD, P. (1973) J. Biol.

4. TENG, C. S., TENG, C. T., AND ALLFREY, V. G. (1971) J. Biol. Chem. 248, 8295-8305 Chem. 246, 3597-3609 31. SHEA, M., AND KLEINSMITH, L. J. (1973) Biochem. Biophys.

5. RICKWOOD, D., RICHES, P. G., AND MACGILLIVRAY, A. J. Res. Commun. 60, 473-477 (1973) Biochim. Biophys. Acta 299, 162-171 32. KAPLOWITZ, P. B., PLATZ, R. D., AND KLEINSMITH, L. J.

6. OLSON, M. 0. J., ORRICK, L. R., JONES, C., AND BUSCH, H. (1971) Biochim. Biophys. Acta 229, 739-748 (1974) J. Biol. Chem. 249, 2823-2827 33. KLEINSMITH, L. J. (1973) J. Biol. Chem. 248, 5648-5653

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Yeou-Jan Kang, Mark O. J. Olson and Harris BuschHepatoma Ascites Cells: EFFECTS OF DIVALENT CATIONS

Phosphorylation of Acid-soluble Proteins in Isolated Nucleoli of Novikoff

1974, 249:5580-5585.J. Biol. Chem. 

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