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Isolation and Purification of RP2-L, a Nuclear Protein Fraction of the Walker 256 Carcinosarcoma* HARRISBUSCH,LUBOMIRS. HNILICA,SU-CHENCHIEN,! JOSEPHR. DAVIS,Õ ANDCHARLESW. TAYLOR (Departments of Biochemistry and Pharmacology, Baylor University College of Medicine, Houston, Texas) SUMMARY One hour after the injection of 5 fie. of L-lysine-U-C14 into each of a group of rats bearing the Walker 256 carcinosarcoma, the acid-soluble proteins were extracted from nuclear preparations of the tumor. The proteins of these extracts were chromatographed on carboxymethylcellulose, with formic acid as the eluting agent. Rechromatography of 150 mg. of RP2-L1 on carboxymethylcellulose resulted in further purification of the proteins as determined by starch gel electrophoresis, amino acid analysis, N-terminal amino acids, and increase in specific activity. Since lysine comprises 14 per cent of the total amino acid residues, the proteins were classifiable as "slightly lysine-rich" histones. As the proteins were purified, the percentage of Nterminal proline increased. From the amino acid composition, sedimentation velocity, and diffusion constant, the molecular weight of the proteins in purified RP2-L was found to be 33,000. Starch gel electrophoresis of the purified product reveals the pres ence of one major and five minor protein bands, emphasizing the need for further im provement in methods for subfractionation of the bands. Recent studies from this and other laboratories (3, 4, 6, 15, 22, 26) have shown that an important pathway in the utilization of amino acids by transplantable rat tumors is the biosynthesis of nuclear proteins, particularly histones, or chromosomalbound nuclear proteins. In the Walker tumor and other tumors 30-55 per cent of the isotope of radioactive lysine which was incorporated into nuclear proteins was found in a cationic nuclear protein fraction tentatively coded as RP2-L (10, 11). This fraction was the second radioactive peak eluted by a gradient increasing to l N formic acid from carboxymethylcellulose columns to which solutions of prelabeled acid-soluble nuclear pro teins had been added. This peak, found initially * These studies were supported in part by grants from the U.S. Public Health Service, the Jane Coffin Childs Fund for Medical Research, and the American Cancer Society. t Predoctoral Trainee in Pharmacology. } Postdoctoral Fellow of the American Cancer Society. Present address: Stritoli College of Medicine, Chicago, Illinois. 1RP2-L was defined in previous studies as the second radio active peak emerging from carboxymethyloellulose columns when a gradient increasing to l N formic acid was added; in these experiments L-lysine-U-C" was used as the tracer (10, 11). Received for publication January 20, 1962. in the Walker tumor, was later shown to be present in chromatograms of the Jensen sarcoma, FlexnerJobling carcinoma, the Ehrlich ascites tumor, and Sarcoma 180, as well as a human malignant mela noma (11). RP2-L was not found in functionally active or growing nontumor tissues, such as re generating rat liver or embryonic rat tissues. Later studies indicated that RP2-L was bound to DNA in the Walker tumor and, hence, was de scribed as a "histone" fraction (7). The possibility that the components of this peak might represent proteins which were uniquely formed in the tumors (1, 9, 11) led to the present studies on isolation and purification of this peak and attempts to characterize the components in terms of amino acid composition, N-terminal amino acids, and molecular weight. In early stud ies on the proteins of the nucleus of tumor cells, it was found that each gram of tumor (wet weight) contained approximately 13 mg. of acid-soluble nuclear proteins and that RP2-L comprised ap proximately 14 per cent of the total weight. Since studies of the properties indicated above require 30-50 mg. of purified protein, initial experiments were carried out on the preparation of large amounts of RP2-L. 637 Downloaded from cancerres.aacrjournals.org on June 11, 2017. © 1962 American Association for Cancer Research. 638 Cancer Research MATERIALS AND METHODS Animals.—The animals used in these experi ments were male rats weighing 180-220 gm., obtained from the Holtzman Rat Company, Hous ton, Texas, and fed ad libitum on Purina Labora tory Chow. The tumor studied was the Walker 256 carcinosarcoma. Each rat was given inocula tions in eight subcutaneous sites which developed approximately 6-10 gm. wet weight of tumor per animal. One hour after injection of 5 /LÕC. of Llysine-U-C14, the animals were anesthetized and exsanguinated;2 the tumors were rapidly excised and dissected free of any hemorrhagic or necrotic tissue in the cold (4°C.). In these experiments, 60-200 gm. of tumor were used. Extraction of cationic nuclear proteins.—In ini tial experiments, nuclear preparations were ob tained by the method previously described (4, 5). Since essentially the same results were obtained with the 600 X g precipitate (3), this simpler method was used in studies with larger quantities of tumors. The acid-soluble nuclear proteins were extracted with 10 ml. of 0.25 N HCl/gm tissue in the cold for 30 minutes with stirring. The extract was then dialyzed against 0.005 M acetic acid.3 The sample was clarified by centrifugation prior to chromatography. Chromatograph]/.—Samples ranging from 1 to 4 gm. of the acid-soluble nuclear proteins were chromatographed on carboxymethylcellulose. A slurry of Selectacel CM-S (Brown Company, Ber lin, New Hampshire) with a capacity of 0.9 meq/ gm was made by stirring 250 gm. of adsorbent in 10.5 1. of distilled water for 1 hour; 0.5 N NaOH (approximately 375 ml.) was added to bring the pH to 10.0. The slurry was blended in a Waring Blendor for 1 minute and immediately poured into glass columns which were 48 cm. in length and 9 cm. in diameter. The adsorbent was packed with air pressure (15 p.s.i.) until a column 45 cm. in height was obtained. Eight liters of 0.05 M ace tate buffer at pH 4.0 were passed through the col umn with the aid of air pressure (10 p.s.i.); the final effluent pH was 4.0. The mixing chamber, an 1In early experiments, 40 ftc. of L-lysine-T monohydrochloride (Volk Radiochemical Company, Chicago) with a spe cific activity of 22 mc/mmole was injected intraperitoneally into each of ten tumor-bearing rats. Because of experimental problems in accurate determination of the specific activities of proteins labeled with tritium by the plating technics employed, all experiments in the present report were carried out with pro teins obtained from rats given injections of 5 /nc. L-lysine-UC». 3To minimize enzymatic hydrolysis of the proteins, dialysis was carried out at pH 8.O.Evidence that substantial enzymatic degradation did not occur is the absence of fast-moving bands on starch gels and the similarity of fractions obtained under these conditions in the text and when DFP was added to the original extracts. Vol. 22, June 1962 Erlenmeyer flask, was completely filled with 3 1. of distilled water; 4 1. of l N formic acid were ini tially added to the reservoir. The flow rate was maintained at 6.0 ml/min by a Minipump®and by adjusting an Ultramax®valve at the bottom of the glass column. Effluent fractions were col lected every 4 minutes with the aid of a fraction collector. Essentially identical chromatograms were also obtained with columns of the same height and a diameter of 5.5 cm.; the mixing flask and flow rate were modified in proportion to the crosssectional area of the column (10, 11). Rechromatography. —For rechromatography, the fractions comprising the various peaks were pooled, dialyzed against 0.005 Macetic acid in the cold for 16 hours, and lyophilized. The dry protein (150 mg.) was dissolved in 10 ml. of cold 0.005 M acetic acid and was added to columns of Selec tacel CM-S, 15 cm. in height and 2 cm. in diame ter. These columns were prepared in the same manner as described above but contained 9 gm. of adsorbent in 600 ml. distilled water; the mixing flask contained 500 ml. of distilled water, and the reservoir contained 1 1. of l N formic acid. The flow rate was 0.5 ml/min. Fractions were collected every 12 minutes. Determination of protein and radioactivity.— Proteins in the eluate were detected spectrophotometrically at 280 m¿t.Aliquots of each fraction were plated as previously described (10), and the radioactivity was determined in an automatic gas-flow counting apparatus (Nuclear-Chicago). N-terminal amino acid analysis.—Samples of the various fractions (20—30mg.) were reacted with l-fluoro-2,4-dinitrobenzene for 12-14 hours at room temperature, according to the technic of Sanger (23) as modified by Phillips (19). Hydrol ysis of the DNP-protein was carried out at 100° C. for 4£hours in 11.4 HC1. The DNP-amino acids were identified by paper chromatography; the moving phases were 2-butanol saturated with 0.05 M phthalate (pH 6) and 1.5 M phosphate at pH 5 (17). For quantitative determinations, the spots were eluted with warm water (40°C.) and measured as described by Fraenkel-Conrat et al. (12). Amino acid analysis.—-The protein samples were hydrolyzed in sealed tubes with 5.7 N HC1 at 110°C.for 22 hours (8). Quantitative assays for amino acids were made with the aid of an auto matic amino acid analyzer (25); tryptophan was determined after alkaline hydrolysis of the pro teins.4 The values for serine and threonine were 4In the alkaline hydrolysis, approximately 40 per cent de struction of tryptophan was found when known quantities of tryptophan were added to protein hydrolysates. Less than 0.5 per cent of tryptophan was found in samples of RP2-L, and, hence, the values are not included in the tables. Downloaded from cancerres.aacrjournals.org on June 11, 2017. © 1962 American Association for Cancer Research. BUSCHet al.—RP2-L, a Historie Fraction from Tumors corrected 10 per cent for destruction during acid hydrolysis (20). The number of amino acid resi dues per minimal molecular weight was obtained by dividing the molar fractions by that number which gave the closest approximation to whole numbers for the various amino acids. From this value and the molecular weight determined by ultracentrifugation, the number of amino acid residues was calculated to the nearest integer per mole of protein. Starch gel eledrophoresis.—Twoprocedures were employed for starch gel electrophoresis of the cationic nuclear proteins—i.e., vertical electro phoresis at various pH ranging from 2 to 5 (18) and horizontal electrophoresis at pH 2-3 (17). In some of these experiments, the gels were cut longi tudinally in half, and the upper halves were stained to show the positions of the proteins. The lower halves were cut into a number of segments corresponding to the protein bands, and the proteins were eluted with 0.25 N HC1. The small amount of solubilized starch was pre cipitated with ethanol sufficient to make a final concentration of 40 per cent. The sample was centrifuged at 30,000 X g for 20 minutes, and the supernatant protein solution was used for deter mination of C14and protein concentration in each of the bands, as well as for amino acid analysis of some of the bands. Sedimentation velocity studies.—Measurements of sedimentation constants of preparations of RP2-L were carried out in a Spinco Model E ana lytical ultracentrifuge at 59,780 r.p.m. The pro teins were in solution in acetate buffers at pH 5, T/2 0.2. The temperature of the rotor was main tained at 20°C. Diffusion measurements.—Reproducible diffu sion constants were obtained by determination of diffusion of protein boundaries in the Spinco Model H electrophoresis apparatus (13). Diffusion constants were also estimated from the boundary spreading observed in the ultracentrifuge and cal culated by the maximal ordinate-area method (14). The buffer used was the same as that for the sedi mentation studies. Molecular weight.—The molecular weights of the proteins were calculated from the Svedberg and Pedersen equation (24). The partial specific volume5 was assumed to be 0.74. RESULTS Chromatography of the initial extract.—-Thechromatographic pattern for the acid-soluble nuclear proteins of the Walker tumor obtained on a pre paratory column is shown in Chart 1. Protein peak 6 For most proteins, partial specific volume has ranged be tween 0.70 and 0.78 ml/gm (34). 639 B, the major protein peak eluted with a gradient increasing to l N formic acid, was found in maxi mal concentration between fractions 270 and 350. The radioactive peak, designated as RP2-L, was found between fraction 270 and 450; this peak followed protein peak B. Another radioactive peak, designated as RP1-L, was eluted before pro tein peak B. For further analyses, the contents of tubes numbered 270-325 were pooled and desig nated as protein peak B; the contents of tubes numbered 326-450 were pooled and designated as crude RP2-L. Table 1 presents the distribution 250 150 WALKER 256 CARCINOSARCOMA PREPARATORY CHROMATOGRAM E280 200 100 ISO 100 50 Z 90 100 200 300 4OO 500 FRACTION NUMBER 600 TOO CHART1.—Chromatographie pattern of radioactivity and protein concentration for the whole acid extract of nuclei of 400 gm. of Walker tumor. The carboxymethylcellulose columns employed were 9 cm. in diameter and 45 cm. in height. Radio activity and protein concentration were assayed in every fifth effluent fraction, and hence points are omitted from the graphs. The conditions for chromatography are described in the text. The data are averages of two experiments. of protein and radioactivity in various fractions obtained by chromatography of acid-soluble nu clear proteins of the Walker tumor. RP2-L con tained approximately 35 per cent of the C14and 32 per cent of the weight of proteins recovered. The specific activity of RP2-L was greater than that of the other fractions when the specific ac tivity was determined as counts/min/mg protein. Rechromatography.—Chart 2 presents the chromatographic pattern obtained by rechromatography of crude RP2-L on carboxymethylcellu lose, when similar conditions of elution were em ployed (see "Materials and Methods"). Following a small "breakthrough" peak, a single protein peak was eluted by a gradient increasing to l N formic acid with a maximum at fraction 95. The distribution of isotope in the chromatogram in dicates that at least two protein fractions were eluted by a gradient increasing to l N formic acid, and another by 8 N formic acid. Table 2 presents Downloaded from cancerres.aacrjournals.org on June 11, 2017. © 1962 American Association for Cancer Research. 640 Vol. 22, June 1962 Cancer Research the results of chromatograms carried out under these conditions and shows that each of the frac tions has been considerably enriched in radio activity by comparison with the fractions obtained in the initial chromatography. The separation between peak B and RP2-L was more difficult to RECHROMAT06RAPHY 0120 0060 IOO 200 300 FRACTION NUMBER 40O CHART2.—Chromatographie pattern of radioactivity and protein concentration for 150 mg. of crude RP2-L on carboxymethylcellulose columns 2 cm. in diameter and 15 cm. in height. The conditions for chromatography are described in the text. Fractions B, RP2-L, and C include fractions num bered 76-100, 100-160, and 160-220, respectively. define in this chromatogram (Chart 2). About 85 per cent of the total isotope was eluted in essential ly a single peak which was divided arbitrarily into fractions B, RP2-L, and C, which included frac tions 76-100, 100-160, and 160-220, respectively.« Table 2 indicates that the specific activity of RP2L is not so great as that of peak E in terms of counts/min/mg. When the specific activities were determined on the basis of counts/mi n//¿mole of lysine, the values for peak E, B, RP2-L, and orig inal extract were 1300, 800, 800, and 650, re spectively. Starch gel electrophoresis of the fractions obtained from initial and rechromatography.—Figure 1 shows that the initial preparations of RP2-L con tained eight reproducible bands on starch gel electrophoresis at pH 2.3 (17). Peak A contained one band which did not move and a diffuse protein 8It is not probable that all the proteins of the fractions ob tained by rechromatography of RP2-L are identical to those obtained initially. The high specific activity of peak E suggests that other proteins are appearing in this region. However, the mobility in starch gel for both the proteins of peak B and peak E obtained by rechromatography was similar to that of the proteins obtained in the initial chromatograms. TABLE 1 SPECIFICACTIVITIESANDRECOVERY OFPROTEINSIN LARGE-SCALE CHROMATOGRAPHIC EXPERIMENTS The conditions for chromatography are described in the text. The table presents the aver ages of recoveries of various fractions and the percentages of the total radioactivity and weight recovered in the various fractions. In these experiments recovery of proteins from the columns ranged from 30 to 40 per cent. The data are averages of two experiments. The results for indi vidual experiments are presented in parentheses. Specific activities are counts/min/mg lyophilized protein. DO.OriginalAB2 Fraction mg.4.7(3.9,5.5)14 cent total counts2.6(2.2,3.0)12.6 cent total (counts/min/mg)530 580)283 (480, E or 3Mg.36 39)103 (33, 19)32 (9, 114)252 (92, (211, 293) (29, 35) 334 (242, 390)Per 39.7(39,40.3)Per 390)446 (176, 14.1)34.7(34,35.4) (11.0, 572)580 (320, (572, 587) 36.6 (35,38.2)S.A. 417 (400, 445) TABLE 2 SPECIFICACTIVITIESANDPER CENTTOTALRECOVERY ON RECHROMATOGRAPHY OFCRUDERP2-L OBTAINEDFROMLARGE-SCALE CHROMATOGRAPHY The conditions for the experiment are presented in the text. The table presents the averages of recoveries of various fractions and the percentages of the total radioactivity and weight re covered in the various fractions. The data are averages of two experiments. The results for individual experiments are presented in parentheses. Specific activities are counts/min/mg lyophilized protein. Fraction no.B mg.18.5 cent total counta16.4 cent total (11.6, 16) RP2-L 33.2(26.5,40) 14.5 (14, 15) CE or 3Mg.13.8 12 (11, 13)Per (17.9, 19) 45 (41, 49) 20 (18.4, 21.6) 16.5(13.5, 19.5)Per (15.3, 17.2) 41.5(40,43) 16 (14, 18) 22 (21, 23.9)S. A. (counts/min/mg)710 (700, 720) 855 (830, 880) 648 (595, 700) 1010 (980, 1032) Downloaded from cancerres.aacrjournals.org on June 11, 2017. © 1962 American Association for Cancer Research. BUSCHet al.—RP2-L, a Historie Fraction from Tumors area which was indistinctly defined. Peak B con tained bands which moved farther than those of crude RP2-L (Fig. 1), suggesting either that some denaturation of protein occurred, that some of the protein was freed from components which im peded electrophoretic mobility, or that B was en riched in fast-moving components. The electro phoretic pattern for RP2-L contained one very heavy band and several smaller bands which were more diffuse and less intense. RP3-L or peak E contained five slow-moving bands, of which the most marked was that which moved the least into the starch gel and approximated the mobility of slow-moving bands of the original starting mate rial. These data along with the data indicated be low with regard to the N-terminal amino acids indicate that purification of RP2-L has been achieved but further procedures are required for 641 jected to re-electrophoresis, a single band was found in the same region (Fig. 2). The results of analytical studies on amounts and specific activi ties of proteins in various fractions of the gels are shown in Table 3. The band coded as RP2-LY contained the largest amount of protein with the highest specific activity of the various fractions studied. Tentatively, it would appear that this Jraction contains the main protein components of RP2-L. The analytical data on amino acids (v.i.) support this suggestion.7 Amino acid analysis.—The amino acid analyses for a number of fractions are shown in Table 4. By the nomenclature employed by Johns et al. (17), RP2-L would be classified as a slightly lysine-rich histone fraction (F2), since lysine comprises ap proximately 14 per cent of the total amino acids present. Glutamic and aspartic acid comprised TABLE3 RECOVERY OFRADIOACTIVITY ANDPROTEIN FOLLOWING STARCH GELELECTROPHORESIS Two to 4 mg. of protein were subjected to electrophoresis on starch gel at pH 5.0. At the end of the run, gels were cut in half. In one half, the protein was stained with Amido Black 10B to position the bands. The bands were cut out of the other half, as described in the text, and the protein and radioactivity were deter mined. Average data for two experiments are presented. Actual values are shown in parentheses. Specific activities are counts/min/mg lyophilized protein. Band1234567CodeRP2-LXRP2-LYRP2-LZMg. protein0.24(0.22,0.25)0.30 protein6.0(5.3,6.6)7.3 cent total protein213 286)249 (140, cent total C" recovered3.4(2.8, 4.0)5.2(3.9,6.4)10.4(8.2, (0.27,0.32)0.5 (7,7.6)13.0(10.5, 318)282 (180, (0.4,0.6)1.92 15.4)48.3 313)417 (250, 12.6)60 1.92)1.02 (1.92, (46.2,50.4)25.5 433)291 (400, (57,62.8)21.2 (0.97, 1.06)Per (20.2, 22.2) (25.4,25.5)Counts/min/mg (280, 303)Per improvement of the purity of the products ob tained thus far. Although the purification was not completely satisfactory, the gel patterns show that purified RP2-L lacks some of the faster moving bands of peak B and some of the slower moving bands of RP3-L. Starch gel electrophoresis at pH 5.O.—The con ditions employed by Neelin and Neelin (16) were used in an effort to obtain sufficient protein for direct amino acid analysis and determination of specific activity of the protein in the bands. The electrophoretic pattern is shown in Figure 2. Only five bands were noted, one very close to the origin, a second faint band which moved at an inter mediate rate, and three rapidly moving bands. The three fast-moving bands were referred to as RP2-LX, RP2-LY, and RP2-LZ, of which RP2LZ moved the farthest. The RP2-LY band was the largest of all the fractions subjected to starch gel electrophoresis. When this fraction was ex tracted from several similar experiments and sub- approximately 13 per cent of the total amino acids, and arginine comprised 8 per cent of the total amino acids. These data suggest that the RP2-L peak is different in amino acid composition from the F2 peak obtained by Johns et al. from calf thymus (17), but, since both groups of proteins are mixtures, it is difficult to establish whether the differences represent the presence of different pro teins, or different compositions within the mixture. Because of the smaller amount of arginine in this peak, the lysine/arginine ratio is 1.71 compared with the 1.34 found for calf thymus (17). On the whole, the similarities of the amino acid analysis of this fraction to the slightly lysine-rich fraction from calf thymus are greater than the differences. Fractions A, B, and RP3-L contained less lysine and arginine than the RP2-L fraction. Peak B con tained more aspartic acid, leucine, and histidine 7Similar studies were not carried out with bands obtained by electrophoresis at pH 2.3 because of the multiplicity and proximity of the bands. Downloaded from cancerres.aacrjournals.org on June 11, 2017. © 1962 American Association for Cancer Research. Cancer Research 642 than RP2-L. RP3-L contained more aspartic acid, glutamic acid, and leucine than RP2-L. The low spe cific activity of peak B and the relatively large amount of histidine suggested the possibility of conCHROMATOGRAPHY OF RAT GLOBIN 0.240 0.120 8N so IOO FRACTION ISO NUMBER A 200 CHART3.—Chromatographie pattern for 150 mg. of rat globin on carboxymethylcellulose columns. The conditions for chromatography were the same as those described for Chart 2, with the exception that a gradient of 8 N formic acid was begun at fraction 170. Vol. 22, June 1962 tamination by proteins of the blood such as globin. Accordingly, the amino acid analysis, Chromato graphie elution pattern, and N-terminal amino acid analysis were determined for rat globin pre pared by the method of Rossi-Fanelli and An tonini (21). The amino acid analysis which is presented for comparison in Table 4 shows that the arginine content is low and the histidine con tent is high by comparison with RP2-L and the whole acid extract. The high level of aspartic acid, leucine, and phenylalanine support the pos sibility that peak B is contaminated with globin. Evidence that proteins other than globin and RP2L are present in peak B emerges from the content of glutamic acid, which is higher in peak B than in either rat globin or RP2-L; other proteins, con taining high concentrations of glutamic acid, must be present. The Chromatographie pattern (Chart 3) and the end-group analysis (v.i.) also support the possibility that globin is present in peak B. The amino acid analysis for the band coded as TABLE 4 AMINOACIDANALYSES OFVARIOUS FRACTIONS OBTAINED BYCHBOMATOGRAPHY ANDRECHROMATOGRAPHY OFACID-SOLUBLE NUCLEAR PROTEINS OFTHEWALKER TUMOR The table presents the percentages of total moles of amino acids recovered by chromatography of protein hydrolysates on Spinco automatic amino acid analyzer. The values are averages of two to five analyses on three separate biological experimeDts. The ranges are presented in parentheses. PeakAlanineArginineAspartic Extract9.6(9.2-9.9)5.6(5.5-5.7)8.6(8.5-8.6)05(0.1-0.9)12.3(11.4-13.2)7.9(7.8-7.9)3.0(2.9-3.1)3.8(3.6-3.9)8.7(8.6-8.7)10.4(10.2-10.5)0.7(0.6-0.8)3.1(3.0-3.3)5.4(5.4-5.5)6.6(6.6-6.6)5.7(5 acid£ CystineGlutamic 3(9.8-12.8)7.8(7.3-8.4)2.1(1.8-2.5)4.2(4.0-4 acidGlycineHistidineIsoleucineLeucineLysineMethioninePhenylalanineProlineSerineThreonineTyrosineValineOrigina] 4)8.5(8.4-8.5)9.3(7.8-10.9)0.6(0.4-0.8)3.4(3.0 6)A9.5(8.4-10.7)4.6(4.4-4.9)8.8(8.0-9.5)0.3(0.2-0.5)11.2(10.0-12.4)7.5(7.2-7.9)3.1(2.4-3.8)4.5(4.1-4.9)9.4(8.8-10.0)10.0( Downloaded from cancerres.aacrjournals.org on June 11, 2017. © 1962 American Association for Cancer Research. BUSCHet cd.—RP2-L, a Histone Fraction from Tumors RP2-LY is also presented in Table 4. The analysis for leucine and methionine differed from those of purified RP2-L, but the analyses for most of the amino acids were essentially identical. The band coded as RP2-LZ contained more arginine, gly cine, and methionine and less lysine, serine, and valine than the corresponding RP2-LY band. N-terminal amino acids.—The results of Nterminal amino acid analyses for the various frac tions isolated from the Walker tumor are presented in Table 5. In the crude extract, ten N-terminal amino acids were found. The lack of equality in quantity indicates that it is unlikely that any single protein species containing two end groups 643 amino acids in this peak would appear to be proline and alanine, with lesser amounts of valine and other amino acids. A correlation between these data and those of starch gel electrophoresis at pH 5 exist when data on percentages of protein are compared with the percentages of N-terminal amino acid. Proline comprised 48.6 per cent of total N-terminal amino acids, and RP2-LY com prised 48.3 per cent of the total protein recovered from the gel. In addition, alanine comprised 33 of the total N-terminal amino acids, and RP2-LZ comprised 25.5 per cent of the total protein. Valine comprised 9.1 per cent of the N-terminal amino acids and RP2-LX comprised 12.9 per cent of the TABLE 5 N-TERMINALAMINOACIDSIN PROTEINSOFTHEVARIOUS FRACTIONS RECOVERED BYCHROMATOGRAPHY ANDRECHROMATOGRAPHY OFACID-SOLUBLE NUCLEARPROTEINSOFTHE WALKERTUMOR The conditions used were those employed by Phillips (17). The table presents average values of from three to five determinations. The ranges are shown in parentheses. The values are percentage of total moles recovered. PeakAlanineAspartic Purified33.3(27.1-40.7)2.4(2.2-2.7)2.9(0.0-5.3)0.9(0.0-2.8)48.6(42.9-54.2)2.4(0.0-4.1)9.1(7.4-10.7)R Crude32.1(27.7-37.2)4.6(4.0-5.5)2.5(2.3-2.8)2.4(2.9-4.3)3.9(2.3-7.0)28.2(21.9-31.9)2.4(1.6-3.8)0.8(0.0-2.3)22.3(19.4-2 extract32.6(30.9-33.6)8.8(6.6-11.5)4.0(2.0-6 B27.0(23.5-30.4)5.0(4.9-5.1)6.7(4.5-8.8)8.2(6.4-10.0)2.8(2.1-3.5)2.2(0.0-4.59.4(7.9-10.9)4.4(0.5-5.5)1.0(0.6-1.3)33.8(28.7-39.0)RP2-L 4(36.0-46.8)5.3(4.6-6.0)5.0(1.3-7.06.1(5.2-7.1)2.6(0.0-6.2)0.0(0.0-2.0)22.8(20.5-2 andglutamic acid acidGlycineLeucine .3)5.6(3.0-8.8)4.8(1.9-7.5)0.6(0.0-1 andisoleucineLysinePhenylalanineProlineSerineThreonineValine 9)17.6(13.1-21.0)13(0.0-3.8)1.6(0.0-4.7)23.0(18.8-25.5)Peak andmethionineWhole on two separate peptide chains accounts for the presence of any two of the N-terminal amino acids found. In the whole acid extract, alanine was the major N-terminal amino acid and, together with proline and valine, comprised almost three-fourths of the N-terminal amino acids present. In peak B, the distribution of N-terminal amino acids was similar to that of the whole extract, al though more glycine, leucine, serine, and valine and less proline, lysine, and aspartic acid were found. In RP2-L, considerably less aspartic acid, glycine, leucine, lysine, phenylalanine, and valine were found by comparison with the whole extract or with peak B. Although the percentage of alanine as an end group was not markedly different in RP2-L from the values for the whole extract, the values for proline were progressively increased as the RP2-L was purified. The major N-terminal total protein recovered. These correlations sug gest that the major component of RP2-L is pro tein in which the N-terminal amino acid is proline. A direct analysis of N-terminal amino acids on band RP2-LY has not been possible because of the low recovery of protein from the gels. PHYSICALCONSTANTS Table 6 presents the results of determinations of the molecular weight and other physical con stants for RP2-L. A homogeneous peak was ob served on ultracentrifugation. The average molec ular weight of the proteins of RP2-L is approxi mately 33,000 on the basis of physical constants and the amino acid analysis. DISCUSSION The proteins containing the radioactivity of peak RP2-L have been purified by rechromatog- Downloaded from cancerres.aacrjournals.org on June 11, 2017. © 1962 American Association for Cancer Research. 644 Cancer Research Vol. 22, June 1962 raphy on carboxymethylcellulose. The product consists of a mixture of proteins of high specific activity which are of the general group denoted as "slightly lysine-rich" histones (16, 17). One major band and several minor bands were noted on starch-gel electrophoresis of the product. The Nterminal amino acid of the major proteins is probably proline, as suggested by the increasing concentration of this N-terminal amino acid as the purity of the proteins increased. One of the acids. It is not possible to state whether the in dividual proteins are qualitatively different from those found in nuclei of normal cells or whether other factors account for the differences which have been found. It is possible that the proportion of the same proteins differs from that found in normal nuclei. It is also possible that the proteins with which histones may complex in nontumor tissues are reduced in amount in tumor tissue and, hence, the proteins of RP2-L emerge earlier in the chromatograms than do corresponding proteins of TABLE6 nontumor tissue. The critical problem confronting such studies is PHYSICAL CONSTANTS ANDMOLECULAR WEIGHTFORRP2-L the need for methods for improvement of the fractionations within this group of proteins. Al The methods for determination of the sedimentation and diffusion constants are described under "Materials and Meth ods." The data are averages of two experiments. The diffusion though it would seem that the simplest approach and sedimentation constants were determined with solutions to this problem would be the development of containing 0.5 per cent and 1 per cent protein in each case. starch-gel technics employing large-scale proce The molecular weight was derived with the aid of the diffusion dures, efforts in this direction in this and a number constant determined in the Tiselius apparatus because of the of other laboratories have been disappointing, greater accuracy of this method. thus far, in the resolution of the proteins in the prod ucts obtained. Another possibility which could ConstantaDSC, be anticipated is the development of ion exchangers with improved resolving power; the possibility w (by free diffusion in Tiselius cm/sec apparatus)DM, exists that weaker exchangers with phenolic hycm/sec1.32X10-'Vsec32,70032,390 2.68X10~7sq droxyls might be useful for this purpose. ultracentrifugation)Sa,, w (by In view of the difficulties in subfractionation WAverage of these proteins by Chromatographie technics, recent studies have been directed toward the use molecular weight by physi constantsAverage cal of a combination of chemical and Chromatographie technics (14, 16). Starch gel electrophoresis of molecular weight from fractions indicates that further purification of the amino acid compositionRPi-L3.32X10-'sq histones may be achieved by this combined pro cedure. At the moment, one fraction which is ap problems in the detection of dinitrophenylproline parently of a higher order of purity has been ob is its relatively great destruction on hydrolysis of tained in this laboratory,8 but further studies are the protein, and hence there is a need to employ a necessary to establish the purity unequivocally. large amount of protein to find the end-group; The need for structural analysis of these pro teins is apparent. If neoplastic cells contain pro this fact accounts for earlier reports from this lab oratory which did not indicate the presence of teins which are different from those of other tis proline as an N-terminal amino acid (2). The pro sues, their structure must be determined not only teins of RP2-L are moderately small in view of for comparative purposes but also for the purpose the molecular weight of approximately 33,000 of directing chemotherapeutic endeavors. It is possible that structural differences could be uti which has been obtained. The primary structure should be analyzable by "fingerprinting" and lized to suggest the synthesis of polypeptide ana other technics once the proteins are isolated in logs containing either cytotoxic groups or amino acids in altered sequences. Such compounds might pure form. These data indicate that RP2-L is a mixture of interfere with templates involved in the biosyn proteins which resemble proteins of calf thymus in thesis of nuclear proteins of tumor cells. amino acid analysis and in N-terminal amino 1L. S. Hnilica and H. Busch, unpublished. FIG. 1.—Starch gel electrophoresis of various fractions ob tained by rechromatography of crude RP2-L. The conditions employed were those used by Johns et al. (17). The letters on the patterns refer to the following: (a) peak B, (6) purified RP2-L, (c) peak C, (d) RP3-L, (e) crude RP2-L, the source material. FIG. 2.—Starch gel electrophoresis of RP2-L at pH 5.0 according to the conditions described by Neelin and Neelin (18). The letters on the patterns refer to the following: (a) pattern of purified RP2-L and (6) pattern for re-electrophoresis of RP2-LY. Downloaded from cancerres.aacrjournals.org on June 11, 2017. © 1962 American Association for Cancer Research. CJ CD O Downloaded from cancerres.aacrjournals.org on June 11, 2017. © 1962 American Association for Cancer Research. BUSCH et al.—RP2-L, a Histone Fraction from Tumors 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. REFERENCES BLACK,M. M.; SPEER, F. D.; and LILLICK,L. Acid-extractable Nuclear Proteins of Cancer Cells. I. Staining with Ammoniacal Silver. J. Nat'l. Cancer Inst., 26:967-89, 1960. BUSCH,H.; BYVOET,P.; and DAVIS,J. R. Nuclear Proteins of Neoplastic Tissues in the Molecular Basis of Neoplasia, pp. 207-24. Univ. of Texas Press, 1962. BCSCH,H.; DAVIS,J. R.; and ANDERSON,D. C. 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