Chapter g
Methods for the Assessment of Histone Methylation PAUL BWOET
AND
DAVID F. SAYRE
Department of Pathology. University of South Florida College of Medicine and Veterans Administration Hospital, Tampa, Florida
C. STUART BAXTER Department of Environmental Health, Kettering Laboratory. University of Cincinnati Medial Center, Cincinnati. Ohio
1. Introduction N-methylation of internal histone lysine residues in nuclear chromatin occurs during the last part of S phase and G , (1-3). It involves specific lysine residues in histones H3 and H4, which have been identified as residues 9 and 27 in calf thymus histone H3, and as residue 20 in H4. The methylation reaction is catalyzed by histone lysine methyltransferase(s) and utilizes Sadenosylmethionine as methyl donor. The enzyme has been characterized and purified from cell nuclei by Paik and Kim (4), who called it methylase 111.
11. Principle Uptake of radiomethyl into histones can be measured in vivo and in vitro. In vitro measurements can be carried out with isolated nuclei or with the isolated histone methyltransferase, which will methylate free histones in the presence of S-adenosylmethionine. This latter system is discussed by Paik and Kim ( 5 ) ; see also this volume, Chapter 7. For in vitro protocols using isolated nuclei, S-adenosylmethionine (Me-”-labeled) should also be used as methyl donor. For in vivo experiments, however, methyl-labeled methionine should 95
96
PAUL BWOET ri a/.
be employed. This label has the disadvantage of being available to the cellular synthetic machinery in the form of labeled methionine, as well as labeled S-adenosylmethionine. Histones (and other proteins) will therefore become labeled owing to incorporation of labeled methionine and/or radiomethyl.
111. In Vivo Assessment of Histone Methylation After administration of the appropriate amount of radioactivity in the form of Me-3Hor '*C-labeled 1-methionine,tissues are isolated or cells are harvested after the desired time interval. Nuclei are isolated according to the usual methods (6)and washed several times with 20-30 volumes of saline-EDTA [0.08M NaC1-0.02 M EDTA (PH 7.4)] by suspension with a Potter-Elvehjem homogenizer and spinning at 2000 g for 10 minutes. The pellet can then be extracted according to the method of Johns (7.8). Whole histones can be prepared by extracting twice with 4-5 volumes of 0.25 N HCl and clarifying the combined extracts at 12,000 g for 15 minutes. Histones are precipitated by addition of 6 volumes of acetone and stored overnight at -2OOC. The precipitate is collected by centrifugation at 2000 g for 15 minutes and subsequently resuspended in acetone-1% HCl and acetone, with centrifugations at 2000 gfor 15minutes, and finally air dried. This histone preparation can then be fractionated by P-60gel chromatography according to von Holt and co-workers (9). This method proved to be very useful in our hands, although alkylation, according to Dixon and Candido (lo), is recommended to prevent aggregation of those H3 (e.g., calf thymus)moleculesthat contain two -SH groups. Histones (up to 20 mg) are dissolved in 0.4 ml of 5 M urea-20 mM dithiothreitol-0.1 M sodium borate buffer (pH 8.9) and incubated at room temperature for 90minutes; then solid iodacetamide is added to a concentration of 0.08 M,and incubation is continued for another 90 minutes.
IV. Uptake of Radiomethyl into Methylated Lysine Derivatives To distinguish radioactivity due to actual incorporation of radiomethyl into methyllysine derivatives from that due to incorporation of the amino acid methionine, some form of amino acid chromatography is required. In
9.
ASSESSMENT OF HISTONE METHYLATION
97
the simplest form, this can be performed on Dowex W-X8in the ammonia form. After hydrolysis of the histone sample as described below, it is charged to the column (0.9 x 3 cm) in dilute acid (HCl, pH 2.0).Neutral and acidic amino acids are then eluted with water (10 ml) and basic amino acids (containing the radiomethyl) with 3 N NH,OH (5 ml). The radioactivity of the water fraction, which stems from methionine, can be useful as a measure of histone synthetic activity (ZI). If, in addition, it is desirable to estimate incorporation of radiomethyl into N-mono-, di-, and trimethyllysine, more extensive chromatography is indicated: Histone fractions are hydrolyzed in 5.9 N HCl at 110°C for 16 hours in N,-flushed, sealed glass ampoules. The HC1 is removed by cryogenic transfer from the hydrolyzates which are then dissolved in dilute HCl of pH 2. A concentrated sucrose solution is added to bring the concentration to 10-20%. The sample is then layered on a0.9 x 50 cm AA-15 resin (Beckman) column, equilibrated with 0.35 N citrate buffer (pH 6.95), which is also the eluting buffer (12). Pressure of elution is 17 atm; flow rate is approximately 30 ml/per hour. The column jacket is kept at 2 8 O +0.02".Fractions can be analyzed by ninhydrin, e.g., by means of the Technicon Automated Amino Acid Analyzer detection system. The effluent can be mixed with toluene; 1:2, 5.5 gm of PPO,0.1 gm of liquid scintillation cocktail (TX-100, POPOP per liter) without noticeable quenching, except in very highly con-
% x
h '
53-
*t
i
2-
I
!IK
& I
,m-
.......,
FIG. 1. A 1 x 21 cm column of Aminex A5 (Bio-Rad Laboratories) was eluted at 22OC with 0.35 N citrate buffer (pH 6.48) at a flow rate of 1 ml/min (13,II).The column effluent was directed through an anthracene crystal scintillation detector cell installed in a twochannel scintillation spectrometer adapted for flow operations (Packard Tricarb Flow System). The rate meter output for both channels, each set at different full-scale ranges, was recorded using a two-channel linear recorder. The first two major peaks to emerge were tentatively identified as methionine and methionine sulfoxide, respectively. The sulfoxide derivative peak was considerably diminished if hydrolyses were performed in an inert atmosphere. The next triad of peaks represents monomethyl-. dimethyl-, and trimethyl lysine, respectively. The last peak, emerging near the position of arginine and at the reported position of p-monomethyl arginine (13), was identified as this monomethyl derivative of arginine.
98
PAUL BWOET ei a/.
centrated fractions. Since the methylated derivatives are present in low concentration, this generally does not present a problem. Alternatively, a stream splitting device can be utilized to divert part of the effluent through an analytical system based on the ninhydrin reaction or one of the more sensitive fluorometric detection procedures now available which utilize Fluorescamhe@ (Roche), or Fluoropa@ (Durrum). Radioactivity can of course also be measured continuously in a flow cell, as demonstrated in Fig. 1 (13,14). The insensitivity of this type of detection, however, requires a high level of radioactivity. In addition to the procedures detailed above, other methods, specifically designed for the separation of methylated lysines, have been described (15-17). Most of these methods can also be used to detect methylarginine and methylhistidine, the presence of which has been reported in histones and nonhistone chromosomal proteins (14,18-20).
V. In Vi/itroAssessment of Histone Methylation The in vitro methylation of histones in isolated nuclei seems for all practical purposes to be very similar to the in vivo process. Incorporation takes place only into H3 and, to a lesser extent, into H4,in the form of mono- di-, and trimethyllysine. The main advantage is that, under these conditions, Sadenosylmethionine can be used as the radiomethyl donor, and thus incorporation of radioactivity into histones is solely due to N-methylation. It is necessary, however, to purify the labeled histones following incubation in the presence of S-adenosylmethionine, since under these conditions, active methylation of DNA and nonhistone proteins is taking place as well. Finally, it should be emphasized that, in a tissue such as rat liver, only a small percentage of the total cell population will incorporate radiomethyl into its histones at any given time, and that the phenomena observed may well be restricted to only those (premitotic) cells in which histone methylation is taking place. The following is a procedure used in the author’s laboratory for measurement of radiomethyl uptake into histones from rat liver nuclei incubated in the presence of methyl-labeled S-adenosylmethionine. Nuclei from 0.5 gm of rat liver, fresh or stored at -60°C with an overlay of 2.1 M sucrose-3 mM CaCl,, are incubated at 37OC with 2.5 pCi S-adenosyl-[methyPH] L-methionine (8 Ci/mmole) for 15 minutes in a mix containing 0.25 M sucrose-0.05 M Tris-HC1-0.02 M KCI-0.01 M MgCI,-0.01 M mercaptoethanol (pH 8.5) (at 37°C) (total volume 0.5 ml).
9.
ASSESSMENT OF HISTONE METHYLATION
99
Nuclei from 10 gm liver are gently suspended in a mixture of 2 ml of quintuply concentrated buffer (i.e., 1.25 M sucrose-0.25 MTrislHC1-O.l M KC1-0.05 M MgC1,-0.05 M mercaptoethanol (pH 8.5) (37°C) and 4 ml of water. For each individual incubation mixture, 300-pl aliquots of this suspension are used, and duplicates are run in each case. Water is added to make the volume up to 400 pl; 50 pl of S-adenosylmethionine solution (100 pCi/2 ml) is added, the mixture is shaken, and the label is rinseddown with 50 pl of water. Mixtures are then incubated with shaking at 37OC for 15 minutes. The reaction is stopped by chilling and addition of 2.5 ml NaCl/EDTA (0.08 :0.02 M )(pH 7.2). The mixtures are centrifuged at 4°C at top speed in a Sorvall GLC-1 centrifuge (3200 rpm = 2000 g) for 10 minutes. The pellet is washed twice more with NaCl/EDTA, centrifuging at 2000 g after washing, and then extracted with 0.25 N HCl(O.6 ml), followed by centrifugation, then extracted further with 0.3 ml of 0.25 N HCl. The extracts are combined, centrifuged, and made 25% trichloroacetic acid by addition of 0.3 ml of 100% (w/v) trichloroacetic acid. The tubes are left overnight at 4”C, centrifuged at 2000 g for 20 minutes; the residue is washed consecutively with 1% HCl in acetone (1 ml), acetone (1 ml), and ether (1 ml), each step being followed by centrifugation at 2000 g for 10 minutes. The residue is dried under vacuum for 30-60 minutes, dissolved in 1 ml of water and stirred vigorously. The tubes are allowed to stand at 4°C for 30 minutes, stirred again (on Vibromixer), and centrifuged at 2000 g for 10 minutes; aliquots are tested for radioactivity by addition to 10 ml of toluene-based scintillation cocktail described above. Protein concentration can be measured by either the Hartree method (21) or fluorometry (22). It is advisable to use the specific radioactivity of histones with respect to radiomethyl if substances other than those listed above are added to the incubation mixture, since they may influence the yield of histone. Some loss of chromatin generally occurs during incubation, probably owing to nuclease activity, and this activity may be inhibited or stimulated by certain conditions. Finally, alternative methods of incubation have been described (23-25)with slight variations from the one detailed above.
REFERENCES 1. Tiddwell, T., Allfrey, V. A., and Mirsky, A. E., J. Biol. Chem. 243,707 (1968). 2. Shepherd, G.R., Hardin, J. M., and Noland, B. J., Arch. Biochem. Biophys. 143,I(1971). 3. Lee, H. W., Paik,W. K., and Borun, T. W., J. Bid. Chem. 248,4194(1973). 4. Paik, W. D., and Kim, S., Science 174, 114 (1971). 5. Paik, W.D., and Kim, S., J. Biol. Chem. 245, 6010 (1970). 6. L. Grossman and K. Moldave, eds., “Methods in Enzymology,” Vol. 12, Part A, Sect. 111. Academic Press, New York, 1%7. 7. Johns, E. W., Biochem. J. 92, 55 (1964).
100
PAUL BYVOET et a/.
8. Oliver, D., Sommer, K. R., Panyim, S., Spiker. S., and Chalkley, R., Biochem. J. 129,349 (1971). 9. Bohm, E. L., Strickland, W. N., Strickland, M.J., Thwaits, B. H.. van der Westhuyzen, D. R., and von Holt, C., FEES Lett. 34,217 (1973). 10. Candido, E. P. M.,and Dixon,G. H., J. Biol. Gem. 247, 3868 (1972). 11. Byvoet, P., Arch. Biochem. Biophys. 152, 887 (1972). 12. Lange, H. W.,Lower, R.,and Hempel, K., J. Gromarogr. 76,252 (1973). 13. Seely, J. H., Edattel, S. R. and Benoiton, N. L., J. Qlromatogr. 44,618 (1969). 14. Byvoet, P., Shepherd, G. R., Hardin, J. M., andNoland, B. J., Arch. Biochem. Biophys. 148, 558 (1972). 15. Duerre, J. A., and Chakrabarty, S., J. Biol. G e m . 250, 8457 (1975). 16. Honda, B. M., Dixon, G. H., and Candido, E. P. M., J. Biol. G e m . 250, 8681 (1975). 17. Deibler, G. E., and Martenson, R. E.,J. Biol. G e m . 248,2387 (1973). 18. Gershey, E. L., Haslett, G. W., Vidali, G., and Allfrey, V. G., J. Biol. Qlem. 244,4871 (1%9). 19. Paik,W. K., and Kim, S., Biochem Biophys. Res. Commun. 40,224 (1970). 20. Byvoet, P., Biochim. Biophys. Ada 238,375a (1971). 21. Hartree, E. F., AMI. Biodtem. 48,422 (1972). 22. Bohlen, P., Stein, S., Dairman, W., and Udenfriend, S., Ardr. Biochem. Biophys. 155,213 (1 973). 23. Lee, C. T., and Duerre, J. A., Nature (London)251,240 (1974).
24. Sekeris, C. E., Sekeris, K. E., and Gallwitz, D., Hoppe-Seyler’s Z. Physiol. Qlem. 348, 1660( 1%7). 25. Thomas, G., Lange, H.W., and Hempel, K., Hoppe-Seyler’s Z. Physiol. G e m . 353, 1423 (1972).
Methods for the Assessment of Histone Methylation PAUL BWOET
AND
DAVID F. SAYRE
Department of Pathology. University of South Florida College of Medicine and Veterans Administration Hospital, Tampa, Florida
C. STUART BAXTER Department of Environmental Health, Kettering Laboratory. University of Cincinnati Medial Center, Cincinnati. Ohio
1. Introduction N-methylation of internal histone lysine residues in nuclear chromatin occurs during the last part of S phase and G , (1-3). It involves specific lysine residues in histones H3 and H4, which have been identified as residues 9 and 27 in calf thymus histone H3, and as residue 20 in H4. The methylation reaction is catalyzed by histone lysine methyltransferase(s) and utilizes Sadenosylmethionine as methyl donor. The enzyme has been characterized and purified from cell nuclei by Paik and Kim (4), who called it methylase 111.
11. Principle Uptake of radiomethyl into histones can be measured in vivo and in vitro. In vitro measurements can be carried out with isolated nuclei or with the isolated histone methyltransferase, which will methylate free histones in the presence of S-adenosylmethionine. This latter system is discussed by Paik and Kim ( 5 ) ; see also this volume, Chapter 7. For in vitro protocols using isolated nuclei, S-adenosylmethionine (Me-”-labeled) should also be used as methyl donor. For in vivo experiments, however, methyl-labeled methionine should 95
96
PAUL BWOET ri a/.
be employed. This label has the disadvantage of being available to the cellular synthetic machinery in the form of labeled methionine, as well as labeled S-adenosylmethionine. Histones (and other proteins) will therefore become labeled owing to incorporation of labeled methionine and/or radiomethyl.
111. In Vivo Assessment of Histone Methylation After administration of the appropriate amount of radioactivity in the form of Me-3Hor '*C-labeled 1-methionine,tissues are isolated or cells are harvested after the desired time interval. Nuclei are isolated according to the usual methods (6)and washed several times with 20-30 volumes of saline-EDTA [0.08M NaC1-0.02 M EDTA (PH 7.4)] by suspension with a Potter-Elvehjem homogenizer and spinning at 2000 g for 10 minutes. The pellet can then be extracted according to the method of Johns (7.8). Whole histones can be prepared by extracting twice with 4-5 volumes of 0.25 N HCl and clarifying the combined extracts at 12,000 g for 15 minutes. Histones are precipitated by addition of 6 volumes of acetone and stored overnight at -2OOC. The precipitate is collected by centrifugation at 2000 g for 15 minutes and subsequently resuspended in acetone-1% HCl and acetone, with centrifugations at 2000 gfor 15minutes, and finally air dried. This histone preparation can then be fractionated by P-60gel chromatography according to von Holt and co-workers (9). This method proved to be very useful in our hands, although alkylation, according to Dixon and Candido (lo), is recommended to prevent aggregation of those H3 (e.g., calf thymus)moleculesthat contain two -SH groups. Histones (up to 20 mg) are dissolved in 0.4 ml of 5 M urea-20 mM dithiothreitol-0.1 M sodium borate buffer (pH 8.9) and incubated at room temperature for 90minutes; then solid iodacetamide is added to a concentration of 0.08 M,and incubation is continued for another 90 minutes.
IV. Uptake of Radiomethyl into Methylated Lysine Derivatives To distinguish radioactivity due to actual incorporation of radiomethyl into methyllysine derivatives from that due to incorporation of the amino acid methionine, some form of amino acid chromatography is required. In
9.
ASSESSMENT OF HISTONE METHYLATION
97
the simplest form, this can be performed on Dowex W-X8in the ammonia form. After hydrolysis of the histone sample as described below, it is charged to the column (0.9 x 3 cm) in dilute acid (HCl, pH 2.0).Neutral and acidic amino acids are then eluted with water (10 ml) and basic amino acids (containing the radiomethyl) with 3 N NH,OH (5 ml). The radioactivity of the water fraction, which stems from methionine, can be useful as a measure of histone synthetic activity (ZI). If, in addition, it is desirable to estimate incorporation of radiomethyl into N-mono-, di-, and trimethyllysine, more extensive chromatography is indicated: Histone fractions are hydrolyzed in 5.9 N HCl at 110°C for 16 hours in N,-flushed, sealed glass ampoules. The HC1 is removed by cryogenic transfer from the hydrolyzates which are then dissolved in dilute HCl of pH 2. A concentrated sucrose solution is added to bring the concentration to 10-20%. The sample is then layered on a0.9 x 50 cm AA-15 resin (Beckman) column, equilibrated with 0.35 N citrate buffer (pH 6.95), which is also the eluting buffer (12). Pressure of elution is 17 atm; flow rate is approximately 30 ml/per hour. The column jacket is kept at 2 8 O +0.02".Fractions can be analyzed by ninhydrin, e.g., by means of the Technicon Automated Amino Acid Analyzer detection system. The effluent can be mixed with toluene; 1:2, 5.5 gm of PPO,0.1 gm of liquid scintillation cocktail (TX-100, POPOP per liter) without noticeable quenching, except in very highly con-
% x
h '
53-
*t
i
2-
I
!IK
& I
,m-
.......,
FIG. 1. A 1 x 21 cm column of Aminex A5 (Bio-Rad Laboratories) was eluted at 22OC with 0.35 N citrate buffer (pH 6.48) at a flow rate of 1 ml/min (13,II).The column effluent was directed through an anthracene crystal scintillation detector cell installed in a twochannel scintillation spectrometer adapted for flow operations (Packard Tricarb Flow System). The rate meter output for both channels, each set at different full-scale ranges, was recorded using a two-channel linear recorder. The first two major peaks to emerge were tentatively identified as methionine and methionine sulfoxide, respectively. The sulfoxide derivative peak was considerably diminished if hydrolyses were performed in an inert atmosphere. The next triad of peaks represents monomethyl-. dimethyl-, and trimethyl lysine, respectively. The last peak, emerging near the position of arginine and at the reported position of p-monomethyl arginine (13), was identified as this monomethyl derivative of arginine.
98
PAUL BWOET ei a/.
centrated fractions. Since the methylated derivatives are present in low concentration, this generally does not present a problem. Alternatively, a stream splitting device can be utilized to divert part of the effluent through an analytical system based on the ninhydrin reaction or one of the more sensitive fluorometric detection procedures now available which utilize Fluorescamhe@ (Roche), or Fluoropa@ (Durrum). Radioactivity can of course also be measured continuously in a flow cell, as demonstrated in Fig. 1 (13,14). The insensitivity of this type of detection, however, requires a high level of radioactivity. In addition to the procedures detailed above, other methods, specifically designed for the separation of methylated lysines, have been described (15-17). Most of these methods can also be used to detect methylarginine and methylhistidine, the presence of which has been reported in histones and nonhistone chromosomal proteins (14,18-20).
V. In Vi/itroAssessment of Histone Methylation The in vitro methylation of histones in isolated nuclei seems for all practical purposes to be very similar to the in vivo process. Incorporation takes place only into H3 and, to a lesser extent, into H4,in the form of mono- di-, and trimethyllysine. The main advantage is that, under these conditions, Sadenosylmethionine can be used as the radiomethyl donor, and thus incorporation of radioactivity into histones is solely due to N-methylation. It is necessary, however, to purify the labeled histones following incubation in the presence of S-adenosylmethionine, since under these conditions, active methylation of DNA and nonhistone proteins is taking place as well. Finally, it should be emphasized that, in a tissue such as rat liver, only a small percentage of the total cell population will incorporate radiomethyl into its histones at any given time, and that the phenomena observed may well be restricted to only those (premitotic) cells in which histone methylation is taking place. The following is a procedure used in the author’s laboratory for measurement of radiomethyl uptake into histones from rat liver nuclei incubated in the presence of methyl-labeled S-adenosylmethionine. Nuclei from 0.5 gm of rat liver, fresh or stored at -60°C with an overlay of 2.1 M sucrose-3 mM CaCl,, are incubated at 37OC with 2.5 pCi S-adenosyl-[methyPH] L-methionine (8 Ci/mmole) for 15 minutes in a mix containing 0.25 M sucrose-0.05 M Tris-HC1-0.02 M KCI-0.01 M MgCI,-0.01 M mercaptoethanol (pH 8.5) (at 37°C) (total volume 0.5 ml).
9.
ASSESSMENT OF HISTONE METHYLATION
99
Nuclei from 10 gm liver are gently suspended in a mixture of 2 ml of quintuply concentrated buffer (i.e., 1.25 M sucrose-0.25 MTrislHC1-O.l M KC1-0.05 M MgC1,-0.05 M mercaptoethanol (pH 8.5) (37°C) and 4 ml of water. For each individual incubation mixture, 300-pl aliquots of this suspension are used, and duplicates are run in each case. Water is added to make the volume up to 400 pl; 50 pl of S-adenosylmethionine solution (100 pCi/2 ml) is added, the mixture is shaken, and the label is rinseddown with 50 pl of water. Mixtures are then incubated with shaking at 37OC for 15 minutes. The reaction is stopped by chilling and addition of 2.5 ml NaCl/EDTA (0.08 :0.02 M )(pH 7.2). The mixtures are centrifuged at 4°C at top speed in a Sorvall GLC-1 centrifuge (3200 rpm = 2000 g) for 10 minutes. The pellet is washed twice more with NaCl/EDTA, centrifuging at 2000 g after washing, and then extracted with 0.25 N HCl(O.6 ml), followed by centrifugation, then extracted further with 0.3 ml of 0.25 N HCl. The extracts are combined, centrifuged, and made 25% trichloroacetic acid by addition of 0.3 ml of 100% (w/v) trichloroacetic acid. The tubes are left overnight at 4”C, centrifuged at 2000 g for 20 minutes; the residue is washed consecutively with 1% HCl in acetone (1 ml), acetone (1 ml), and ether (1 ml), each step being followed by centrifugation at 2000 g for 10 minutes. The residue is dried under vacuum for 30-60 minutes, dissolved in 1 ml of water and stirred vigorously. The tubes are allowed to stand at 4°C for 30 minutes, stirred again (on Vibromixer), and centrifuged at 2000 g for 10 minutes; aliquots are tested for radioactivity by addition to 10 ml of toluene-based scintillation cocktail described above. Protein concentration can be measured by either the Hartree method (21) or fluorometry (22). It is advisable to use the specific radioactivity of histones with respect to radiomethyl if substances other than those listed above are added to the incubation mixture, since they may influence the yield of histone. Some loss of chromatin generally occurs during incubation, probably owing to nuclease activity, and this activity may be inhibited or stimulated by certain conditions. Finally, alternative methods of incubation have been described (23-25)with slight variations from the one detailed above.
REFERENCES 1. Tiddwell, T., Allfrey, V. A., and Mirsky, A. E., J. Biol. Chem. 243,707 (1968). 2. Shepherd, G.R., Hardin, J. M., and Noland, B. J., Arch. Biochem. Biophys. 143,I(1971). 3. Lee, H. W., Paik,W. K., and Borun, T. W., J. Bid. Chem. 248,4194(1973). 4. Paik, W. D., and Kim, S., Science 174, 114 (1971). 5. Paik, W.D., and Kim, S., J. Biol. Chem. 245, 6010 (1970). 6. L. Grossman and K. Moldave, eds., “Methods in Enzymology,” Vol. 12, Part A, Sect. 111. Academic Press, New York, 1%7. 7. Johns, E. W., Biochem. J. 92, 55 (1964).
100
PAUL BYVOET et a/.
8. Oliver, D., Sommer, K. R., Panyim, S., Spiker. S., and Chalkley, R., Biochem. J. 129,349 (1971). 9. Bohm, E. L., Strickland, W. N., Strickland, M.J., Thwaits, B. H.. van der Westhuyzen, D. R., and von Holt, C., FEES Lett. 34,217 (1973). 10. Candido, E. P. M.,and Dixon,G. H., J. Biol. Gem. 247, 3868 (1972). 11. Byvoet, P., Arch. Biochem. Biophys. 152, 887 (1972). 12. Lange, H. W.,Lower, R.,and Hempel, K., J. Gromarogr. 76,252 (1973). 13. Seely, J. H., Edattel, S. R. and Benoiton, N. L., J. Qlromatogr. 44,618 (1969). 14. Byvoet, P., Shepherd, G. R., Hardin, J. M., andNoland, B. J., Arch. Biochem. Biophys. 148, 558 (1972). 15. Duerre, J. A., and Chakrabarty, S., J. Biol. G e m . 250, 8457 (1975). 16. Honda, B. M., Dixon, G. H., and Candido, E. P. M., J. Biol. G e m . 250, 8681 (1975). 17. Deibler, G. E., and Martenson, R. E.,J. Biol. G e m . 248,2387 (1973). 18. Gershey, E. L., Haslett, G. W., Vidali, G., and Allfrey, V. G., J. Biol. Qlem. 244,4871 (1%9). 19. Paik,W. K., and Kim, S., Biochem Biophys. Res. Commun. 40,224 (1970). 20. Byvoet, P., Biochim. Biophys. Ada 238,375a (1971). 21. Hartree, E. F., AMI. Biodtem. 48,422 (1972). 22. Bohlen, P., Stein, S., Dairman, W., and Udenfriend, S., Ardr. Biochem. Biophys. 155,213 (1 973). 23. Lee, C. T., and Duerre, J. A., Nature (London)251,240 (1974).
24. Sekeris, C. E., Sekeris, K. E., and Gallwitz, D., Hoppe-Seyler’s Z. Physiol. Qlem. 348, 1660( 1%7). 25. Thomas, G., Lange, H.W., and Hempel, K., Hoppe-Seyler’s Z. Physiol. G e m . 353, 1423 (1972).
