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Chapter 15. Chromosomal Protein Phosphorylation on Basic Amino Acids
Chapter
15
Chromosomal Protein Phosphotylation on Basic Amino Acids ROBERTS A. SMITH, RICHARD M. HALPERN, BERNDT B. BRUEGGER, ALBERT K. DUNLAP, AND OSCAR FRICKE Department of Chemistty, University of California. Los Angeles, Los Angeles. California
I. General Considerations Phosphorylation of chromosomal proteins has generally been measured either after isolation of phosphorylated chromosomal proteins from the nucleus or after the action of a chromosomal protein kinase in an in vitro system. Almost totally, these procedures involve one or the other step carried out at an acidic pH value such that the only surviving phosphoryl linkage is found on the hydroxyl group of serine or threonine. Usually the protein is precipitated, generally by acid, followed by suitable washing or extraction to remove nonprotein phosphorus components. Sometimes the protein is dissolved or suspended in a suitable medium followed by removal of nonprotein phosphorus components by ion exchange or extraction procedures. Almost invariably these procedures involve the use of dilute mineral acid solutions in order to avoid chromosomal protein aggregation and/or proteolytic degradation. Such procedures obviously preclude the detection and isolation of phosphorylated components labile to the acidic pH values employed. N-phosphorylated compounds have been recognized as phosphoryl donors since Fiske and SubbaRow ( 1 ) identified N-phosphorylcreatine. The occurrence of N-phosphoryl linkages in proteins was first discovered by Boyer and his colleagues (2) with their isolation of N-phosphorylated succinyl-CoA synthase from mitochondria. The very rapid identification of the N-phosphorylated species as 3-phosphohistidine was quickly followed 153
154
ROBERTS A. SMITH et al.
by the demonstration of other N-phosphorylated enzymes, such as the phosphoenolpyruvate transferase of Roseman (3) and the phosphoramidatehexose transferase system described by Stevens-Clark et al. (4). The single most outstanding property of N-phosphorylated compounds is their extreme sensitivity to acidic pH and their relative stability under basic conditions (5-7). Most N-phosphorylated species have half-lives of less than a few minutes in 10--2 M mineral acid at room temperature, while they are virtually stable at alkaline pH values. This property is in contrast to that of phosphorylated peptide-bound serine, which is quite stable at normally employed acidic pH values, but undergoes rapid pelimination at alkaline pH values (8). Free phosphoserine, on the other hand, is relatively more base stable (9). It is this difference in properties between the O-phosphoryl and N-phosphoryl linkages in proteins that forms the basis of procedures we have used thus far for isolation and demonstration of such N-posphoryl linkages in histones (10, 11). The procedures described herein permit the detection of N-phosphoryl amino acids in chromosomal proteins based upon isolation of 32P-labeled N-phosphoryl substances. The 3zP-labeledchromosomal proteins are isolated either after enzymic reactions have taken place utilizing y-32P-labeled ATP as phosphoryl donor or directly from whole animal tissues excised and fractionated after rapid sacrifice of animals following administration of 32Piintraperitoneally. We have used the procedures directly most with rat liver preparations and/or rat mammary tumor Walker-256 carcinosarcoma or directly from enzyme reaction mixtures.
11. Phenol Extraction Procedure A. Principles Chromosomal proteins and lipids are extracted into phenol, while 32Pi and/or [y3zP]ATPare removed by careful washing of the phenol layer. 3zP left in the phenol extract gives a measure of the total covalently bound protein and lipid 32P.For further elucidation of the nature of the phosphorylated material, the radioactivity can be precipitated from phenol by acetone and the water-insoluble lipid phosphate extracted from the precipitate by organic solvents. The phosphate subsequently released by acid hydrolysis is a measure of the N-phosphorylated amino acid, and phosphoserine and phosphothreonine are measured as the acid-stable fraction. The following procedure is slightly modified from that of Boyer and Bieber (12).
IS.
PHOSPHORYLATION OF BASIC AMINO ACIDS
155
B. Procedure Incubation mixtures containing from 0.5 to 20 mg of chromosomal proteins in 1-2 ml were stopped with 2 ml of 88% phenol (adjusted to pH 8 with 0.01 M sodium phosphate buffer). Each mixture was added to a 40-ml centrifuge tube containing an additional 8 ml of the same solution. The solution was mixed thoroughly with 25 ml of a washing buffer [0.01 M EDTA4.01 M sodium pyrophosphate (PH 8.3)] and centrifuged at about 2500 rpm in a swinging-bucket clinical centrifuge. The upper aqueous layer was removed by careful aspiration. Wash buffer was again added, and the washing procedure was repeated six times. The 12P-containingproteins were then precipitated by the rapid addition of about 5 volumes of cold acetone. The mixture was usually stored at - 20° C for several hours or overnight, and the precipitate was ultimately collected by centrifugation. Any lipids were removed by successive extraction with about 5 ml of chloroform-methanol (1 :1, v:v), 5 ml chloroform-methanol-water (20: 10: 1, v:v:v), and 5 ml of methanol, in each case centrifuging in a clinical centrifuge to separate the residue from the organic solvents. The protein residue can be treated with 3 ml of 0.3 N trichloroacetic acid-1 mM sodium phosphate for 3 minutes in a boiling water bath. The mixture is cooled and the proteins removed by centrifugation in a clinical centrifuge. An aliquot of the supernatant fluid is counted, giving a measure of the acid-labile N-phosphoryl protein phosphate. The protein residue is washed twice with about 10 ml of phosphate buffer, and the 12Premaining with the precipitate is used as a measure of the acid-stable phosphoprotein. In our experience, very little 32P-phospholipidis formed in the short enzymic incubation mixtures, and often we dispense with the solvent extraction procedure for such mixtures. In such cases, phosphoserine and phosphothreonine are eliminated, since we usually stop the reaction by making the mixture 0.15 M in sodium hydroxide and heating it to 6OoC for 15 minutes prior to the phenol extraction. Thus, 12Premaining in the phenol layer is a direct measure of the amount of N-phosphorylated protein formed.
111. Isolation of ["PI Phosphohistones from Whole Animal Tissues Rats were sacrificed by decapitation between 1 and 3 hours after the intraperitoneal administration of IzPi in 0.9% NaCl solution. The tissues of choice (usually liver, regenerating liver, or Walker-256 carcinosarcoma)
156
ROBERTS A. SMITH cr al.
were rapidly excised and placed in ice cold 0.25 Msucrose-0.05 MTris. HCl (pH 7.5)-0.05 M NaCl. After the adhering connective tissues were removed, the nuclei were isolated by a modification of the method of Chauveau et al. (13). All procedures were performed at ice bath temperatures. The tissues were minced with scissors in an isotonic solution containing 0.25 Msucrose and 1 mM MgCl,. The minced tissue was homogenized with a PotterElvehjem homogenizer using a motor-driven pestle (0.005-0.007 inch clearance) for 40 strokes. The homogenate was filtered through one layer of coarse cheesecloth, followed successively by filtration through one layer of fine cheesecloth and eight layers of fine cheesecloth. The nuclei werepelleted ,by centrifugation at 1000 g for 10 minutes, and then resuspended in a solution containing 1 mM MgCl, and 2.2 M sucrose. The suspension was centrifuged at 40,OOO g for 90minutes. The pellet was washed twice with a solution of 1 M sucrose-1 mM MgCl, by homogenation with a loose-fitting Teflon pestle, followed by recentrifugation at 3000 g for 10 minutes. Chromatin was prepared from the nuclei by washing them with a0.15 MNaCl solution. The chromatin was subsequently washed 3 times with 0.05 M TriseHCl buffer (pH 7.5) followed by three washings with 0.15 M NaCl. The total chromosomal proteins were isolated by dissociation of the purified chromatin for 4 hours with constant stirring in an ice bath in 2.5 M guanidinium hydrochloride-0.1 M sodium phosphate buffer (pH 7). The DNA was removed by centrifugation at 90,OOO g for 10 hours. In separate aliquots, the phosphoserine linkages were almost totally removed by treatment at 6OoC for 10 minutes withO.l NNaOH (theacid0.4
0
0
I
I
1
I
!-.-.I
!
0.3 -
1
FRACTION
40 8
5 0
230-6 0
x
NUMBER
FIG. 1. Chromatography of acid-labile histone phosphates and acidic proteins on Bio-Rex 70 resin. Incorporation of radioactive inorganic phosphate, isolation of histones and acidic proteins, removal of acid-stable protein phosphates, and chromatographic separation were performed as described (11). Calf thymus histonts (3 rng) were added as carriers during column chromatography. - - -, OD,,, (Cl, CCOOH turbidity); -, radioactivity (,,P incorporated in vivo).
15.
PHOSPHORYLATION OF BASIC AMINO ACIDS
157
labile phosphoryl linkages were eliminated by treatment of an aliquot at 60°C for 10 minutes with 1 N HCl). This treatment was followed by dialysis overnight against two changes of 2 liters each of 0.01 M sodium phosphate buffer (pH 7). Separation of the chromosomal proteins was carried out on a Bio-Rex 70 column (0.6 x 60 cm). The elution was performed with a total of 50 ml of a linear gradient of 8-13% guanidinium HC1 in 0.1 M phosphate buffer (pH 6.8) prior to elution with 40% guanidinium hydrochloride in the same buffer essentially as described by Bonner et al. (14). For most of our experiments, we also added 3 mg of whole calf thymus histone to the column so that the appropriate histone peak could easily be identified by the TCA turbidity method (14). Radioactivity was determined by liquid scintillation spectrometry. A typical result showing the distribution of radioactivity associated with the chromosomal proteins eluted from a Bio-Rex 70 resin as described above is shown in Fig. 1.
IV. Degradation of Phosphoproteins and Demonstration of N-Phosphoryl Amino Acids
The separate peaks from the Bio-Rex 70 column after a whole tissue run or from an in vitro kinase reaction were pooled and dialyzed against two changes of 1 liter each ofO.O1 MTris. HCl buffer (pH 8)and concentrated by lyophilization. The residue was usually suspended in 0.1% NaHCO,(pH 8.5) and incubated with trypsin (on a weight-to-weight ratio of 100: 1, protein sample to proteolytic enzyme) for 2 hours at 37OC. After the trypsin incubation, Pronase was added to the reaction mixture (45 proteolytic units per milligram of Pronase) on a 25 :1 weight-to-weightprotein sample to Pronase ratio, and the incubation was continued at 37OC for 24 hours. This treatment was usually sufficient to accomplish complete hydrolysis to the component amino acids without apparent degradation of P-N bonds. Alternatively, samples were occasionally hydrolyzed with 3 M KOH as follows. To the lyophilized protein sample (usually less than 1 mg), 1 ml of 3 M KOH was added and the reaction mixture was sealed in a glass test tube, placed in an autoclave at 15 psi pressure for 3 hours. After cooling and removal of the sample from the test tube, the potassium ions were precipitated by the dropwise addition of 10%perchloric acid to a pH of 7.5. The pH was never allowed to go below that value. The mixture was centrifuged to remove the potassium perchlorate precipitate, and the precipitate was washed once with a small volume of cold water. Both phosphohistidine
158
ROBERTS A. SMITH et al.
and eaminophospholysine survive this procedure, whereas phosphoarginine is destroyed by the basic hydrolysis. All three N-phosphoryl amino acids survive the enzymic proteolysis described above.
V.
Chromatographic Identification of N-Phosphoryl Amino Acids
Paper partition chromatography was usually performed with Whatman No. 1 or Whatman 3 MM paper, either ascending or descending in a solvent system consisting of isopropanol, ethanol, water, triethylamine (30: 30 :39 :1, v: v). The Rfvalues for the phosphoryl amino acids are reported as a function of phospholysine, which has been assigned a value of 1. They are 0-phosphoserine, 1.10-1.25; phosphoarginine, 0.90-0.95; 3-phosphohistidine, 0.60-0.62; and 1-phosphohistidine, 0.45. In addition, since phosphoarginine and phospholysine do not separate
-
iOLVENT I t - BuOH :Methyl Ethyl Ketone :(CH3)2CO: MeOH: H20 : N H 4 0 H (10 : 20:20:5 : 40 : 5 ) iOLVENT
-
II:
Isopropanol : HCOOH : H 2 0 (2O:l:S)
Q
P- Hist
0
0
P-Arg
P-Ser
0
P- Lys
t
I
-
-It
sample
FIG. 2. Two-dimensional thin-layer chromatography of phosphoamino acids on ICN-44613 gel glass plates.
15.
PHOSPHORYLATION OF BASIC AMINO ACIDS
159
well by any of the many paper chromatographic methods we haveattempted, a two-dimensional thin-layer chromatographic system has been developed; it is as follows (Fig. 2). The sample is applied to a silica gel glass plate (ICN404613, 20 x 20 cm) and developed in the first direction with t-butanol, methyl ethyl ketone, acetone, methanol, water, conc. NH,OH (10: 20:20: 5 :40:5, v: v), allowed to dry and developed in the second dimension with isopropyl alcohol, formic acid, H,O (20 :1 :5, v :v). A grid is constructed on the plate with pencil, and the dried matrix is removed for counting N-phosphoryl amino acids distribution as shown in Fig. 2. REFERENCES
1. Fiske, C. H., and SubbaRow, Y.,J. Eiol. Chem. 81,629 (1929). 2. Boyer. P. D., Hultquist, D. E., Peter, J. B., Kreil, G., Mitchell, R. A., DeLuca, M., Hinkson, J. W., Butler, L. G., and Moyer, R. W., Fed. Proc.. Fed. Am. SOC.Exp. Eiol. 22, 1080 (1963). 3. Kundig, W., Ghosh, S., and Roseman, S., Proc. Nurl. Acud. Sci. U.S.A.52, 1067 (1964). 4. Stevens-Clark, J. R., Conklin, K. A., Fujimoto, A., and Smith, R. A., J. Eiol. Chem. 243, 4474 (1968). 5. Halmann, M., Lapidot, A., and Samuel, D., J. Chem. SOC.p. 1299 (1963). 6. Ratlev, T., and Rosenberg, T., Arch. Eiochem. Eiophys. 65, 319 (1956). 7. Chanley, J. D., and Feageson, E., J. Am. Chem. SOC.85, 1181 (1963). 8. Plimmer, R. H. A., and Bayliss, W. M., J. Physiol. (London)33,439 (1906). 9. Plimmer, R. H. A., Eiochem. J. 35, 461 (1941). 10. Smith, D. L. Chen, C.-C., Bruegger, B. B., Holz, S. L., Halpern, R.M., and Smith, R. A., Eiochemisrry 13, 3780 (1974). I / . Chen, C.-C., Smith, D. L., Bruegger, B. B., Halpern, R. M., and Smith, R. A., Biochemistry 13, 3785 (1974). 12. Boyer, P. D., and Bieber, L. L., in “Methods in Enzymology” (R. W. Estabrook and M. E. Pullman, eds.), Vol. 10, p. 768. Academic Press, New York, (1967). 13. Chauveau, J., Moule, Y.,and Rouiller, C., Exp. Cell Res. 11, 317 (1956). 14. Bonner, J., Chalkley, G. R., Dahmus, M., Fambrough, D., Fujimura, F.. Huang, R. C., Huberman, J., Jensen, R., Marushige, K., Ohlenbusch, H., Oliver% B. M., and Wilholm, J., in “Methods in Enzymology” (L. Grossman and K.Moldave, eds.), Vol. 12, Part B, p. 3. Academic Press, New York, (1968).
15
Chromosomal Protein Phosphotylation on Basic Amino Acids ROBERTS A. SMITH, RICHARD M. HALPERN, BERNDT B. BRUEGGER, ALBERT K. DUNLAP, AND OSCAR FRICKE Department of Chemistty, University of California. Los Angeles, Los Angeles. California
I. General Considerations Phosphorylation of chromosomal proteins has generally been measured either after isolation of phosphorylated chromosomal proteins from the nucleus or after the action of a chromosomal protein kinase in an in vitro system. Almost totally, these procedures involve one or the other step carried out at an acidic pH value such that the only surviving phosphoryl linkage is found on the hydroxyl group of serine or threonine. Usually the protein is precipitated, generally by acid, followed by suitable washing or extraction to remove nonprotein phosphorus components. Sometimes the protein is dissolved or suspended in a suitable medium followed by removal of nonprotein phosphorus components by ion exchange or extraction procedures. Almost invariably these procedures involve the use of dilute mineral acid solutions in order to avoid chromosomal protein aggregation and/or proteolytic degradation. Such procedures obviously preclude the detection and isolation of phosphorylated components labile to the acidic pH values employed. N-phosphorylated compounds have been recognized as phosphoryl donors since Fiske and SubbaRow ( 1 ) identified N-phosphorylcreatine. The occurrence of N-phosphoryl linkages in proteins was first discovered by Boyer and his colleagues (2) with their isolation of N-phosphorylated succinyl-CoA synthase from mitochondria. The very rapid identification of the N-phosphorylated species as 3-phosphohistidine was quickly followed 153
154
ROBERTS A. SMITH et al.
by the demonstration of other N-phosphorylated enzymes, such as the phosphoenolpyruvate transferase of Roseman (3) and the phosphoramidatehexose transferase system described by Stevens-Clark et al. (4). The single most outstanding property of N-phosphorylated compounds is their extreme sensitivity to acidic pH and their relative stability under basic conditions (5-7). Most N-phosphorylated species have half-lives of less than a few minutes in 10--2 M mineral acid at room temperature, while they are virtually stable at alkaline pH values. This property is in contrast to that of phosphorylated peptide-bound serine, which is quite stable at normally employed acidic pH values, but undergoes rapid pelimination at alkaline pH values (8). Free phosphoserine, on the other hand, is relatively more base stable (9). It is this difference in properties between the O-phosphoryl and N-phosphoryl linkages in proteins that forms the basis of procedures we have used thus far for isolation and demonstration of such N-posphoryl linkages in histones (10, 11). The procedures described herein permit the detection of N-phosphoryl amino acids in chromosomal proteins based upon isolation of 32P-labeled N-phosphoryl substances. The 3zP-labeledchromosomal proteins are isolated either after enzymic reactions have taken place utilizing y-32P-labeled ATP as phosphoryl donor or directly from whole animal tissues excised and fractionated after rapid sacrifice of animals following administration of 32Piintraperitoneally. We have used the procedures directly most with rat liver preparations and/or rat mammary tumor Walker-256 carcinosarcoma or directly from enzyme reaction mixtures.
11. Phenol Extraction Procedure A. Principles Chromosomal proteins and lipids are extracted into phenol, while 32Pi and/or [y3zP]ATPare removed by careful washing of the phenol layer. 3zP left in the phenol extract gives a measure of the total covalently bound protein and lipid 32P.For further elucidation of the nature of the phosphorylated material, the radioactivity can be precipitated from phenol by acetone and the water-insoluble lipid phosphate extracted from the precipitate by organic solvents. The phosphate subsequently released by acid hydrolysis is a measure of the N-phosphorylated amino acid, and phosphoserine and phosphothreonine are measured as the acid-stable fraction. The following procedure is slightly modified from that of Boyer and Bieber (12).
IS.
PHOSPHORYLATION OF BASIC AMINO ACIDS
155
B. Procedure Incubation mixtures containing from 0.5 to 20 mg of chromosomal proteins in 1-2 ml were stopped with 2 ml of 88% phenol (adjusted to pH 8 with 0.01 M sodium phosphate buffer). Each mixture was added to a 40-ml centrifuge tube containing an additional 8 ml of the same solution. The solution was mixed thoroughly with 25 ml of a washing buffer [0.01 M EDTA4.01 M sodium pyrophosphate (PH 8.3)] and centrifuged at about 2500 rpm in a swinging-bucket clinical centrifuge. The upper aqueous layer was removed by careful aspiration. Wash buffer was again added, and the washing procedure was repeated six times. The 12P-containingproteins were then precipitated by the rapid addition of about 5 volumes of cold acetone. The mixture was usually stored at - 20° C for several hours or overnight, and the precipitate was ultimately collected by centrifugation. Any lipids were removed by successive extraction with about 5 ml of chloroform-methanol (1 :1, v:v), 5 ml chloroform-methanol-water (20: 10: 1, v:v:v), and 5 ml of methanol, in each case centrifuging in a clinical centrifuge to separate the residue from the organic solvents. The protein residue can be treated with 3 ml of 0.3 N trichloroacetic acid-1 mM sodium phosphate for 3 minutes in a boiling water bath. The mixture is cooled and the proteins removed by centrifugation in a clinical centrifuge. An aliquot of the supernatant fluid is counted, giving a measure of the acid-labile N-phosphoryl protein phosphate. The protein residue is washed twice with about 10 ml of phosphate buffer, and the 12Premaining with the precipitate is used as a measure of the acid-stable phosphoprotein. In our experience, very little 32P-phospholipidis formed in the short enzymic incubation mixtures, and often we dispense with the solvent extraction procedure for such mixtures. In such cases, phosphoserine and phosphothreonine are eliminated, since we usually stop the reaction by making the mixture 0.15 M in sodium hydroxide and heating it to 6OoC for 15 minutes prior to the phenol extraction. Thus, 12Premaining in the phenol layer is a direct measure of the amount of N-phosphorylated protein formed.
111. Isolation of ["PI Phosphohistones from Whole Animal Tissues Rats were sacrificed by decapitation between 1 and 3 hours after the intraperitoneal administration of IzPi in 0.9% NaCl solution. The tissues of choice (usually liver, regenerating liver, or Walker-256 carcinosarcoma)
156
ROBERTS A. SMITH cr al.
were rapidly excised and placed in ice cold 0.25 Msucrose-0.05 MTris. HCl (pH 7.5)-0.05 M NaCl. After the adhering connective tissues were removed, the nuclei were isolated by a modification of the method of Chauveau et al. (13). All procedures were performed at ice bath temperatures. The tissues were minced with scissors in an isotonic solution containing 0.25 Msucrose and 1 mM MgCl,. The minced tissue was homogenized with a PotterElvehjem homogenizer using a motor-driven pestle (0.005-0.007 inch clearance) for 40 strokes. The homogenate was filtered through one layer of coarse cheesecloth, followed successively by filtration through one layer of fine cheesecloth and eight layers of fine cheesecloth. The nuclei werepelleted ,by centrifugation at 1000 g for 10 minutes, and then resuspended in a solution containing 1 mM MgCl, and 2.2 M sucrose. The suspension was centrifuged at 40,OOO g for 90minutes. The pellet was washed twice with a solution of 1 M sucrose-1 mM MgCl, by homogenation with a loose-fitting Teflon pestle, followed by recentrifugation at 3000 g for 10 minutes. Chromatin was prepared from the nuclei by washing them with a0.15 MNaCl solution. The chromatin was subsequently washed 3 times with 0.05 M TriseHCl buffer (pH 7.5) followed by three washings with 0.15 M NaCl. The total chromosomal proteins were isolated by dissociation of the purified chromatin for 4 hours with constant stirring in an ice bath in 2.5 M guanidinium hydrochloride-0.1 M sodium phosphate buffer (pH 7). The DNA was removed by centrifugation at 90,OOO g for 10 hours. In separate aliquots, the phosphoserine linkages were almost totally removed by treatment at 6OoC for 10 minutes withO.l NNaOH (theacid0.4
0
0
I
I
1
I
!-.-.I
!
0.3 -
1
FRACTION
40 8
5 0
230-6 0
x
NUMBER
FIG. 1. Chromatography of acid-labile histone phosphates and acidic proteins on Bio-Rex 70 resin. Incorporation of radioactive inorganic phosphate, isolation of histones and acidic proteins, removal of acid-stable protein phosphates, and chromatographic separation were performed as described (11). Calf thymus histonts (3 rng) were added as carriers during column chromatography. - - -, OD,,, (Cl, CCOOH turbidity); -, radioactivity (,,P incorporated in vivo).
15.
PHOSPHORYLATION OF BASIC AMINO ACIDS
157
labile phosphoryl linkages were eliminated by treatment of an aliquot at 60°C for 10 minutes with 1 N HCl). This treatment was followed by dialysis overnight against two changes of 2 liters each of 0.01 M sodium phosphate buffer (pH 7). Separation of the chromosomal proteins was carried out on a Bio-Rex 70 column (0.6 x 60 cm). The elution was performed with a total of 50 ml of a linear gradient of 8-13% guanidinium HC1 in 0.1 M phosphate buffer (pH 6.8) prior to elution with 40% guanidinium hydrochloride in the same buffer essentially as described by Bonner et al. (14). For most of our experiments, we also added 3 mg of whole calf thymus histone to the column so that the appropriate histone peak could easily be identified by the TCA turbidity method (14). Radioactivity was determined by liquid scintillation spectrometry. A typical result showing the distribution of radioactivity associated with the chromosomal proteins eluted from a Bio-Rex 70 resin as described above is shown in Fig. 1.
IV. Degradation of Phosphoproteins and Demonstration of N-Phosphoryl Amino Acids
The separate peaks from the Bio-Rex 70 column after a whole tissue run or from an in vitro kinase reaction were pooled and dialyzed against two changes of 1 liter each ofO.O1 MTris. HCl buffer (pH 8)and concentrated by lyophilization. The residue was usually suspended in 0.1% NaHCO,(pH 8.5) and incubated with trypsin (on a weight-to-weight ratio of 100: 1, protein sample to proteolytic enzyme) for 2 hours at 37OC. After the trypsin incubation, Pronase was added to the reaction mixture (45 proteolytic units per milligram of Pronase) on a 25 :1 weight-to-weightprotein sample to Pronase ratio, and the incubation was continued at 37OC for 24 hours. This treatment was usually sufficient to accomplish complete hydrolysis to the component amino acids without apparent degradation of P-N bonds. Alternatively, samples were occasionally hydrolyzed with 3 M KOH as follows. To the lyophilized protein sample (usually less than 1 mg), 1 ml of 3 M KOH was added and the reaction mixture was sealed in a glass test tube, placed in an autoclave at 15 psi pressure for 3 hours. After cooling and removal of the sample from the test tube, the potassium ions were precipitated by the dropwise addition of 10%perchloric acid to a pH of 7.5. The pH was never allowed to go below that value. The mixture was centrifuged to remove the potassium perchlorate precipitate, and the precipitate was washed once with a small volume of cold water. Both phosphohistidine
158
ROBERTS A. SMITH et al.
and eaminophospholysine survive this procedure, whereas phosphoarginine is destroyed by the basic hydrolysis. All three N-phosphoryl amino acids survive the enzymic proteolysis described above.
V.
Chromatographic Identification of N-Phosphoryl Amino Acids
Paper partition chromatography was usually performed with Whatman No. 1 or Whatman 3 MM paper, either ascending or descending in a solvent system consisting of isopropanol, ethanol, water, triethylamine (30: 30 :39 :1, v: v). The Rfvalues for the phosphoryl amino acids are reported as a function of phospholysine, which has been assigned a value of 1. They are 0-phosphoserine, 1.10-1.25; phosphoarginine, 0.90-0.95; 3-phosphohistidine, 0.60-0.62; and 1-phosphohistidine, 0.45. In addition, since phosphoarginine and phospholysine do not separate
-
iOLVENT I t - BuOH :Methyl Ethyl Ketone :(CH3)2CO: MeOH: H20 : N H 4 0 H (10 : 20:20:5 : 40 : 5 ) iOLVENT
-
II:
Isopropanol : HCOOH : H 2 0 (2O:l:S)
Q
P- Hist
0
0
P-Arg
P-Ser
0
P- Lys
t
I
-
-It
sample
FIG. 2. Two-dimensional thin-layer chromatography of phosphoamino acids on ICN-44613 gel glass plates.
15.
PHOSPHORYLATION OF BASIC AMINO ACIDS
159
well by any of the many paper chromatographic methods we haveattempted, a two-dimensional thin-layer chromatographic system has been developed; it is as follows (Fig. 2). The sample is applied to a silica gel glass plate (ICN404613, 20 x 20 cm) and developed in the first direction with t-butanol, methyl ethyl ketone, acetone, methanol, water, conc. NH,OH (10: 20:20: 5 :40:5, v: v), allowed to dry and developed in the second dimension with isopropyl alcohol, formic acid, H,O (20 :1 :5, v :v). A grid is constructed on the plate with pencil, and the dried matrix is removed for counting N-phosphoryl amino acids distribution as shown in Fig. 2. REFERENCES
1. Fiske, C. H., and SubbaRow, Y.,J. Eiol. Chem. 81,629 (1929). 2. Boyer. P. D., Hultquist, D. E., Peter, J. B., Kreil, G., Mitchell, R. A., DeLuca, M., Hinkson, J. W., Butler, L. G., and Moyer, R. W., Fed. Proc.. Fed. Am. SOC.Exp. Eiol. 22, 1080 (1963). 3. Kundig, W., Ghosh, S., and Roseman, S., Proc. Nurl. Acud. Sci. U.S.A.52, 1067 (1964). 4. Stevens-Clark, J. R., Conklin, K. A., Fujimoto, A., and Smith, R. A., J. Eiol. Chem. 243, 4474 (1968). 5. Halmann, M., Lapidot, A., and Samuel, D., J. Chem. SOC.p. 1299 (1963). 6. Ratlev, T., and Rosenberg, T., Arch. Eiochem. Eiophys. 65, 319 (1956). 7. Chanley, J. D., and Feageson, E., J. Am. Chem. SOC.85, 1181 (1963). 8. Plimmer, R. H. A., and Bayliss, W. M., J. Physiol. (London)33,439 (1906). 9. Plimmer, R. H. A., Eiochem. J. 35, 461 (1941). 10. Smith, D. L. Chen, C.-C., Bruegger, B. B., Holz, S. L., Halpern, R.M., and Smith, R. A., Eiochemisrry 13, 3780 (1974). I / . Chen, C.-C., Smith, D. L., Bruegger, B. B., Halpern, R. M., and Smith, R. A., Biochemistry 13, 3785 (1974). 12. Boyer, P. D., and Bieber, L. L., in “Methods in Enzymology” (R. W. Estabrook and M. E. Pullman, eds.), Vol. 10, p. 768. Academic Press, New York, (1967). 13. Chauveau, J., Moule, Y.,and Rouiller, C., Exp. Cell Res. 11, 317 (1956). 14. Bonner, J., Chalkley, G. R., Dahmus, M., Fambrough, D., Fujimura, F.. Huang, R. C., Huberman, J., Jensen, R., Marushige, K., Ohlenbusch, H., Oliver% B. M., and Wilholm, J., in “Methods in Enzymology” (L. Grossman and K.Moldave, eds.), Vol. 12, Part B, p. 3. Academic Press, New York, (1968).