Structure of the Glycine-Rich, Arginine-Rich Histone of the Novikoff Hepatoma'

(CANCER RESEARCH 30, 2942â€”2951,December 1970J Structure of the Glycine-rich, Arginine-rich Histone of the Novikoff Hepatoma'

R. KeithWilson,2WesleyC.Starbuck,CharlesW.Taylor, JohnJordan,andHarrisBusch Department ofPharmacology, Baylor CollegeofMedicine, Houston, Texas 77025

SUMMARY the carboxyl-terminal peptide produced by cleavage with cyanogen bromide were esseniially the same. It then became The glycine-nich, arginine-nich histone of Novikoff of interest to compare the remaining portion of the GAR hepatoma was hydrolyzed with trypsin, chymotrypsin, and histone of 1 neoplastic cell line with the known sequence of thermolysin. The peptides obtained were analyzed and normal calf thymus GAR histone. In this study, the Novikoff compared to similar peptides from the calf thymus hepatoma GAR histone is compared with calf thymus GAR glycine-nich, arginine-nich histone (14). histone. Thirteen tnyptic peptides were analyzed, and three were The approach for sequencing the Novikoff hepatoma GAR sequenced. Six chymotryptic and seven thermolysin peptides histone was the same as that used in this laboratory for the were analyzed. calf thymus GAR histone (14). The cyanogen bromide On the basis of similarity of the peptides isolated from cleavage study of the carboxyl terminal was done by Desai Ct Novikoff hepatoma and calf thymus glycine-nich, arginine-nich aL (8), as mentioned. Tryptic digestion was then carried out, histones, it seems evident that the two histone structures are because of the specificity of this enzyme (17), and the success essentially identical. of tryptic hydrolysis with the GAR histone of calf thymus, in order to obtain multiple peptides from the protein. The INTRODUCTION chymotryptic digestion was performed to obtain overlaps with the tryptic peptides for the carboxyl-terminal half of the Although the precise role of histones in regulation of gene molecule. The thermolysin digestion was performed to obtain activity is not defined (1 , 3), Stedman and Stedman (20) peptides overlapping tryptic peptides on the amino-terminal proposed that histones were indeed different in various cell half of the molecule. Three tryptic peptides were sequenced types and could thus serve as specific gene regulators. Studies completely. However, because other peptides agreed by amino have been made on histones of cancer cells with the hope of acid content with corresponding peptides in the calf thymus finding different histones in cancer cells that had derepressed GAR histone and their positions in the histone structure cor specific genes, resulting in cellular reproduction without the responded, they were not sequenced. usual types of control. Early work from this laboratory On the basis of the comparative information obtained, it showed that a nuclear protein complex (RP2-L) was present in seems clear that the linear sequence of amino acids in these 2 the Walker and other tumors (4), but later work indicated that histones is the same. histones in this complex may not differ from those of other tissues (10). More definitive studies ofhistones have shown no MATERIALS AND METHODS grossdifferencesbetweentumorandnontumorhistonesin amino acid analysis, NH2 terminals, or peptide maps (1 1, 12). The Novikoff hepatoma GAR histone was prepared as With the development of improved characterization described previously for calf thymus (19). The identity and procedures, further studies on the structures of histones of purity of the histone were determined by amino acid composi tumors and other cells have become possible. tion and polyacrylamide gel electrophoresis (13). After exclusion chromatography techniques developed by Tryptic Hydrolysis. Thirty pmoles of histone were dissolved Maunitzen et aL (13) and Starbuck et al. (19) provided a in deionized water to a concentration of 1 pmole/2 ml. The method for obtaining highly purified GAR3 histone, the GAR pH was adjusted and maintained at 8.0 with 0.1 N NaOH with histone of calf thymus was purified and sequenced the use of a pH-stat. The reaction was carried out for 2 hr at independently by 2 different groups (6, 14). When Desai et aL 37Â°. Trypsin (0.1 mg/pmole of protein) was added at the (8) comparedthecarboxyl-terminalportionsofhistonesfrom beginning of the reaction and again after 1 hr. The reaction bovine lymphosancoma, fetal calf thymus, and Novikoff was stopped by reducing the pH to 3 .0 with glacial acetic acid. hepatoma GAR histone, they found that the 18 amino acids of The mixture was reduced to 5 ml and applied to a column of Aminex A-S. I This work was supported in part by Welch Foundation Grant Chymotryptic Hydrolysis. Thirty j.zmoles of histone were Q-272, American Cancer Society Grant P-369, and Cancer Research Center Grant CA-10893. dissolved in deionized water to a concentration of 1 j@mole/2

2 Predoctoral trainee of the USPHS Grant CA-S 154. ml. The rest of the digestion was the same as that for the 3The abbreviation used is: GAR, glycine-nich, arginine-nich. tryptic hydrolysis. The mixture was applied to a column of Received June 25, 1970; accepted September 2, 1970. Sephadex G-25.

2942 CANCER RESEARCH VOL. 30

Table 1 10 Amino acidanalysisofthe GAR histone ofthe Novikoff Acety lSer-.Gly-Argâ€”Gly-Lys-Gly-Glyâ€”Lys-Gly-Leu hepatomaand calf thymus 20 The values are mole % of total amino acids recovered. Gly-Lys-Gly.-Gly-A 1a-@y@(Ac )-Arg-His-Arg-Lys (Me)â€” 30 Novikoff hepatoma Calf thymus va 1-Leu-Arg-Asp-Asn-I le-Gln-Gly-I le-Thr-.Lys-Pro Amino acid GAR histone GAR histone 40 Lysine 10.5 9.8 Ala- I le-Arg-Arg-Leu-A la-Argâ€”Arg-Gly-Gly-Va 1-Lys N@-methyllysine 1.0 0.2 50 N@-dimethyllysine 0.6 Arg- I le-Ser-Gly-Leuâ€” I le-Tyr-Glu-Glu-Thrâ€”Argâ€”G ly Histidine 2.4 2.2 Arginine 12.6 13.0 60 Aspartic acid 5.7 5.4 va 1-Leuâ€”Lys-Va1-Phe-Leu-Glu-Asn-Va 1- I leâ€”Arg-Asp 6.5 6.8 Threonine 80 Serine 2.2 2.4 Ala-Va lâ€”Thrâ€”Tyrâ€”Thrâ€”Glu-Hisâ€”Alaâ€”Lys-Argâ€”Lysâ€”Thr Glutamic acid 6.9 6.7 Proline 1.5 1.0 90 Glycine 16.1 16.8 Va l-Thr-Ala-Met-Asp-Va 1-Va l-Tyr-Ala-Leu-Lys-Arg Alanine 7.6 7.8 100 Cystine (half) 0.0 0.0 Gln-Gly-Arg-Thr-Leu-Tyr-Gly-Phe-Gly-G1yCOOH Valine 7.9 7.7 Methionine 0.8 1.0 Isoleucine 5.0 5.3 Chart 1. Primary sequence of the call thymus GAR histone (14). The Leucine 7.9 8.5 Tyrosine 2.8 3.1 underlined regions contain clusters of basic amino acids. Phenylalamne 2.2 2.0 Amide ammonia 6.9 4.7 @ I'@â€”__1h10 â€”p-i-. 1119 iPwI @ L-@-â€”â€”[email protected],Gly.Gly.Lys.GIy.Leu.Gly.lys.Gly.Gly.Ala.lys.Arg.His.Arg.lys(Me).Val.leu.Aâ€¢rg. 27S@*.T25T24 I22â€”IP@[4T18@IPrI 1-@ Th2 @lorI

Asx.Asx,lie.Clx.Gly.lie.Thr.Lys.Pro.Ala,[email protected] @ I @- @â€”117 @Tl644.....T15

@....Th1

lie. Ser. Gly. Leu. lie, Tyr. Clx. Clx. Thr, Arg. Giy-VakLeuLys.Vai. Phe. Leu. Clx, Asx. Val. lie. Arg. Asx-AIa-Vai-Thr-Tyr-Thr-Glu@His-Aia-1ys @@ T8___@@ @,uI, 16

@ â€”C8 â€”p C5 * Cl0

C9 4 C6

@ I ThC Iiâ€”niB._4o____IhAâ€”k

@ Arg. lys. Thr. Val. Thr. Ala.

4 T4 @4 T1

C5 @p,I

Chart 2. Primary sequence of the Novikoff hepatoma GAR histone. Peptides are designated as tryptic (7), chymotryptic (C), and thermolysin (Th) derived.Arrows, sequenceanalysisby Edman degradation.

DECEMBER 1970 2943

720 840 EFFLUENTVOLUME(ml) Chart 3. Elution pattern obtained by chromatography of 30 @.zmolesofatryptic hydrolysate of the GAR histone on a column (1.2 x 50 cm) of Aminex A-Sresin. Temperature, 55Â°;flow rate, 60 mI/hr. A stepwisegradient of pynidine:formatebuffers was used as indicated.

Table 2 Amino acid composition ofpeptides isolatedfrom the tryptic digestsof the Novikoffhepatoma GAR histone Chart No.Peakpeptides37TThr,No.PeptideNo.Novikoff hepatoma GAR histone tryptic

1.0316T4Asx, 1.0; Gly, 2.6; Tyr, 0.6; Phe, 0.7; Leu, 1.S;Thr, 1.7;Glx, 1.2;Gly, 1.2;Ala, 1.9;Val,2.4;Met,0.6; 1.036T6Asx, Leu, 1.2; Tyr, 0.6; Arg, 1.7; Lys, 1.0;Thr, 1.7;Glx, 1.0;Ala, 1.7;Val,0.8;Tyr,0.7;His,0.8; 1.0313T8Gly, Lys, 1.0317T1,Ala,1.0;Leu,1.1;Arg,1.0318T,6Arg,1.035T17Asx,1.0;Val,0.8;Leu,0.8;Lys,

1.9;Thr,0.9;Glx,0.9;Pro,0.7;Gly,1.0;Ala,0.9;Ile,2.8; 1.0327T Arg,0.9;Lys, 1.038T22Gly,2.3;Ala,1.0;Arg,1.0;Lys,1.0330T24Gly,8Val, 1.0; Leu, 1.1 ; Lys(Me), 0.7; Arg, 1.0312T25Gly,2.2;Lys,1.0314Gly,2.1 ; Leu, 1.0; Lys,

1.032T28Ser,0.8;Gly,1.4;Arg,1.0 1.0; Lys,

Thermolysin Hydrolysis. Thirty @zmolesof histone were dis (SO x 1.2 cm) of Aminex A-S resin (Bio-Rad Laboratories, solved in 2 mM CaC12 to a concentration of 1 jimole/ml. The Richmond, Calif.) was used for ion-exchange chromatography. pH was adjusted and maintained at 8.0 with 0.1 N NaOH The column was packed and run at 55Â°.A flow rate of 60 titrated by hand. Thermolysin (1 mg/lOO mg of histone) was mi/hr was used. The column was equilibrated with 0.2 M dissolved in as small a volume as possible of 0.05 M Tnis pynidine formate buffer, pH 3.1 . Stepwise gradients of pynidine chloride (pH 8.0) containing 2 mM CaCl2 . The reaction was formate buffer were used for peptide elution. The effluent was carried out for 3 hr at 40Â°,and thermolysin was added at the monitored by performing alkaline hydrolysis and ninhydrin start of the reaction and after I .5 hr. Digestion was stopped by assay (16) on an aliquot from every other fraction. decreasing the pH to 3 .0 with glacial acetic acid. The mixture Chromatography on Carboxymethyl Cellulose. The very was reduced to a volume of 5 ml and applied to a column of basic thermolysin peptides were fractionated on a 0.9- x 55-cm Sephadex G-25. column of carboxymethyl cellulose (CM-32 Whatman Exclusion Chromatography. Exclusion chromatography of microgranular) (2, 14). The sample was applied in and eluted the chymotryptic and thermolysin digests was accomplished with a solution of0.1 M sodium acetate, pH 6.0. After elution with a column of Sephadex G-25 (145 x 5.0 cm). The eluent with 180 ml of the sodium acetate buffer, a gradient was run was 0.2 N acetic acid, and the flow rate was 30 mi/hr. The from 0.1 M sodium acetate, pH 6.0, to 0.1 M sodium acetate, absorbance of the effluent was read at 230 mj.z. pH 6.5, and 1.0 M sodium chloride. The effluent was Ion-Exchange Chromatography on Aminex A-5. A column monitored by alkaline hydrolysis and ninhydrin assay.

2944 CANCER RESEARCH VOL. 30

Ion-Exchange Chromatography on Dowex 1-X8. A 1.2- x Edman Degradation. Edman degradation was carried out 55-cm column of Dowex 1-X8 was prepared as described pre essentially as described by Dopheide et aL (9). The peptides viously (15). The column was equilibrated with 03 M (0.5 to 1.5 pmoles) were dissolved in 1 ml of buffer that pyridine:formate buffer, pH 8.7. A stepwise gradient of contained pyridine :water.N-ethyhnorpholine (1@0:100:29), pyndine:formate buffers was used for elution. which was adjusted to pH 8.5 with glacial acetic acid. To this

Table 3 @ Sequencesoft@ypticpeptides 7', , T8, and T, , determined by Edman degradation

Peptide@â€˜677):Asx

LysComposition:0.82.41.22.21.01.40.71.0Step!Ã§[email protected].!1.01.20.71.0Step- Ala - Val - Thr - Tyr - Thr - Glu - His - Ala -

.01.0Step41.30.1130.81.00.91.0StepS31 [email protected]

1.0Stepl1.00.40.50.81.0Step81.30.21.0Step61.4 1.01.3 Q40.11.3 0.80.7 0.71.0

Peptide @â€˜8(56â€”59): Gly â€”Val â€”Leu â€”Lys

Composition: 1.0 0.8 0.8 1.0 Step 1 Q@ 1.2 0.9 1.0 Step 2 0.5 Q@ 0.9 1.0 Peptide @: Leu â€”Ala â€”Arg

Composition: 0.9 0.9 1.0 Step 1 Q@ 0.9 1.0

a Underlined numbers, step in which the amino acid decreased significantly.

I,., [email protected] Iâ€”

C.,

0 â€˜I,

VoidDiscard 300ml EFFLUENT VOLUME (ml) Chart 4. Elution pattern obtained by exclusion chromatography of 30 @smolesofa chymotryptic hydrolysate of the GAR histone on a column (145 x 2.5 cm) of Sephadex G-25 . The eluent was 0.2 N acetic acid.

DECEMBER 1970 2945

Wilson, Starbuck, Taylor, Jordan, and Busch

were added 50 ml of phenylisothiocyanate, and the tube was toluene bath for 20 hr. Aliquots of peptides were hydrolyzed thoroughly flushed with prepurified nitrogen. The mixture was in S times their volume of constant boiling HC1. After hydro allowed to react at 37Â°for 2.5 hr with continuous shaking. lysis, the HC1was diluted with deionized water and removed in The excess reagent was removed by S extractions with 2.5 ml a Rotary Evapomix (Buchier Instruments, Inc., Fort Lee, of benzene saturated with water. The aqueous phase was N. J.). The hydrolysates were then dissolved in 1 ml of sodium lyophilized and completely dried under vacuum in a desiccator citrate buffer (pH 2.2), filtered through a 0.22-p Milhipore containing P2 O@. filter, analyzed on a Spinco 120 automatic amino acid The residue was dissolved in 100 jil of anhydrous tnifluoro analyzer, and calculated as described previously (18). acetic acid. The tube was flushed with prepunified nitrogen, Peptide Mapping. Preparative paper chromatography and and the solution was maintained at 25Â° for 1 hr. Trifluoro electrophoresis were performed on Whatman No. 3MM paper acetic acid was removed with a stream of nitrogen, and the (19). Descending chromatography was run for 16 hr in residue was dissolved in 1 ml of 0.4 M acetic acid. This solu 1-butanol:pyridine :acetic acid :water (1 5 :12 :3 :10) with phe tion was then extracted 3 times with 2 ml of benzene satur nol red as an external marker (5). Electrophoresis was carried ated with 0.4 M acetic acid. An aliquot corresponding to 0.05 out as described earlier (13) with arginine as an external to 0.1 j.imole was taken for hydrolysis and amino acid analysis. marker (5). Amino Acid Analysis. Samples for amino acid analysis were Polyacrylamide Electrophoresis. Polyacrylamide electro prepared as described previously (19) and hydrolyzed in a phoresis was carried out as described previously (13).

Table 4 Amino acid composition ofpeptides isolatedfrom chymot,ypric digestsof the NovikoffhepatomaGARhistone

Chart No.PeakpeptidesS14C5Thr,2.6;Lys,!.6;His,0.8;Arg,1.0;Asx,1.0;Glx,1.!;Ala,1.7;No.PeptideNo.Novikoff hepatoma GAR histone chymotryptic

1.0SSC6Leu, Val,2.6;Met, 1.0;Tyr, 1.0;Arg, 1.!;Asx,2.2;Thr, 1.0;Glx, 1.4;Ala, 1.0;Val, 1.6; 0.9S10C8Leu,1.0;Lys,0.8;Arg,0.8;Asx,1.9;Thr,1.3;Glx,1.4;Ala,!.3;Ile, 0.5 ; Tyr,

1.0S8C9Leu, Val, 2.6; Ile, 0.5; Tyr, 1.2; Phe, 0.8S9Leu, 1.0; Arg, 0.7; Thn, 1.0; Gix, 1.6; Gly, 0.7; Val, 1.0; Arg, 0.7; Thr, 0.9; Gix, 1.7; Gly, 0.6; Val, 0.8; lie, 0.5; 0.8S15C,1Leu, Tyr, 1.0;Lys,0.8;Arg,2.8;Asx,1.7;Thr,0.9;Glx,0.9;Pro, 1.2; Gly, 0.7; Ala, 0.9; Ile, 2.8

0. 30

@I(@l @I @l ! @â€˜ 0. 20 $@t @ E I @r

C-, 10 9

Iâ€• 11 1213 14 0 0.10 ) â€˜I,

Cl â€” @IfL-@ 300 600 900 1200 1500 EFFLUENT VOLUME (ml) Chart 5 . Elution pattern obtained by chromatography of Peak D (Chart 4) on a column (1 .2 x SO cm) of Aminex A-S resin. Conditions were the same as in Chart 3.

2946 CANCER RESEARCH VOL. 30

C-)z

Void Discard 400ml EFFLUENT VOLUME rn)) Chart 6. Elution pattern obtained by chromatography of 30 j.imoles01 thermolysin digest of the GAR histone on a column (150 x 2.5 cm) of Sephadex G-25. The eluent was 0.2 N acetic acid.

1.50

I-.

C-, z C C C 0 0 v, 1', @I 0.75 200 C-, 4â€”. Void Discard 100ml EFFLUENT VOLUME ml) r 0 Chart 7. Elution pattern obtained by chromatography of Peak C I', (Chart 6) on a column (55 x 0.9 cm) of carboxymethyl cellulose (CM-32, Whatman). Sodium acetate (0.1 M) was the eluent for the first 280 ml, after which a gradient was used (from 0.! M sodium acetate, pH 6.0, to 0.1 M sodium acetate, pH 6.5, and 1.0 M NaCl). A iA'@AAAAAAx,@A'@ Void 200 300 400 500 Discard EFFLUENT VOLUME (ml) Nomenclature. Tryptic peptides, chymotryptic peptides, 100ml and thermolysin bear the prefixes T, C, and Th, respectively. Chart 8. Elution pattern obtained by chromatography of Peak D Except for 3 thermolysin peptides that were not found in calf (Chart 6) on a column (55 x 0.9 cm) of carboxymethyl cellulose. The thymus GAR histone, all peptides bear the same designation in conditions were the same as those for Chart 7. both Novikoff hepatoma and calf thymus GAR histone. obtained from the Novikoff hepatoma GAR histone and that RESULTS agree either by sequence or by amino acid content with comes ponding peptides from calf thymus GAR histone (14, 16). The total amino acid content of Novilcoff hepatoma GAR Tryptic Peptides. The tryptic digest was fractionated on histone and calf thymus GAR histone, as determined in this Aminex A-S (Chart 3). Thirteen peptides were isolated from laboratory, is shown in Table 1. the tryptic hydrolysate. The amino acid composition for each The primary amino acid sequence of calf thymus GAR peptide is shown in Table 2. The presence ofglutamic acid or histone is shown in Chart 1 (6, 7, 14). In Chart 2, the glutamine and of aspai@ticacid or asparagine was not differen proposed structure for Novikoff hepatoma GAR histone is tiated, except for the 18 amino acids of the carboxyl-terminal shown. The peptides indicated in Chart 2 are those that were cyanogen bromide peptide (8); therefore, these amino acids

DECEMBER 1970 2947

C-) z

Void Discard lOOm) EFFLUENT VOLUME (mo Chart 9. Elution pattern obtained by chromatography of Peak E (Chart 6) on a column (55 x 0.9 cm) of carboxymethyl cellulose. The conditions were the same as those for Chart 7.

1. from Peak D (Chart 5). The amino acid composition for each of the 6 peptides is shown in Table 4. Thermolysin Peptides. The themmolysin hydrolysate was chromatographed on Sephadex G-25 , as shown in Chart 6. Thirteen peaks, A through M, were obtained. Peaks A and B were discarded. Peak C (Chart 7), Peak D (Chart 8), Peak E C (Chart 9), and Peaks F and G combined (Chart 10) were 0 chromatographed on carboxymethyl cellulose. Peptide Th9 was isolated from Peak C, Peptide Th2 was isolated from Peak C-, D, and Peptides o and Th@were isolated from Peak E. The z amino acid analyses of these peptides are shown along with other thermolysin peptides in Table @. 0 Peaks F and G combined were eluted from carboxymethyl cellulose as 1 major peak of acidic and neutral amino acids (Peak 1). The peak was collected, reduced in volume, and chromatographed on Dowex l-X8 (Chart 1 1). Peptide Th1 was obtained from the Dowex column, and the analysis of this peptide is shown together with other thermolysin peptides in Discard Table 5. 100ml Peaks H, I, J, and K combined (Chart 12) and Peaks L and Chart 10. Elusion pattern obtained by chromatography of combined M combined (Chart 13) were chromatographed on Aminex Peaks F and G (Chart 6) on a column (55 x 0.9 cm) of carboxymethyl A-S . Peptides ThA and ThB were isolated both from Peaks H, cellulose. The conditions were the same as those for Chart 7. I, J, and K combined from Peaks L and M combined. The analyses of ThA and ThB are shown along with other thermo will be designated respectively as Gix and Asx in all tables. lysin peptides in Table 5. Three tryptic peptides were sequenced by the Edman pro cedure, and the results are shown in Table 3. DISCUSSION Chymotryptic Peptides. The pattern obtained by chr omatography of the chymotryptic hydrolysate on Sephadex The 18 amino acids of the carboxyl-terminal peptide pro G-25 is shown in Chart 4. Four major peaks, B, C, D, and E, duced by cyanogen bromide cleavage of the Novikoffhepatoma were obtained. Peaks B, C, and E were discarded. Peak D was GAR histone were sequenced earlier (8). The sequence was rechromatographed on Aminex A-S. Six peptides were isolated. found to agree with that of calf thymus GAR histone.

CANCER RESEARCH VOL.30 2948

TableS Amino acid composition ofpeptides isolatedfrom the digestion with thermolysin ofthe Novikoffhepatoma GAR histone

Chart No.Peakpeptides11STh1Ile,i.0;Ser,!.l;Gly,1.2813Th,Leu, No.PeptideNo.Novikoff hepatoma GAR histone thermolysin

0.972Th9Leu, 1.0; Lys, 1.0; Arg, 2.6 ; Gly, 2.0; Ala, 0.9 ; Val, !.0;Lys,2.0;His, 1.0;Arg, 1.9;Gly,3.1;Ala, 1.1; 1.29 Lys (Me), @ !.0; Sen. 1.1; Gly, 5.2; Lys, 2.1 13 9 ThA Phe, 1.0; Gly, 1.8 13 4 ThB Leu, 1.0; Gly, 1.1; Tyr, 0.6 97 9Th1 ThcArg.Leu, 1.0; Gly, 1.3; GIx, 0.9; Thr, 0.9; Lys, 1.2; Ang, 1.7

C 0

C-) z

0 â€œ1

80 160 280 320 400 480 EFFLUENT VOLUME rn)) Chart 11. Elution pattern of Peak 1 (Chart 10) chromatographed on a column (50 x 0.9 cm) of Dowex 1-X8.The temperature was 550 and the flow rate was 80 mI/hr. A stepwisegradient of pyridine:formate buffers was used as indicated.

Thirteen tryptic peptides of Novikoff hepatoma GAR tryptic peptides. Because of the identity of the amino acid histone were isolated and examined in this study. Three content of the peptides and the overlaps that were found, the @ peptides, T6 , T8 , and T1 , were sequenced with the use of the other tryptic peptides were not sequenced. Edman procedure. The sequences agreed with the come Six chymotryptic peptides of Novikoff hepatoma GAR sponding peptides from calf thymus GAR histone. The other histone were isolated and analyzed (C5 , C6 , C8 , C9 , C1o , and @ trypticpeptides(T1,T4,T16,T17,T18,T22,T24,T25,T27, 1 )@ The peptides were found to have amino acid composi and@@ ) were found to have amino acid compositions iden tions identical with those of corresponding peptides from calf tical with those of the corresponding calf thymus GAR histone thymus GAR histone. In addition, the chymotryptic peptides provided overlaps with tryptic peptides in the carboxyl 4 Peptide ,@ was found to contain serine, glycine, and arginine. Its terminal end of the molecule (Chart 2). elution position from Aminex A-4 column was identical to that found for N-acetylserylglycyiarginineand was unique for the tryptic peptides. Seven thermolysin peptides from Novikoff hepatoma GAR This peptide was previously shown to strain weakly with ninhydnin but histone were isolated and analyzed. Four of the peptides (Th1, strongly with the Rydon-Smith stain (19). Peptide 28 had the same Th2 â€˜ @,andTh1 @)agreed in amino acid content with the RF's and staining properties as the N-acetylserylglycylarginine in the corresponding peptides from calf thymus GAR histone. In @ â€œfingerprintingâ€•systemused, and, accordingly, it was presumed to be addition, 3 new peptides, ThA ThB and Th@, were isolated. N-acetylserylglycylarginine. Since no other peptide was found that corresponded in composition, the amino-terminal amino acid of the These 3 peptides were found to fit overlaps with the tryptic Novikoff hepatoma is presumably completely acetylated. and chymotryptic peptides found in the carboxyl-terminal

DECEMBER 1970 2949

Wilson, Starbuck, Taylor, Jordan, and Busch

0.4

â€” 0 0 0 0 0 0 @ â€˜@: â€˜@ Lr@ Lt@% E @. - @- Q. 0. @. 2 @l@l @l @l @l @l @@@ Lt@% @f\ ,,@ 0 0 @ c; c; d c; @ t t t 10;@217 20

@ 2 A 16 18

4@f/Th\j@I@A A1A I __ 0 100 200 300 400 EFFLUENT VOLUME (ml) Chart 12. Elution pattern obtained by chromatography of combined Peaks H, I, J, and K (Chart 6) on a column (SO x 1.2 cm) of Aminex A-S resin. The conditions were the same as those for Chart 3.

0. 20

C 0 â€˜a.'0.10

C-) z

EFFLUENT VOLUME (ml) Chart 13. Elution pattern obtained by chromatography of combined Peaks L and M (Chart 6) on a column (SO x 1.2 cm) of Aminex A-S resin. The conditions were the same as those for Chart 3.

portion of both the calf thymus and Novikoff hepatoma GAR sponding peptides in calf thymus GAR histone (14). As seen in molecule (Chart 2). Chart 2, amino acids from the amino terminal to arginine 23 Final evidence presented for establishing identity of the have been located in both tryptic and thermolysine peptides. Novikoff hepatoma and calf thymus GAR histones is the Thus, by 2 different hydrolytic procedures, peptides were placing of the various peptides isolated. The procedure for isolated with identical amino acid content to those peptides alignment of overlapping peptides of the calf thymus GAR from Amino Acids 1 through 23 in calf thymus GAR histone. histone has been described earlier (16). Alignment of over From aspartate 24 through leucine 37, tryptic and chymo lapping peptides was made by comparison with the come tryptic peptides were isolated. These peptides also agreed by

2950 CANCER RESEARCH VOL.30

amino acid content with corresponding peptides from calf Pea Seedling Histone IV; Comparison with the Homologous Calf thymus GAR histone. From alanine 38 through arginine 45, all Thymus Histone. J. Biol. Chem., 244: 5669â€”5679, 1969. amino acids were again accounted for 2 separate procedures, 8. Desai, L., Ogawa, Y., Mauritzen, C. M., Taylor, C. W., and Star i.e., from thermolysin and tryptic peptides. Amino Acids 46 buck, W. C. Carboxyl-terminal Sequence of the Glycine-Arginine rich Histone from Bovine Lymphosarcoma, Novikoff Hepatoma, through 48 were accounted for in only a thermolysin peptide. and Fetal Calf Thymus. Biochim. Biophys. Acta, 181: 146â€”153, Leucine 49 was positioned by difference. The peptide corre 1969. sponding to calf thymus GAR histone T9 was not isolated. 9. Dopheide, T. A. A., Moore, S., and Stein, W. H. The Carboxyl However, as seen in Table 1, the total number of leucines in terminal Sequence of Porcine Pepsin. J. Biol. Chem., 242: both calf thymus and Novikoff hepatoma GAR histones is 1833â€”1837, 1967. essentially the same. In addition, because of the specificity of 10. Hidvegi, E. J., Arky, I., Antoni, F., and Varteresz, V. Studies on thermolysin and chymotrypsin for enzymatic cleavages to the Heterogeneity and Metabolic Activity of Histones from Rabbit form peptides Th1 and o , leucine was placed in Position 49. Bone Marrow Cells. Bnit. J. Cancer, 1 7: 377â€”380,1963. From isoleucine 50 to the carboxyl terminal, all amino acids 11. Hnilica, L. S., Taylor, C. W., and Busch, H. Analysis of Peptides of have been located in peptides obtained by at least 2 hydrolytic the Moderately Lysine-rich Histone Fraction f2b of the Walker procedures. In addition, carboxyl-terminal amino acids from Tumor and Other Tissues. Exptl. Cell Res. Suppi., 9: 367â€”375, 1963. aspartate 85 through the carboxyl terminal were sequenced by 12. Leclenc, J., Martinage, A., Moschetto, Y., and Biserte, G. A Com @ Desai et a!. (8). Finally, Tryptic Peptides T6 , T8 , and parative Study of Histone Fraction F2(a)1 and F2(a)2. European J. were sequenced completely and were found to agree with the Biochem., 11: 261â€”266,1969. corresponding peptides from calf thymus GAR histone. 13. Maunitzen, C. M., Starbuck, W. C., Saroja, I. S., Taylor, C. W., and On the basis of the agreement in structure of peptides from Busch, H. The Fractionation of Arginine-nich Histones from Fetal tryptic, chymotryptic, and thermolysin digestion, total protein Calf Thymus by Exclusion Chromatography. J. Biol. Chem., 242: amino acid analysis, and specificity of enzymatic cleavage, it 2240â€”2245,1967. appears the linear sequence of Novikoff hepatoma and calf 14. Ogawa, Y., Quagliarotti, G., Jordan, J., Taylor, C. W., Starbuck, W. thymus GAR histones is essentially identical. C., and Busch, H. Structural Analysis of the Glycine-rich, Arginine-rich Histone. III. Sequence of the Amino-terminal Half of the Molecule Containing the Modified Lysine Residues and the REFERENCES Total Sequence. J. Biol. Chem., 244: 4387â€”4392, 1969. 15. Rombauts, W. A., and Raffery, M. A. A Device for Automatic 1. Allfrey, V. G., Littau, V. C., and Minsky, A. E. On the Role of Gradient or Stepwise Chromatography. Anal. Chem., 37: Histones in Regulating Ribonucleic Acid Synthesis in the Cell 1611â€”1614,1965. Nucleus. Proc. Nail. Acad. Sci. U. S., 49: 414â€”421, 1963. 16. Sautiere, P., Starbuck, W. C., Roth, C., and Busch, H. Structural 2. Ando, T., and Suzuki, K. The Amino Acid Sequence of the Third Analysis of the Glycine-rich, Arginine-rich Histone. I. Sequence of Component of Clupeine. Biochim. Biophys. Acta, 140: 375â€”377, the Carboxyl-terminal Portion. J. Biol. Chem., 243: 5899â€”5905, 1967. 1968. 3. Busch, H. Histones and Other Nuclear Proteins. New York: 17. Starbuck, W. C. The Determination of the Sequence of Amino AcademicPress,Inc., 1965. Acids in Proteins. In: H. Busch (ed.), Methods in Cancer Research, 4. Busch, H., Hnilica, L. S., Chien, S. C., Davis,J. R., and Taylor, C. Vol. 5, pp. 251â€”351.New York: Academic Press, Inc., 1970. W. Isolation and Purification of RP2-L,a NuclearProtein Fraction 18. Starbuck, W. C., Maunitzen, C. M., McClimans, C., and Busch, H. of the Walker Carcinosarcoma. Cancer Res., 22: 637â€”645,1962. A Computer Program for the Calculation of Amino Acid Analysis 5. Canfield, R. E. Peptides Derived from Tryptic Digestion of Egg Data. Anal. Biochem., 20: 439â€”462, 1967. White Lysozyme. J. Biol. Chem., 238: 2691â€”2697, 1963. 19. Starbuck, W.C., Maunitzen,C. M., Taylor, C. W.,Saroja, I. S., and 6. DeLange, R. J., Fambrough, D. M., Smith, E. L., and Bonner, J. Busch, H. A Lange Scale Procedure for Isolation of the Glycine Calf and Pea Histone IV. II. The Complete Amino Acid Sequence rich, Arginine-nich Histone and the Arginine-nich, Lysine-nich of Calf Thymus Histone IV; Presence of e-N-Acetyllysine.J. Biol. Histone in a Highly Purified Form. J. Biol. Chem., 243: Chem., 244: 319â€”334,1969. 2038â€”2047,1968. 7. DeLange, R. J., Fambrough, D. M., Smith, E. L., and Bonner, J. 20. Stedman, E., and Stedman, E. Cell Specificity of Histones. Nature, Calf and Pea Histone IV. III. Complete Amino Acid Sequence of 166: 780â€”781,1950.

DECEMBER 1970 2951

R. Keith Wilson, Wesley C. Starbuck, Charles W. Taylor, et al.

Cancer Res 1970;30:2942-2951.

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