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Rapid isolation of total acidic proteins from chromatin of various chick tissues
Biochimica et Biophysica Acta, 322 (I973) 1 4 5 - I 5 4 © E l s e v i e r Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m - P r i n t e d in The N e t h e r l a n d s
BBA 36502 RAPID ISOLATION OF TOTAL ACIDIC PROTEINS FROM CHROMATIN OF VARIOUS CHICK TISSUES
E L I Z A B E T H M. W I L S O N a• AND T H O M A S C. S P E L S B E R G b'*
a Department of Biochemistry, Vanderbilt University, Nashville, Tenn. 37232 and b Department of Obstetrics and Gynecology, Vanderbilt University School of Medicine, Nashville, Tenn. 37232
(u.s.A.) ( R e c e i v e d F e b r u a r y 28th, 1973)
SUMMARY
A method is described for the rapid isolation of the nuclear acidic proteins of chromatin. Bovine pancreatic deoxyribonuelease I is used to hydrolyze DNA of dehistonized chromatin into acid-soluble fragments. The acidic proteins are recovered with more than 95% yield. Characterization of the isolated acidic proteins by sodium dodecyl sulfate pglyacrylamide gel electrophoresis reveals reproducible heterogeneous banding patterns which are specific for the following chick tissues: liver, spleen, erythrocyte and heart, as well as oviduct at various stages of development. [~H]Ovalbumin added to dehistonized chromatin is not degraded throughout the procedure demonstrating that the multiple bands are not due to proteolytic activity.
I NTRODUCTION
The increasing awareness of the role of the acidic chromatin proteins as specific gene regulators has been obscured by difficulties inherent in their isolation and characterization 1. The acidic proteins not only have great tenacity for DNA, but also, when dissociated by high salt and detergents, tend to self-aggregate and adhere to surfaces. It has been the objective of many workers to obtain an efficient method for isolating the total acidic proteins free of DNA so that they may be further separated by polyacrylamide gel electrophoresis and column chromatography. Fractions such as the phosphoproteins, which constitute anywhere from 30 to 60% of tile total acidic proteins 1, can be selectively recovered using buffered phenol, as reported by Shelton and Allfrey2. Further attempts have been made to fractionate the total acidic proteins using hydroxylapatite 3 and ion exchange chromatography4, 5. In these and other reports 6-s, either recoveries were not reported or were low. Also, controls * E. M. W i l s o n is an N D E A p r e d o c t o r a l fellow. ** T. C. S p e l s b e r g is a fellow of t h e N a t i o n a l Genet i c s F o u n d a t i o n . P r e s e n t a d d r e s s : D e p a r t m e n t of E n d o c r i n e Research, M a y o Clinic, R o c h e s t e r , Minn. 559oi , U.S.A.
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to check for possible proteolytic activity were often not carried out and generally long dialysis and centrifugation steps were required. This report describes a rapid, reproducible, and efficient isolation of the total acidic proteins utilizing bovine pancreatic deoxyribonuclease I. Separation is accomplished by sodium dodecyl sulfate polyacrylamide gel electrophoresis. METHODS
Assays Protein is determined by the procedure of Lowry et al. 9 using bovine serum albumin and calf thymus histones as standards for acidic proteins and histones, respectively. DNA is determined using the diphenylamine method of Burton 1°, with calf thymus DNA as control. Addition of 2% sodium dodeeyl sulfate and o.oi M sodium phosphate buffer (pH 7-5) to protein and DNA standards had only minimal effects on color development. Deoxyribonuclease procedure Chromatin was isolated as previously describedS, 11 from nuclei of various tissues from chicks (Rhode Island Reds) at least 7 days old which had received daily injections (subcutaneous) of 5 mg dietbylstilbestrol for 15 days unless specified otherwise. I m g chromatin in I ml of o.I mM EDTA and 2 mM Tris-HC1 (pH 7.5) is thawed, rehomogenized with a Teflon pestle-glass homogenizer and dehistonized by adding H2SO 4 to give 12 ml of a 0.2 M H2SO 4 solutionl~, 13. After standing for 15 rain at 4 °C, the solution is centrifuged at 20o0 × g for IO rain. The supernatant containing histones is decanted and the pellet is resuspended in IO ml of cold 0.2 M H2SO~ and centrifuged again. The tubes are thoroughly drained and the pellet is homogenized in 2 ml of a buffer containing 2 mM MgC12, 2 mM CaCI~, and o.I M Tris-HC1 (pH 7.5). To the dehistonized chromatin is added 25 ~g bovine pancreatic deoxyribonuclease [ (EC 3.1,4. 5, code DPFF-electrophoretically purified, Worthington, Freehold, N.J.) in 25 #1 of the Tris-MgC12-CaC12 buffer. It is important to check the pH and adjust if necessary to 7-5. The suspension is routinely incubated for 30 min at 30 °C, with occasional shaking. The proteins are pelleted by adding IO ml of 0. 4 M HCI04, incubating IO rain at 4 °C, and centrifuging at 2000 × g for IO rain. Tile pellet is resuspended in 1-2 ml of 20/0 sodium dodecyl sulfate and o.oi M sodium phosphate (pH 7.5) for analysis of protein and DNA, and for separation of the nuclear acidic proteins by gel electrophoresis. The proteins may also be resuspended directly into 30/0 sodium dodecyl sulfate containing o.14 M 2-mercaptoethanol and o.oi M sodium phosphate (pH 7.5) and dialyzed as described below. Sodium dodecyl sulfate polyaerylamide gel electrophoresis A polyacrylamide gel system similar to that of Laemmli 14 is used, except 4 M urea is added to the upper and lower gels as described by MaeGillivray et al. 3The gels consist of a stacking gel of 30/0 acrylamide, and two separating gel phases of 5 and 8.7% aerylamide. The protein samples are dialyzed overnight against a o.oi M phosphate buffer (pH 7.5) containing o.14 M 2-mercaptoethanol, lO°/0 glycerol (v/v) and 1% sodium dodecyl sulfate, and stored at --2o °C. Directly before eleetrophoresis, the samples are heated to 6o °C for 20 min to enhance solubilization and o.I vol. of
ACIDIC PROTEINS FROM CHROMATIN
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0.05% b r o m o p h e n o l is added to each as a t r a c k i n g dye. Electrophoresis is carried out for 4-5 h at 2 m A per gel. The gels are s u b s e q u e n t l y stained with a solution of o.25% Coomasie blue in 5 0 % m e t h a n o l (v/v) a n d lO% (v/v) acetic acid for 2 h. T h e y are destained b y soaking overnight in the same solution lacking the stain a n d are t h e n eleetrophoretically destained a n d stored in 7 % acetic acid. RESULTS I t is generally k n o w n t h a t the removal of D N A from c h r o m a t i n preparations enhances the resolution of the acidic proteins on sodium dodeeyl sulfate polyacrylamide gels. We also f o u n d t h a t electrophoresis of whole c h r o m a t i n results in considerable streaking a n d faint b a n d s (Fig. I, Gel I). I n c u b a t i o n of bovine serum a l b u m i n with s t a n d a r d calf t h y m u s D N A followed b y electrophoresis on gels confirmed t h a t D N A was p r o b a b l y causing the poor b a n d i n g patterns. I n addition, diffuse protein b a n d p a t t e r n s were seen when the D N A fragments were n o t removed subs e q u e n t to deoxyribonuclease t r e a t m e n t b y H C l Q precipitation. I n order to establish the o p t i m u m conditions for rapid D N A hydrolysis a n d m i n i m u m proteolytic activity, various parameters were tested. We found t h a t a
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Fig. I. Sodium dodecyl sulfate polyacrylamide gel electrophoretic patterns of whole chromatin and the acidic proteins and histones separated from DNA. The following gels are represented: ~, undissociated oviduct chromatin (ioo ~,g protein); 2, total acidic proteins (ioo/~g) of oviduct chromatin isolated by the deoxyribonuclease procedure (cf. Methods) ; 3, histones (5o/~g) extracted from oviduct chromatin with acid; and 4, bovine pancreatic deoxyribonuclease I (5°/~g). Whole chromatin was resuspended directly in the 3% sodium dodecyl sulfate solution described below. Histone was isolated from oviduct chromatin using 0.2 M H,SO 4 as described in Methods. The histone solution was dialyzed against deionized water and lyophilized. Each protein preparation and the whole chromatin were resuspended in 3% sodium dodeeyl sulfate, o.14 M 2-mercaptoethanol, o.oi M sodium phosphate buffer (pH 7.5) to make a solution of I mg protein per ml, and subsequently dialyzed overnight against IOO vol. of a buffer containing 1% sodium dodecyl sulfate, o.14 M 2-mercaptoethanol, lO% glycerol (v/v), and o.oi M sodium phosphate (pH 7.5). The molecular weights of some standard proteins are indicated at their characteristic position of migration.
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E. M. WILSON, T. C. SPELSBERG
deoxyribonuclease I concentration of at least I2 #g per 0.9 mg dehistonized chromatin DNA in 2 ml buffer was required when the incubation was carried out for 30 min (Fig. 2). The time of incubation can be shortened to io rain when 25 pg deoxyribonuclease were used for 0. 9 mg dehistonized chromatin DNA (Fig. 3). Addition of Ca 2+ to the Tris buffer was found to enhance DNA hydrolysis by deoxyribonuclease, as previously reported ~5. Precipitation of the protein subsequent to deoxyribonuclease treatment was found to be most complete with 0. 4 M HCIO4. Visual loss of protein bands on the gels and a lower yield of protein were observed when 0.2 M H2SO a was used for this step. 0.8 C3 OA ._J cn
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Fig. 2. Effect of deoxyribonuclease concentration on the e x t e n t of D N A hydrolysis. Oviduct chrom a t i n (IO mg) from chicks stimulated for io days w i t h 5 mg injections of diethylstilbestrol was dehistonized as described in Methods using o.2 M H2SO 4. The pellet was rinsed and resuspended in 12 ml of 2 mM MgC12, 2 mM CaC1 v o.i M T r i s - H C l (pH 7.5). To i-ml aliquots of this dehistonized c h r o m a t i n is added increasing a m o u n t s of deoxyribonuclease. The solutions are incubated for 3 ° min at 3° °C. Protein was precipitated with o. 4 M HC10 a and resuspended in 2°J; sodium dodecyl sulfate and o.oi M sodium p h o s p h a t e buffer (pH 7.5) for q u a n t i t a t i o n of DNA and protein. Fig. 3. Effect of varying the time of incubation with deoxyribonuclease on removal of a c i d - w e cipitable DNA. Oviduct c h r o m a t i n was dehistonized as described in Methods. Dehistonized chrom a t i n (o.9 rag) in 2 ml 2 mM MgC12, 2 mM CaCI~, o.i M Tris-HC1 (pH 7.5) was incubated at 3 ° °C with 25 fig deoxyribonuclease for v a r y i n g lengths of time. Protein was precipitated with o. 4 M HCIO 4 (@ Methods) and resuspended in 2°,/o sodium dodecyl sulfate and o.o~ M sodium p h o s p h a t e (pH 7.5) for analysis of DNA.
Proteolytic activity during acidic protein isolation seemed nonexistent (Fig. 4)This was shown by adding [3Hlovalbumin to dehistonized chromatin and completing the isolation procedure (cf. Methods). After electrophoresis, 2-ram slices were counted for radioactivity. Since the number of counts and the relative mobility of [aH~ovalbumin were unchanged, it seems that no proteolysis either due to endogenous activity among the acidic proteins or possible contamination in the deoxyribonuclease preparation occurred. The effectiveness of tile deoxyribonuclease procedure described in Methods can be seen in Fig. I, Gel 2. All protein band smearing has been eliminated. Also shown are the electrophoresis patterns of the histone components of chromatin (Gel 3) and bovine pancreatic deoxyribonuclease I used in the isolation procedure (Gel 4). A comparison was made between the banding patterns of the acidic proteins when the histones were removed by two different methods. Fig. 5 shows that salt extraction using 2 M NaCl, 2 mM phosphate buffer (pH 6) (Gel I) results in the loss of some bands when compared with histone extraction with o.2 M H2SO 4 (Gel 2).
ACIDIC PROTEINS FROM CHROMATIN
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GEL SLICE Fig. 4. Control for proteolytic activity of chromatin acidic proteins and deoxyribonuclease, o. 5 mg [~H]ovalbumin was added to i mg 14 day dehistonized oviduct chromatin DNA which is in 2 ml of a solution containing 2 mM MgC1v 2 mM CaC1v and o.i M Tris-HC1 (pH 7.5). Deoxyribonuclease (25 fig) is added and the solution is incubated for 3° min at 3° °C. Saturated (NHa)2SOa (2.0 ml) is added and the mixture is stirred for l h at 4 °C and centrifuged at 12 ooo × g for 5 min [(NH4)2SO4 was used to precipitate the acidic proteins in the initial experiments]. The pellet was resuspended in 3% sodium dodecyl sulfate, o.14 M 2-mercaptoethanol, o.oI M phosphate (pH 7.4) and dialyzed overnight against I °/o sodium dodecyl sulfate, o.14 M 2-mercaptoethanol, lO% glycerol, o.oi M phosphate (pH 7.5) for electrophoresis. Then ioo-#1 aliquots of this dialysate containing about I5oo cpm (o.o5 mg) [3Hlovalbumin, o.oo3 mg deoxyribonuclease, and o.o6 mg acidic protein were applied to the gels. After electrophoresis, the gels were sliced into 2-mm sections which were incubated separately with o.5 ml NCS (Amersham-Searle) overnight at 37 °C. IO ml of a scintillation fluid containing 2o ml spectrafluor PPO-POPOP (Amersham-Searle) per I pint of Baker's toluene was added. All vials were dark adapted and counted in a Beckman LS23o liquid scintillation counter. - - , [3H]ovalbumin + deoxyribonuclease -~ acidic proteins; __--, [3H]ovalbumin. Acid removal of histones reportedly involves a loss of less t h a n 5 ~o acidic protein 16. Of the procedures formulated in the past for recovering portions of the acidic proteins, Shelton a n d Allfrey~ describe a m e t h o d using phenol to recover primarily the acidic phosphoproteins of chromatin. Fig. 6 compares the p a t t e r n of these proteins o b t a i n e d b y their (Gel 2) a n d the deoxyribonuclease (Gel I) procedures. It is rather clear t h a t significantly more protein species are visible on gels, particularly in the high molecular weight region, when the deoxyribonuclease procedure is used which recovers all of the acidic proteins. The m a j o r b a n d s which do appear on both procedures seem to have similar relative mobilities. Application of the deoxyribonuclease procedure to various chick tissues resulted in the complete removal of DNA a n d the following recovery of protein: liver, 123 o/0 ; spleen, 9 5 % ; erythrocyte, 92~o; heart, IO3~o; a n d oviduct, 9 2 % (Table I). These recoveries represent the average of four d e t e r m i n a t i o n s , with values r a n g i n g from 85 to 125%. The only loss appears to be due to h a n d l i n g of the sample a n d adherence to glass surfaces. Fig. 7 shows t h a t the acidic protein p a t t e r n s are heterogeneous a n d tissue specific. The p a t t e r n s are reproducible when different preparations of c h r o m a t i n from the same tissue are used.
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Fig. 5. Sodium dodecyl sulfate polyacrylamide gel p a t t e r n s of oviduct c h r o m a t i n in which histones were r e m o v e d by (i) salt extraction 22, or (2) t r e a t m e n t w i t h o.2 M H~SO~ (cf. Methods). I n i, 2 ml of a solution containing 3.o M NaC1 and I mM sodium p h o s p h a t e buffer (pH 6.o) was added to o.5-I.O mg c h r o m a t i n D N A in i ml of o. i mM E D T A and 2.o mM Tris-HC1 (pH 7.4) to r e m o v e histones. After shaking vigorously, the solution is allowed to s t a n d for several h o u r s at o °C. I t is t h e n centrifuged at i IO ooo x g for 2 4 h at o °C. The s u p e r n a t a n t containing the histones is decanted. The pellet is rinsed and resuspended in I ml of 2 mM MgC12, 2 mM CaC12, and o.I M T r i s HC1 (pH 7.5) and treated w i t h deoxyribonuclease as described in Methods,
Top o£ 5% g e l ~
Top of 8.7% gel
Fig. 6. Sodium dodecyl sulfate polyacrylamide gel p a t t e r n s of total acidic proteins and phenolsoluble acidic proteins. O v i d u c t c h r o m a t i n from chicks which received daily injections of diethylstilbestrol for 15 days was dehistonized with o.2 M H~SO 4 (cf. Methods) and the acidic proteins isolated by (I) the deoxyribonuclease procedure (cf. Methods), and (2) b y the phenol procedure according to Shelton and Allfrey 2. ioo #g protein was applied to each gel.
ACIDIC PROTEINS FROM CHROMATIN
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TABLE I A N A L Y S I S OF C H R O M A T I N A N D T O T A L A C I D I C P R O T E I N S THE DEOXYRIBONUCLEASE
PROCEDURE
DESCRIBED
ISOLATED FROM VARIOUS CHICK TISSUES BY
I N ~¢~ETHODS
The values represent the average of 4 experiments. Tissue
Liver Spleen Erythrocyte Heart Oviduct
Chromatin (Acidie protein/DNA )
o. 52 0.48 0.44 5.75 i .42
Acidic protein ( % recovery) A cidie protein
DNA
123 95 92 i o3 92
o o o o o
The deoxyribonuclease procedure was also applied to chick oviduct c h r o m a t i n at various stages of development. W h e n the estrogenic hormone diethylstilbestrol is a d m i n i s t e r e d daily to 7-day-old i m m a t u r e chicks, the r u d i m e n t a r y oviduct is induced to develop gradually over a period of 15 days, at which time it is fully developed 17. The sodium dodecyl sulfate polyacrylamide gel p a t t e r n s of the total acidic proteins of o v i d u c t c h r o m a t i n isolated at various stages of development, i.e. o, 4, 7, 12, a n d 19 days are shown in Fig. 8. A gradual increase in the n u m b e r of protein b a n d s is observed d u r i n g development, p a r t i c u l a r l y in the very high a n d low molecular weight regions. Variations occur b o t h in the b a n d intensities a n d the relative mobilities. The proteins of i n t e r m e d i a t e molecular weight (60 o o o - I o o ooo) seem to be di m i n i s h e d at the latter stages of development.
Fig. 7- Sodium dodecyl sulfate polyacrylamide gel patterns of various chick tissues. 7-day-old chicks received diethylstilbestrol for 15 days prior to removal of the liver, spleen, oviduct, and heart. Erythrocytes were obtained from 2-year-old hens. Total acidic proteins were isolated by the deoxyribonuclease procedure. The following chick tissues are represented: I, liver; 2, spleen, 3, erythrocyte; 4, heart; and 5, oviduct. About ioo/zg protein per gel was applied.
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Fig. 8. S o d i u m dodecyl sulfate p o l y a c r y l a m i d e gel p a t t e r n s of t h e t o t a l acidic p r o t e i n s (deoxyribonuclease m e t h o d ) of t h e c h r o m a t i n of o v i d u c t at v a r i o u s stages of d e v e l o p m e n t . C h r o m a t i n w a s o b t a i n e d from o v i d u c t of chicks w h i c h were (I) n o t injected, or were injected with diethylstilbestrol for (2) 4 days, (3) 7 days, (4) i2 days, a n d (5) 19 d a y s . A b o u t IOO g g p r o t e i n was applied to each gel.
DISCUSSION
We have demonstrated that bovine pancreatic deoxyribonuclease l, an endonuclease requiring divalent cations, can be used to quantitatively recover more than 95% of the acidic proteins of chromatin from several chick tissues. By three simple steps--acid dehistonization, short incubation with bovine pancreatic deoxyribonuclease I, and acid precipitation--the protein can be quickly and quantitatively separated in bulk from DNA. After dialysis against a sodium dodecyl sulfate buffer, the nuclear acidic proteins are separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Addition of !3Hlovalbumin to the chromatin preparations indicates that no proteolytic breakdown occurs during the deoxyribonuclease treatment and dialysis against the buffered sodium dodecyl sulfate solution. As previously reported for mammalian systems using other isolation methods~, ls-21, tissue-specific, heterogeneous protein band patterns are observed for the chick tissues studied. Furthermore, changes in the gel patterns of the acidic proteins during diethylstilbestrol-induced development of the chick oviduct support the theory that alterations in the acidic protein species of chromatin may be part of the initial events required for the dynamic processes of changing gene expression during cytodifferentiation. Several parameters were investigated to obtain the optimal conditions for the removal of DNA and recovery of protein. It was found that addition of Ca 2+ to the deoxyribonuelease incubation mixture greatly facilitated DNA hydrolysis relative to the extent which occurred in the presence of only Mg2+. This enhancement is in agreement with previous work which reports that the presence of Ca z+ not only maintains the active conformation and protection of the deoxyribonuclease enzyme from trypsin ~, but also promotes the simultaneous cleavage of both strands of
ACIDIC PROTEINS FROM CHROMATIN
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DNA 15. In contrast, Mg2+ seems to initiate only one break per encounter of enzyme. Since the fragmented DNA could not be removed by dialysis, it appears that the DNA was not completely broken down into mononucleotides. The most efficient separation was precipitation of protein with HC10 4. Although use of dilute H2SO 4 m a y be preferred since it was used to dehistonize chromatin, it was avoided because loss of protein occurred due to solubility in the acid. The main drawback of the procedure described here is the denaturing effects of acid treatment, which have been shown to alter the immunochemical response and circular dichroism spectra of chromatin, and hence the secondary structure of its protein 2a. Although this is of little concern when analyzing the proteins by sodium dodecyl sulfate gel electrophoresis, it m a y limit biological applications to the isolated proteins. Although sodium dodecyl sulfate polyacrylamide gel electrophoresis can serve as a helpful tool when examining proteins of different molecular weights, a degree of caution must be taken in interpreting results of studies involving the acidic chromatin proteins. For instance, some difficulties persist in getting all the applied material into gels. This is a rather common problem when using polyacrylamide and m a y represent nonspecific aggregation of some of the protein. Also, one has to be aware that some of the bands seen on the gels m a y be caused by contaminants which m a y vary from tissue to tissue. In order to minimize this latter possibility, chromatin was isolated only from those nuclei which displayed good microscopic purity. Thus, a procedure is presented which allows for optimal recovery of the acidic proteins of chromatin free of DNA and histones. With close adherence to the specified conditions, it m a y well be applicable to m a n y systems and to further characterization b y column chromatography. ACKNOWLEDGMENTS
The excellent technical assistance of Mrs Gayle Cashion is appreciated. This research is supported by Public Health Service Grants CA 13o65-Ol and CA 1492O-Ol from the National Cancer Institute. T.C.S. is a member of the Center for Population Research (USPHSHD 05797) at Vanderbi]t University School of Medicine.
REFERENCES 1 Spelsberg, T. C., Wilhelm, J. A. and Hnilica, L. S. (1972) Sub-Cell. Biochem. I, lO7-145 2 Shelton, K. R. and Allfrey, V. G. (197 o) Nature 228, 132-134 3 MacGillivray, A. J., Cameron, A., Krauze, R. J., Richwood, D. and Paul, J. (1972) Biochim. Biophys. Acta 277, 384-402 4 Levy, S., Simpson, R. T. and Sober, H. A. (1972) Biochemistry 11, 1547-1554 5 Van den Brock, H. W. J., Nooden, L. D., Sevall, J. S. and Bonner, J. (1973) Biochemistry 12, 229-236 6 Elgin, S. C. and Bonner, J. (1972) Biochemistry i i , 772-781 7 Bhorjee, J. S. and Pederson, T. (1972) Proc. Natl. 3cad. Sei. U.S. 69, 3345-3349 8 Spelsberg, T. C., Steggles, A. W. and O'Malley, B. W. (1971) J. Biol. Chem. 246, 4186-4197 9 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 IO Burton, K. (1956) Biochem. J. 62, 315-323 i i Spelsberg, T. C. and Hnilica, L. S. (1971) Biochim. Biophys. Acta 228, 2o2-211 12 Johns, E. W. (1964) Biochem. J. 92, 55-59 13 Murray, K. (1966) J. Mol. Biol. 15, 4o9-419
154 14 15 16 17 18 19 20 21 22 23
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Laemmli, V. K. (i97 o) Nature 227, 68o-685 Poulos, T. L. and Price, P. A. (i972) J. Biol. Chem. 247, 2900-2904 Wilhelm, J. A., Spelsberg, T. C. and Hnilica, L. S. (1971) Sub-Cell. Biochem. (1971) i, 39-65 O'Malley, B. W., McGuire, \¥. L., Nohler, P. O. and K o r e n m a n , S. (;. (1969) Rec. Prog Hormone Res. 25, lO5-16o Benjamin, W. and Gellhorn, A. (1968) Proc. Natl. Acad. Sci. U.S. 59, 262-268 Shaw, L. M. J. and Huang, R. C. C. (197 o) Biochemistry 9, 453o-4542 Loeb, J. E. and Creuzet, C. (197 o) Bull. Soc. Chim. Biol. 52, lOO7-1o2o MacGillivray, A. J., Carrol, D. and Paul, J. (1971) F E B S Lett. 13, 204-208 Price, P. A., Liu, T. Y., Stein, W. H. and Moore, S. (1969) J. Biol. Chem. 244, 917-923 Spelsberg, T. C., Mitchell, W. M. and Chytil, F. (1973) Mol. Cell. Biochem. I, 1- 4.
BBA 36502 RAPID ISOLATION OF TOTAL ACIDIC PROTEINS FROM CHROMATIN OF VARIOUS CHICK TISSUES
E L I Z A B E T H M. W I L S O N a• AND T H O M A S C. S P E L S B E R G b'*
a Department of Biochemistry, Vanderbilt University, Nashville, Tenn. 37232 and b Department of Obstetrics and Gynecology, Vanderbilt University School of Medicine, Nashville, Tenn. 37232
(u.s.A.) ( R e c e i v e d F e b r u a r y 28th, 1973)
SUMMARY
A method is described for the rapid isolation of the nuclear acidic proteins of chromatin. Bovine pancreatic deoxyribonuelease I is used to hydrolyze DNA of dehistonized chromatin into acid-soluble fragments. The acidic proteins are recovered with more than 95% yield. Characterization of the isolated acidic proteins by sodium dodecyl sulfate pglyacrylamide gel electrophoresis reveals reproducible heterogeneous banding patterns which are specific for the following chick tissues: liver, spleen, erythrocyte and heart, as well as oviduct at various stages of development. [~H]Ovalbumin added to dehistonized chromatin is not degraded throughout the procedure demonstrating that the multiple bands are not due to proteolytic activity.
I NTRODUCTION
The increasing awareness of the role of the acidic chromatin proteins as specific gene regulators has been obscured by difficulties inherent in their isolation and characterization 1. The acidic proteins not only have great tenacity for DNA, but also, when dissociated by high salt and detergents, tend to self-aggregate and adhere to surfaces. It has been the objective of many workers to obtain an efficient method for isolating the total acidic proteins free of DNA so that they may be further separated by polyacrylamide gel electrophoresis and column chromatography. Fractions such as the phosphoproteins, which constitute anywhere from 30 to 60% of tile total acidic proteins 1, can be selectively recovered using buffered phenol, as reported by Shelton and Allfrey2. Further attempts have been made to fractionate the total acidic proteins using hydroxylapatite 3 and ion exchange chromatography4, 5. In these and other reports 6-s, either recoveries were not reported or were low. Also, controls * E. M. W i l s o n is an N D E A p r e d o c t o r a l fellow. ** T. C. S p e l s b e r g is a fellow of t h e N a t i o n a l Genet i c s F o u n d a t i o n . P r e s e n t a d d r e s s : D e p a r t m e n t of E n d o c r i n e Research, M a y o Clinic, R o c h e s t e r , Minn. 559oi , U.S.A.
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E. M. W I L S O N , T. C. S P E L S B E R G
to check for possible proteolytic activity were often not carried out and generally long dialysis and centrifugation steps were required. This report describes a rapid, reproducible, and efficient isolation of the total acidic proteins utilizing bovine pancreatic deoxyribonuclease I. Separation is accomplished by sodium dodecyl sulfate polyacrylamide gel electrophoresis. METHODS
Assays Protein is determined by the procedure of Lowry et al. 9 using bovine serum albumin and calf thymus histones as standards for acidic proteins and histones, respectively. DNA is determined using the diphenylamine method of Burton 1°, with calf thymus DNA as control. Addition of 2% sodium dodeeyl sulfate and o.oi M sodium phosphate buffer (pH 7-5) to protein and DNA standards had only minimal effects on color development. Deoxyribonuclease procedure Chromatin was isolated as previously describedS, 11 from nuclei of various tissues from chicks (Rhode Island Reds) at least 7 days old which had received daily injections (subcutaneous) of 5 mg dietbylstilbestrol for 15 days unless specified otherwise. I m g chromatin in I ml of o.I mM EDTA and 2 mM Tris-HC1 (pH 7.5) is thawed, rehomogenized with a Teflon pestle-glass homogenizer and dehistonized by adding H2SO 4 to give 12 ml of a 0.2 M H2SO 4 solutionl~, 13. After standing for 15 rain at 4 °C, the solution is centrifuged at 20o0 × g for IO rain. The supernatant containing histones is decanted and the pellet is resuspended in IO ml of cold 0.2 M H2SO~ and centrifuged again. The tubes are thoroughly drained and the pellet is homogenized in 2 ml of a buffer containing 2 mM MgC12, 2 mM CaCI~, and o.I M Tris-HC1 (pH 7.5). To the dehistonized chromatin is added 25 ~g bovine pancreatic deoxyribonuclease [ (EC 3.1,4. 5, code DPFF-electrophoretically purified, Worthington, Freehold, N.J.) in 25 #1 of the Tris-MgC12-CaC12 buffer. It is important to check the pH and adjust if necessary to 7-5. The suspension is routinely incubated for 30 min at 30 °C, with occasional shaking. The proteins are pelleted by adding IO ml of 0. 4 M HCI04, incubating IO rain at 4 °C, and centrifuging at 2000 × g for IO rain. Tile pellet is resuspended in 1-2 ml of 20/0 sodium dodecyl sulfate and o.oi M sodium phosphate (pH 7.5) for analysis of protein and DNA, and for separation of the nuclear acidic proteins by gel electrophoresis. The proteins may also be resuspended directly into 30/0 sodium dodecyl sulfate containing o.14 M 2-mercaptoethanol and o.oi M sodium phosphate (pH 7.5) and dialyzed as described below. Sodium dodecyl sulfate polyaerylamide gel electrophoresis A polyacrylamide gel system similar to that of Laemmli 14 is used, except 4 M urea is added to the upper and lower gels as described by MaeGillivray et al. 3The gels consist of a stacking gel of 30/0 acrylamide, and two separating gel phases of 5 and 8.7% aerylamide. The protein samples are dialyzed overnight against a o.oi M phosphate buffer (pH 7.5) containing o.14 M 2-mercaptoethanol, lO°/0 glycerol (v/v) and 1% sodium dodecyl sulfate, and stored at --2o °C. Directly before eleetrophoresis, the samples are heated to 6o °C for 20 min to enhance solubilization and o.I vol. of
ACIDIC PROTEINS FROM CHROMATIN
147
0.05% b r o m o p h e n o l is added to each as a t r a c k i n g dye. Electrophoresis is carried out for 4-5 h at 2 m A per gel. The gels are s u b s e q u e n t l y stained with a solution of o.25% Coomasie blue in 5 0 % m e t h a n o l (v/v) a n d lO% (v/v) acetic acid for 2 h. T h e y are destained b y soaking overnight in the same solution lacking the stain a n d are t h e n eleetrophoretically destained a n d stored in 7 % acetic acid. RESULTS I t is generally k n o w n t h a t the removal of D N A from c h r o m a t i n preparations enhances the resolution of the acidic proteins on sodium dodeeyl sulfate polyacrylamide gels. We also f o u n d t h a t electrophoresis of whole c h r o m a t i n results in considerable streaking a n d faint b a n d s (Fig. I, Gel I). I n c u b a t i o n of bovine serum a l b u m i n with s t a n d a r d calf t h y m u s D N A followed b y electrophoresis on gels confirmed t h a t D N A was p r o b a b l y causing the poor b a n d i n g patterns. I n addition, diffuse protein b a n d p a t t e r n s were seen when the D N A fragments were n o t removed subs e q u e n t to deoxyribonuclease t r e a t m e n t b y H C l Q precipitation. I n order to establish the o p t i m u m conditions for rapid D N A hydrolysis a n d m i n i m u m proteolytic activity, various parameters were tested. We found t h a t a
Pyruw
Ovalb
Cho ....
c
Ly, oz
Fig. I. Sodium dodecyl sulfate polyacrylamide gel electrophoretic patterns of whole chromatin and the acidic proteins and histones separated from DNA. The following gels are represented: ~, undissociated oviduct chromatin (ioo ~,g protein); 2, total acidic proteins (ioo/~g) of oviduct chromatin isolated by the deoxyribonuclease procedure (cf. Methods) ; 3, histones (5o/~g) extracted from oviduct chromatin with acid; and 4, bovine pancreatic deoxyribonuclease I (5°/~g). Whole chromatin was resuspended directly in the 3% sodium dodecyl sulfate solution described below. Histone was isolated from oviduct chromatin using 0.2 M H,SO 4 as described in Methods. The histone solution was dialyzed against deionized water and lyophilized. Each protein preparation and the whole chromatin were resuspended in 3% sodium dodeeyl sulfate, o.14 M 2-mercaptoethanol, o.oi M sodium phosphate buffer (pH 7.5) to make a solution of I mg protein per ml, and subsequently dialyzed overnight against IOO vol. of a buffer containing 1% sodium dodecyl sulfate, o.14 M 2-mercaptoethanol, lO% glycerol (v/v), and o.oi M sodium phosphate (pH 7.5). The molecular weights of some standard proteins are indicated at their characteristic position of migration.
I48
E. M. WILSON, T. C. SPELSBERG
deoxyribonuclease I concentration of at least I2 #g per 0.9 mg dehistonized chromatin DNA in 2 ml buffer was required when the incubation was carried out for 30 min (Fig. 2). The time of incubation can be shortened to io rain when 25 pg deoxyribonuclease were used for 0. 9 mg dehistonized chromatin DNA (Fig. 3). Addition of Ca 2+ to the Tris buffer was found to enhance DNA hydrolysis by deoxyribonuclease, as previously reported ~5. Precipitation of the protein subsequent to deoxyribonuclease treatment was found to be most complete with 0. 4 M HCIO4. Visual loss of protein bands on the gels and a lower yield of protein were observed when 0.2 M H2SO a was used for this step. 0.8 C3 OA ._J cn
dU3
D
_1
0.4
Z
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E
<
5 o.8[
O 10 20 30 ~g DEOXYRIBONUCLEASE PER REACTION
z o.4 ~ qQ E
• O
10
20
30
MINUTES
Fig. 2. Effect of deoxyribonuclease concentration on the e x t e n t of D N A hydrolysis. Oviduct chrom a t i n (IO mg) from chicks stimulated for io days w i t h 5 mg injections of diethylstilbestrol was dehistonized as described in Methods using o.2 M H2SO 4. The pellet was rinsed and resuspended in 12 ml of 2 mM MgC12, 2 mM CaC1 v o.i M T r i s - H C l (pH 7.5). To i-ml aliquots of this dehistonized c h r o m a t i n is added increasing a m o u n t s of deoxyribonuclease. The solutions are incubated for 3 ° min at 3° °C. Protein was precipitated with o. 4 M HC10 a and resuspended in 2°J; sodium dodecyl sulfate and o.oi M sodium p h o s p h a t e buffer (pH 7.5) for q u a n t i t a t i o n of DNA and protein. Fig. 3. Effect of varying the time of incubation with deoxyribonuclease on removal of a c i d - w e cipitable DNA. Oviduct c h r o m a t i n was dehistonized as described in Methods. Dehistonized chrom a t i n (o.9 rag) in 2 ml 2 mM MgC12, 2 mM CaCI~, o.i M Tris-HC1 (pH 7.5) was incubated at 3 ° °C with 25 fig deoxyribonuclease for v a r y i n g lengths of time. Protein was precipitated with o. 4 M HCIO 4 (@ Methods) and resuspended in 2°,/o sodium dodecyl sulfate and o.o~ M sodium p h o s p h a t e (pH 7.5) for analysis of DNA.
Proteolytic activity during acidic protein isolation seemed nonexistent (Fig. 4)This was shown by adding [3Hlovalbumin to dehistonized chromatin and completing the isolation procedure (cf. Methods). After electrophoresis, 2-ram slices were counted for radioactivity. Since the number of counts and the relative mobility of [aH~ovalbumin were unchanged, it seems that no proteolysis either due to endogenous activity among the acidic proteins or possible contamination in the deoxyribonuclease preparation occurred. The effectiveness of tile deoxyribonuclease procedure described in Methods can be seen in Fig. I, Gel 2. All protein band smearing has been eliminated. Also shown are the electrophoresis patterns of the histone components of chromatin (Gel 3) and bovine pancreatic deoxyribonuclease I used in the isolation procedure (Gel 4). A comparison was made between the banding patterns of the acidic proteins when the histones were removed by two different methods. Fig. 5 shows that salt extraction using 2 M NaCl, 2 mM phosphate buffer (pH 6) (Gel I) results in the loss of some bands when compared with histone extraction with o.2 M H2SO 4 (Gel 2).
ACIDIC PROTEINS FROM CHROMATIN
149
160
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GEL SLICE Fig. 4. Control for proteolytic activity of chromatin acidic proteins and deoxyribonuclease, o. 5 mg [~H]ovalbumin was added to i mg 14 day dehistonized oviduct chromatin DNA which is in 2 ml of a solution containing 2 mM MgC1v 2 mM CaC1v and o.i M Tris-HC1 (pH 7.5). Deoxyribonuclease (25 fig) is added and the solution is incubated for 3° min at 3° °C. Saturated (NHa)2SOa (2.0 ml) is added and the mixture is stirred for l h at 4 °C and centrifuged at 12 ooo × g for 5 min [(NH4)2SO4 was used to precipitate the acidic proteins in the initial experiments]. The pellet was resuspended in 3% sodium dodecyl sulfate, o.14 M 2-mercaptoethanol, o.oI M phosphate (pH 7.4) and dialyzed overnight against I °/o sodium dodecyl sulfate, o.14 M 2-mercaptoethanol, lO% glycerol, o.oi M phosphate (pH 7.5) for electrophoresis. Then ioo-#1 aliquots of this dialysate containing about I5oo cpm (o.o5 mg) [3Hlovalbumin, o.oo3 mg deoxyribonuclease, and o.o6 mg acidic protein were applied to the gels. After electrophoresis, the gels were sliced into 2-mm sections which were incubated separately with o.5 ml NCS (Amersham-Searle) overnight at 37 °C. IO ml of a scintillation fluid containing 2o ml spectrafluor PPO-POPOP (Amersham-Searle) per I pint of Baker's toluene was added. All vials were dark adapted and counted in a Beckman LS23o liquid scintillation counter. - - , [3H]ovalbumin + deoxyribonuclease -~ acidic proteins; __--, [3H]ovalbumin. Acid removal of histones reportedly involves a loss of less t h a n 5 ~o acidic protein 16. Of the procedures formulated in the past for recovering portions of the acidic proteins, Shelton a n d Allfrey~ describe a m e t h o d using phenol to recover primarily the acidic phosphoproteins of chromatin. Fig. 6 compares the p a t t e r n of these proteins o b t a i n e d b y their (Gel 2) a n d the deoxyribonuclease (Gel I) procedures. It is rather clear t h a t significantly more protein species are visible on gels, particularly in the high molecular weight region, when the deoxyribonuclease procedure is used which recovers all of the acidic proteins. The m a j o r b a n d s which do appear on both procedures seem to have similar relative mobilities. Application of the deoxyribonuclease procedure to various chick tissues resulted in the complete removal of DNA a n d the following recovery of protein: liver, 123 o/0 ; spleen, 9 5 % ; erythrocyte, 92~o; heart, IO3~o; a n d oviduct, 9 2 % (Table I). These recoveries represent the average of four d e t e r m i n a t i o n s , with values r a n g i n g from 85 to 125%. The only loss appears to be due to h a n d l i n g of the sample a n d adherence to glass surfaces. Fig. 7 shows t h a t the acidic protein p a t t e r n s are heterogeneous a n d tissue specific. The p a t t e r n s are reproducible when different preparations of c h r o m a t i n from the same tissue are used.
150
E . M . WILSON, T. C. SPELSBERG
Fig. 5. Sodium dodecyl sulfate polyacrylamide gel p a t t e r n s of oviduct c h r o m a t i n in which histones were r e m o v e d by (i) salt extraction 22, or (2) t r e a t m e n t w i t h o.2 M H~SO~ (cf. Methods). I n i, 2 ml of a solution containing 3.o M NaC1 and I mM sodium p h o s p h a t e buffer (pH 6.o) was added to o.5-I.O mg c h r o m a t i n D N A in i ml of o. i mM E D T A and 2.o mM Tris-HC1 (pH 7.4) to r e m o v e histones. After shaking vigorously, the solution is allowed to s t a n d for several h o u r s at o °C. I t is t h e n centrifuged at i IO ooo x g for 2 4 h at o °C. The s u p e r n a t a n t containing the histones is decanted. The pellet is rinsed and resuspended in I ml of 2 mM MgC12, 2 mM CaC12, and o.I M T r i s HC1 (pH 7.5) and treated w i t h deoxyribonuclease as described in Methods,
Top o£ 5% g e l ~
Top of 8.7% gel
Fig. 6. Sodium dodecyl sulfate polyacrylamide gel p a t t e r n s of total acidic proteins and phenolsoluble acidic proteins. O v i d u c t c h r o m a t i n from chicks which received daily injections of diethylstilbestrol for 15 days was dehistonized with o.2 M H~SO 4 (cf. Methods) and the acidic proteins isolated by (I) the deoxyribonuclease procedure (cf. Methods), and (2) b y the phenol procedure according to Shelton and Allfrey 2. ioo #g protein was applied to each gel.
ACIDIC PROTEINS FROM CHROMATIN
151
TABLE I A N A L Y S I S OF C H R O M A T I N A N D T O T A L A C I D I C P R O T E I N S THE DEOXYRIBONUCLEASE
PROCEDURE
DESCRIBED
ISOLATED FROM VARIOUS CHICK TISSUES BY
I N ~¢~ETHODS
The values represent the average of 4 experiments. Tissue
Liver Spleen Erythrocyte Heart Oviduct
Chromatin (Acidie protein/DNA )
o. 52 0.48 0.44 5.75 i .42
Acidic protein ( % recovery) A cidie protein
DNA
123 95 92 i o3 92
o o o o o
The deoxyribonuclease procedure was also applied to chick oviduct c h r o m a t i n at various stages of development. W h e n the estrogenic hormone diethylstilbestrol is a d m i n i s t e r e d daily to 7-day-old i m m a t u r e chicks, the r u d i m e n t a r y oviduct is induced to develop gradually over a period of 15 days, at which time it is fully developed 17. The sodium dodecyl sulfate polyacrylamide gel p a t t e r n s of the total acidic proteins of o v i d u c t c h r o m a t i n isolated at various stages of development, i.e. o, 4, 7, 12, a n d 19 days are shown in Fig. 8. A gradual increase in the n u m b e r of protein b a n d s is observed d u r i n g development, p a r t i c u l a r l y in the very high a n d low molecular weight regions. Variations occur b o t h in the b a n d intensities a n d the relative mobilities. The proteins of i n t e r m e d i a t e molecular weight (60 o o o - I o o ooo) seem to be di m i n i s h e d at the latter stages of development.
Fig. 7- Sodium dodecyl sulfate polyacrylamide gel patterns of various chick tissues. 7-day-old chicks received diethylstilbestrol for 15 days prior to removal of the liver, spleen, oviduct, and heart. Erythrocytes were obtained from 2-year-old hens. Total acidic proteins were isolated by the deoxyribonuclease procedure. The following chick tissues are represented: I, liver; 2, spleen, 3, erythrocyte; 4, heart; and 5, oviduct. About ioo/zg protein per gel was applied.
152
E. M. WILSON, T. C. SPELSBERG
Fig. 8. S o d i u m dodecyl sulfate p o l y a c r y l a m i d e gel p a t t e r n s of t h e t o t a l acidic p r o t e i n s (deoxyribonuclease m e t h o d ) of t h e c h r o m a t i n of o v i d u c t at v a r i o u s stages of d e v e l o p m e n t . C h r o m a t i n w a s o b t a i n e d from o v i d u c t of chicks w h i c h were (I) n o t injected, or were injected with diethylstilbestrol for (2) 4 days, (3) 7 days, (4) i2 days, a n d (5) 19 d a y s . A b o u t IOO g g p r o t e i n was applied to each gel.
DISCUSSION
We have demonstrated that bovine pancreatic deoxyribonuclease l, an endonuclease requiring divalent cations, can be used to quantitatively recover more than 95% of the acidic proteins of chromatin from several chick tissues. By three simple steps--acid dehistonization, short incubation with bovine pancreatic deoxyribonuclease I, and acid precipitation--the protein can be quickly and quantitatively separated in bulk from DNA. After dialysis against a sodium dodecyl sulfate buffer, the nuclear acidic proteins are separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Addition of !3Hlovalbumin to the chromatin preparations indicates that no proteolytic breakdown occurs during the deoxyribonuclease treatment and dialysis against the buffered sodium dodecyl sulfate solution. As previously reported for mammalian systems using other isolation methods~, ls-21, tissue-specific, heterogeneous protein band patterns are observed for the chick tissues studied. Furthermore, changes in the gel patterns of the acidic proteins during diethylstilbestrol-induced development of the chick oviduct support the theory that alterations in the acidic protein species of chromatin may be part of the initial events required for the dynamic processes of changing gene expression during cytodifferentiation. Several parameters were investigated to obtain the optimal conditions for the removal of DNA and recovery of protein. It was found that addition of Ca 2+ to the deoxyribonuelease incubation mixture greatly facilitated DNA hydrolysis relative to the extent which occurred in the presence of only Mg2+. This enhancement is in agreement with previous work which reports that the presence of Ca z+ not only maintains the active conformation and protection of the deoxyribonuclease enzyme from trypsin ~, but also promotes the simultaneous cleavage of both strands of
ACIDIC PROTEINS FROM CHROMATIN
153
DNA 15. In contrast, Mg2+ seems to initiate only one break per encounter of enzyme. Since the fragmented DNA could not be removed by dialysis, it appears that the DNA was not completely broken down into mononucleotides. The most efficient separation was precipitation of protein with HC10 4. Although use of dilute H2SO 4 m a y be preferred since it was used to dehistonize chromatin, it was avoided because loss of protein occurred due to solubility in the acid. The main drawback of the procedure described here is the denaturing effects of acid treatment, which have been shown to alter the immunochemical response and circular dichroism spectra of chromatin, and hence the secondary structure of its protein 2a. Although this is of little concern when analyzing the proteins by sodium dodecyl sulfate gel electrophoresis, it m a y limit biological applications to the isolated proteins. Although sodium dodecyl sulfate polyacrylamide gel electrophoresis can serve as a helpful tool when examining proteins of different molecular weights, a degree of caution must be taken in interpreting results of studies involving the acidic chromatin proteins. For instance, some difficulties persist in getting all the applied material into gels. This is a rather common problem when using polyacrylamide and m a y represent nonspecific aggregation of some of the protein. Also, one has to be aware that some of the bands seen on the gels m a y be caused by contaminants which m a y vary from tissue to tissue. In order to minimize this latter possibility, chromatin was isolated only from those nuclei which displayed good microscopic purity. Thus, a procedure is presented which allows for optimal recovery of the acidic proteins of chromatin free of DNA and histones. With close adherence to the specified conditions, it m a y well be applicable to m a n y systems and to further characterization b y column chromatography. ACKNOWLEDGMENTS
The excellent technical assistance of Mrs Gayle Cashion is appreciated. This research is supported by Public Health Service Grants CA 13o65-Ol and CA 1492O-Ol from the National Cancer Institute. T.C.S. is a member of the Center for Population Research (USPHSHD 05797) at Vanderbi]t University School of Medicine.
REFERENCES 1 Spelsberg, T. C., Wilhelm, J. A. and Hnilica, L. S. (1972) Sub-Cell. Biochem. I, lO7-145 2 Shelton, K. R. and Allfrey, V. G. (197 o) Nature 228, 132-134 3 MacGillivray, A. J., Cameron, A., Krauze, R. J., Richwood, D. and Paul, J. (1972) Biochim. Biophys. Acta 277, 384-402 4 Levy, S., Simpson, R. T. and Sober, H. A. (1972) Biochemistry 11, 1547-1554 5 Van den Brock, H. W. J., Nooden, L. D., Sevall, J. S. and Bonner, J. (1973) Biochemistry 12, 229-236 6 Elgin, S. C. and Bonner, J. (1972) Biochemistry i i , 772-781 7 Bhorjee, J. S. and Pederson, T. (1972) Proc. Natl. 3cad. Sei. U.S. 69, 3345-3349 8 Spelsberg, T. C., Steggles, A. W. and O'Malley, B. W. (1971) J. Biol. Chem. 246, 4186-4197 9 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 IO Burton, K. (1956) Biochem. J. 62, 315-323 i i Spelsberg, T. C. and Hnilica, L. S. (1971) Biochim. Biophys. Acta 228, 2o2-211 12 Johns, E. W. (1964) Biochem. J. 92, 55-59 13 Murray, K. (1966) J. Mol. Biol. 15, 4o9-419
154 14 15 16 17 18 19 20 21 22 23
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Laemmli, V. K. (i97 o) Nature 227, 68o-685 Poulos, T. L. and Price, P. A. (i972) J. Biol. Chem. 247, 2900-2904 Wilhelm, J. A., Spelsberg, T. C. and Hnilica, L. S. (1971) Sub-Cell. Biochem. (1971) i, 39-65 O'Malley, B. W., McGuire, \¥. L., Nohler, P. O. and K o r e n m a n , S. (;. (1969) Rec. Prog Hormone Res. 25, lO5-16o Benjamin, W. and Gellhorn, A. (1968) Proc. Natl. Acad. Sci. U.S. 59, 262-268 Shaw, L. M. J. and Huang, R. C. C. (197 o) Biochemistry 9, 453o-4542 Loeb, J. E. and Creuzet, C. (197 o) Bull. Soc. Chim. Biol. 52, lOO7-1o2o MacGillivray, A. J., Carrol, D. and Paul, J. (1971) F E B S Lett. 13, 204-208 Price, P. A., Liu, T. Y., Stein, W. H. and Moore, S. (1969) J. Biol. Chem. 244, 917-923 Spelsberg, T. C., Mitchell, W. M. and Chytil, F. (1973) Mol. Cell. Biochem. I, 1- 4.