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Enzymatic methylation of histones
ARCHIVES
OF
BIOCHEMISTRY
AND
Enzymatic WOON Fels Research
Institute
134, 632-637
BIOPHYSICS
Methylation KI PAIK
of Histones SANGDUK
AND
and Department of Biochemistry, Philadelphia, Pennsylvania Received
(1969)
September
KIM
Temple University 19140
School
of Medicine,
4, 1969
Various proteins were methylated by purified protein methylase I. Since the enzyme also methylates its endogenous histones, detailed studies on the protein methylation by the purified protein methylase I were carried out with added histone. Exogenous histone methylated enzymatically gave rise to guanidyl-methylated and &V-methyl, guanidyl-methylated arginine on acid hydrolysis.
We have recently identified an enzyme which methylates its endogenous protein (1). The enzyme was mainly located in cytosol, and was designated as protein methylase I. An analysis of the endogenous methylated protein showed that approximately 70% of the total radioactivity was in histone, and methylation occurred indiscriminately in the various histone fractions. In the present paper, we report that various added exogenous proteins, particularly histones, were methylated by protein methylase I. MATERIALS
AND
METHODS
Materials. S-Adenosyl-n-methionine-methyli4C (specific activity, 29.9 mC/mmole in H&04, pH 2) was purchased from Tracerlab. Various histones and other proteins used were obtained from Sigma Chemical Co., St. Louis, MO. Purified protein methylase I was prepared according to the method described previously (1). Enzymatic assay. Detailed description of enzymatic assay was as described previously (1); 0.1 ml of enzyme preparation, 0.1 ml of 0.5 M phosphate buffer at pH 7.2, 0.1 ml of S-adenosyl-nmethionine-methyl-i% (3.07 X lo6 cpm; 4.78 mpmoles; SAM) and 0.2 ml of protein suspension (water in the case of control) were incubated for 10 min at 37” and the reaction was terminated by the addition of 0.5 ml of 30% trichloroacetic acid (TCA). The experimental procedures for the removal of nucleic acids and phospholipids, and for counting the radioactivity were described elsewhere (2). PuriJication of commercial his&ones. When his-
tone type II or anginine-rich histone from Sigma Chemical Co. was analyzed by the method described (3), all four subfractions were detected in varying amounts depending on the preparations (one arginine-rich histone preparation contained F,:lysine -rich:slightly lysine-rich:arginine-rich histone = 12:39:35:14). Therefore, for more detailed studies (Table I), purification of the commercial histone was carried out: 300 mg of histone type II was fractionated by CM-cellulose column (1.8 X 20 cm) according to the method of Phillips and Johns (3). Fractions corresponding to each histone were pooled. Two and one-half volumes of acetone were added into the fractions of F, and lysine-rich histone, and the precipitate after centrifugation was dialyzed against 0.1 N acetic acid. On the other hand, slightly lysine-rich and arginine-rich histone fractions were concentrated in reduced pressure at about 30”, and the concentrated suspensions were dialyzed against 0.1 N acetic acid as before. The samples were lyophilized. Amino acid analysis. Protein residues, which were treated to remove nucleic acids and phospholipids, were hydrolyzed in 6 N HCl at 110’ for 16 hr under reflux. Conditions for the amino acid analysis by the Beckman automatic amino acid analyzer were as described previously (4). Protein concentration was determined by the method of Lowry et al. (5), and the enzyme activity was expressed as pprnoles methyl transferred/mini mg protein. RESULTS
Effect of histone on methylation by crude and puri$ed protein methylase I. The effect of 632
ENZYMATIC TABLE
METHYLATION
I
EFFECT OF VARIOUS PROTEINS ON THE METHYLATION ACTIVITY OF PROTEIN METHYLASE Ia Protein
added
None Histone type II* FX Lysine-rich histone Slightly lysine-rich histone Arginine-rich histone r-Globulin, bovine serum Trypsin inhibitor Globulin, egg white Ribonuclease, pancreatic Urease Protamine sulfate” Lysozyme Deoxyribonuclease Polyglutamic acid RNA Albuminc, serum Polylysinec Polyleucine DNA Trypsin
Per cent enzyme activity
100 224 222 281 265 209 247 218 184 160 158 157 155 126 110 107 105 98 84 71 0
OF
633
HISTONES
to an increased availability of methyl acceptor protein. More detailed studies will be presented below. E$ect of concentration of protein methylase I. One of the characteristics of the purified protein methylase I was that specific activity (ppmoles SAM/min/mg protein) increased as the concentration of the enzyme increased (1). This is again shown in Fig. 2, together with the data on the relationship between the concentration of the enzyme and the effect of exogenous added histone. It is seen that, at the low concentrations of the purified protein methylase I, the effect of addition of the histone is apparent. However, the effect is abolished at the high concentrations of the enzyme. This suggests that the purified enzyme is saturated with the
1
400-
a Two tenths milliliter of protein suspension containing 3 mg dry weight was added to give a total 0.5 ml of incubation mixture. One hundred per cent enzyme activity indicated 3.46 PImole SAM-methyl-W transferred/min/mg protein. Detailed procedures for enzymatic assay are described under Methods. * Since histone type II of Sigma Chemical Co. was a mixture of various histones, this commercial histone was purified into four subfractions as described under Methods. c Difficulties were observed during washing with TCA.
4
2
0
different amounts of histone type II on the methyl&ion of crude and purified protein methylase I is shown in Fig. 1. The exogenous added histone markedly increased the methylation activity of both preparations. However, as shown in this paper later, effect of the histone on the purified enzyme is dependent upon the concentration of the enzyme. Since approximately 70% of the total radioactivity incorporated into the endogenous proteins was found in histones (I), it was highly likely that the increased methylation by the added histone was due
1
---------4
Mg
histone
6
added
FIG. 1. Effect of amounts of histone on the methylation activity of the crude and purified protein methylase I. Experimental conditions for the enzymatic assay are described under Methods, The crude (0.83 mg of protein) and the purified (0.06 mg of protein) protein methylase I were used, and the histone type II. The crude enzyme indicates the supernatant fraction of calf thymus whole homogenate at 105,OOOg for 60 min. One hundred per cent activity was 0.9 PMmole SAM/ min/mg protein for the crude, and 3.46 wmole SAM/min/mg protein for the purified enzyme.
I
634
PAIK
AND
KIM
evident from the foregoing results that exogenous histone increasesthe methylation activity of the purified protein methylase I. However, since various histones comprise approximately 70 % of the total radioactivity incorporated into the endogenous proteins (I), we attempted to prove that exogenous added histone, indeed, served as methyl acceptor. 3-chymotrypsin 125000)
Mg
protein
lys;;o”;e
methylase1
FIG. 2 Relationship between the concentration of protein methylase I and increase in methylation activity by added histone. Three milligrams of histone type II of Sigma Chemical Co. was used. Detailed experimental procedures are described under Met,hods.
endogenous methyl acceptor proteins at the high concentrations. Effect of various exogenousproteins rm the methylation of protein methylase I. Table I lists the effect of various purified commercial proteins on the methylation activity of the purified protein methylase I. It is striking to notice that basic proteins such as histones, r-globulin, and trypsin inhibitor increase the methylation activity. In the presenceof trypsin, the methylation activity is completely abolished in a lo-min incubation period. This is most likely due to the hydrolytic action of trypsin on both the protein methylase I and the methyl acceptor protein. Since arginine-rich histone and histone type II of Sigma Chemical Co. were found to be a mixture of F,, lysine-rich histone, slightly lysine-rich histone as well as arginine-rich histone, the histone type II was further fractionated. As shown in Table I, purified lysine-rich histone is most effective in increasing the methylation activity. E$ect of added exogenoushistone vn the methulation of endoaenous nroteins. It is -
I
Y
1
ml effluent FIG. 3. Chromatographic analysis of the effect of added histone on Bio-Gel P-200. Two tenths milliliter of SAM-methy-1% (9.56 mmoles), 0.2 ml of 0.5 M phosphate buffer at pH 7.2, 0.2 ml of the purified protein methylase I (0.52 mg of prot,ein), and 6 mg of histone type II in a total volume of 1.0 ml were incubated at 37” for 2 hr. Control contained no added histone. The incubation mixture was charged on Bio-Gel P-200 (0.8 X 60 cm) which had been equilibrated with 0.01 M phosphate buffer at pH 7.2, and 1 ml of each fraction was collected by eluting the column with the above buffer. Chromatographic analysis was carried on simultaneously with both the assay and the control with two identical separate columns. Each fraction was treated to remove TCA-soluble, nucleic acid and phospholipid fraction, and the residue counted for radioactivity according to the method described (2). To determine the elution position of a-chymotrypsin or lysozyme, 9.0 mg of each protein in 1.0 ml of the above buffer were analyzed as above.
ENZYMATIC
-11
METHYLATION
volume
OF
of effluent
HISTONES
635
(MI)
FIG. 4. Identification of methylated amino acid of histone. The incubation mixture contained the following; 0.5 ml of the protein methylase I (0.3 mg of protein), 0.5 ml of SAM-methyl-14C (23.9 nqumoles), 0.5 ml of 0.5 M phosphate buffer at pH 7.2, and 1.0 ml of histone suspension (15 ml of histone type II of Sigma). They were incubated for 4 hr at 37”. Histone was isolated from the incubation mixture by the method described (2). After removal of nucleic acids and phospholipids, the residue was dialyzed against water at room temperature. The sample was hydrolyzed in 6 N HCl and one-third of the final sample was analyzed by the Beckman automatic amino acid analyzer. Five milliliters of each fraction were collected from the analyzer, and l-ml portions were assayed for radioactivity. More detailed procedures are described under Methods. Almost complete recovery of the charged radioact,ivit,y was observed (98%). All the neutral and acidic amino acids were eluted before lysine.
As illustrated by the control in Fig. 3, the size of endogenous methylated protein has a molecular weight of over 200,000, as determined by elution on Bio-Gel P-200 in 0.01 &I phosphate buffer at pH 7.2. Since the molecular weight of histone is around 15,000 (6), this indicates that endogenous histone (possibly other proteins) is in complexed state with some endogenous macromolecules. However, when methylation reaction was carried out in the presence of exogenous added histone and the reaction mixture was chromatographed on the column, there appeared two radioactivity peaks; one of which completely overlapped with the control peak, but was enhanced. The second peak, which was not found in the appeared between the elution control, position of ar-chymotrypsin (mol u-t 25,000) and that of lysozyme (mol wt 15,000). Therefore, these results strongly indicate that the exogenous added histone was itself
methylated. It is very difficult to account for the increased radioactivity in the first peak. Two alternate hypotheses could be offered, one of which was that exogenous histone was first methylated and some of them complexed with endogenous macromolecule, or that histone simply stimulated the methylation of endogenous histone as well as other proteins by some unknown mechanism. Methylation products. When freshly isolated calf thymus nuclei were methylated with SAM-methyl-14C, analysis of the acid hydrolyzate by the Beckman amino acid analyzer of the residual protein after removal of nucleic acids and phospholipids gave rise to four radioactivity peaks in the basic region (2). Two of these peaks coincided with those of e-N-methyllysine and E-Ndimethyllysine, respectively. One of the remaining peaks appeared before arginine, and this was tentatively identified as
636
PAIK
AND
KIM
A I
I
3.1
3.2
I
I
3.3
3.4
XlO’3/“K FIG.
methylase
5. Effect of added histone on activation I (0.06 mg) was used. The experimental
energy of the protein methylase I. The purified conditions are described under Methods.
guanidino-methylated arginine (l).’ The remaining peak, which appeared after arginine and whose identity is less certain than the former, was assigned to be a-N-methyl, guanidino-methylated arginine. On the other hand, the endogenous methylated protein of the purified protein methylase I gave rise to the last two peaks on acid hydrolysis. Figure 4 shows chromatogram of acid hydrolyzate of histone isolated from the incubation mixture which was carried out in the presenceof exogenous added histone. Even though two methylated lysine derivatives exist, these comprise only a small fraction and approximately 90% of the radioactivity is associated with the methylated arginine derivatives. E$ect of added histone on activation energy of protein methylase I. Figure 5 illustrates Arrhenius plot of the protein methylase I in the presence and in the absence of added histone type II. The activation energies for the presence and for the absence of added histone are the same at low temperatures (12.7 kcal). However, the curve of the added histone has an inflexion point at about 43’, and it is possiblethat added histone rendered the enzyme less stable at high temperature. 1 Recently, we have elucidated be w-l\i-monomethyl arginine (to
the structure be published).
to
protein
DISCUSSION
Histones are a group of basic proteins which are soluble in acidic solution. They are found in the nucleus. However, evidence indicates that they are synthesized in the cytoplasm of HeLa cells (7), and the presence of extranuclear histone in amphibian oocytes has also been reported (8). Although biological significance of histone in vivo has not yet been well characterized, it has an antibacterial and antitumor function (9-11). It has stimulatory effect on albumin uptake by a sarcoma monolayer (12), affects mitochondrial adenosine triphosphatase activity (13), and has an inhibitory action on DNAdependent RNA polymerase (14). Recently, histone has been reported to be bound to carcinogen or its metabolites (15). Evidence presented here indicates that the added histone was methylated itself by the purified protein methylase I at the guanidino group of arginine residues. Since methyl substitution on the amino group greatly influence the pK values of the compound (16), and histone exist in situ in conjugation with DNA or RNA, modification of histone molecule by enzymatic methylation might be expected to have profound effect on the action of the nucleic acids. However, no experimental evidence has yet been presented
ENZYMATIC
METHYLATION
to indicate a direct relationship between the methylation of histone and the function of the nucleic acids. Studying the kinetics of in vivo methylation of e-amino group of lysine residues of histone during hepatic regeneration, Tidwell et al. (17) did not find any correlation between the methylation of histone and an increase in DNA template activity for RNA synthesis, or with DNA synthesis. We are now, however, in the process of investigating biological significance of methylation of arginine residues during various physiological conditions such as thyroxine-induced amphibian metamorphosis or hepatic regeneration. ACKNOWLEDGMENT This work was supported by research grants from the Institute of Arthritis and Metabolic Diseases (AM-O9602-03), National Science Foundation (GB-6019)) and National Cancer Institute (CA 10439). REFERENCES 1. PAIK, 243, 2. PAX, Res.
W. K., AND KIM, S., J. Biol. Chem. 2108 (1968). W. K., AND KIM, S., Biochem. Biophys. Commun. 29, 14 (1967).
OF
HISTONES
637
3. PHILLIPS, 11. M. P., AND JOHNS, E. W., Biothem. J. 74, 538 (1959). 4. PAIK, W. K., AND KIM, S., Biochem. Biophys. Res. Commun. 27, 479 (1967). 5. LOWRY, 0. H., ROSYTBROVGH, N. J., FARR, A. L., AND RANDALL, Ii. J., J. Biol. Chem. 193, 265 (1951). 6. KELLER, 11. C., KINKADA, J. M., AND COLE, R. Il., Biochem. Biophys. Res. Commun. 236, 673 (1961). 7. RORBINS, E., AND BORUN, T. W., Proc. Null. Acad. Sci. U. 6’. 6’7, 409 (1967). 8. HORN, E. C., Proc. Natl. Acad. Sci. U. S. 40, 257 (1962). 9. HIRSCH, J. G., J. Ezptl. Med. 198, 925 (1958). 10. BISERTE, G., TACQUET, A., LECLERC, H., AND SAUTIERE, P., Compt. Rend. Soe. Biol. 43, 1790 (1959). 11. BECKER, F. F., AND GREEN, H., Exptl. Cell Res. 19, 361 (1966). 12. RYSER, H. J.-P., AND HANCOCK, R., Science 150, 591 (1965). 13. SCHWARTZ, A., J. Biol. Chew. 240, 944 (1965). 14. HUANG, R. C., AND BONNER, J., Proc. Natl. Acad. Sci. U. S. 48, 1216 (1962). 15. BARRY, E. J., OVECHKA, C. A., AND GUTMANN, H. R., J. Biol. Chem. 243. 51 (1968). 16. FIESER, L. F. AND FIESER, M., “Advanced Organic Chemistry,” p. 493. Reinhold, New York (1963). 17. TIDWELL, T., ALLFREY, V. G., AND MIR~KY. A. E., 1. Biol. Chem. 243, 707 (1968).
OF
BIOCHEMISTRY
AND
Enzymatic WOON Fels Research
Institute
134, 632-637
BIOPHYSICS
Methylation KI PAIK
of Histones SANGDUK
AND
and Department of Biochemistry, Philadelphia, Pennsylvania Received
(1969)
September
KIM
Temple University 19140
School
of Medicine,
4, 1969
Various proteins were methylated by purified protein methylase I. Since the enzyme also methylates its endogenous histones, detailed studies on the protein methylation by the purified protein methylase I were carried out with added histone. Exogenous histone methylated enzymatically gave rise to guanidyl-methylated and &V-methyl, guanidyl-methylated arginine on acid hydrolysis.
We have recently identified an enzyme which methylates its endogenous protein (1). The enzyme was mainly located in cytosol, and was designated as protein methylase I. An analysis of the endogenous methylated protein showed that approximately 70% of the total radioactivity was in histone, and methylation occurred indiscriminately in the various histone fractions. In the present paper, we report that various added exogenous proteins, particularly histones, were methylated by protein methylase I. MATERIALS
AND
METHODS
Materials. S-Adenosyl-n-methionine-methyli4C (specific activity, 29.9 mC/mmole in H&04, pH 2) was purchased from Tracerlab. Various histones and other proteins used were obtained from Sigma Chemical Co., St. Louis, MO. Purified protein methylase I was prepared according to the method described previously (1). Enzymatic assay. Detailed description of enzymatic assay was as described previously (1); 0.1 ml of enzyme preparation, 0.1 ml of 0.5 M phosphate buffer at pH 7.2, 0.1 ml of S-adenosyl-nmethionine-methyl-i% (3.07 X lo6 cpm; 4.78 mpmoles; SAM) and 0.2 ml of protein suspension (water in the case of control) were incubated for 10 min at 37” and the reaction was terminated by the addition of 0.5 ml of 30% trichloroacetic acid (TCA). The experimental procedures for the removal of nucleic acids and phospholipids, and for counting the radioactivity were described elsewhere (2). PuriJication of commercial his&ones. When his-
tone type II or anginine-rich histone from Sigma Chemical Co. was analyzed by the method described (3), all four subfractions were detected in varying amounts depending on the preparations (one arginine-rich histone preparation contained F,:lysine -rich:slightly lysine-rich:arginine-rich histone = 12:39:35:14). Therefore, for more detailed studies (Table I), purification of the commercial histone was carried out: 300 mg of histone type II was fractionated by CM-cellulose column (1.8 X 20 cm) according to the method of Phillips and Johns (3). Fractions corresponding to each histone were pooled. Two and one-half volumes of acetone were added into the fractions of F, and lysine-rich histone, and the precipitate after centrifugation was dialyzed against 0.1 N acetic acid. On the other hand, slightly lysine-rich and arginine-rich histone fractions were concentrated in reduced pressure at about 30”, and the concentrated suspensions were dialyzed against 0.1 N acetic acid as before. The samples were lyophilized. Amino acid analysis. Protein residues, which were treated to remove nucleic acids and phospholipids, were hydrolyzed in 6 N HCl at 110’ for 16 hr under reflux. Conditions for the amino acid analysis by the Beckman automatic amino acid analyzer were as described previously (4). Protein concentration was determined by the method of Lowry et al. (5), and the enzyme activity was expressed as pprnoles methyl transferred/mini mg protein. RESULTS
Effect of histone on methylation by crude and puri$ed protein methylase I. The effect of 632
ENZYMATIC TABLE
METHYLATION
I
EFFECT OF VARIOUS PROTEINS ON THE METHYLATION ACTIVITY OF PROTEIN METHYLASE Ia Protein
added
None Histone type II* FX Lysine-rich histone Slightly lysine-rich histone Arginine-rich histone r-Globulin, bovine serum Trypsin inhibitor Globulin, egg white Ribonuclease, pancreatic Urease Protamine sulfate” Lysozyme Deoxyribonuclease Polyglutamic acid RNA Albuminc, serum Polylysinec Polyleucine DNA Trypsin
Per cent enzyme activity
100 224 222 281 265 209 247 218 184 160 158 157 155 126 110 107 105 98 84 71 0
OF
633
HISTONES
to an increased availability of methyl acceptor protein. More detailed studies will be presented below. E$ect of concentration of protein methylase I. One of the characteristics of the purified protein methylase I was that specific activity (ppmoles SAM/min/mg protein) increased as the concentration of the enzyme increased (1). This is again shown in Fig. 2, together with the data on the relationship between the concentration of the enzyme and the effect of exogenous added histone. It is seen that, at the low concentrations of the purified protein methylase I, the effect of addition of the histone is apparent. However, the effect is abolished at the high concentrations of the enzyme. This suggests that the purified enzyme is saturated with the
1
400-
a Two tenths milliliter of protein suspension containing 3 mg dry weight was added to give a total 0.5 ml of incubation mixture. One hundred per cent enzyme activity indicated 3.46 PImole SAM-methyl-W transferred/min/mg protein. Detailed procedures for enzymatic assay are described under Methods. * Since histone type II of Sigma Chemical Co. was a mixture of various histones, this commercial histone was purified into four subfractions as described under Methods. c Difficulties were observed during washing with TCA.
4
2
0
different amounts of histone type II on the methyl&ion of crude and purified protein methylase I is shown in Fig. 1. The exogenous added histone markedly increased the methylation activity of both preparations. However, as shown in this paper later, effect of the histone on the purified enzyme is dependent upon the concentration of the enzyme. Since approximately 70% of the total radioactivity incorporated into the endogenous proteins was found in histones (I), it was highly likely that the increased methylation by the added histone was due
1
---------4
Mg
histone
6
added
FIG. 1. Effect of amounts of histone on the methylation activity of the crude and purified protein methylase I. Experimental conditions for the enzymatic assay are described under Methods, The crude (0.83 mg of protein) and the purified (0.06 mg of protein) protein methylase I were used, and the histone type II. The crude enzyme indicates the supernatant fraction of calf thymus whole homogenate at 105,OOOg for 60 min. One hundred per cent activity was 0.9 PMmole SAM/ min/mg protein for the crude, and 3.46 wmole SAM/min/mg protein for the purified enzyme.
I
634
PAIK
AND
KIM
evident from the foregoing results that exogenous histone increasesthe methylation activity of the purified protein methylase I. However, since various histones comprise approximately 70 % of the total radioactivity incorporated into the endogenous proteins (I), we attempted to prove that exogenous added histone, indeed, served as methyl acceptor. 3-chymotrypsin 125000)
Mg
protein
lys;;o”;e
methylase1
FIG. 2 Relationship between the concentration of protein methylase I and increase in methylation activity by added histone. Three milligrams of histone type II of Sigma Chemical Co. was used. Detailed experimental procedures are described under Met,hods.
endogenous methyl acceptor proteins at the high concentrations. Effect of various exogenousproteins rm the methylation of protein methylase I. Table I lists the effect of various purified commercial proteins on the methylation activity of the purified protein methylase I. It is striking to notice that basic proteins such as histones, r-globulin, and trypsin inhibitor increase the methylation activity. In the presenceof trypsin, the methylation activity is completely abolished in a lo-min incubation period. This is most likely due to the hydrolytic action of trypsin on both the protein methylase I and the methyl acceptor protein. Since arginine-rich histone and histone type II of Sigma Chemical Co. were found to be a mixture of F,, lysine-rich histone, slightly lysine-rich histone as well as arginine-rich histone, the histone type II was further fractionated. As shown in Table I, purified lysine-rich histone is most effective in increasing the methylation activity. E$ect of added exogenoushistone vn the methulation of endoaenous nroteins. It is -
I
Y
1
ml effluent FIG. 3. Chromatographic analysis of the effect of added histone on Bio-Gel P-200. Two tenths milliliter of SAM-methy-1% (9.56 mmoles), 0.2 ml of 0.5 M phosphate buffer at pH 7.2, 0.2 ml of the purified protein methylase I (0.52 mg of prot,ein), and 6 mg of histone type II in a total volume of 1.0 ml were incubated at 37” for 2 hr. Control contained no added histone. The incubation mixture was charged on Bio-Gel P-200 (0.8 X 60 cm) which had been equilibrated with 0.01 M phosphate buffer at pH 7.2, and 1 ml of each fraction was collected by eluting the column with the above buffer. Chromatographic analysis was carried on simultaneously with both the assay and the control with two identical separate columns. Each fraction was treated to remove TCA-soluble, nucleic acid and phospholipid fraction, and the residue counted for radioactivity according to the method described (2). To determine the elution position of a-chymotrypsin or lysozyme, 9.0 mg of each protein in 1.0 ml of the above buffer were analyzed as above.
ENZYMATIC
-11
METHYLATION
volume
OF
of effluent
HISTONES
635
(MI)
FIG. 4. Identification of methylated amino acid of histone. The incubation mixture contained the following; 0.5 ml of the protein methylase I (0.3 mg of protein), 0.5 ml of SAM-methyl-14C (23.9 nqumoles), 0.5 ml of 0.5 M phosphate buffer at pH 7.2, and 1.0 ml of histone suspension (15 ml of histone type II of Sigma). They were incubated for 4 hr at 37”. Histone was isolated from the incubation mixture by the method described (2). After removal of nucleic acids and phospholipids, the residue was dialyzed against water at room temperature. The sample was hydrolyzed in 6 N HCl and one-third of the final sample was analyzed by the Beckman automatic amino acid analyzer. Five milliliters of each fraction were collected from the analyzer, and l-ml portions were assayed for radioactivity. More detailed procedures are described under Methods. Almost complete recovery of the charged radioact,ivit,y was observed (98%). All the neutral and acidic amino acids were eluted before lysine.
As illustrated by the control in Fig. 3, the size of endogenous methylated protein has a molecular weight of over 200,000, as determined by elution on Bio-Gel P-200 in 0.01 &I phosphate buffer at pH 7.2. Since the molecular weight of histone is around 15,000 (6), this indicates that endogenous histone (possibly other proteins) is in complexed state with some endogenous macromolecules. However, when methylation reaction was carried out in the presence of exogenous added histone and the reaction mixture was chromatographed on the column, there appeared two radioactivity peaks; one of which completely overlapped with the control peak, but was enhanced. The second peak, which was not found in the appeared between the elution control, position of ar-chymotrypsin (mol u-t 25,000) and that of lysozyme (mol wt 15,000). Therefore, these results strongly indicate that the exogenous added histone was itself
methylated. It is very difficult to account for the increased radioactivity in the first peak. Two alternate hypotheses could be offered, one of which was that exogenous histone was first methylated and some of them complexed with endogenous macromolecule, or that histone simply stimulated the methylation of endogenous histone as well as other proteins by some unknown mechanism. Methylation products. When freshly isolated calf thymus nuclei were methylated with SAM-methyl-14C, analysis of the acid hydrolyzate by the Beckman amino acid analyzer of the residual protein after removal of nucleic acids and phospholipids gave rise to four radioactivity peaks in the basic region (2). Two of these peaks coincided with those of e-N-methyllysine and E-Ndimethyllysine, respectively. One of the remaining peaks appeared before arginine, and this was tentatively identified as
636
PAIK
AND
KIM
A I
I
3.1
3.2
I
I
3.3
3.4
XlO’3/“K FIG.
methylase
5. Effect of added histone on activation I (0.06 mg) was used. The experimental
energy of the protein methylase I. The purified conditions are described under Methods.
guanidino-methylated arginine (l).’ The remaining peak, which appeared after arginine and whose identity is less certain than the former, was assigned to be a-N-methyl, guanidino-methylated arginine. On the other hand, the endogenous methylated protein of the purified protein methylase I gave rise to the last two peaks on acid hydrolysis. Figure 4 shows chromatogram of acid hydrolyzate of histone isolated from the incubation mixture which was carried out in the presenceof exogenous added histone. Even though two methylated lysine derivatives exist, these comprise only a small fraction and approximately 90% of the radioactivity is associated with the methylated arginine derivatives. E$ect of added histone on activation energy of protein methylase I. Figure 5 illustrates Arrhenius plot of the protein methylase I in the presence and in the absence of added histone type II. The activation energies for the presence and for the absence of added histone are the same at low temperatures (12.7 kcal). However, the curve of the added histone has an inflexion point at about 43’, and it is possiblethat added histone rendered the enzyme less stable at high temperature. 1 Recently, we have elucidated be w-l\i-monomethyl arginine (to
the structure be published).
to
protein
DISCUSSION
Histones are a group of basic proteins which are soluble in acidic solution. They are found in the nucleus. However, evidence indicates that they are synthesized in the cytoplasm of HeLa cells (7), and the presence of extranuclear histone in amphibian oocytes has also been reported (8). Although biological significance of histone in vivo has not yet been well characterized, it has an antibacterial and antitumor function (9-11). It has stimulatory effect on albumin uptake by a sarcoma monolayer (12), affects mitochondrial adenosine triphosphatase activity (13), and has an inhibitory action on DNAdependent RNA polymerase (14). Recently, histone has been reported to be bound to carcinogen or its metabolites (15). Evidence presented here indicates that the added histone was methylated itself by the purified protein methylase I at the guanidino group of arginine residues. Since methyl substitution on the amino group greatly influence the pK values of the compound (16), and histone exist in situ in conjugation with DNA or RNA, modification of histone molecule by enzymatic methylation might be expected to have profound effect on the action of the nucleic acids. However, no experimental evidence has yet been presented
ENZYMATIC
METHYLATION
to indicate a direct relationship between the methylation of histone and the function of the nucleic acids. Studying the kinetics of in vivo methylation of e-amino group of lysine residues of histone during hepatic regeneration, Tidwell et al. (17) did not find any correlation between the methylation of histone and an increase in DNA template activity for RNA synthesis, or with DNA synthesis. We are now, however, in the process of investigating biological significance of methylation of arginine residues during various physiological conditions such as thyroxine-induced amphibian metamorphosis or hepatic regeneration. ACKNOWLEDGMENT This work was supported by research grants from the Institute of Arthritis and Metabolic Diseases (AM-O9602-03), National Science Foundation (GB-6019)) and National Cancer Institute (CA 10439). REFERENCES 1. PAIK, 243, 2. PAX, Res.
W. K., AND KIM, S., J. Biol. Chem. 2108 (1968). W. K., AND KIM, S., Biochem. Biophys. Commun. 29, 14 (1967).
OF
HISTONES
637
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