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

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Structure of the Glycine-Rich, Arginine-Rich Histone of the Novikoff Hepatoma' (CANCER RESEARCH 30, 2942—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 37°. 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 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1970 American Association for Cancer Research. NovikoffHepatoma GAR Histone Table 1 10 Amino acidanalysisofthe GAR histone ofthe Novikoff Acety lSer-.Gly-Arg—Gly-Lys-Gly-Gly—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)— 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-Arg—Arg-Gly-Gly-Va 1-Lys N@-methyllysine 1.0 0.2 50 N@-dimethyllysine 0.6 Arg- I le-Ser-Gly-Leu— I le-Tyr-Glu-Glu-Thr—Arg—G ly Histidine 2.4 2.2 Arginine 12.6 13.0 60 Aspartic acid 5.7 5.4 va 1-Leu—Lys-Va1-Phe-Leu-Glu-Asn-Va 1- I le—Arg-Asp 6.5 6.8 Threonine 80 Serine 2.2 2.4 Ala-Va l—Thr—Tyr—Thr—Glu-His—Ala—Lys-Arg—Lys—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'@—__1h10 —p-i-. 1119 iPwI @L-@-——[email protected],Gly.Gly.Lys.GIy.Leu.Gly.lys.Gly.Gly.Ala.lys.Arg.His.Arg.lys(Me).Val.leu.A•rg. 27S@*.T25T24 I22—IP@[4T18@IPrI 1-@ Th2 @lorI Asx.Asx,lie.Clx.Gly.lie.Thr.Lys.Pro.Ala,[email protected] @ I @- @—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 @ —C8 —p C5 * Cl0 C9 4 C6 @ I ThC Ii—niB._4o____IhA—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 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1970 American Association for Cancer Research. Wilson, Starbuck, Taylor, Jordan, and Busch 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, 55°;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 55°.A flow rate of 60 titrated by hand.
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