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Isolation and partial purification of non-histone chromosomal proteins from rat liver, thymus, and kidney
.ZRCHIVES
OF
HIOCHEMISTRY
Isolation
and
.\ND
Partial
Proteins
Purification
from
K. HARTilIUT Physiologisch-Chemisches
148, 44-53 (1972)
RIOPHYSICS
Rat Liver,
RICHTER Institut
Received
of Non-Histone
July
ANI)
Thymus,
and
CONSTANTIX
der Philipps-Universitiit, 26, 1971; accepted
Chromosomal Kidney E. SEKERIS
355 MarburglLahn, October
Lahnberge
5, 1971
1. Chromosomal proteins were solubilized with a high salt-urea buffer. After separat,ion of DNA by ultracentrifugation and binding of the histones on Bio-Rex 70 the non-histone proteins were fractionated on QAE-Sephadex-A 25. 2. The non-histone proteins are shown to be heterogeneous as seen from the QAEelution-profile and from polyacrylamide gels in the presence of sodillm dodecylsulfate. RNase treat)ment did not decrease this heterogeneity. 3. Polyacrylamide gels of fractionated non-histone proteins from rat kidney, thymus, and liver revealed many bands common to all organs, however, several prot,ein components restricted to only one tissue could be det,ected. 4. The phosphate and thiol group content as well as the labeling pattern after applicat,ion of [~-la] tyrosine of various fractions was determined.
and can be used for in oitro studies exploring t,heir potential significance in the control of gene activity.
The main components of the interphase chromosomal material called chromatin (1) are DNA, RNA and proteins, the relative amounts of which depend on species (2), developmental stage (3), t,issue (1, 2), and functional activity (4, 5). On the basis of their extractability by dilut,e mineral acids, the chromosomal proteins have been mainly separated into the acid soluble histones and an acid insoluble fraction usually called non-h&one or acidic proteins. The histones have been amply characterized, the primary structures of some fract,ions have been determined (6) and there is reasonable hope that the complete sequence of all histone fractions will soon be established. On the other hand, the status of our knowledge wit,h regard to the non-histone proteins is very poor. Due to the increasing evidence of a possible involvement in the control of chromosomal functions (7, S), interest in this class of proteins has been rapidly increasing. In t#his paper we present the results of our studies on the isolation and separation of the non-histone chromosomal proteins into reproducible fractions which are amenable to chemical characterization
MATERIALS
@ 1972 by Academic
Press,
In@.
METHODS
1 Abbreviations: SDS is sodium dodecyl TCA is trichloroacetic acid. 44
Copyright
AND
QAE-Sephadex-A 25 and Sephadex G-25 were obtained from Pharmacia, Uppsala, Bio-Rex 70 (Na form) from Bio-Rad, Munich. Enzite-RNaae was a product of Miles-Seravac, Maidenhead, England. Sodium dodecylsulfate (SDS)’ 99% was a product of Schuchardt, Munich. All other chemicals were of analytical grade. Silica-gel plates (F 254) were obtained from Merck. Wistar BR II male rats weighing 120-140 g were used throughout. Protein, RNA, DNA, phosphate, and chloride were determined according to Lowry et al. (Q), or to Warburg and Christian (lo), Ogur and Rosen (ll), Burton (12), Barlett (13), and Mohr (14), respectively. Analytical gel electrophoresis of the various protein fractions was performed according to Shapiro et al. (15), aa modified by Weber and Osborn (16) in 0.6 X 7.4 cm 10% gels for 12 hr. The bands were stained with 0.25% Coomassie blue in 9.2y0 acetic acid: 45.5% methanol for 3 hr and destained with 7.570 acetic acid: 5% methanol for 36 hr at 40”. Gel electrophoresis was also carried out according to Benjamin and Gellhorn sulfate,
NON-HISTONE
CHROMOSOMAL
(17) with 10% gels, for 3 hr at O-4” and wit)h a current of 4 mA per gel. The N-terminal groups of proteins were determined according to Stahl (18) using Sangers fluorodinitjrobenzene method (19). The protein fractions were precipitated with 12% trichloroacetic acid (TCA)‘, washed with TCA, ethanol, and ether, and dried over PZOs. They were then oxidized with performic acid, precipitated with TCA, and washed with ethanol and ether. The protein was finely suspended in a solution of 0.05 M KC1 of pH 9 and treated with an ethanolic solution of fluorodinitrobenzene for 3 hr at 40” and further, as described in reference (18). The dinitrophenylated amino acids were identified by thin layer chromatography on silica-gel plates. The gel was equilibrated with the lower phase of 0.8 N toluene-pyridine-ethylene-chlorohydrin NH3 (1003@6&60). Then the two dimensional chromatogram of the ether soluble fractions was developed, first with the upper phase of the mixture, followed by the second run with chloroform-benzyl alcohol-acetic acid (70-30-3). The ether insoluble fractions were run with n-propanol 34% NH:, (70-30). Determinatieon of the thiol content of the protein fractions was performed by treating with 3H-labeled iodoacetate as described in reference (20). Thirty micrograms of protein in 1 ml 30 mM Mg-acetate and 30 mM Tris-acetate pH 8. 8 were treated with 50 &i iodoacetate-2-T (149 mCi/ mmole) in 50 rrl ethanol for 12 hr at 37”. The incorporated radioactivity was determined on filter paper dis,cs as described in reference (20). The radioactive discs were counted in a solution composed of 5 g PPO and 200 mg POPOP in 1 liter toluene in a Nuclear Chicago Mark I scintillation counter. RNase-treatment of the protein fractions was carried out aa follows: 100 mg of Enzite-RNase was added to :285 ml of the Bio-Rex 70 filtrate (definition see below), containing 0.14 mg protein per milliliter, and slowly stirred at 25” for 9 hr. The enzyme was then removed from the protein solution by filtration. Preparation of nuclei from liver, kidney, and thymus was, performed essentially according to Dukes et al. (21) aa modified in Ref. (22). Chromatin from liver, kidney, and thymus W&B prepared starting from purified nuclei. The method is that of Bonner et al. (1) as modified by Beato et al. (23).
Preparatim of non-h&one chromosomal proteins Chromatin derived from 20 rat livers was suspended in an end volume of 200 ml of a buffer
PROTEINS
FROM
RAT
ORGANS
45
consisting of 2 M NaCl, 5 M urea (pure tryst.), 1 mM mercaptoethanol, 0.5 mM MgCl, , 1 rnM EDTA and 10 mM Tris-HCl, pH 8.3 (Medium A). This is the solution described by Paul and Gilmour (7). The suspension was stirred for one hr. The solution wa8 then centrifuged for 24 hr, at 65000 rpm (average 269,000g) in a Beckman L2 65 ultracentrifuge. The sediment (DNP is deoxyribonucleoprotein) was extracted in the same way a second time for further investigations. The supernatant solution obtained was passed through filter papers to separate floating material and the NaCl concentration w&s decreased to 0.05 M by passing it over a Sephadex G-25 column (V = 900 ml) equilibrated with Medium A cont,aining 0.05 M NaCl instead of 2 M. The protein-containing fractions (280 ml) were passed through a Bio-Rex 70 (Na form) column (5 X 2 cm) in order to remove the histones which remained tightly bound to the resin. The histone-free solution was dialyzed three times against 2 liters of Medium A without NaCl (Medium B), so that the end concentration of chloride in the dialyzed solution reached concentrations lower than 0.009 M. The solution was then submitted to chromatography on QAE-SephadexA 25 equilibrated with Medium B. After washing the column with 100 ml of the same buffer, fractionation was performed with a salt gradient of 0.01-0.60 M (either 200/200 or lOO/lOO ml with fractions of either 5 or 2 ml respectively). The histones were partially recovered from the Bio-Rex 70 column by washing with Medium A. RESULTS
Using the extraction procedure described in Methods (see Table I), it was possible to obtain the bulk of the chromosomal proteins in solution. The elimination of the histones on Bio-Rex 70, under conditions which excluded precipitation, was the prerequisite for a successful fractionation of the nonhistone proteins on QAE-Sephadex. As seen from Fig. 1 the elution profile of the protein fractions is heterogeneous, proteins appearing at the NaCl concent,ration of 0.02 M and throughout the gradient up to a molarity of 0.34 ;cI. The uv-absorbing material eluting at 0.45 M NaCl is Lowry-negative showing a typical nucleic acid spectrum and a positive Burton reaction, identifying this fraction as DNA. In order to exclude the possibility that the heterogeneous behavior of the non-histone proteins is due to degradation
Ii ICHTER
4G
AND TABLE
PREPARATION SCHEME OF NON-HISTONE
SEKEKIS I
PROTEINS FROM RAT LIVER
CHROMATIN
extraction in medium A ultracentrifugation (269000g/24h)
sedime
A&T
Sephadex
G 25 155 mg protein O.l2nq
Bio -Rex elution
RNA
70
with
66rng protein
40 mg protein 0.12 mg RNA dialysis I QAE-chromatography 0.01 -0.60 M NaCl
26 mg protein
gradient
zm-
1 . !60-
/ /
/
/
/
/
/
/
/
/
/
/
; o-
Y)-
FIG. 1. Chromatography of non-h&one proteins on QAE-Sephadex-A 25. Forty milligrams of non-h&one prot.eins were bound on a QAE-Sephadex-A 25 column (44 cm X 1.8 cm) in a buffer consisting of 5 M urea, 1 mM mercaptoethanol, 0.5 mM MgCl2 , 1 mM EDTA, and 10 mM Tris-HCl, pH 8.3. Chloride concentration did not exceed 9 mM. After washing the column with 100 ml Medium B elution was performed with a NaCl-gradient of O.Ol0.60 M (200/200 ml). Fractions of 5 ml were collected.
NON-HISTONE
CHROMOSOMAL
of the proteins during the fractionation procedure, IV-terminal amino acid analysis of total protein extracted from chromatin immediately and after maintenance for 48 hr at room temperature was performed. No significa,nt difference between the two preparations was detected, making it unlikely that degradative processes had taken place. Acrylamide gel electrophoresis of the histones recovered from the Bio-Rex 70 column showed no difference to electrophoresis of acid-extracted histones, point’ing also to a similar conclusion. The high degree of heterogeneity is further demonstrated by submitting the different protein QAE-Sephadex fractions (see Fig. 1) to SDS-acrylamidc gel electrophoresis. Each of these fractions is shown to be a highly heterogeneous mixture of proteins (Fig. 2). Due to the fact that the protein preparations contain substantial amounts of nucleic acids, we considered the possibility t,hat heterogeneit:y could be due to the nucleic acid components of a relatively homogeneous protein population. We therefore submitted the proteins to RNase digestion as described in Methods. Treatment with RNase did not diminish the heterogeneous electrophoretic and chromatographic behavior of the proteins. A comparison was then conducted of the non-histone proteins derived from chromatin of liver and that of thymus, and kidney. The different steps of the preparation and fractionation of non-histone proteins from the three organs were identical. As seen from Fig. 3 the elution profiles of the three preparations of non-histone proteins from QAESephadex columns show similarities. The liver preparation contains much more protein in the regions 2 and 3 of the chromatogram. Further analysis of the different regions of -the chromatogram by SDSacrylamide gel electrophoresis (see Fig. 4) revealed many bands common to all three organs. However, several protein components restricted only to one tissue could be detected. The differences in the non-histone proteins components between the various organs became more pronounced by submitting the fractions to acrylamide gel electrophoresis according to Benjamin and Gellhorn (see Fig. 5). This is possibly due to
PROTEINS
a
47
FROM RAT ORGANS
b
c
d
e
Fro. 2. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate of QAEfractions of non-histone proteins. Fractions of the QAE-Sephadex-eluate were pooled ss indicated in Fig. 1. The electrophoresis was performed with samples of 5&80 pg protein on 10% gels (7.5 cm X 0.6 cm) for 12 hr and 8 mA/tube. Direction of the electrophoresis was from the top to the bottom.
the absence of SDS in the buffer, resulting in a partial retention of the native quaternary structure of the proteins. The thiol and phosphate content of the different protein fractions (see Table II and III) was subsequently determined. The distribution of thiol groups was very similar in the three preparations; higher SH values however were found in the liver fractions (see Table II). The phosphate content of liver and thymus preparations showed a similar distribution. However, the kidney fractions show significant differences with respect to the other two preparations (see Table III). In order to examine the metabolic status of the non-histone proteins 10 rats were
48
RICHTER
AND
SEKERIS
0.6 i / / .s-k, /
0.1a--
i
/
/
/
/
i
MS-
El,/ --20
-0.4
,/’ /
/
/ A
/
c
/
0 40
/
-0.2
AL50
G 80
100
120
Ml
NclCI(Y)
- 0.6
-0.4
-0.2
20
40
60 FRACTIONS
10
100
I 120
FIG. 3. Chromatography of non-hi&one proteins from rat. kidney, thymus and liver on QAE-Sephadex-A 25. Approximately equal amounts (20 mg) of non-h&one proteins of the three organs were bound on QAE-Sephadex-A 25 columns (44 cm X 1.4 cm) under conditions as described at Fig. 1. Elution was carried out with a NaCl-gradient of 0.01 to 0.60 M (lOO/lOO ml). Fractions of 2 ml were collected.
NON-H [ISTONE
CHROMOSOMAL
PROTEINS
FROM
RAT
ORGANS
49
cio
RICHTER
AND
SEKERIS
5
2
FIG. 5. Polyacrylamide gel elect.rophoresis according to Benjamin and Gellhorn of corresponding non-hi&one protein fractions of various rat, organs. Fractions of the three QAESephadex-chromatograms were pooled as indicated in Fig. 3 by the arrows (l-5). The electrophoresis was performed with samples of about 50 fig on 10% gels (7.5 cm X 0.6 cm) for 3 hr and 4 mA/tube at 4”. Direction of the electrophoresis was from the top t,o bottom. The order of the corresponding fractions is from left to right: kidney-thymris-liver.
TABLE THIOL
TABLE
II
CONTENT OF QAE-SEPHADEX FRACTIONS OF NON-HISTONE PROTEINS IN pmols/ 10 g PROTEINS
Fraction
A B C D E F G
Kidney
2 21 12 11 5 2
Thymus
7 14 9 7 4 -
Liver
8 33 27 24 14 6 2
a Determination of the thiol content in corresponding fractions of non-histone proteins from different rat organs. Fractions of the QAESephadex-chromatograms of the non-histone proteins from the examined rat organs were pooled as indicated in Fig. 3 (A-G) and the thiol content was determined in aliquots of 3Opg protein by reaction with 5OpCi iodoacetate-2-T (149 mCi/ maa) at pH 8.8 for 12 hr at 37”. Thiol content expressed in fimole/lO g protein.
PHOSPHATE
Fraction A
B C D E F G
III
CONTENT OF QAE-SEPHADEX FRACTIONS OF NON-HISTONE PROTEINS* Kidney
Thymus
Liver
2.55 0.80 0.20 0.35 0.05
-
-
0.15 0.40 1.35 -
1.00 1.25 1.10 0.20
8 Determination of the phosphate content in corresponding fractions of non-histone proteins from different rat organs. Fractions of the QAESephadex-chromatograms of the nonhistone proteins from the examined rat organs were pooled as indicated in Fig. 3 (A-G) and the phosphate content was determined after washing with aliquots of 250 rg protein. Phosphate content expressed in y0 (w/w).
NON-HISTONE TABLE LABELING
CHROMOSOMAL IV
017 RAT LIVER PROTEINS INJECTION OF 3H-T~~~~~~~ (ONE HOUR PULSE)~ Fraction
CPdW
cytoso1 G 254itrate Bio-Rex-filtrate Histones DNP QAE-fraction
AFTER
a b I e
19000 2900 4900 2700 12000 - 18000 2aoo 10000 7500 5509 2509
a Determination of the specific radioactivity of cytoplasmic proteins and various chromosomal proteins from rat liver labeled with [L-3Hl tyrosine. Ten rats were each injected with 0.5 mCi[L-3H] tyrosine (1 Ci/mM) i.p. and after one hour the distribution of radioactivity was determined. Specific activities of proteins are expressed in counts/min per mg.
each injected with 0.5 mCi [L-3H] tyrosine (1 Ci/rmr in 2 % ethanol) i.p., and after one hour the distribution of radioactivity in soluble cytoplasmic proteins, histones and non-histone proteins was followed (see Table IV). The highest specific activity was found in the soluble cytoplasmic proteins, followed by the non-histone proteins and, with lowest incorporation, by the histones. The distribution of radioactivity in the non-histone proteins is very heterogeneous, which most probably reflects the difference in the turnover rate of the various protein fractions. As seen from Table IV label is also found in the DNP-fraction. This is due to the presence of 34% protein tightly bound to the DNA which incorporates more tyrosine than the non-histone proteins. Holoubek and Cracker also reported the high labeling of proteins tightly bound to DNA (24). The characteristics of these proteins will be reported in a, following paper (25). DISCUSSION
One of the first methods used to obtain non-histone proteins from cell nuclei was to
PROTEINS
FROM
RAT
ORGANS
51
solubilize the residue after acid extraction of nuclei or of chromatin (26). However, only a very small fraction of the proteins could be brought into solution and due to the very drastic procedures applied, further utilization of the obtained proteins for functional studies was limited. Another approach, giving significantly better results, was to extract nuclei or chromatin with high salt solutions thus dissociating the proteins from the DNA and then reducing the salt concentration by the addition of dilute buffers so as to reassociate the histones with the DNA, leaving the non-histone fraction in solution (27). However some of these proteins do reassociate with the DNA whereas aggregation of histones to non-histone proteins regularly occurs. Independent of its shortcomings, this method was the first applicable method to obtain non-histone proteins suitable for functional studies. Paul and Gilmour extracted chromatin with high salt-urea solutions then spun down the DNA leaving the proteins in solution. They then separated the histones from the non-hrstone proteins by QAE-Sephadex treatment. In a recent paper (as), which came to our attention during the preparation of our manuscript, Paul and collaborators submitted chromatin dissolved in a salturea solution to hydroxylapatite chromatography thus separating the histones, the non-histone proteins and the DNA in the same run. The method we have applied utilizes the extraction conditions of Paul and Gilmour (7). The DNA, to which proteins are still attached, is quantitatively spun down by centrifugation at 269000g for 24 hr which is very important for the success of the further preparation. One additional step of importance is the elimination of the histones by absorption to Bio-Rex 70, leading to a nonhist,one protein fraction without even traces of histone. The retention also in dilute aqueous media of the non-histone proteins constitutes a major advantage of this method. As is well-known, even traces of histones would lead to aggregation and precipitation of non-histone proteins.
52
RICHTER
AND
Chromatography on QAE-Sephadex-A 25 separates the non-histone proteins into several fractions which are very reproducible. The separation procedure is easily performed and, due to its reproducibilit,y, can serve as a first att)empt toward a systematic classification of the non-histone proteins. The further analysis of the different QAESephadex fractions by acrylamide gel electrophoresis reveals their high degree of heterogeneity. The possibility that the heterogeneous behavior of the non-histone proteins may be due t,o degradation has been rendered unlikely by the results of the end group determination according to Sanger. The presence also of ribonucleic acid in these preparations, manifested by the high 260/280 o.d. ratios, is not the cause of the heterogeneity, for treatment with RNase does not change t’he elect,rophoretic or chromatographic behavior of these proteins to any significant extent. From the data of Wang (27), Langan (29), Kleinsmith (30), and others it is known that a part of these proteins cont’ains phosphate groups in significant amounts. The work of Stocken and Ord (31) has focused attention on the presence of thiol groups in these proteins. We have therefore determined the content of phosphate and thiol in the different regions of the QAESephadex chromatogram. It is interesting to note that the proteins with a high phosphate content had low amount’s of t’hiol groups and vice versa. This distribution of thiol and phosphate was generally similar in preparations of non-histone proteins from liver, thymus, and kidney tissue. Due to the implication of phosphoproteins (32) and of the state of thiol/disulfide (33, 20) in the control of gene activity, the knowledge of the distribution of phosphate and thiol groups in the various fractions of the QAESephadex chromatogram should be of potential value for the use of these fractions in the various functional studies. The work of Gilmour and Paul has drawn attention to this class of proteins as imparting specificity to the in vitro transcribed RNA (7, 34). It was therefore of interest to compare the non-histone proteins derived from different organs. The chromatographic profiles of the non-histone proteins derived
SEKERIS
from liver, thymus, and kidneys were, to a great extent, very similar with slight qualitative and quantitative differences. Further analysis by acrylamide gel electrophoresis, either according to Shapiro et al., or to Benjamin and Gellhorn revealed the presence of unique bands parallel to the existence of pronounced similarities in the electrophoretic pattern for each of the organs studied. This is in agreement with t,he findings of Loeb and Creuzet (35), and of Dastugue et al. (36), partially wit,h the results of Elgin and Bonner (a), and in cont,rast to those of MacGillivray et al. (28). Whether t’his variability is connected to t’he determination of specificity of transcription by these proteins remains to be seen. When considering differences or similarities in the non-histone proteins between different organs, it should be mentioned that many enzymes are tightly bound to chromatin such as the DNA- (37), and RNA-polymerase (38), the nucleoside triphosphatases (39), the histone acetylases (40, 41), and methylases (42)) non-histone phosphokinases (43, 44), as well as dephosphorylat’ing enzymes. As regards histone transacetylases, differences in the behavior between the enzymes derived from liver, thymus, and kidney on DEAE-cellulose have recently been demonstrated (45) so that heterogeneity in the non-histone proteins of t’his kind must also be considered. The prerequisite for tackling problems involving the functional significance of t’he non-histone proteins is the availability of sufficient amounts of defined non-histone protein fractions soluble in dilute aqueous solutions. The method described above offers such a possibility and should be regarded as a first step towards this goal. ACKNOWLEDGMENTS We thank Professor P. Karlson for his encouraging interest in our work and we are grateful to Miss Froehlich, and Mrs. Pfeiffer for their skillful assistance. This work was supported by the Deutsche Forschungsgemeinschaft. REFERENCES 1. BONNER,
J., DAHMUS, M., FAMBROUGH, D., R. C., MARUSHIGE, K., AND TUAN, D., Scielzce 169, 47 (1968). HUANG,
NON-HISTONE
CHROMOSOMAL
2. ELGIN, S. C. R., .?ND BONNER, J., Biochemistry 4440 (1970) . 3. MARUSHIGE,, K., AND OZAKI, H., Develop. Biol. 16, 474 (1967). 4. TENG, C. S., AND HAMILTON, T. H., Proc. Nut. Acad. Sci. U. S. A. 63,465 (1969). 5. HOLT, T. K. H., Chromosoma 32, 64 (1970). 6. DELANGE, R. J., SMITH, E. L., FAMBROUGH, D. M., AND BONNER, J., J. Biol. Chem. 244, 5669 (1969). 7. PAUL, J., AND GILMOUR, R. S., J. Mol. Biol. 34, 305 (1968). 8. BEKHOR, I., KUNG, G. M., AND BONNER, J., J. Mol. Biol. 39, 351 (1969). 9. LO~VRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, J. R., J. Biol. Chem. 193, 265 (1951). 10. WARBURG, O., AND CHRISTIAN, W., Biochem. z. 310, 384 (1942). 11. OGUR, M., I~ND ROSEN, G., Arch. Biochem. 26. 262 (1950). 12. BURTON, K.., Biochem. J. 63, 315 (1956). 13. BARTLETT, G. R., J. Biol. Chem. 234, 466 (1959). 14. MOHR F.. 14nn. Chsm. Ph,nrm.. 97. 335 (1856). 15. SHAPIRO, A. L., VINUELA, E., AND M~IZEL, J. V., B&hem. Biophys. Res. Commun. 20, 815 (1967). 16. WEBER, K., AND OSBORN, M., J. Biol. Chem. 244, 4406 (1969). 17. BENJAMIN, W., AND GELLHORN, A., Proc. Nat. Acad. Sci. U. S. A. 69, 262 (1968). 18. STAHL, E., “Dtinnschichtchromatographie”, Springer Verlag, Berlin, Giittingen, Heidelberg (1963). 19. SANGER, F., B&hem. J. 39, 507 (1945). 20. SEKERIS, C. E., BEATO, M., HOMOKI, J., AND CONGOTE, L. F., Hoppe Seyler. Z. Physiol. Chem. 349, 857 (1968). 21. DUKES, P. P., SEKERIS, C. E., AND SCHMID, W., Biochim. Biophys. Acta 123, 126 (1966). 22. BEATO, M., HOMOKI, J., AND SEKERIS, C. E., Exp. Gel2 Res. 66, 107 (1969). 23. BE~TO, M., SEIFART, K. H., AND SEICERIS, C. E., Arch. Biochem. Biophys. 136, 272 (1970). 24. HOLOUBEK, V., .~ND CROCKER, T. T., Biochim. Biophys. llcta 167, 352 (1968). 25. ROE~EKAMF~, W., RICHTER, K. H., AND SEKERIS, C. E., in preparation.
PROTEINS
FROM
RAT
ORGANS
53
26. DOUNCE, A. L., AND HILGSRTNER, C. A., Exp. Cell Res. 36, 228 (1964). 27. WANG, T. Y., J. Biol. Chem. 243, 1225 (1967). 28. MACGILLIVRAY, A. J., CAROLL, D., AND PAUL, J., Fed. Eur. Biochem. Sot. Lett. 13, 204 (1971). T. A., in “Regulatory Mechanisms 29. LaNGaN, in Nucleic Acid and Protein Biosynthesis” (V. V. Koenigsberger, and L. Bosch, eds.), Vol. 10, p. 233. BBA Library Series, Elsevier, Amsterdam (1967). 30. KLEINSMITH, L. J., .~ND ALLFREY, V. G., Biochim. Biophys. Acta 176, 123 (1969). 31. STOCKEN, L. A., AND ORD, M. G., “The Cell Nucleus -Metabolism and Radiosensitivity”, p. 141. Tylor and Francis Ltd., London (1966). 32. KLEINSMITH, L. J., ALLFREY, V. G., AND MIRSKY, A. E., Proc. Nat. Acad. Sci. U. S. A. 66, 1182 (1966). 33. ORD, M. G., AND STOCKEN, L. A., Biochem. J. 107, 403 (1968). 34. GILMOUR, R. S., AND PAUL, J., Fed. Eur. Biochem. Sot. Lett. 9, 242 (1970). 35. LOEB, J., AND CREUZET, C., Fed. Eur. Biochem. Sot. Lett. 6, 37 (1969). 36. DASTUGUE, B., TICHONICKY, L., PENITSORIA, J., BND KRUH, J., Bull. Sot. Chim. Biol. &, 391 (1970). 37. HOOK, R., AND WANG, T. Y., Arch. Biochem. Biophys. 133, 238 (1969). 38. SEIF.IR~‘, K. H., AND SEKERIS, C. E., Eur. J. Biochem. 7, 408 (1969). 39. SCHILTZ, E., AND SEKERIS, C. E., Hoppe Seylers Z. Physiol. Chem. 360, 317 (1969). 40. GALLWITZ, D., .*ND SEKENS, C. E., unpubpublished observations. 41. RACEY, L. A., :~ND BYVOET, P., Exp. Cell Res. 64, 366 (1971). 42. SEKERIS, C. E., SEKERI, K. E., AND GALL~ITZ, D., Hoppe Seyler. Z. Physiol. Chem. 349, 1660 (1967). 43. SCHILTZ, E., AND SEKERIS, C. E., Experientia 27, 30 (1971). 44. T~KEDA, M., YAM.\MURA, H., .~ND OHG.4, Y., Biochem. Biophys. Res. Commun. 42, 103 (1971). 45. GALLWTIZ, D., Fed. Eur. Biochem. Sot. Lett. 13, 306 (1971).
OF
HIOCHEMISTRY
Isolation
and
.\ND
Partial
Proteins
Purification
from
K. HARTilIUT Physiologisch-Chemisches
148, 44-53 (1972)
RIOPHYSICS
Rat Liver,
RICHTER Institut
Received
of Non-Histone
July
ANI)
Thymus,
and
CONSTANTIX
der Philipps-Universitiit, 26, 1971; accepted
Chromosomal Kidney E. SEKERIS
355 MarburglLahn, October
Lahnberge
5, 1971
1. Chromosomal proteins were solubilized with a high salt-urea buffer. After separat,ion of DNA by ultracentrifugation and binding of the histones on Bio-Rex 70 the non-histone proteins were fractionated on QAE-Sephadex-A 25. 2. The non-histone proteins are shown to be heterogeneous as seen from the QAEelution-profile and from polyacrylamide gels in the presence of sodillm dodecylsulfate. RNase treat)ment did not decrease this heterogeneity. 3. Polyacrylamide gels of fractionated non-histone proteins from rat kidney, thymus, and liver revealed many bands common to all organs, however, several prot,ein components restricted to only one tissue could be det,ected. 4. The phosphate and thiol group content as well as the labeling pattern after applicat,ion of [~-la] tyrosine of various fractions was determined.
and can be used for in oitro studies exploring t,heir potential significance in the control of gene activity.
The main components of the interphase chromosomal material called chromatin (1) are DNA, RNA and proteins, the relative amounts of which depend on species (2), developmental stage (3), t,issue (1, 2), and functional activity (4, 5). On the basis of their extractability by dilut,e mineral acids, the chromosomal proteins have been mainly separated into the acid soluble histones and an acid insoluble fraction usually called non-h&one or acidic proteins. The histones have been amply characterized, the primary structures of some fract,ions have been determined (6) and there is reasonable hope that the complete sequence of all histone fractions will soon be established. On the other hand, the status of our knowledge wit,h regard to the non-histone proteins is very poor. Due to the increasing evidence of a possible involvement in the control of chromosomal functions (7, S), interest in this class of proteins has been rapidly increasing. In t#his paper we present the results of our studies on the isolation and separation of the non-histone chromosomal proteins into reproducible fractions which are amenable to chemical characterization
MATERIALS
@ 1972 by Academic
Press,
In@.
METHODS
1 Abbreviations: SDS is sodium dodecyl TCA is trichloroacetic acid. 44
Copyright
AND
QAE-Sephadex-A 25 and Sephadex G-25 were obtained from Pharmacia, Uppsala, Bio-Rex 70 (Na form) from Bio-Rad, Munich. Enzite-RNaae was a product of Miles-Seravac, Maidenhead, England. Sodium dodecylsulfate (SDS)’ 99% was a product of Schuchardt, Munich. All other chemicals were of analytical grade. Silica-gel plates (F 254) were obtained from Merck. Wistar BR II male rats weighing 120-140 g were used throughout. Protein, RNA, DNA, phosphate, and chloride were determined according to Lowry et al. (Q), or to Warburg and Christian (lo), Ogur and Rosen (ll), Burton (12), Barlett (13), and Mohr (14), respectively. Analytical gel electrophoresis of the various protein fractions was performed according to Shapiro et al. (15), aa modified by Weber and Osborn (16) in 0.6 X 7.4 cm 10% gels for 12 hr. The bands were stained with 0.25% Coomassie blue in 9.2y0 acetic acid: 45.5% methanol for 3 hr and destained with 7.570 acetic acid: 5% methanol for 36 hr at 40”. Gel electrophoresis was also carried out according to Benjamin and Gellhorn sulfate,
NON-HISTONE
CHROMOSOMAL
(17) with 10% gels, for 3 hr at O-4” and wit)h a current of 4 mA per gel. The N-terminal groups of proteins were determined according to Stahl (18) using Sangers fluorodinitjrobenzene method (19). The protein fractions were precipitated with 12% trichloroacetic acid (TCA)‘, washed with TCA, ethanol, and ether, and dried over PZOs. They were then oxidized with performic acid, precipitated with TCA, and washed with ethanol and ether. The protein was finely suspended in a solution of 0.05 M KC1 of pH 9 and treated with an ethanolic solution of fluorodinitrobenzene for 3 hr at 40” and further, as described in reference (18). The dinitrophenylated amino acids were identified by thin layer chromatography on silica-gel plates. The gel was equilibrated with the lower phase of 0.8 N toluene-pyridine-ethylene-chlorohydrin NH3 (1003@6&60). Then the two dimensional chromatogram of the ether soluble fractions was developed, first with the upper phase of the mixture, followed by the second run with chloroform-benzyl alcohol-acetic acid (70-30-3). The ether insoluble fractions were run with n-propanol 34% NH:, (70-30). Determinatieon of the thiol content of the protein fractions was performed by treating with 3H-labeled iodoacetate as described in reference (20). Thirty micrograms of protein in 1 ml 30 mM Mg-acetate and 30 mM Tris-acetate pH 8. 8 were treated with 50 &i iodoacetate-2-T (149 mCi/ mmole) in 50 rrl ethanol for 12 hr at 37”. The incorporated radioactivity was determined on filter paper dis,cs as described in reference (20). The radioactive discs were counted in a solution composed of 5 g PPO and 200 mg POPOP in 1 liter toluene in a Nuclear Chicago Mark I scintillation counter. RNase-treatment of the protein fractions was carried out aa follows: 100 mg of Enzite-RNase was added to :285 ml of the Bio-Rex 70 filtrate (definition see below), containing 0.14 mg protein per milliliter, and slowly stirred at 25” for 9 hr. The enzyme was then removed from the protein solution by filtration. Preparation of nuclei from liver, kidney, and thymus was, performed essentially according to Dukes et al. (21) aa modified in Ref. (22). Chromatin from liver, kidney, and thymus W&B prepared starting from purified nuclei. The method is that of Bonner et al. (1) as modified by Beato et al. (23).
Preparatim of non-h&one chromosomal proteins Chromatin derived from 20 rat livers was suspended in an end volume of 200 ml of a buffer
PROTEINS
FROM
RAT
ORGANS
45
consisting of 2 M NaCl, 5 M urea (pure tryst.), 1 mM mercaptoethanol, 0.5 mM MgCl, , 1 rnM EDTA and 10 mM Tris-HCl, pH 8.3 (Medium A). This is the solution described by Paul and Gilmour (7). The suspension was stirred for one hr. The solution wa8 then centrifuged for 24 hr, at 65000 rpm (average 269,000g) in a Beckman L2 65 ultracentrifuge. The sediment (DNP is deoxyribonucleoprotein) was extracted in the same way a second time for further investigations. The supernatant solution obtained was passed through filter papers to separate floating material and the NaCl concentration w&s decreased to 0.05 M by passing it over a Sephadex G-25 column (V = 900 ml) equilibrated with Medium A cont,aining 0.05 M NaCl instead of 2 M. The protein-containing fractions (280 ml) were passed through a Bio-Rex 70 (Na form) column (5 X 2 cm) in order to remove the histones which remained tightly bound to the resin. The histone-free solution was dialyzed three times against 2 liters of Medium A without NaCl (Medium B), so that the end concentration of chloride in the dialyzed solution reached concentrations lower than 0.009 M. The solution was then submitted to chromatography on QAE-SephadexA 25 equilibrated with Medium B. After washing the column with 100 ml of the same buffer, fractionation was performed with a salt gradient of 0.01-0.60 M (either 200/200 or lOO/lOO ml with fractions of either 5 or 2 ml respectively). The histones were partially recovered from the Bio-Rex 70 column by washing with Medium A. RESULTS
Using the extraction procedure described in Methods (see Table I), it was possible to obtain the bulk of the chromosomal proteins in solution. The elimination of the histones on Bio-Rex 70, under conditions which excluded precipitation, was the prerequisite for a successful fractionation of the nonhistone proteins on QAE-Sephadex. As seen from Fig. 1 the elution profile of the protein fractions is heterogeneous, proteins appearing at the NaCl concent,ration of 0.02 M and throughout the gradient up to a molarity of 0.34 ;cI. The uv-absorbing material eluting at 0.45 M NaCl is Lowry-negative showing a typical nucleic acid spectrum and a positive Burton reaction, identifying this fraction as DNA. In order to exclude the possibility that the heterogeneous behavior of the non-histone proteins is due to degradation
Ii ICHTER
4G
AND TABLE
PREPARATION SCHEME OF NON-HISTONE
SEKEKIS I
PROTEINS FROM RAT LIVER
CHROMATIN
extraction in medium A ultracentrifugation (269000g/24h)
sedime
A&T
Sephadex
G 25 155 mg protein O.l2nq
Bio -Rex elution
RNA
70
with
66rng protein
40 mg protein 0.12 mg RNA dialysis I QAE-chromatography 0.01 -0.60 M NaCl
26 mg protein
gradient
zm-
1 . !60-
/ /
/
/
/
/
/
/
/
/
/
/
; o-
Y)-
FIG. 1. Chromatography of non-h&one proteins on QAE-Sephadex-A 25. Forty milligrams of non-h&one prot.eins were bound on a QAE-Sephadex-A 25 column (44 cm X 1.8 cm) in a buffer consisting of 5 M urea, 1 mM mercaptoethanol, 0.5 mM MgCl2 , 1 mM EDTA, and 10 mM Tris-HCl, pH 8.3. Chloride concentration did not exceed 9 mM. After washing the column with 100 ml Medium B elution was performed with a NaCl-gradient of O.Ol0.60 M (200/200 ml). Fractions of 5 ml were collected.
NON-HISTONE
CHROMOSOMAL
of the proteins during the fractionation procedure, IV-terminal amino acid analysis of total protein extracted from chromatin immediately and after maintenance for 48 hr at room temperature was performed. No significa,nt difference between the two preparations was detected, making it unlikely that degradative processes had taken place. Acrylamide gel electrophoresis of the histones recovered from the Bio-Rex 70 column showed no difference to electrophoresis of acid-extracted histones, point’ing also to a similar conclusion. The high degree of heterogeneity is further demonstrated by submitting the different protein QAE-Sephadex fractions (see Fig. 1) to SDS-acrylamidc gel electrophoresis. Each of these fractions is shown to be a highly heterogeneous mixture of proteins (Fig. 2). Due to the fact that the protein preparations contain substantial amounts of nucleic acids, we considered the possibility t,hat heterogeneit:y could be due to the nucleic acid components of a relatively homogeneous protein population. We therefore submitted the proteins to RNase digestion as described in Methods. Treatment with RNase did not diminish the heterogeneous electrophoretic and chromatographic behavior of the proteins. A comparison was then conducted of the non-histone proteins derived from chromatin of liver and that of thymus, and kidney. The different steps of the preparation and fractionation of non-histone proteins from the three organs were identical. As seen from Fig. 3 the elution profiles of the three preparations of non-histone proteins from QAESephadex columns show similarities. The liver preparation contains much more protein in the regions 2 and 3 of the chromatogram. Further analysis of the different regions of -the chromatogram by SDSacrylamide gel electrophoresis (see Fig. 4) revealed many bands common to all three organs. However, several protein components restricted only to one tissue could be detected. The differences in the non-histone proteins components between the various organs became more pronounced by submitting the fractions to acrylamide gel electrophoresis according to Benjamin and Gellhorn (see Fig. 5). This is possibly due to
PROTEINS
a
47
FROM RAT ORGANS
b
c
d
e
Fro. 2. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate of QAEfractions of non-histone proteins. Fractions of the QAE-Sephadex-eluate were pooled ss indicated in Fig. 1. The electrophoresis was performed with samples of 5&80 pg protein on 10% gels (7.5 cm X 0.6 cm) for 12 hr and 8 mA/tube. Direction of the electrophoresis was from the top to the bottom.
the absence of SDS in the buffer, resulting in a partial retention of the native quaternary structure of the proteins. The thiol and phosphate content of the different protein fractions (see Table II and III) was subsequently determined. The distribution of thiol groups was very similar in the three preparations; higher SH values however were found in the liver fractions (see Table II). The phosphate content of liver and thymus preparations showed a similar distribution. However, the kidney fractions show significant differences with respect to the other two preparations (see Table III). In order to examine the metabolic status of the non-histone proteins 10 rats were
48
RICHTER
AND
SEKERIS
0.6 i / / .s-k, /
0.1a--
i
/
/
/
/
i
MS-
El,/ --20
-0.4
,/’ /
/
/ A
/
c
/
0 40
/
-0.2
AL50
G 80
100
120
Ml
NclCI(Y)
- 0.6
-0.4
-0.2
20
40
60 FRACTIONS
10
100
I 120
FIG. 3. Chromatography of non-hi&one proteins from rat. kidney, thymus and liver on QAE-Sephadex-A 25. Approximately equal amounts (20 mg) of non-h&one proteins of the three organs were bound on QAE-Sephadex-A 25 columns (44 cm X 1.4 cm) under conditions as described at Fig. 1. Elution was carried out with a NaCl-gradient of 0.01 to 0.60 M (lOO/lOO ml). Fractions of 2 ml were collected.
NON-H [ISTONE
CHROMOSOMAL
PROTEINS
FROM
RAT
ORGANS
49
cio
RICHTER
AND
SEKERIS
5
2
FIG. 5. Polyacrylamide gel elect.rophoresis according to Benjamin and Gellhorn of corresponding non-hi&one protein fractions of various rat, organs. Fractions of the three QAESephadex-chromatograms were pooled as indicated in Fig. 3 by the arrows (l-5). The electrophoresis was performed with samples of about 50 fig on 10% gels (7.5 cm X 0.6 cm) for 3 hr and 4 mA/tube at 4”. Direction of the electrophoresis was from the top t,o bottom. The order of the corresponding fractions is from left to right: kidney-thymris-liver.
TABLE THIOL
TABLE
II
CONTENT OF QAE-SEPHADEX FRACTIONS OF NON-HISTONE PROTEINS IN pmols/ 10 g PROTEINS
Fraction
A B C D E F G
Kidney
2 21 12 11 5 2
Thymus
7 14 9 7 4 -
Liver
8 33 27 24 14 6 2
a Determination of the thiol content in corresponding fractions of non-histone proteins from different rat organs. Fractions of the QAESephadex-chromatograms of the non-histone proteins from the examined rat organs were pooled as indicated in Fig. 3 (A-G) and the thiol content was determined in aliquots of 3Opg protein by reaction with 5OpCi iodoacetate-2-T (149 mCi/ maa) at pH 8.8 for 12 hr at 37”. Thiol content expressed in fimole/lO g protein.
PHOSPHATE
Fraction A
B C D E F G
III
CONTENT OF QAE-SEPHADEX FRACTIONS OF NON-HISTONE PROTEINS* Kidney
Thymus
Liver
2.55 0.80 0.20 0.35 0.05
-
-
0.15 0.40 1.35 -
1.00 1.25 1.10 0.20
8 Determination of the phosphate content in corresponding fractions of non-histone proteins from different rat organs. Fractions of the QAESephadex-chromatograms of the nonhistone proteins from the examined rat organs were pooled as indicated in Fig. 3 (A-G) and the phosphate content was determined after washing with aliquots of 250 rg protein. Phosphate content expressed in y0 (w/w).
NON-HISTONE TABLE LABELING
CHROMOSOMAL IV
017 RAT LIVER PROTEINS INJECTION OF 3H-T~~~~~~~ (ONE HOUR PULSE)~ Fraction
CPdW
cytoso1 G 254itrate Bio-Rex-filtrate Histones DNP QAE-fraction
AFTER
a b I e
19000 2900 4900 2700 12000 - 18000 2aoo 10000 7500 5509 2509
a Determination of the specific radioactivity of cytoplasmic proteins and various chromosomal proteins from rat liver labeled with [L-3Hl tyrosine. Ten rats were each injected with 0.5 mCi[L-3H] tyrosine (1 Ci/mM) i.p. and after one hour the distribution of radioactivity was determined. Specific activities of proteins are expressed in counts/min per mg.
each injected with 0.5 mCi [L-3H] tyrosine (1 Ci/rmr in 2 % ethanol) i.p., and after one hour the distribution of radioactivity in soluble cytoplasmic proteins, histones and non-histone proteins was followed (see Table IV). The highest specific activity was found in the soluble cytoplasmic proteins, followed by the non-histone proteins and, with lowest incorporation, by the histones. The distribution of radioactivity in the non-histone proteins is very heterogeneous, which most probably reflects the difference in the turnover rate of the various protein fractions. As seen from Table IV label is also found in the DNP-fraction. This is due to the presence of 34% protein tightly bound to the DNA which incorporates more tyrosine than the non-histone proteins. Holoubek and Cracker also reported the high labeling of proteins tightly bound to DNA (24). The characteristics of these proteins will be reported in a, following paper (25). DISCUSSION
One of the first methods used to obtain non-histone proteins from cell nuclei was to
PROTEINS
FROM
RAT
ORGANS
51
solubilize the residue after acid extraction of nuclei or of chromatin (26). However, only a very small fraction of the proteins could be brought into solution and due to the very drastic procedures applied, further utilization of the obtained proteins for functional studies was limited. Another approach, giving significantly better results, was to extract nuclei or chromatin with high salt solutions thus dissociating the proteins from the DNA and then reducing the salt concentration by the addition of dilute buffers so as to reassociate the histones with the DNA, leaving the non-histone fraction in solution (27). However some of these proteins do reassociate with the DNA whereas aggregation of histones to non-histone proteins regularly occurs. Independent of its shortcomings, this method was the first applicable method to obtain non-histone proteins suitable for functional studies. Paul and Gilmour extracted chromatin with high salt-urea solutions then spun down the DNA leaving the proteins in solution. They then separated the histones from the non-hrstone proteins by QAE-Sephadex treatment. In a recent paper (as), which came to our attention during the preparation of our manuscript, Paul and collaborators submitted chromatin dissolved in a salturea solution to hydroxylapatite chromatography thus separating the histones, the non-histone proteins and the DNA in the same run. The method we have applied utilizes the extraction conditions of Paul and Gilmour (7). The DNA, to which proteins are still attached, is quantitatively spun down by centrifugation at 269000g for 24 hr which is very important for the success of the further preparation. One additional step of importance is the elimination of the histones by absorption to Bio-Rex 70, leading to a nonhist,one protein fraction without even traces of histone. The retention also in dilute aqueous media of the non-histone proteins constitutes a major advantage of this method. As is well-known, even traces of histones would lead to aggregation and precipitation of non-histone proteins.
52
RICHTER
AND
Chromatography on QAE-Sephadex-A 25 separates the non-histone proteins into several fractions which are very reproducible. The separation procedure is easily performed and, due to its reproducibilit,y, can serve as a first att)empt toward a systematic classification of the non-histone proteins. The further analysis of the different QAESephadex fractions by acrylamide gel electrophoresis reveals their high degree of heterogeneity. The possibility that the heterogeneous behavior of the non-histone proteins may be due t,o degradation has been rendered unlikely by the results of the end group determination according to Sanger. The presence also of ribonucleic acid in these preparations, manifested by the high 260/280 o.d. ratios, is not the cause of the heterogeneity, for treatment with RNase does not change t’he elect,rophoretic or chromatographic behavior of these proteins to any significant extent. From the data of Wang (27), Langan (29), Kleinsmith (30), and others it is known that a part of these proteins cont’ains phosphate groups in significant amounts. The work of Stocken and Ord (31) has focused attention on the presence of thiol groups in these proteins. We have therefore determined the content of phosphate and thiol in the different regions of the QAESephadex chromatogram. It is interesting to note that the proteins with a high phosphate content had low amount’s of t’hiol groups and vice versa. This distribution of thiol and phosphate was generally similar in preparations of non-histone proteins from liver, thymus, and kidney tissue. Due to the implication of phosphoproteins (32) and of the state of thiol/disulfide (33, 20) in the control of gene activity, the knowledge of the distribution of phosphate and thiol groups in the various fractions of the QAESephadex chromatogram should be of potential value for the use of these fractions in the various functional studies. The work of Gilmour and Paul has drawn attention to this class of proteins as imparting specificity to the in vitro transcribed RNA (7, 34). It was therefore of interest to compare the non-histone proteins derived from different organs. The chromatographic profiles of the non-histone proteins derived
SEKERIS
from liver, thymus, and kidneys were, to a great extent, very similar with slight qualitative and quantitative differences. Further analysis by acrylamide gel electrophoresis, either according to Shapiro et al., or to Benjamin and Gellhorn revealed the presence of unique bands parallel to the existence of pronounced similarities in the electrophoretic pattern for each of the organs studied. This is in agreement with t,he findings of Loeb and Creuzet (35), and of Dastugue et al. (36), partially wit,h the results of Elgin and Bonner (a), and in cont,rast to those of MacGillivray et al. (28). Whether t’his variability is connected to t’he determination of specificity of transcription by these proteins remains to be seen. When considering differences or similarities in the non-histone proteins between different organs, it should be mentioned that many enzymes are tightly bound to chromatin such as the DNA- (37), and RNA-polymerase (38), the nucleoside triphosphatases (39), the histone acetylases (40, 41), and methylases (42)) non-histone phosphokinases (43, 44), as well as dephosphorylat’ing enzymes. As regards histone transacetylases, differences in the behavior between the enzymes derived from liver, thymus, and kidney on DEAE-cellulose have recently been demonstrated (45) so that heterogeneity in the non-histone proteins of t’his kind must also be considered. The prerequisite for tackling problems involving the functional significance of t’he non-histone proteins is the availability of sufficient amounts of defined non-histone protein fractions soluble in dilute aqueous solutions. The method described above offers such a possibility and should be regarded as a first step towards this goal. ACKNOWLEDGMENTS We thank Professor P. Karlson for his encouraging interest in our work and we are grateful to Miss Froehlich, and Mrs. Pfeiffer for their skillful assistance. This work was supported by the Deutsche Forschungsgemeinschaft. REFERENCES 1. BONNER,
J., DAHMUS, M., FAMBROUGH, D., R. C., MARUSHIGE, K., AND TUAN, D., Scielzce 169, 47 (1968). HUANG,
NON-HISTONE
CHROMOSOMAL
2. ELGIN, S. C. R., .?ND BONNER, J., Biochemistry 4440 (1970) . 3. MARUSHIGE,, K., AND OZAKI, H., Develop. Biol. 16, 474 (1967). 4. TENG, C. S., AND HAMILTON, T. H., Proc. Nut. Acad. Sci. U. S. A. 63,465 (1969). 5. HOLT, T. K. H., Chromosoma 32, 64 (1970). 6. DELANGE, R. J., SMITH, E. L., FAMBROUGH, D. M., AND BONNER, J., J. Biol. Chem. 244, 5669 (1969). 7. PAUL, J., AND GILMOUR, R. S., J. Mol. Biol. 34, 305 (1968). 8. BEKHOR, I., KUNG, G. M., AND BONNER, J., J. Mol. Biol. 39, 351 (1969). 9. LO~VRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, J. R., J. Biol. Chem. 193, 265 (1951). 10. WARBURG, O., AND CHRISTIAN, W., Biochem. z. 310, 384 (1942). 11. OGUR, M., I~ND ROSEN, G., Arch. Biochem. 26. 262 (1950). 12. BURTON, K.., Biochem. J. 63, 315 (1956). 13. BARTLETT, G. R., J. Biol. Chem. 234, 466 (1959). 14. MOHR F.. 14nn. Chsm. Ph,nrm.. 97. 335 (1856). 15. SHAPIRO, A. L., VINUELA, E., AND M~IZEL, J. V., B&hem. Biophys. Res. Commun. 20, 815 (1967). 16. WEBER, K., AND OSBORN, M., J. Biol. Chem. 244, 4406 (1969). 17. BENJAMIN, W., AND GELLHORN, A., Proc. Nat. Acad. Sci. U. S. A. 69, 262 (1968). 18. STAHL, E., “Dtinnschichtchromatographie”, Springer Verlag, Berlin, Giittingen, Heidelberg (1963). 19. SANGER, F., B&hem. J. 39, 507 (1945). 20. SEKERIS, C. E., BEATO, M., HOMOKI, J., AND CONGOTE, L. F., Hoppe Seyler. Z. Physiol. Chem. 349, 857 (1968). 21. DUKES, P. P., SEKERIS, C. E., AND SCHMID, W., Biochim. Biophys. Acta 123, 126 (1966). 22. BEATO, M., HOMOKI, J., AND SEKERIS, C. E., Exp. Gel2 Res. 66, 107 (1969). 23. BE~TO, M., SEIFART, K. H., AND SEICERIS, C. E., Arch. Biochem. Biophys. 136, 272 (1970). 24. HOLOUBEK, V., .~ND CROCKER, T. T., Biochim. Biophys. llcta 167, 352 (1968). 25. ROE~EKAMF~, W., RICHTER, K. H., AND SEKERIS, C. E., in preparation.
PROTEINS
FROM
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ORGANS
53
26. DOUNCE, A. L., AND HILGSRTNER, C. A., Exp. Cell Res. 36, 228 (1964). 27. WANG, T. Y., J. Biol. Chem. 243, 1225 (1967). 28. MACGILLIVRAY, A. J., CAROLL, D., AND PAUL, J., Fed. Eur. Biochem. Sot. Lett. 13, 204 (1971). T. A., in “Regulatory Mechanisms 29. LaNGaN, in Nucleic Acid and Protein Biosynthesis” (V. V. Koenigsberger, and L. Bosch, eds.), Vol. 10, p. 233. BBA Library Series, Elsevier, Amsterdam (1967). 30. KLEINSMITH, L. J., .~ND ALLFREY, V. G., Biochim. Biophys. Acta 176, 123 (1969). 31. STOCKEN, L. A., AND ORD, M. G., “The Cell Nucleus -Metabolism and Radiosensitivity”, p. 141. Tylor and Francis Ltd., London (1966). 32. KLEINSMITH, L. J., ALLFREY, V. G., AND MIRSKY, A. E., Proc. Nat. Acad. Sci. U. S. A. 66, 1182 (1966). 33. ORD, M. G., AND STOCKEN, L. A., Biochem. J. 107, 403 (1968). 34. GILMOUR, R. S., AND PAUL, J., Fed. Eur. Biochem. Sot. Lett. 9, 242 (1970). 35. LOEB, J., AND CREUZET, C., Fed. Eur. Biochem. Sot. Lett. 6, 37 (1969). 36. DASTUGUE, B., TICHONICKY, L., PENITSORIA, J., BND KRUH, J., Bull. Sot. Chim. Biol. &, 391 (1970). 37. HOOK, R., AND WANG, T. Y., Arch. Biochem. Biophys. 133, 238 (1969). 38. SEIF.IR~‘, K. H., AND SEKERIS, C. E., Eur. J. Biochem. 7, 408 (1969). 39. SCHILTZ, E., AND SEKERIS, C. E., Hoppe Seylers Z. Physiol. Chem. 360, 317 (1969). 40. GALLWITZ, D., .*ND SEKENS, C. E., unpubpublished observations. 41. RACEY, L. A., :~ND BYVOET, P., Exp. Cell Res. 64, 366 (1971). 42. SEKERIS, C. E., SEKERI, K. E., AND GALL~ITZ, D., Hoppe Seyler. Z. Physiol. Chem. 349, 1660 (1967). 43. SCHILTZ, E., AND SEKERIS, C. E., Experientia 27, 30 (1971). 44. T~KEDA, M., YAM.\MURA, H., .~ND OHG.4, Y., Biochem. Biophys. Res. Commun. 42, 103 (1971). 45. GALLWTIZ, D., Fed. Eur. Biochem. Sot. Lett. 13, 306 (1971).