ANALYTICAL
BIOCHEMISTRY
90,
262-272 (1978)
Preparation of Radioactive A/-Methylated Lysines Involving Elaborate Organic Synthesis WOON
Kr PAIK, PETER DIMARIA,
Fels Research
Institute
EARL
PEARSON,
and Department of Biochemistry, Medicine, Philadelphia. Pennsylvania
Not
AND SANCDUK
Temple 19140
University
KIM
School
of
Received May 3, 1978 A simple method to prepare small quantities of radioactive l -N-methylated lysines for use as analytical standard markers, which does not involve elaborate organic synthesis, is highly desired in numerous research applications. The method in this report involved growing Neurospora crassa or Salmonella typhimurium in the presence of uniformly labeled [U-W] lysine or reductively methylating a protein with [W]formaldehyde at pH 9.0. The radiolabeled proteins were then isolated and hydrolyzed in 6 N HCI. Finally, the radioactive methylated Iysines in the acid hydrolysates were isolated by Dowex 50 column chromatography.
Protein methylation is an ubiquitous biochemical reaction occurring in all the organisms examined thus far (1). Enzymatic methylation of lysine residues is observed in various proteins such as histone, myosin, actin, cytochrome c, ribosomal proteins, and opsin. Indeed, methylation of histone-lysine residues has been suggested to play an important role during conjugation of nucleic acids and histones in eukaryotic cells (2,3). Furthermore, recent evidence strongly indicates that the proteolytic product of e-N-trimethylated protein, e-N-trimethyllysine, is used for the synthesis of carnitine (4,5). While investigating the protein methylation reaction, it is often desired to have authentic standard methylated lysines. Unlabeled E-N-monomethylL-lysine, e-N-dimethyl-L-lysine, and e-N-trimethyl-L-lysine are now available from various commercial sources such as Sigma Chemical Company (St. Louis, MO.), Calbiochem (La Jolla, Calif.), or Cycle Chemical (Los Angeles, Calif.). However, radiolabeled l -N-methylated lysine standards are not yet available on the market. This paper describes a method to prepare in a simple way all three of the methylated lysines by growing microorganisms with [U- 14C]lysine. MATERIALS
AND METHODS
Materials
Uniformly labeled [14C]lysine (sp act 286 mCi/mmol) and [14C]formaldehyde (sp act 59 mCi/mmol) were purchased from New England Nuclear, 0003-2697/78/0901-0262$02.00/O Copyright All rights
0 1918 by Academic Press, Inc. of reproduction in any form reserved.
262
LABELED
l -N-METHYLLYSINES
263
Boston, Massachusetts. Dowex 5OW hydrogen form (200-400 mesh; 8% crosslinking) and L-histidine were obtained from Sigma Chemical Company. The Neurospora crassa used in these experiments was originally a gift from Dr. L. Brodsky, Hahnemann Medical College, Philadelphia, Pennsylvania and stock cultures were maintained on slants of Neurospora culture agar (Difco, Detroit, Mich.). Salmonella typhimurium SL 870 strain was Jla+ nml+ his- (6). The remaining chemicals were obtained from various local sources and were of the highest grade of purity. In Vivo Labeling of Neurospora crassa Protein n’ith [.?I-14C]Lysine
Five hundred milliliters of Vogel’s Neurospora medium (7) was mixed with approximately 30 PCi of [U-14C]lysine (7.1 x 10’ cpm) and then divided into two 250-ml portions which were placed in two 500-ml capacity Erlenmyer flasks. The flasks were then sterilized. Innoculation of the flasks was accomplished by first introducing 3 ml of deionized water into a Neurospora agar slant contained in a 25ml capacity test tube. After vortexing for 1 min, 0.5-ml aliquots were introduced into the above sterilized Erlenmyer flasks. The flasks were shaken for 62 h in a Dubnoff incubator at 30°C. The mycelia were harvested by filtration on a Buchner funnel, washed a few times with water, and then suction dried. Three grams of the still wet mycelia pad was homogenized in 50 ml of 15% trichloroacetic acid with the aid of a glass-glass homogenizer, and the whole homogenate was centrifuged at 39,000g for 10 min. The precipitate was further washed twice by homogenization. The pellet was suspended in 30 ml of 15% trichloroacetic acid and the suspension was heated at 90°C for 15 min and then pelleted by centrifugation. This step was repeated once more. The above two procedures with trichloroacetic acid remove any radioactivity arising from free L-[U-14C]lysine and nucleic acids. The above-treated pellet was now treated once with a mixture of chloroform:ethanol:ether (at ratio of 1:2: 1) at 40°C for 10 min, and finally with ethanol at 70°C for 10 min. These procedures should extract phospholipids. The final preparation was air-dried at room temperature overnight. The dried sample was then hydrolyzed in 6 N HCl at 110°C for 48 h. The HCl hydrolysate was strongly black in color. Thus, by treating with charcoal and celite, most of the black material was removed. The filtrate and washings were combined, and the combined solution was concentrated under reduced pressure. The residue was washed a few times with water in order to remove as much HCl as possible. The total radioactivity recovered was 3.26 x lo7 cpm in this hydrolysate (46% recovery). In Vivo Labeling of Salmonella typhimurium with L-[UJ4C]Lysine
One hundred microcuries L-[U-“Cllysine (2.24 x 10’ cpm) was mixed with 1000 ml ofSalmonella typhimurium medium [S.T. medium (8) without
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PAIK
ET AL
the glucose] supplemented with 0.11 mM of L-histidine in a 2-liter capacity Erlenmyer flask, and the flask was autoclaved. Approximately 20 ml of glucose solution containing 5 g of glucose, which had been autoclaved separately, was added to the above S.T. medium. Ten milliliters of inoculums of the SL 870 strain (6) were grown in YA broth (8 g of nutrient broth plus 3 g of yeast extract per liter) at 37°C for about 24 h. This was then added into the above L-[U-14C]lysine containing S.T. medium. The entire flask was incubated at 34 to 37°C by shaking in a Dubnoff incubator for 12 h. One hundred grams of solid trichloroacetic acid was added to the above culture medium after the incubation, and the mixture was stirred for 10 min with the aid of a magnetic stirrer. The mixture was centrifuged at 39,000g for 10 min, and the pellet was washed three times with 100 ml each of 15% trichloroacetic acid at 39,000g for 10 min. The obtained pellet was subsequently treated with hot trichloroacetic acid, a mixture of chloroform:ethanol:ether, and ethanol by procedures as described for the Neurospora crussa experiment. A 0.7-g dried sample with 1.33 x lo* cpm (59% radioactivity recovery in the protein fraction) was observed. The protein was hydrolyzed in 6 N HCl and the hydrolysate was treated as in the case of the Neurospora described above. Reductive Methylation
of Protein with [14C]Formaldehyde
Reductive methylation of protein with formaldehyde and sodium borohydride at pH 9.0 results in the specific methylation of the e-amino group of lysine residues (9,lO). Since histones contain a relatively large amount of lysine residues, 1 g of histone type II-A (Sigma Chemical Co.) was dissolved in 12 ml of water. Six milliliters of 0.2 M borate buffer at pH 9.0 and 10 mg of sodium borohydride were added to the above histone suspension in ice. Aliquots offormaldehyde (0.010 ml) were added at 5-min intervals while mixing until a total of 0.050 ml was added. The formaldehyde was prepared by diluting commercially obtained [14C]formaldehyde with an equal volume of nonradioactive 37% formaldehyde (10 M). After completing the addition, the mixture was left at 0°C for another 5 min and was then dialyzed against 6 liters of water in the cold room. Dialysis was continued for 24 h while changing the water three times. After lyophilization of the dialyzed sample, the material was hydrolyzed in 10 ml of 6 N HCl for 48 h. Chromatographic Isolation of Various [Methyl-14C]Lysines by Dowex 50 Column Preliminary separation of basic amino acids. The above prepared [U-14C]lysine or methyl-14C-labeled HCl hydrolysates were individually charged onto a Dowex 50 H+ column (3 x 24 cm). The column was eluted
LABELED
265
E-N-METHYLLYSINES
successively with 1000 ml of water and 4000 ml of 0.1 N pyridine. These elutions remove inorganic salts and acidic and neutral amino acids, and thus these eluents were discarded. The column was finally eluted with 1000 ml of 1.5 N NH,OH in order to elute basic amino acids (11). Practically all of the radioactivity was eluted in this fraction. The NH,OH eluate was concentrated to dryness under reduced pressure, and the residue was washed a few times with water to eliminate as much ammonia as possible. Dowex 50 NH,+ column lysines. The above NHIOH
chromatographic
separation
of methylated
eluate from a Dowex 50 H+ column was dissolved in 5 ml of water, and the suspension was charged on a Dowex 50 NH4+ column (2.2 x 66 cm) which had been equilibrated with water. The column was eluted with a linear gradient formed by 1000 ml of 0.1~ NH,OH and 1000 ml of 1.5 N NH,OH. Fractions (3 to 3.70 ml) were collected and O.OlO- to O.lO-ml aliquots were examined for radioactivity. Radioactivity was counted in 10 ml of scintillation solution (Formula-963, New England Nuclear Product) with a Packard Tri-Carb liquid scintillation spectrophotometer with an efficiency of approximately 85%.
Preparation
of Authentic
Radiolabeled
Methyllysines
The radiolabeled authentic methylated lysine standards were derived from enzymatically methylated horse heart cytochrome c that had been methylated with the highly purified Neurospora crassa cytochrome c-specific protein-lysine methyltransferase recently described (12). The reaction mixture contained 30 mg of horse heart cytochrome c (Sigma Chemical Co.), 400 nmol of S-adenosyl-L-[methyl-14C]methionine (50 cpmpmol), 10 pg of the highly purified cytochrome c-specific methyltransferase, and 0.1 M final concentration of Tris-HCl buffer, pH 9.0, all in a total volume of 10 ml. This mixture was incubated at 37°C for 1 h and then stopped by adding an equivolume of 15% trichloroacetic acid. The mixture was then heated at 90°C for 15 min and then cooled on ice. This was then centrifuged at 10,OOOg for 10 min in a Sorvall centrifuge. The precipitate was washed three times with 40-ml portions of 7.5% trichloroacetic acid. The precipitate was finally washed in a similar manner with absolute ethanol to remove the residual trichloroacetic acid. The precipitate was then hydrolyzed in 6 N HCl for 48 h at 110°C in vacua. The pure [methyl-14C] lysines were prepared from the hydrolysate as follows. First, a small aliquot (approximately one-twentieth of the sample) of the hydrolysate was analyzed by a Perkin-Elmer automatic amino acid analyzer (KLA-3B) attached to a flow cell for constant radioactivity monitoring (1). The column (Bio-Rad A-5 resin, a particle size of 13 2 2 Km, and 0.9 x 47 cm) was eluted with 0.38 N sodium citrate buffer with a flow rate of 45 ml/h at 27°C. The following elution times were used here and throughout the study for identification of various unknown amino acid peaks:
266
PAIKET AL.
Radiogram elution time
Ninhydrin elution time
Lysine
4 h 42 min
5 h 12 min
l -N-Monomethyllysine l -N-Dimethyllysine
6 h 50 min
7 h 20 min
7h
7 h 30 min
e-N-Trimethyllysine
7 h 30 min
8h
Amino acids
After the above elution times were determined, pure samples of e-N-mono-, l -N-di-, and e-N-trimethyllysine were prepared preparatively by charging half of the remaining hydrolysate on the amino acid analyzer attached to a fraction collector (without reacting with ninhydrin solution on the analyzer, but by monitoring the radioactivity). The peak fractions corresponding to the various radioactivity peaks were pooled individually. These pooled fractions were then concentrated under reduced pressure and consequently desalted on the Dowex 50 H+ column procedure described earlier in this section. The final samples were rechecked on the amino acid analyzer for purity. Identification of Peaks from Dowex 50 NH,+ Column Chromatography of Various Basic Amino Acid Mixtures
All peaks were identified by the amino acid analyzer using the conditions and the elution times described above. First, an initial identification of the radioactive peak in question was made on the amino acid analyzer using elution time. Further proof of identity was obtained by the co-elution of the radiolabeled sample with its standard nonlabeled counterpart. RESULTS Chromatographic Separation of Various e-N-Methylated L-Lysine
Lysines and
Figure 1 illustrates that, when a mixture of radioactive e-N-monomethyllysine, l -N-dimethyllysine, e-N-trimethyllysine, and L-lysine was applied on a Dowex 50 NH,+ column and the column was eluted with a linear gradient formed by 1000 ml of 0.1 N NH,OH and 1000 ml of 1.5 N NH,OH, these four compounds are very well separated. The sequence of elution indicates that the more the e-amino group is substituted with the methyl group, the earlier the compound eluted. This pattern of elution is in absolute opposite to that observed with pH 5.84 citrate buffer on an automatic amino acid analyzer (13). We are at a loss to explain this difference in the elution patterns.
LABELED
e-A’-METHYLLYSINES
267
TM1 6
'I I I
MM1 DML
FRACTION
NUMBER
FIG. 1. Separation of various radioactive methylated lysines on a Dowex 50 NH,+ column. Authentic radiolabeled methylated lysines and L-lysine were applied on a Dowex 50 NH,+ column (2.2 cm i.d. x 66 cm), and the column was eluted with a linear gradient formed by 1000 ml of 0.1 NNH,OH and 1000 ml of 1.S NNH,OH. At the rate of approximately six fractions per hour 3.7-m] frations were collected at room temperature. One-tenth-milliliter aliquots were used for determining radioactivity. TML. DML, MML, and L represent l -N-trimethyllysine, c-N-dimethyllysine, c-N-monomethyllysine, and Iysine, respectively. More detailed experimental procedures are described under Materials and Methods.
Production of l -N-Methylated of Salmonella typhimurium
[U-14C]Lysine
by Use of in Vivo Labeling
After establishing the above separation method, we grew Salmonella typhimurium in the presence of 2.24 x lOa cpm of [U- 14C]lysine and found that 59% (1.33 x lOa cpm) of the added radioactivity was incorporated into protein fraction. Furthermore, when the acid hydrolysate of this protein was analyzed by Dowex 50 H+ column chromatography (see Materials and Methods), 93% of the incorporated radioactivity (1.24 x lOa cpm) was found in basic amino acids. Figure 2 illustrates Dowex 50 NH,+ column chromatographic separation of various e-N-methylated lysines from the above basic amino acid mixture. Although l -N-di- and e-N-monomethyllysine are prominent, production of l -N-trimethyllysine by this organism is evidently rather poor. Furthermore, total radioactivity found in three e-N-methylated lysines comprises only 0.95% of the total incorporated to the protein. Thus, we searched for an organism which would provide a better yield.
268
PAIK ET AL.
DML
MML R
TML
0kiF7v
I
i
1
80
I
I
100
80
FRACTION
NUMBER
FIG. 2. Chromatographic analysis of acid hydrolysate of radioactive Salmonellu typhimurium protein. Acid hydrolysate of Salmonella typhimurium protein which had been labeled with [CJ-WIlysine was analyzed on a Dowex 50 NH,+ column. Fractions corresponding to each of the methylated lysines were pooled separately. These pooled samples were concentrated under reduced pressure and were washed a few times with water to remove as much ammonia as possible. Since the elution position of each of the methylated lysines on the Dowex 50 NH,+ column varied from sample to sample, these pooled samples were further analyzed on an automatic amino acid analyzer for confirmation of the identities with authentic nonlabeled amino acid standards. For this purpose, a Perkin-Elmer automatic amino acid analyzer (KLA-3B) with an attached flow cell for constant radioactivity monitoring was used. Further details are described under Materials and Methods. Ofthe total radioactivity incorporated into protein, 0.95% was found in the methylated lysines at the ratio of TML:DML:MML at 1.O:1.9: 1.4. All other conditions and detailed experimental procedures are as described under Fig. 1 and Materials and Methods.
Production of l -N-Methylated of Neurospora crassa
[U-14C]Lysine by Use of in Vivo Labeling
Neurospora crassa was grown in the presence of 30 &i of [ U-‘4C]lysine (7.1 x lo7 cpm) for 62 h at 30°C by shaking, the protein fraction was
LABELED
269
e-N-METHYLLYSINES
subsequently isolated, and 46% of the radioactivity initially present in the culture medium (3.26 x lo7 cpm) was found to be incorporated into protein. When the acid hydrolysate of this [U-‘4C]lysine-labeled protein was analyzed by Dowex 50 H+ column chromatography, practically all of the incorporated radioactivity was recovered as basic amino acid fraction. When one-fourth of the radioactivity (recovered as basic amino acids) was applied on a Dowex 50 NH,+ column, the elution pattern shown in Fig. 3 was obtained. Three methylated lysines (E-N-mono-, l -N-di-, and l -N-trimethyllysine) were obtained with much higher yield than that observed with Salmonella typhimurium; 1.58% of the total radioactivity in the protein hydrolysate was recovered as methylated lysines.
8-
1 1 L
o-
FRACTION
NUMBER
FIG. 3. Chromatographic separation of acid hydrolysate of radioactive Neurospora crassa protein. One-fourth of the acid hydrolysate of Neurospora crassa protein which had been labeled with [UL4C]lysine according to the method described under Materials and Methods was analyzed on a Dowex 50 NH,+ column. Of the total radioactivity incorporated into protein, 1.58% was found to exist in the various methylated lysines at the ratio of TML:DML:MML at 1.O:1.12:0.36. All the conditions are the same as in Fig. 1 and the rest of the detailed experimental procedures are described under Materials and Methods.
270
PAIK
ET AL.
Reductive Methylution of Protein as a Means of Producing Radiolabeled e-N-Methylated Lysines
Acid-hydrolyzed reductively methylated [methylJ4C]protein(3.42 x lo6 cpm) was charged on Dowex 50 H+ column. Of this, approximately 95% (3.24 x lo6 cpm) of the radioactivity was recovered in the basic amino acid fraction (in 1.5 N NH,OH eluant). Figure 4 illustrates the elution pattern of this basic amino acid fraction on a Dowex 50 NH,’ column. Approximately 94% of the charged radioactivity (3.05 x lo6 cpm) was recovered in the E-Ndimethyllysine and e-N-monomethyllysine peaks. As shown previously (9,10), reductive methylation of protein with formaldehyde and sodium borohydride did not produce E-N-trimethyllysine.
FRACTION
NUMBER
FIG. 4. Chromatographic separation of acid hydrolysate of histone which had been reductively methylated with [‘Tlformaldehyde. Detailed experimental procedures are the same as in Fig. I. and the rest of the procedures are described under Materials and Methods. Of the total charged radioactivity, 94.1% was recoverable in these two major peaks, and the ratio of radioactivity of DML:MML was 1: 1.21.
LABELED
e-N-METHYLLYSINES
271
DISCUSSION
Methods for synthesizing E-N-mono-, e-N-di-, or l -N-trimethyl-r-lysine have been known for some time (14-16). In addition, these compounds (unlabeled) are easily available from various commercial sources. However, when an investigator is lacking in experience in organic synthesis, and yet needs small amounts of these radioactive methylated lysines, he (or she) is confronted with a serious technical problem. In this paper, we have presented a method to synthesize small quantities of these compounds possessing radioactive labeling. The method has the following advantages; (i) by a simple procedure, three methylated lysines (e-N-mono-, l -N-di-, and e-N-trimethyllysine) can be synthesized biologically or chemically to possess radioactivity; (ii) the synthesized compounds could be separated in pure form; (iii) the specific activity of the compounds synthesized by either organisms or by reductive methylation of protein with [‘4C]formaldehyde could be dependent on the amounts of radioactive precursor compound used; and finally, (iv) the method also suggests the possibility of preparing methylated arginine or methylhistidine in a similar fashion. Among the various ways to prepare the methylated lysines, the Neurospora system appears to be superior to that of the Salmonella system, in that the overall yield of radioactivity found in the methylated lysines is much higher and that the system produces much higher amounts of c-N-trimethyllysine relative to r-N-mono- and e-N-dimethyllysine. When only e-N-mono- and l -N-dimethyllysine is needed, the reductive methylation method seems to be the preferred method. It is possible that in the future an organism(s) might be found that produces the three methylated lysines in a more favorable way than the two systems examined in this study. ACKNOWLEDGMENTS This work was supported by research Grants AM 09602 from the National Institute of Arthritis, Metabolism, and Digestive Diseases, CA 10439 and CA 12226 from the National Cancer Institute. and GM 20594 from the National Institute of General Medical Sciences.
REFERENCES 1. Paik, W. K., and Kim. S. (1975) in Advances in Enzomology (Meister, A., ed.). Vol. 42. p. 227. Wiley, New York. 2. Paik. W. K., and Kim. S. (1975) in Post-Synthetic Modification of Macromolecules (Antoni. F.. and Farago. A.. eds.). North-Holland/American Elsevier; FEB.$, 34, 127. 3. Paik. W. K. (1977) in The Biochemistry of S-Adenosylmethionine (Salvatore. F.. Borek, E.. Zappia, V., William-Ashman. H. G., and Schlenk. F., eds.). Columbia Univ. Press, New York. 4. LaBadie. J. H.. Dunn, W. A., and Aronson. N. N. (1976) Biochem. J. 160, 85. 5. Home, D. W., and Broquist, H. P. (1973) J. Bid. Chent. 248, 2170. 6. Tronick, S. R.. and Martinez. R. J. (1971) J. Brrcteriol. 105, 21 I.
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7. Davis, R. H., and DeSerres, F. J. (1970) in Methods in Enzymology (Tabor, H., and Tabor, C. W., eds.), Vol. 172, pp. 79, Academic Press, New York. 8. Co&on, J. W., and Kapoor, M. (1973) Canad. J. Microbial. 19, 427. 9. Means, G. E., and Feeney, R. E. (1968) Biochemistry 7, 2192. 10. Paik, W. K., and Kim, S. (1972) Biochemistry 11, 2589. 11. Markiw, R. T. (1975) Biochem. Med. 13, 23. 12. Durban, E., Nochumson, S., Kim. S., Paik, W. K., and Chan, S-K. (1978)5. Biol. Chem. 253, 1427. 13. Paik, W. K., and Kim, S. (1967) Biochem. Biophys. Res. Commun. 27, 479. 14. Benoiton, L. (1964) Canad. J. Chem. 42, 2043. 15. Neuberger, A., and Danger, F. (1944) Biochem. J. 38, 125. 16. DeLange, R. J., Glazer, A. N., and Smith, E. L. (1969) J. Biol. Chem. 244, 1385.
BIOCHEMISTRY
90,
262-272 (1978)
Preparation of Radioactive A/-Methylated Lysines Involving Elaborate Organic Synthesis WOON
Kr PAIK, PETER DIMARIA,
Fels Research
Institute
EARL
PEARSON,
and Department of Biochemistry, Medicine, Philadelphia. Pennsylvania
Not
AND SANCDUK
Temple 19140
University
KIM
School
of
Received May 3, 1978 A simple method to prepare small quantities of radioactive l -N-methylated lysines for use as analytical standard markers, which does not involve elaborate organic synthesis, is highly desired in numerous research applications. The method in this report involved growing Neurospora crassa or Salmonella typhimurium in the presence of uniformly labeled [U-W] lysine or reductively methylating a protein with [W]formaldehyde at pH 9.0. The radiolabeled proteins were then isolated and hydrolyzed in 6 N HCI. Finally, the radioactive methylated Iysines in the acid hydrolysates were isolated by Dowex 50 column chromatography.
Protein methylation is an ubiquitous biochemical reaction occurring in all the organisms examined thus far (1). Enzymatic methylation of lysine residues is observed in various proteins such as histone, myosin, actin, cytochrome c, ribosomal proteins, and opsin. Indeed, methylation of histone-lysine residues has been suggested to play an important role during conjugation of nucleic acids and histones in eukaryotic cells (2,3). Furthermore, recent evidence strongly indicates that the proteolytic product of e-N-trimethylated protein, e-N-trimethyllysine, is used for the synthesis of carnitine (4,5). While investigating the protein methylation reaction, it is often desired to have authentic standard methylated lysines. Unlabeled E-N-monomethylL-lysine, e-N-dimethyl-L-lysine, and e-N-trimethyl-L-lysine are now available from various commercial sources such as Sigma Chemical Company (St. Louis, MO.), Calbiochem (La Jolla, Calif.), or Cycle Chemical (Los Angeles, Calif.). However, radiolabeled l -N-methylated lysine standards are not yet available on the market. This paper describes a method to prepare in a simple way all three of the methylated lysines by growing microorganisms with [U- 14C]lysine. MATERIALS
AND METHODS
Materials
Uniformly labeled [14C]lysine (sp act 286 mCi/mmol) and [14C]formaldehyde (sp act 59 mCi/mmol) were purchased from New England Nuclear, 0003-2697/78/0901-0262$02.00/O Copyright All rights
0 1918 by Academic Press, Inc. of reproduction in any form reserved.
262
LABELED
l -N-METHYLLYSINES
263
Boston, Massachusetts. Dowex 5OW hydrogen form (200-400 mesh; 8% crosslinking) and L-histidine were obtained from Sigma Chemical Company. The Neurospora crassa used in these experiments was originally a gift from Dr. L. Brodsky, Hahnemann Medical College, Philadelphia, Pennsylvania and stock cultures were maintained on slants of Neurospora culture agar (Difco, Detroit, Mich.). Salmonella typhimurium SL 870 strain was Jla+ nml+ his- (6). The remaining chemicals were obtained from various local sources and were of the highest grade of purity. In Vivo Labeling of Neurospora crassa Protein n’ith [.?I-14C]Lysine
Five hundred milliliters of Vogel’s Neurospora medium (7) was mixed with approximately 30 PCi of [U-14C]lysine (7.1 x 10’ cpm) and then divided into two 250-ml portions which were placed in two 500-ml capacity Erlenmyer flasks. The flasks were then sterilized. Innoculation of the flasks was accomplished by first introducing 3 ml of deionized water into a Neurospora agar slant contained in a 25ml capacity test tube. After vortexing for 1 min, 0.5-ml aliquots were introduced into the above sterilized Erlenmyer flasks. The flasks were shaken for 62 h in a Dubnoff incubator at 30°C. The mycelia were harvested by filtration on a Buchner funnel, washed a few times with water, and then suction dried. Three grams of the still wet mycelia pad was homogenized in 50 ml of 15% trichloroacetic acid with the aid of a glass-glass homogenizer, and the whole homogenate was centrifuged at 39,000g for 10 min. The precipitate was further washed twice by homogenization. The pellet was suspended in 30 ml of 15% trichloroacetic acid and the suspension was heated at 90°C for 15 min and then pelleted by centrifugation. This step was repeated once more. The above two procedures with trichloroacetic acid remove any radioactivity arising from free L-[U-14C]lysine and nucleic acids. The above-treated pellet was now treated once with a mixture of chloroform:ethanol:ether (at ratio of 1:2: 1) at 40°C for 10 min, and finally with ethanol at 70°C for 10 min. These procedures should extract phospholipids. The final preparation was air-dried at room temperature overnight. The dried sample was then hydrolyzed in 6 N HCl at 110°C for 48 h. The HCl hydrolysate was strongly black in color. Thus, by treating with charcoal and celite, most of the black material was removed. The filtrate and washings were combined, and the combined solution was concentrated under reduced pressure. The residue was washed a few times with water in order to remove as much HCl as possible. The total radioactivity recovered was 3.26 x lo7 cpm in this hydrolysate (46% recovery). In Vivo Labeling of Salmonella typhimurium with L-[UJ4C]Lysine
One hundred microcuries L-[U-“Cllysine (2.24 x 10’ cpm) was mixed with 1000 ml ofSalmonella typhimurium medium [S.T. medium (8) without
264
PAIK
ET AL
the glucose] supplemented with 0.11 mM of L-histidine in a 2-liter capacity Erlenmyer flask, and the flask was autoclaved. Approximately 20 ml of glucose solution containing 5 g of glucose, which had been autoclaved separately, was added to the above S.T. medium. Ten milliliters of inoculums of the SL 870 strain (6) were grown in YA broth (8 g of nutrient broth plus 3 g of yeast extract per liter) at 37°C for about 24 h. This was then added into the above L-[U-14C]lysine containing S.T. medium. The entire flask was incubated at 34 to 37°C by shaking in a Dubnoff incubator for 12 h. One hundred grams of solid trichloroacetic acid was added to the above culture medium after the incubation, and the mixture was stirred for 10 min with the aid of a magnetic stirrer. The mixture was centrifuged at 39,000g for 10 min, and the pellet was washed three times with 100 ml each of 15% trichloroacetic acid at 39,000g for 10 min. The obtained pellet was subsequently treated with hot trichloroacetic acid, a mixture of chloroform:ethanol:ether, and ethanol by procedures as described for the Neurospora crussa experiment. A 0.7-g dried sample with 1.33 x lo* cpm (59% radioactivity recovery in the protein fraction) was observed. The protein was hydrolyzed in 6 N HCl and the hydrolysate was treated as in the case of the Neurospora described above. Reductive Methylation
of Protein with [14C]Formaldehyde
Reductive methylation of protein with formaldehyde and sodium borohydride at pH 9.0 results in the specific methylation of the e-amino group of lysine residues (9,lO). Since histones contain a relatively large amount of lysine residues, 1 g of histone type II-A (Sigma Chemical Co.) was dissolved in 12 ml of water. Six milliliters of 0.2 M borate buffer at pH 9.0 and 10 mg of sodium borohydride were added to the above histone suspension in ice. Aliquots offormaldehyde (0.010 ml) were added at 5-min intervals while mixing until a total of 0.050 ml was added. The formaldehyde was prepared by diluting commercially obtained [14C]formaldehyde with an equal volume of nonradioactive 37% formaldehyde (10 M). After completing the addition, the mixture was left at 0°C for another 5 min and was then dialyzed against 6 liters of water in the cold room. Dialysis was continued for 24 h while changing the water three times. After lyophilization of the dialyzed sample, the material was hydrolyzed in 10 ml of 6 N HCl for 48 h. Chromatographic Isolation of Various [Methyl-14C]Lysines by Dowex 50 Column Preliminary separation of basic amino acids. The above prepared [U-14C]lysine or methyl-14C-labeled HCl hydrolysates were individually charged onto a Dowex 50 H+ column (3 x 24 cm). The column was eluted
LABELED
265
E-N-METHYLLYSINES
successively with 1000 ml of water and 4000 ml of 0.1 N pyridine. These elutions remove inorganic salts and acidic and neutral amino acids, and thus these eluents were discarded. The column was finally eluted with 1000 ml of 1.5 N NH,OH in order to elute basic amino acids (11). Practically all of the radioactivity was eluted in this fraction. The NH,OH eluate was concentrated to dryness under reduced pressure, and the residue was washed a few times with water to eliminate as much ammonia as possible. Dowex 50 NH,+ column lysines. The above NHIOH
chromatographic
separation
of methylated
eluate from a Dowex 50 H+ column was dissolved in 5 ml of water, and the suspension was charged on a Dowex 50 NH4+ column (2.2 x 66 cm) which had been equilibrated with water. The column was eluted with a linear gradient formed by 1000 ml of 0.1~ NH,OH and 1000 ml of 1.5 N NH,OH. Fractions (3 to 3.70 ml) were collected and O.OlO- to O.lO-ml aliquots were examined for radioactivity. Radioactivity was counted in 10 ml of scintillation solution (Formula-963, New England Nuclear Product) with a Packard Tri-Carb liquid scintillation spectrophotometer with an efficiency of approximately 85%.
Preparation
of Authentic
Radiolabeled
Methyllysines
The radiolabeled authentic methylated lysine standards were derived from enzymatically methylated horse heart cytochrome c that had been methylated with the highly purified Neurospora crassa cytochrome c-specific protein-lysine methyltransferase recently described (12). The reaction mixture contained 30 mg of horse heart cytochrome c (Sigma Chemical Co.), 400 nmol of S-adenosyl-L-[methyl-14C]methionine (50 cpmpmol), 10 pg of the highly purified cytochrome c-specific methyltransferase, and 0.1 M final concentration of Tris-HCl buffer, pH 9.0, all in a total volume of 10 ml. This mixture was incubated at 37°C for 1 h and then stopped by adding an equivolume of 15% trichloroacetic acid. The mixture was then heated at 90°C for 15 min and then cooled on ice. This was then centrifuged at 10,OOOg for 10 min in a Sorvall centrifuge. The precipitate was washed three times with 40-ml portions of 7.5% trichloroacetic acid. The precipitate was finally washed in a similar manner with absolute ethanol to remove the residual trichloroacetic acid. The precipitate was then hydrolyzed in 6 N HCl for 48 h at 110°C in vacua. The pure [methyl-14C] lysines were prepared from the hydrolysate as follows. First, a small aliquot (approximately one-twentieth of the sample) of the hydrolysate was analyzed by a Perkin-Elmer automatic amino acid analyzer (KLA-3B) attached to a flow cell for constant radioactivity monitoring (1). The column (Bio-Rad A-5 resin, a particle size of 13 2 2 Km, and 0.9 x 47 cm) was eluted with 0.38 N sodium citrate buffer with a flow rate of 45 ml/h at 27°C. The following elution times were used here and throughout the study for identification of various unknown amino acid peaks:
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PAIKET AL.
Radiogram elution time
Ninhydrin elution time
Lysine
4 h 42 min
5 h 12 min
l -N-Monomethyllysine l -N-Dimethyllysine
6 h 50 min
7 h 20 min
7h
7 h 30 min
e-N-Trimethyllysine
7 h 30 min
8h
Amino acids
After the above elution times were determined, pure samples of e-N-mono-, l -N-di-, and e-N-trimethyllysine were prepared preparatively by charging half of the remaining hydrolysate on the amino acid analyzer attached to a fraction collector (without reacting with ninhydrin solution on the analyzer, but by monitoring the radioactivity). The peak fractions corresponding to the various radioactivity peaks were pooled individually. These pooled fractions were then concentrated under reduced pressure and consequently desalted on the Dowex 50 H+ column procedure described earlier in this section. The final samples were rechecked on the amino acid analyzer for purity. Identification of Peaks from Dowex 50 NH,+ Column Chromatography of Various Basic Amino Acid Mixtures
All peaks were identified by the amino acid analyzer using the conditions and the elution times described above. First, an initial identification of the radioactive peak in question was made on the amino acid analyzer using elution time. Further proof of identity was obtained by the co-elution of the radiolabeled sample with its standard nonlabeled counterpart. RESULTS Chromatographic Separation of Various e-N-Methylated L-Lysine
Lysines and
Figure 1 illustrates that, when a mixture of radioactive e-N-monomethyllysine, l -N-dimethyllysine, e-N-trimethyllysine, and L-lysine was applied on a Dowex 50 NH,+ column and the column was eluted with a linear gradient formed by 1000 ml of 0.1 N NH,OH and 1000 ml of 1.5 N NH,OH, these four compounds are very well separated. The sequence of elution indicates that the more the e-amino group is substituted with the methyl group, the earlier the compound eluted. This pattern of elution is in absolute opposite to that observed with pH 5.84 citrate buffer on an automatic amino acid analyzer (13). We are at a loss to explain this difference in the elution patterns.
LABELED
e-A’-METHYLLYSINES
267
TM1 6
'I I I
MM1 DML
FRACTION
NUMBER
FIG. 1. Separation of various radioactive methylated lysines on a Dowex 50 NH,+ column. Authentic radiolabeled methylated lysines and L-lysine were applied on a Dowex 50 NH,+ column (2.2 cm i.d. x 66 cm), and the column was eluted with a linear gradient formed by 1000 ml of 0.1 NNH,OH and 1000 ml of 1.S NNH,OH. At the rate of approximately six fractions per hour 3.7-m] frations were collected at room temperature. One-tenth-milliliter aliquots were used for determining radioactivity. TML. DML, MML, and L represent l -N-trimethyllysine, c-N-dimethyllysine, c-N-monomethyllysine, and Iysine, respectively. More detailed experimental procedures are described under Materials and Methods.
Production of l -N-Methylated of Salmonella typhimurium
[U-14C]Lysine
by Use of in Vivo Labeling
After establishing the above separation method, we grew Salmonella typhimurium in the presence of 2.24 x lOa cpm of [U- 14C]lysine and found that 59% (1.33 x lOa cpm) of the added radioactivity was incorporated into protein fraction. Furthermore, when the acid hydrolysate of this protein was analyzed by Dowex 50 H+ column chromatography (see Materials and Methods), 93% of the incorporated radioactivity (1.24 x lOa cpm) was found in basic amino acids. Figure 2 illustrates Dowex 50 NH,+ column chromatographic separation of various e-N-methylated lysines from the above basic amino acid mixture. Although l -N-di- and e-N-monomethyllysine are prominent, production of l -N-trimethyllysine by this organism is evidently rather poor. Furthermore, total radioactivity found in three e-N-methylated lysines comprises only 0.95% of the total incorporated to the protein. Thus, we searched for an organism which would provide a better yield.
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PAIK ET AL.
DML
MML R
TML
0kiF7v
I
i
1
80
I
I
100
80
FRACTION
NUMBER
FIG. 2. Chromatographic analysis of acid hydrolysate of radioactive Salmonellu typhimurium protein. Acid hydrolysate of Salmonella typhimurium protein which had been labeled with [CJ-WIlysine was analyzed on a Dowex 50 NH,+ column. Fractions corresponding to each of the methylated lysines were pooled separately. These pooled samples were concentrated under reduced pressure and were washed a few times with water to remove as much ammonia as possible. Since the elution position of each of the methylated lysines on the Dowex 50 NH,+ column varied from sample to sample, these pooled samples were further analyzed on an automatic amino acid analyzer for confirmation of the identities with authentic nonlabeled amino acid standards. For this purpose, a Perkin-Elmer automatic amino acid analyzer (KLA-3B) with an attached flow cell for constant radioactivity monitoring was used. Further details are described under Materials and Methods. Ofthe total radioactivity incorporated into protein, 0.95% was found in the methylated lysines at the ratio of TML:DML:MML at 1.O:1.9: 1.4. All other conditions and detailed experimental procedures are as described under Fig. 1 and Materials and Methods.
Production of l -N-Methylated of Neurospora crassa
[U-14C]Lysine by Use of in Vivo Labeling
Neurospora crassa was grown in the presence of 30 &i of [ U-‘4C]lysine (7.1 x lo7 cpm) for 62 h at 30°C by shaking, the protein fraction was
LABELED
269
e-N-METHYLLYSINES
subsequently isolated, and 46% of the radioactivity initially present in the culture medium (3.26 x lo7 cpm) was found to be incorporated into protein. When the acid hydrolysate of this [U-‘4C]lysine-labeled protein was analyzed by Dowex 50 H+ column chromatography, practically all of the incorporated radioactivity was recovered as basic amino acid fraction. When one-fourth of the radioactivity (recovered as basic amino acids) was applied on a Dowex 50 NH,+ column, the elution pattern shown in Fig. 3 was obtained. Three methylated lysines (E-N-mono-, l -N-di-, and l -N-trimethyllysine) were obtained with much higher yield than that observed with Salmonella typhimurium; 1.58% of the total radioactivity in the protein hydrolysate was recovered as methylated lysines.
8-
1 1 L
o-
FRACTION
NUMBER
FIG. 3. Chromatographic separation of acid hydrolysate of radioactive Neurospora crassa protein. One-fourth of the acid hydrolysate of Neurospora crassa protein which had been labeled with [UL4C]lysine according to the method described under Materials and Methods was analyzed on a Dowex 50 NH,+ column. Of the total radioactivity incorporated into protein, 1.58% was found to exist in the various methylated lysines at the ratio of TML:DML:MML at 1.O:1.12:0.36. All the conditions are the same as in Fig. 1 and the rest of the detailed experimental procedures are described under Materials and Methods.
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PAIK
ET AL.
Reductive Methylution of Protein as a Means of Producing Radiolabeled e-N-Methylated Lysines
Acid-hydrolyzed reductively methylated [methylJ4C]protein(3.42 x lo6 cpm) was charged on Dowex 50 H+ column. Of this, approximately 95% (3.24 x lo6 cpm) of the radioactivity was recovered in the basic amino acid fraction (in 1.5 N NH,OH eluant). Figure 4 illustrates the elution pattern of this basic amino acid fraction on a Dowex 50 NH,’ column. Approximately 94% of the charged radioactivity (3.05 x lo6 cpm) was recovered in the E-Ndimethyllysine and e-N-monomethyllysine peaks. As shown previously (9,10), reductive methylation of protein with formaldehyde and sodium borohydride did not produce E-N-trimethyllysine.
FRACTION
NUMBER
FIG. 4. Chromatographic separation of acid hydrolysate of histone which had been reductively methylated with [‘Tlformaldehyde. Detailed experimental procedures are the same as in Fig. I. and the rest of the procedures are described under Materials and Methods. Of the total charged radioactivity, 94.1% was recoverable in these two major peaks, and the ratio of radioactivity of DML:MML was 1: 1.21.
LABELED
e-N-METHYLLYSINES
271
DISCUSSION
Methods for synthesizing E-N-mono-, e-N-di-, or l -N-trimethyl-r-lysine have been known for some time (14-16). In addition, these compounds (unlabeled) are easily available from various commercial sources. However, when an investigator is lacking in experience in organic synthesis, and yet needs small amounts of these radioactive methylated lysines, he (or she) is confronted with a serious technical problem. In this paper, we have presented a method to synthesize small quantities of these compounds possessing radioactive labeling. The method has the following advantages; (i) by a simple procedure, three methylated lysines (e-N-mono-, l -N-di-, and e-N-trimethyllysine) can be synthesized biologically or chemically to possess radioactivity; (ii) the synthesized compounds could be separated in pure form; (iii) the specific activity of the compounds synthesized by either organisms or by reductive methylation of protein with [‘4C]formaldehyde could be dependent on the amounts of radioactive precursor compound used; and finally, (iv) the method also suggests the possibility of preparing methylated arginine or methylhistidine in a similar fashion. Among the various ways to prepare the methylated lysines, the Neurospora system appears to be superior to that of the Salmonella system, in that the overall yield of radioactivity found in the methylated lysines is much higher and that the system produces much higher amounts of c-N-trimethyllysine relative to r-N-mono- and e-N-dimethyllysine. When only e-N-mono- and l -N-dimethyllysine is needed, the reductive methylation method seems to be the preferred method. It is possible that in the future an organism(s) might be found that produces the three methylated lysines in a more favorable way than the two systems examined in this study. ACKNOWLEDGMENTS This work was supported by research Grants AM 09602 from the National Institute of Arthritis, Metabolism, and Digestive Diseases, CA 10439 and CA 12226 from the National Cancer Institute. and GM 20594 from the National Institute of General Medical Sciences.
REFERENCES 1. Paik, W. K., and Kim. S. (1975) in Advances in Enzomology (Meister, A., ed.). Vol. 42. p. 227. Wiley, New York. 2. Paik. W. K., and Kim. S. (1975) in Post-Synthetic Modification of Macromolecules (Antoni. F.. and Farago. A.. eds.). North-Holland/American Elsevier; FEB.$, 34, 127. 3. Paik. W. K. (1977) in The Biochemistry of S-Adenosylmethionine (Salvatore. F.. Borek, E.. Zappia, V., William-Ashman. H. G., and Schlenk. F., eds.). Columbia Univ. Press, New York. 4. LaBadie. J. H.. Dunn, W. A., and Aronson. N. N. (1976) Biochem. J. 160, 85. 5. Home, D. W., and Broquist, H. P. (1973) J. Bid. Chent. 248, 2170. 6. Tronick, S. R.. and Martinez. R. J. (1971) J. Brrcteriol. 105, 21 I.
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7. Davis, R. H., and DeSerres, F. J. (1970) in Methods in Enzymology (Tabor, H., and Tabor, C. W., eds.), Vol. 172, pp. 79, Academic Press, New York. 8. Co&on, J. W., and Kapoor, M. (1973) Canad. J. Microbial. 19, 427. 9. Means, G. E., and Feeney, R. E. (1968) Biochemistry 7, 2192. 10. Paik, W. K., and Kim, S. (1972) Biochemistry 11, 2589. 11. Markiw, R. T. (1975) Biochem. Med. 13, 23. 12. Durban, E., Nochumson, S., Kim. S., Paik, W. K., and Chan, S-K. (1978)5. Biol. Chem. 253, 1427. 13. Paik, W. K., and Kim, S. (1967) Biochem. Biophys. Res. Commun. 27, 479. 14. Benoiton, L. (1964) Canad. J. Chem. 42, 2043. 15. Neuberger, A., and Danger, F. (1944) Biochem. J. 38, 125. 16. DeLange, R. J., Glazer, A. N., and Smith, E. L. (1969) J. Biol. Chem. 244, 1385.
