Proc. Nat. Acad. Sci. USAVol. 70, No. 5, pp. 1316-1320, May 1973

Comparison of Nucleolar Proteins of Normal Rat Liver and Novikoff HepatomaAscites Cells by Two-Dimensional Polyacrylamide Gel Electrophoresis

(acid-extracted nucleolar proteins)

LARRY R. ORRICK, MARK 0. J. OLSON, AND HARRIS BUSCH

Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77025

Communicated by Daniel Mazia, February 22, 1973

ABSTRACT A two-dimensional polyacrylamide gelelectrophoresis technique is presented for separation ofacid-extracted proteins of isolated nucleoli of normal ratliver and Novikoff hepatoma ascites cells. About 100 dis-tinct protein spots were resolved. Comparison of the pat-terns for normal liver and Novikoff hepatoma revealed ninespots with markedly different intensities in the two pat-terns. Two spots were found that were unique to the nor-mal liver pattern, and one spot was found that was uniqueto the hepatoma pattern.

Although considerable information on the possible functionalroles and metabolism of nonhistone nuclear proteins is nowaccumulating (1, 2), understanding of the chemistry of thesemolecules has advanced slowly because of the uncertainty asto the number and types of these proteins (3-5). The im-pression that there is a substantial number of these proteinshas emerged from studies by polyacrylamide disc-gel electro-phoresis on nuclear and nucleolar proteins associated withchromatin (1, 4, 5). However, very little information is avail-able regarding the number and characteristics of proteins ofextracts of whole nuclei and nucleoli (6, 7).Inasmuch as the development of two-dimensional electro-

phoresis for separation of ribosomal proteins providedexcellent resolution of these molecules (8-10), which have asimilar overall composition to nucleolar proteins (7), itseemed possible that this approach could have special valuefor studies on nucleolar proteins. The nucleolar acid-extract-able proteins had solubility characteristics that preventedthe use of the systems used for studies on ribosomal proteins.In the present study, these problems have been resolved, and ahigh-resolution two-dimensional acrylamide gel electro-phoresis technique has been developed that is suitable forapplication to nucleolar extracts as well as for extracts ofwhole nuclei.The extraction procedure used 0.4 N H2SO4, which had

been used previously, to permit comparison of the resultswith those on histones (6, 7). To determine whether themethod had discriminatory capability for tissue function, theresults are compared for two tissues, normal liver and Novikoffhepatoma.

MATERIALS AND METHODS

Nucleoli of Novikoff hepatoma ascites cells and normalrat-liver cells were isolated (6, 7) and subsequently extractedwith 0.4 N H2SO4 as reported (6). The ethanol-precipitated,vacuum-dried protein sample was dissolved in a concentrationof 10 mg/ml in a solution containing 10 M urea-0.9 N aceticacid-i mM dithiothreitol.

Electrophoresis in the first dimension was performed in12-cm tubes with an inner diameter of 5 mm. The solutionsfor the polyacrylamide gel of the first dimension contained thefollowing: (a) 40% acrylamide, 1.4% bisacrylamide, 4 Murea; (b) 4.0% TEMED (N,N,N',N'-tetramethylenediamine)in 2 M urea; and (c) 0.21% ammonium persulfate, 3.6 Nacetic acid, 6 M urea. These solutions were combined in aratio of 1 volume of (a), 1 volume of (b), and 2 volumes of (c)to yield a gel composition of 10% acrylamide, 0.35% bis-acrylamide, 0.1% persulfate, 1% TEMED, 4.5 M urea. Thepolymerization time was about 1 hr.

After pre-electrophoresis for 2 hr at 120 V to constantamperage with 0.9 M acetic acid as the electrolyte, a 20- to50-,u sample was loaded on the gel. It was subjected toelectrophoresis for 5 hr at 120 V and about 2.5 mA per geltube.The gels of the first dimension were then removed from the

tubes and bisected longitudinally with a taut wire cutter. Halfthe gel was stained in 1% Buffalo Black-7% acetic acid(45 min) and destained in 10% methanol-7% acetic acid.The second half of the gel was then adapted for the seconddimension.The gel for electrophoresis in the sodium dodecyl sulfate

second dimension was prepared by dialysis in a series ofthree adaptation solutions: (d) 1% Na dodecyl sulfate, 0.1 Mphosphate buffer (pH 7.1), 6 M urea; (e) 1% Na dodecylsulfate, 0.01 M phosphate buffer (pH 7.1), 6 M urea; and(f) 0.1% Na dodecyl sulfate, 0.01 M phosphate buffer (pH7.1), 6 M urea. Perforated tubes containing the gel halveswere immersed and vigorously stirred in the adaptationsolutions which were maintained at 40-45° throughout thedialysis, 30 min in the first bath and 25 min each in the othertwo baths.The slab-gel electrophoresis in the second dimension was

performed with an Ortec (Ortec, Inc., Oak Ridge, Tenn.)electrophoresis tank and cell. The glass cells used form a gel100-mm across by 95-mm long and 3 mm in width. The slabgels formed in these cells had a volume of 25 ml. The solutionsfor the slab were: (g) 20% acrylamide, 0.52% bisacrylamide,8 M urea; (h) 0.2% TEMED in 0.4 M phosphate buffer(pH 7.1) and 0.4% Na dodecyl sulfate; and (i) 0.5% ammo-nium persulfate, 8 M urea. The solutions were combined in theproportions: 0.6 parts of (g) to 0.25 parts of (h) to 0.15 parts of(i). This combination produced a gel containing 12% acryl-amide, 0.31% bisacrylamide, 0.05% TEMED, 0.10% Nadodecyl sulfate, 0.075% persulfate, 6 M urea, and 0.1 Mphosphate buffer (pH 7.1). The phosphate buffer contained

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Electrophoresis of Nucleolar Proteins 1317

132 mM NaH2PO4 H20-268 mM Na2HPO47 H20-0.4% Nadodecyl sulfate.The adapted portion of the bisected first-dimension gel was

immersed for 2 min in a cap gel mixture identical in com-

position to the slab gel except that the phosphate buffer was

0.01 M. The cap gel solution was made by substitution of stockbuffer diluted 1:10 in solution (h) in place of the full-strengthbuffer. The soaked gel was then placed on the previouslypolymerized slab and overlayered with sufficient cap gel tofill the slab (about 5 ml). The cap gel layer was then over-

layered with water and allowed to polymerize for 10 min.Two 12% slabs were run in one buffer tank for 14 hr at a

constant current of 50 mA per slab gel and an initial voltageof about 40 V. The electrolyte solution was stock phosphatebuffer diluted 1:4 to give 0.1 M phosphate-0.1% Na dodecylsulfate. After removal from the cells, the slab gels were stainedfor 3 hr in 0.25% Coomassie blue dissolved in 1 part water-1 part methanol-0.2 parts acetic acid. The gels were thendestained in several changes of a solution containing 5%methanol-10% glacial acetic acid.

RESULTS

97 Stained protein spots were found both in the patterns forNovikoff hepatoma samples (Fig. la and b) and in samples ofnormal liver (Fig. 2a and b). The gel slabs were divided intoregions A, B, and C, and the components were numbered inorder of decreasing mobility in both dimensions. Regions A,B, and C contained 31, 41, and 25 spots, respectively, in thenormal liver pattern. In the hepatoma pattern there were 30,36, and 31 spots, respectively, in the A, B, and C regions.The reproducibility of the relative intensities and positions

of spots for samples of nucleolar proteins from individualpreparations of Novikoff hepatoma and normal liver was

excellent both for duplicates in a single run and among runs

of separate series. Optimal resolution of areas of high proteindensity of the gel was found with a sample of 125 ,ug per slabgel, while regions of intermediate and low protein densitywere best resolved with sample sizes ranging from 250-500Mg per slab gel.The mobilities of some of these spots corresponded with

those of known histone samples. The position of the GARhistone, also referred to as the f2al histone or histone IV, isshown in Figs. 1 and 2. In addition, spot Al is probably theAL histone. Spot A2 is probably the N-proline histone or f2bhistone; spot A4 is the F3 histone or histone III; and spotsA17, A18, and A19 are very lysine-rich or fl histones (2, 3).There were differences between the patterns of the nucleolar

proteins of normal liver and Novikoff hepatoma. Strikingdifferences were observed in the relative staining intensities ofcertain spots (Table 1). For example, A1OL was much denserthan A10 in the normal liver pattern, while A10 was muchlarger than A1OL in the hepatoma pattern. Similarly, in theB region, quantitative differences were found for spots Biand B18, which were consistently denser spots in the liverpattern compared with the hepatoma pattern. In the Cregion of the liver pattern, spot C11 was a dense spot, C8 was

very small, and C9 was absent. Conversely, in the hepatomapattern C8 constituted a dense spot, C9 was a small definitespot, and Cl 1 was only a minor spot.Some qualitative differences were also found in the patterns.

In the B region two protein spots, B4L and B5L, were foundin the liver pattern but not in the hepatoma pattern. In the

hepatoma pattern, C9 was present but was not found in thenormal liver pattern.

DISCUSSION

The present studies provide a practical procedure for sepa-ration of a large number of nuclear proteins by two-dimen-sional gel electrophoresis. The reproduction of the results onthe gels by photography or by mapping is not as satisfactoryas direct visualization of the gels. However, the results arehighly reproducible from preparation to preparation, and thedifferences found for liver and nucleoli patterns are con-sistent from experiment to experiment and readily visualizedby examination of photographs of the gels (Figs. 1 and 2).By light and electron microscopy and by enzymatic

analysis, there was no appreciable contamination of theisolated nucleoli with membranes, extranucleolar chromatin,or cytoplasmic particles (7). In studies in progress, most ofthe protein spots found in the nucleolar two-dimensional gelshave also been found among the protein spots in the two-dimensional gel patterns for acid-extractable proteins ofnuclei obtained by the citric acid procedure (7). Since thecitric acid nuclei are essentially devoid of cytoplasm, theseresults make it unlikely that nonnucleolar proteins areabsorbed from the cytoplasm during these preparations. Onthe other hand, it is possible that highly soluble nucleolarproteins may be extracted during the isolation procedure.The demonstration of several quantitative differences in

spot size and intensity of staining for the tumor and liverpreparations was unexpected. However, the prominence ofA19 in the very lysine-rich histone group of the hepatomapattern as compared with the diminution of this spot in thenormal liver pattern may reflect the difference in phos-phorylation of lysine-rich histones that was correlated withgrowth rate (11). One of the more striking quantitativedifferences is the greater density of spot A10 in the Novikoffhepatoma pattern as compared to that in the normal liverpattern. On the other hand, the dense spot A1OL in thenormal liver pattern was hardly visible in the tumor pattern.The presence of spots of different size having similar migrationin the second dimension (Na dodecyl sulfate) suggests thepossibility that these may reflect different chemical modi-fications of the same parent protein.

TABLE 1. Spot density differences for thehepatoma and liver patterns

Density of spot

Spot number Hepatoma Liver

A10 ++++ +A1OL ++ +++All + +++Bi + ++++B4L Absent +B5L Absent +B18 ++ +++C8 +++ ++C9 + AbsentClH + ++

Tabulation of the spots with major differences in density be-tween the nucleolar protein patterns of normal liver and Novikoffhepatoma. The criteria for the differences are spot density andsize.

Proc. Nat. Acad. Sci. USA 70 (1973)

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1318 Biochemistry: Orrick et al. Proc. Nat. Acad. Sci. USA 70 (1978)

BIBio,

laa

AIO AIOL

b ~~~~~~~~~~~~~~~~~~~~~25e027A 1.,22q9I C 2

I 29~~~14 26'3 3

16~~~~~~~~~~~~~~~~~~~~~~1

GAR- IOLQ I~~~~~~~2-01

A A10 B I

33e 40 12 t826A7

68 3 24 A 12 13

,_>~~~~~~~~~~~~14A

_~~~~~~~~10 I II

1

FIG. 1. (a) Two-dimensional polyacrylamide gel electrophoresis of 250 jig of Novikoff hepatoma nucleolar proteins. Samples were firstloaded on tube gels of 10% acrylamide-6 M urea and run in the first dimension for 5 hr at 120 V constant voltage. For the second dimen-sion, a 12% acrylamide-0.1% Na dodecyl sulfate slab gel was run for 14 hr at 50 mA constant amperage and then stained withCoomassie blue. The numbered spots are different in size and density from those of the pattern of normal rat liver.

(b) Diagrammatic representation of the pattern of Fig. la showing the numbering system for the spots. The most-dense spots%re black, the less-dense spots are cross-hatched, and even less-dense spots are open circles. Minor spots are shown as broken circles.

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Electrophoresis of Nucleolar Proteins 1319

C_

All

BlAq~ 81_ 18

_t t Bs1 *~~~~~~~~~~~~~~~- BDo

w i_ ~B4L.

AID AIOL

I 24ajr

_r~~~~~~~~~~~9B5L

2Sda31 25e

Z9AS' 030 W

2% I¶~34110 C1.2

.*s~~~~~~~~~~~~~~~~~19

A10 2480L

5_11|17t 1 3s867811eFS 2 WO 1

.'-}~~~~~~1 24Aw

4A|100 AIO4 I

GAR l 1

A B C

I52

Fia. 2. (a) Two-dimensional electrophoresis of nucleolar proteins of normal liver. Conditions were identical to those in Fig. la.(b) Diagrammatic representation of the two-dimensional electrophoretic pattern of nucleolar proteins of normal rat liver, numbered

asshowninFig. lb.

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1320 Biochemistry: Orrick et al.

The method used for isolation of the nucleoli providessatisfactory products from both Novikoff hepatoma andnormal liver (7), and it was anticipated that because thenucleoli are primarily involved in synthesis of preribosomalproducts that the proteins present in these nucleoli would beessentially the same (7). As listed in Table 1, in addition tothe quantitative differences, some protein spots were foundin normal liver nucleoli that are not found in tumor nucleoli.One possibility is that these spots might constitute some genemodulator or other control proteins that may account for thelower rates of synthesis of nucleolar products in liver com-pared with tumors. If this were the case, such proteins wouldbe of special interest from the point of view of the control ofthe nucleolus and rDNA activities. On the other hand, theobserved quantitative differences may be due to differentialrates of synthesis of certain proteins as a result of a greaterproportion of tumor cells undergoing cell division.These results have shown that protein fingerprinting

methods may be of special value in distinguishing significantchanges in protein complements of nuclear and nucleolarextracts of tissues in different functional states. In particular,the possible functional differences of proteins B4L and B5L

and the possible interrelation of A10 and A1OL are of interestfor further investigation.

These studies were supported by the American Cancer SocietyGrant NP-21G, the USPHS Grant CA-10893,P-3 and the USPHSGrant GM/670. L.R.O. is a Predoctoral Trainee.

1. Wilhelm, J. A., Anseven, A. T., Johnson, A. W. & Hnilica,L. S. (1972) Biochim. Biophys. Acta 272, 220-230.

2. Hnilica, L. (1972) in Structure and Biological Function ofHistones (CRC Press, Cleveland), pp. 151-159.

3. Busch, H. (1965) in Histones and Other Nuclear Proteins(Academic Presg Inc., New York), pp. 197-226.

4. Teng, C. S., Teng, C. T. & Allfrey, V. A. (1971) J. Biol.Chem. 246, 3597-3609.

5. Elgin, S. C. & Bonner, J. (1970) Biochemistry 9, 4440-4447.6. Knecht, M. & Busch, H. (1971) Life Sci. 10, 1297-1309.7. Busch, H. & Smetana, K. (1970) in The Nucleolus (Academic

Press, Inc., New York), pp. 531-538.8. Martini, 0. H. W. & Gould, H. J., (1971) J. Mol. Biol. 62,

403-405.9. Kaltschmidt, E. & Wittmann, H. G. (1970) Anal. Biochem.

36, 401-412.10. Sherton, C. C. & Wool, I. G. (1972) J. Biol. Chem. 247,4460-

4467.11. Balhorn, R. B. M., Morris, H. P. & Chalkley, R. (1972)

Cancer Res. 32, 1775-1784.

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