Effects of various ionic media on extraction of soluble nuclear proteins and on nuclear ultrastructure

Experimental

Cell Research 85 (1974) 191-204

EFFECTS OF VARIOUS IONIC MEDIA ON EXTRACT1 NUCLEAR PROTEINS AND ON NUCLEAR ULT

LE

M. KELLERMAYER,l M. 0. J. OLSQN, K. SMETANA, I. DASKAL and H. BUSCH Depaviment of Pharmacology, Baylor College of Medi’cinnez Houston, Tex. 77025, USA

SUMMARY Isolated liver nuclei were extracted 3 times at pN 7.2 with solutions containing either (1) monovalent cations, (2) both mono- and divalent cations, or (3) sucrose solutions containing only divalent cations. The extracted proteins were analysed by two-dimensional acrylamide gel electrophoresis and the ultrastructural alterations of the treated nuclei were examined by electron microscopy. The solutions containing Na+ or K+ monovalent and Ca2’ and MgZ+ divalent ions extracted the same amount (18-22 %) of the nucIear nroteins. The two-dimensional gel electrophoretic patterns of these extracts were nearly identical and the structures of the nuclearcomponents were well preserved even after 3 times repeated extractions. The solution containing only Na+ extracted less protein (14-15 X) than the solutions containing both mono- and divalent cations. Extraction with isotonic NaCl solution altered the nuclear and nucleolar morphology; unlike the other solutions employed, this solution extracted some DNA and histones. The isotonic sucrose solution containing only divalent cations extracted less protein than the other solutions (9-ll %) and produced marked condensation of the chromatin. These analytical and electron microscopic studies showed that mono-and divalent cations play a role in structural organization of chromatin.

Methods for isolation of nuclear producis with the use of various types of salt solutions have indirectly shown that ionic bonds exist between various nucleic acids and proteins [6, 7, 12, 141. Moreover, both light and electron microscopic changes accompanying the various extractions have been useful in descriptions of the structural organization of deoxyribonucieoproteins and ribonucleoprotein particles ]l, 4, 20, 21, 27, 311. Generally, the first step in the fractionation of the cell nuclei is the extraction with bufferred isotonic saline [35, 371.The basisof this step is that at an ionic strength of 0.15, the nucleohistone or DNP complexes are insoluble [6, 14, 15, 41). Although 0.15 M NaCl does not extract much DNA or histone, it

doesextract many nuclear proteins 13, 12,291~ Since the characteristics of tbe nuclear proteins extracted with 0.15 M salt solutions are not completely defined, the present studies were initially designed to determine t terns of two-dimensional gel electrophoresis of these proteins. Inasmuch as earlier studies showed t divalent cations decrease the extractability of histones 119, 4O], a second goal of the studies was to make a comparison of the proteins extracted with 0.15 M NaCl with sob tions containing Ca2+ and MgZ+ ions. Such studies may have relevance to the structures and functions of nuclear components in living cells.

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et al.

MATERIALS

AND METHODS

Isolation of nuclei. The cell nuclei were isolated from liver cells of male Holtzman rats (200-240 g). The livers were perfused with Hanks solution [18] by way of the portal vein, then removed and minced with a tissue press. The homogenization of the cells has been carried out by a Tissumizer (Tekmar SD-45K Super Dispax System) in a solution of 2.2 M sucrose and 3.3 mM calcium acetate [8, 10, 361. The isolated nuclei were centrifuged at 16 000 g for 90 min. The pelleted nuclei were resuspended in the 2.2 M sucrose and 3.3 mM calcium acetate solution and centrifuged again at the same speed and time. The isolated nuclei were examined for purity by phase contrast microscopy after staining with azure C [9]. Extraction of proteins from isolated nuclei. The nuclei were extracted 3 times for 30 min each at 0-4°C with continuous stirring in the following solutions: (a) TN (0.15 M NaCl, 0.01 M Tris-HCl, pH 7.2); (b) TNCM (0.15 M NaCI, 0.01 M Tris-HCI, pH 7.2, 3.8 mM CaCl, and 12 mM MgCQ; (c) TKCM (0.15 M KCI, 0.01 M Tris-HCl, pH 7.2, 3.8 mM CaCl, and 12 mM MgCl,); (d) TSCM (0.25 M sucrose, 0.01 Tris-HCl, pH 7.2, 3.8 mM CaCl, and 12 mM MgC&). In all cases the extractant was in a 10 : 1 (v:v) ratio with the sample. From the first nuclear suspension, a samule was analyzed for total nuclear DNA [5] and protein [26]. After extraction for 30 min, the nuclei were centrifuged at 300 g for 10 min to minimize damage by centrifugation. The first and second pellet was resuspended again in fresh solution and reextracted for 30 min. From the last pellet after the third incubation, a sample was taken for electron microscopy. In all cases, the 300 g supernatants were further centrifuged at 17 000 g for 10 min to remove nuclear fragments; aliquots of the supernatants were analysed for protein and DNA. In some cases the TNCM extracted nuclei were re-extracted twice for 30 min with TN solution and the resulting supernatants were treated in the same manner as the other extracts. Amino acid anlysis of TNCM and TN extracts was done with a Beckman Model 121 amino acid analyzer [34]. Treatment of the extracts. After centrifugation at 17 000 a for 10 min. the 3 consecutive extracts were made to 8 M urea, ‘0.9 M acetic acid and 5 mM /3mercaptoethanol to minimize degradation: The samples were then pooled and centrifuged at 100 000 g for 1 h. The dilute protein extracts were concentrated in an Amicon cell equipped with a UM-2 Diaflo ultrafiltration membrane (Amicon Inc., Lexington, Mass.) to 7-10 mg/ml protein content. Before the electrophoretic analysis, the concentrated samples were dialysed against a sample buffer composed of 10 M urea, 0.9 M acetic acid and 5 mM /3-mercaptoethanol. Electrophoretic analysis. The two-dimensioual polyacrylamide electrophoretic method developed ‘&this laboratory [28, 431 was used in these studies. qhe numbers l-100 represent the acid-soluble nucleolar proteins [28]. The- letters represent the acid-soluble nuclear proteins [44] and the numbers from 101 up are the acid-insoluble “cytonucleoproteins” [18]. Exptl Cell Res 85 (1974)

Electron microscopic analysis. Small portions of the nuclear pellets were fixed in glutaraldehyde and postfixed in osmium tetroxide [9, 331. The specimens dehydrated in ethanol containing uranyl acetate were embedded in Epon-Araldite mixture [9, 331 and sectioned with a Porter Blum II ultramicrotome. The ultrathin sections were stained with uranyl acetate, followed by lead citrate, and observed with a Philips 200 electron microscope.

RESULTS Protein and DNA content of the extracts

The total amount of the nuclear proteins extracted from jsolated rat liver nuclei was greater (i.e. 20%) with TNCM and TKCM than with TN or TSCM solutions (table 1). The three consecutive incubations of the isolated nuclei in TN solution removed 14-15 % of the total nuclear protein (table 1). With the first treatment, only 7-9 % protein was extracted but the protein content of the second (4 “/*) and third extracts (3 %) was also relatively high. With both TNCM and TKCM the first and second extractions removed most of the extractable proteins and the third extract contained very little protein. Although TSCM removed less protein, most was extracted with first treatments. The TN solution released 2-5 % of the DNA of the nuclei during the repeated extractions (table 1). The TNCM, TKCM and Table

1. Protein and DNA content o,f the

extracts % of total Extracts

Protein

DNA

T&M TKCM TSCM

1415 19-22 18-20 9-11

2-5 il
Protein and DNA content of three consecutive extracts related to the total protein and DNA of isolated nuclei. Protein determined by the Lowry method [26]; DNA according to the Burton method [5]. Prior to this analysis, the samples were centrifuged at 17 000 g for 10 min to remove nuclear fragments.

Soluble nuclear proteim Table 2. Number ele~t~o~hores~~

of separated protein spots from different

A Region

B Region

extracts

in

~~~-~~~~%~~5~~~

193 gel

C Region

Extracts

Dense

Faint

Dense

Faint

Dense

Faint

Total

TN TNCM TKCM I-SCM

11 7 8 3

17 19 25 5

14 27 22 9

22 23 27 18

21 31 28 17

10 3 10 5

95 110 120 57

-

Proteins separated by two-dimensional polyacrylamide gel electrophoresis from 250 ,ug of the extracted proteins. “Dense” represents those protein spots characterized on the maps (figs 1 b, 2b, 3 b) by black and crosshatched spots. “Faint” corresponds to the open and broken circles.

TSCM solutions which contained divalent cations extracted very little if any DNA from the isolated nuclei (table 1). Two-dimensional electrophoretic separation of extracted nuclear proteins

y two-dimensional acrylamide gel electrophoresis, 95 protein spots were found in the TN extract (fig. I, table 2). Although extracts with solutions containing mono- and divalent ions (TNCM and TKCM) contained more protein spots, those with TSCM contained remarkably fewer proteins (figs 2, 3, table 2). The size and intensity of the protein spots are denoted on figs 26, 3 b and 4b by the convention adopted earlier [28]. In the TN extract, the GAR histone, and proteins Al, A2, A4, A17 and Al8 were dense spots (fig. 1, table 3); Al, A2 and A4 contain the AL, F2b and F3 histones respectively and A 17 and A 18 contain the lysinerich histones [28]. Spot A24, which is not a histone, is extracted with TN solution. The other solutions employed extracted few of these proteins (table 3). t is notable that spots As-r, B24, C4, C5, , c-?, Cll,

c13, CI4, C2I, c104, c107,

C 108 and C 112 were very dense in all the studied. Moreover, spots B 18, Bu, 718, C10, C23, C24, Cl09 and

Cl10 were all very dense in the patterns for the TN, TNCM an concentrations were extracts indicating that the monovalent cation exerted a significant effect on their extraction The TSCM extract containe spots than any of the other solutions employed; thus, AllO, C24 and C 111 proteins spots in the TNCM extract were TSCM extract (figs 2, 3, table 3). It is noteworthy that the solutions containing botb the mono- and divalent cations, TNCM and TKCM, extracted A 110, B7, B 121, C23, C24 and C B1I to a greater extent than the TN solution containing only monovalent cations (table 3). The differences between the electro patterns of TN and TNCM extracts that re-extraction of the TNCM treated nucEei with TN solution removes some histones, The second treatment with TN solution extracted 5 % more protein which was mainly histones (fig. 4, table 4)* The electrophoretic analysis of nuclear proteins extracted by TN in this way also showed that solubilization of histones (GAR, Al, A2, A4, Al7 and A18) and the A24 nonhistone protein is characteristic only for the TN solution (figs 4). Furt lack of protein spots in the high and C regions showed that the

194 Kellermayer

et al.

Table 3. Comparison

of some protein spots in

different extracts Protein spots GAR Al 2 All A18 A24 All0 Bl Bu B121 Cl7 C23 C24 Cl11

Extracts TN

TNCM

TKCM

TSCM

++++ ++++ ++++ ++++ ++++ ++++ ++++ + i+ ++++

+ + + + + + + ++++ ++++ ++++

+ + + + + + + ++++ ++++ -I-+++

Absent Absent Absent Absent Absent Absent Absent Absent Absent

++++ ++++ ++++ ++++ ++++

t+ ++ ++++ ++++ ++

Absent Absent Absent Absent

Absent ++++ ++ ++ +

The table contains the proteins spots which showed the most prominent differences of the four extracts. + + -t + represents black, + + represents crosshatched and + represents faint spots on the maps made according to the electrophorograms of the extracted proteins.

TNCM extracted all of the salt-soluble ‘nuclear sap’ proteins.

or

Electron microscopic analysis

The isolated, untreated nuclei (fig. 5) were characterized by the presence of large clusters of condensed chromatin, clumping of the interchromatin granules and filamentous protein structures of the interchromatin areas. The nucleoli were compact and the nucleolar ribonucleoprotein components were less distinct. It was difficult to differentiate the perinucleolar chromatin from the periphery of the nucleolar body. The perichromatin granules were preserved; their ultrastructure and localization in the nucleus were similar

to previous descriptions [38]. The nuclear envelope was composed of the usual two dense layers (table 5). The incubation of nuclei in 0.15 M NaCl in the absence of Ca2+ and Mg2+ produced a marked alteration of their ultrastructure (fig. 6, table 5). The chromatin was loosely organized and more dispersed although a greater number of chromatin fibrils were still present near the nuclear membrane and in the chromocenters. The perinucleolar chromatin seemed to be dispersed and the separation of the nucleolar body from the rest of the nucleus was less distinct. The nucleolus contained distinct fibrillar components. The perichromatin granules were prominent. There was a striking change in the interchromatin granules; both their density and number seemed to be markedly reduced. The outer layer of the nuclear envelope was largely free of adjacent ribosomes. In nuclei incubated in TNCM (fig. 7), the chromatin at the nuclear envelope was condensed. The chromatin around the nucleolus and of the chromocenters was also condensed. The interchromatin areas contained clumps of interchromatin granules embedded in less dense, amorphous matrix. The nucleolus was composed of the characteristic nucleolar components and the nucleolar body was clearly separated from the perinucleolar chromatin. The perichromatin granules were usually present at the periphery of the condensed chromatin. The outer layer of the nuclear envelope was preserved along with its adjacent ribosomes (fig. 7, table 5). Lacuna-like spaces were notable in the condensed chromatin adjacent to the nuclear membrane.

Fig. 1 (a) Two-dimensional polyacrylamide gel electrophoresis of rat liver nuclear proteins extracted by TN salt solution. 500 pg protein was initially loaded on the first-dimension gel. The second-dimension slab gel was stained with Coomassie blue and analysed after destaining; (b) diagrammatic representation of the gel pattern of fig. 1 a. The designating system using numbers and small letters for the characterization of the protein spots was mentioned in the Materials and Methods. The intensive spots are black, the less dense spots are crosshatched and the faint spots are open or broken circles. Exptl Cell Res 85 (1974)

F&q. 2. (a) Two-dimensional electrophoresis of nuclear proteins extracted by the TNCM solution. The conditions were identical with those employed in fig. 1 a; (b) diagrammatic representation of the slab gel pattern prepared from the TNCM extract, fig. 2~. Designating system was the same as fig. 1 b. Exptl

Cell Res 85 (1974)

Fig. 3. (ce>Two-dimensional gel electrophoresis of TSCM extract. The method applied was the same as for the TN extract; @> diagram of the slab gel of the TSCM extract, fig. 3 a. Exptl Cdl Res M (1974)

198 Kellermayer

et al. DISCUSSION Quantitative analysis of the protein extracts showed that the divalent cation-free TN solution extracted less protein than the TNCM or TKCM solutions containing Ca2+ and Mg2+ ions at the same concentration as in the cytoplasm of rat liver cells [22, 321. (Both mono- and divalent cation-free isotonic sucrose solution extracted 10% of the total nuclear protein, less than TNCM, TKCM or even TN solution [19]). The smaller amount of protein in the TN extract suggests an incomplete extraction of the ‘nuclear sap’ proteins after TN treatment which might be the result of aggregation and clump formation of nuclei in this solution. The divalent cations prevented the clump-

Fig. 4. Two-dimensional electrophoresis of nuclear proteins extracted by the TN solution from isolated liver nuclei preextracted by TNCM. The electrophoretie conditions were the same as those employed in fig. la, but the amount of total protein on the firstdimension gel was 200 pg.

The ultrastructure of nuclei incubated in TKCM solution did not differ substantially from that of nuclei incubated in TNCM with the presence of Ca2+ and Mg2+ divalent ions (fig. 8, table 5). The ultrastructure of nuclei incubated in 0.25 M sucrose containing Ca and Mg ions (TSCM) was characterized by a very dense perinucleolar chromatin which was sharply demarcated from the periphery of the nucleolar body (fig. 9, table 5). Although the presence of divalent and monovalent cations in the incubation media (TNCM and TKCM) caused some condensation of chromatin (figs 7, S), incubation of nuclei in TSCM which contained divalent but not monovalent cations produced a hypercondensation of all chromatin structures (fig. 9).

Table 4. Amino acid composition and TN extracts Amino acids

TNCM

Corrected

Cell Res 85 (1974)

TNa

mole percentages

;z 3:57 6.88 0.17 3.64

13.40 1.88 7.68 5.19 5.69 6.95 8.29 3.98 9.19 12.11 0.00 7.81 1.39 4.05 7.19 3.09 2.12

1.58

0.59

23.52 14.91

13.48 22.96

1.17

LYS

His

2.01 5.13 11.25 4.93 7.13 12.27 9.88 10.47 7.12 0.66

Arg Asp Thr Ser Glu Pro GUY Ala CYl2 Val Met Ileu Leu Tyr Phe Ratio

Glu -I-Asp/Lys + His + Arg Sums

(mole percentages)

Glu, Asp Lys, His, Arg

a The TN extraction followed the TNCM of nuclei. Exptl

of TNCM

extraction

Fig. 5. A nucleus from a preparation in 2.2 M sucrose containing 3.3 mM Ca2+. Note that the separation of the nucleolar body from the perinucleolar chromatin is not distinct (white pointers). The interchromatin areas contam large clusters of interchromatin granules (black pointer). Perichromatin granule (arrow), ribosomes adjacent to the dense external layer of the nuclear envelope (small arrow). x 45 000.

ing and aggregation of nuclei which enhanced a complete extraction of soluble nuclear proteins. That the extraction of the ‘nuclear sap’ proteins was essentially complete by TNCM or TMCM extraction was supported by the Table 5. Alteration

of the nuclear ultrastructureproducedby

Ultrastructural alteration

Control nuclei

Cbromatin density Perinucleolar chromatin density Nuclear body and perinucleolar chromatin separation Preservation of external membrane of nuclear envelope

++ ++

The

protein estimation on consecutive extra~t~~~s and the reextraction of TNC with TN solution (fig. 4). treatment, the TN solution extracted histones and only trace amounts of non-l&stone prs-

TN

incubation of nuclei in various media TNCM

TKCM

TSCM

0 ++

L

+++

j-

+++

+

.

marks ( + C) + ) 0) arose from examinations of numerous EM pictures of the nuclei.

Exptl Cd

Res

85 (1974)

200

Kellermayer

et al.

Fig. 6. A nucleus incubated three times for 30 min each in 0.15 M NaCl in the absence of divalent ions (TN solution). The interchromatin granules are very faint. The demarcation of the nucleolus is indistir 1ct (t )lack pointers). The prominent fibrillar components organized in nucleolonemas (F), perichromatin granules (arrcd, the perichromatin granule associated with a less dense strand (crossed arrow), interchromatin grar iules in a polyribosomelike configuration (crossed small arrow), chromocenter (long thin arrow), fragment of th e extcernal layer of the nuclear envelope (small arrow), ribosome (small black pointer). x 62 000.

Exptl Cell Res 85 (1974)

Fig. 7. A nucleus incubated 3 times for 30 min each in TNCM solution. The nucleolar body is well separated from the perinucleolar chromatin (white pointers), a cluster of interchromatin granules (black pointer), perichromatin granule (arrow), chromocenter (long thin arrow), ribosomes at the external dense layer of the nuclear membrane (small arrow). x 33 750. Fig. 8. A nucleus incubated three times in TKCM solution. The nucleolar body (white pointers), perichromatin granule (arrow), small cluster of interchromatin granules (black pointer), ch omocenter (long thin arrow), ribosomes at the nuclear membrane (small arrow). x 50 600. Exptl

Cell Rm 85 (1974)

202

Kellermayer

et al.

Fig. 9. A nucleus after incubation 3 times (30 min each) in TSCM solution containing 0.25 M sucrose, 3.8 mM NaCl and 12 mM MgC12, 0.1 M Tris-HCl (pH 7.2). The nucleolar body is well separated from the perinucleolar chromatin which is highly condensed (white pointers). Clump of interchromatin granules (black pointer), perichromatin granules (arrows). ribosomes at the external layer of the nuclear envelope (small black arrow). x26760. ~ “

teins. Thus, the divalent cation containing TNCM or TKCM solutions have a great advantage over the often used 0.15 M NaCl (TN) solution for removal of the nuclear sap proteins for studies of chromatin composition. The main difference in the proteins extracted with the divalent cation-free TN solution (fig. 1) and Ca2+, Mg2+ containing TNCM and the TKCM solutions (fig. 2) is that the former solubilized some histones while the latter solubilized little or none. Since 2-5% of the DNA was solubilized with the TN solution, it would appear that an approximately equal amount of histone was solubilized. Although it is not possible to Exptl Cell Res 85 (1974)

specify the mechanism of the extraction of DNA by the TN solution it is possible that this fraction is the less condensed euchromatin or ‘active chromatin’ that may be more sensitive to mechanical shear, nuclease digestion or more loosely bound to histones than the remainder of the chromatin. It would be of interest to determine whether the histones released by the TN solution have same modifications as those that remain unextracted or are extracted from the nuclear envelope in preference to the central regions of the nucleus. The comparison between TSCM and TNCM or TKCM extracted proteins provides new information about the combined effects

of divalent and monovalent cations. The osmolarity, p , and the divalent cation concentration were similar in the three solutions but the TSCM was free of monovalent cations. The solutions containing both monoand divalent cations extracted both more and different proteins. The TNCM or TKCM extracts contained all of the proteins extracted by TSCM solution and many additional proteins as well (tables 2, 3). Cations at 0.15 ionic strength apparently cleave aminocarboxyl salt linkages [24], which suggests that the proteins present in the TNCM or TKCM extract and absent from the TSCM extract may bind to histones by carboxyl-amino salt linkages. Such looselybound proteins are included in the nuclear sap proteins. The acidic character of the TNCM extracted proteins may also support ionic interactions among the loosely bound nuclear sap proteins and histones. There is some evidence for binding of histones to saline soluble proteins in cell nuclei. The Fast-green binding property of histones increased after 0.15 M salt treatment in isolated thymocyte nuclei and HeLa cell nuclei [B6]. Auer [2] found a decreased Fastgreen stainability of histones of activated cell nuclei suggesting that the newly synthesized and transported proteins in the nuclei are bound to the ammo groups of histones. An a~~urn~latio~ of soluble or cytonucleoprotems in cell nuclei accompanies gene activation [2, 131. Also, alteration of proteinhistone interactions may modify the relationships between histones and DNA and us influence gene activity [I, 2, 11, 14, 39, lectron microscopic examinations corwith the protein analysis. Along with removal of the histones with TN solutions, ere was extensive dispersion of the chromatin. On the other hand, nuclei incubated with solutions containing divalent cations were

mor~~o~og~cal~y well pres condensation was found in treated nuclei, but, in general, their ulrrastructure was similar to contra! nuclei, In cell nuclei extracted three tames with solution, all of the chromatin strxtures were highly condensed, similar ?o the heteroehromatin of small ~y~m~ho~yte~ or chicken erythrocyte nuclei [4,43]. Apparently the removal of K+ and Nat cations during the repeated extractions permitted this intense condensation and even separation from adjacent structutres. Accordingly, mono- and divalent cations may play an important role in chromatIn condensation or in interconversion of hetero- and euchromatin [4, 20, 21, 23, 27, 437. These analyticai and moq&ological data support the idea that both pn teins and cations in the nuclear sap play important roles in structural organization ef chromatin as well as in gene regdarory that some of the differences in the extracts -with these various solutions may result from their d~~~~~e~~ionic

the other hand, there are some d~ff~~e~~~~ which are not accounted for strengths. For example, more proteins are

solution are the most results with TK relevant to the usual p~ysi~~ogica~ condlrians in cells.

This study 10893, P-l, Foundation M. K. is Chemistry,

was supported by USPHS (grants CAIcb P-3 and P-5) and The Robert A. Q-212 grant. on leave from the Department of Cknical Medical University of Pets, Pets, Hungary.

204 Kellermayer et al.

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Received August 29, 1973 Revised version received November 6, 1973