Proc. Natd. Acad. Sci. USA
Vol. 74, No. 8, pp.,3226-3229, August 1977 Biochemistry
Nucleotide phosphotransferase of Escherichia coli: Purification by affinity chromatography (affinity resins/phosphorylation of ribonucleosidonucleotides/positionsal specificities regarding hydroxyls)
ELINOR F. BRUNNGRABER AND ERWIN CHARGAFF* Cell Chemistry Laboratory, The Roosevelt Hospital, New York, New York 10019
Contributed by Erwin Chargaff, May 19,1977
ABSTRACT Improved extraction and purification procedures permit the isolation from Eschenchia coli W cells of much larger quantities and of more highly purified preparations of nucleotide phosphotransferase. Of various affinity resins tested for efficiency of purification, columns of agarose/5'-AMP (AGAMP), type 3, proved the best. In this way a 300- to 450-fold purification of the enzyme was achieved in a few steps. The enzyme, which, as reported before, transfers organically bound phosphate to the 2' or 3' hydroxyls of nucleosides and nucleotides, was tested in its behavior toward a series of ribonucleosidonucleotides, namely, CpC, ApA, CpA, and ApC. All were phosphate acceptors, but a detailed comparative study of adenosine and cytidine, 5'-AMP and 5'-CMP, and ApA and ApC revealed peculiar specificities in the relative distribution of the phosphorylated products.
We have reported previously the discovery (1) and partial purification (2) of a nucleotide phosphotransferase capable of transferring phosphoric acid from an organic phosphate donor to the 2' or 3' hydroxyls of nucleosides and nucleotides. The enzyme preparations then available were used in a search for the possible biological functions of this interesting enzyme. It was found that deoxyribooligothymidylic acid preparations (2) and NAD (3) could also serve as phosphate acceptors, but it proved impossible to test oligoribonucleotides as acceptors, since the enzyme specimens were contaminated with a ribonuclease or ribophosphodiesterase. The present paper explores the use of affinity chromatography in order to overcome this difficulty. The resin agarose/adenylic acid (AGAMP) proved especially valuable for the purification of the transferase. This procedure is presented here, together with the application of preparations thus purified to the phosphorylation of ribonucleosidonucleotides. MATERIALS AND METHODS Escherichia coli W, grown on a high peptone medium and harvested in the early logarithmic phase, served as the enzyme source. Frozen preparations of these organisms were supplied by General Biochemicals, Chagrin Falls, OH, and by Grain Processing Corp., Muscatine, IA. The enzymes used in the isolation of the phosphotransferase and the investigation of the enzymic products, pancreatic deoxyribonuclease I (EC 3.1.4.5), spleen phosphodiesterase II (EC 3.1.4. 18), and lysozyme (EC 3.2.1.17), were obtained commercially, as was the phosphate donor p-nitrophenylphosphate. The various nucleosides, nucleotides, and nucleosidonucleotides tested as acceptors came from P-L Biochemicals, Inc., Milwaukee, WI. DEAE-cellulose Type 40 (Schleicher and Schuell) had a capacity of 0.93 mequiv/g. The affinity resins, agarose/adenylic The costs of publication of this article were defrayed in part by the payment of page charges from funds made available to support the research which is the subject of the article. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
acid (AGAMP) and agarose/nicotinamide adenine dinucleotide (AGNAD), were also supplied by P-L Biochemicals. Procedures described previously (1, 2) were followed consistently for the assay of the activity of nucleotide phosphotransferase and the study of acceptor specificity. The release of p-nitrophenol was estimated through the reading of the absorbance at 410 nm. Gel electrophoresis was performed with 7.5% gels at pH 4.3 and at pH 8.9. For the analysis of the products of the enzymic phosphate transfer, the incubation mixture was streaked on Whatman no. 1 paper and developed in one of two solvents: solvent 1, n-propyl alcohol/concentrated ammonia/water/, 11:2:7, vol/vol/vol; solvent 2, n-propyl alcohol/concentrated ammonia/water/, 11:7:2, vol/vol/vol. When nucleosidonucleotides served as acceptors, the dinucleotides produced by phosphorylation were eluted from the paper with water and the extracts were concentrated in a vacuum; this was followed by hydrolysis, either with 0.3 M KOH (300, 18 hr) or with spleen phosphodiesterase (p. 245 of ref. 4). The hydrolysis products were separated on paper (solvent 2) and the appropriate zone was then analyzed by liquid chromatography in the Varian Aerograph LCS 1000 under the conditions described before (1). ISOLATION OF ENZYME In a typical run, 50 g of E. coli W cells were suspended at room temperature in 100 ml of water with the aid of a Sorvall Omnimixer at low speed. The mixture was poured into 100 ml of 0.02 M Tris.HCl, pH 7.5, containing 0.01 M EDTA and 150 mg of lysozyme.t After 10 min the mixture was adjusted to 0.015 M MgCl2 by the addition of an 1 M solution, and 10 ml of a 0.1% solution of deoxyribonuclease was added. The mixture was stirred in the Omnimixer for 30 sec, kept for 10 min, and centrifuged (10,000 X g, 1 hr, 40). All of the following procedures were performed in the cold. The supernatant solution represents step 1 in Table 1. This solution was brought to 25% saturation by the addition of solid ammonium sulfate. A small precipitate was removed (20,000 X g, 20 min) and the supernatant was adjusted to 65% saturation with respect to ammonium sulfate. After 1 hr the
Abbreviations: AGAMP resin, agarose/hexane/adenosine 5'-phosphate resin; AGNAD resin, agarose/hexane/nicotinamide adenine dinucleotide resin. * To whom correspondence should be addressed. t We are grateful to J. G. Stavrianopoulos for suggesting this procedure. Occasionally batches of bacteria were encountered on which lysozyme appeared to act less effectively, as was shown by the small change in viscosity when the cell suspension was added to the lysozyme. This was especially apparent when the volume of homogenizing medium used per gram of cells was smaller than specified here. By avoiding the use of too dense a cell suspension or by washing the cells with the Tris-HCl buffer before exposing them to lysis, this difficulty could be overcome.
3226
Biochemistry: Brunngraber and Chargaff
Proc. Nati. Acad. Sci. USA 74 (1977)
3227
Table 1. Purification of nucleotide phosphotransferase of E. coli by affinity chromatography* Vol., Protein, ml S.A.t Procedure % Step mg/ml 1 2 3 4
Lysis (NH4)2SO4 DEAE-cellulose AGAMP type 3
130 51 122 10
17.4 35.2 0.85 0.32
2.2 2.2 37.6 660
100 79 78 42
U~~~~~~~~~~~~
10
* In this experiment 50 g of E. coli W cells were processed. (S.A.) was determined with 5'-thymidylic acid as acceptor (2); it is expressed as gmol of thymidine 3',5'-diphosphate produced in 1 hr by 1 mg of protein in 1 ml of assay mixture.
-O
t The specific activity
precipitate was spun down (20,000 X g, 20 min) and resuspended in about 50 ml of water, and the solution was dialyzed overnight, first against 1 liter of water and then against the same volume of 0.005 M Tris-HCI, pH 7.2. This represents step 2. The transferase activity then was adsorbed on a column (2.5 X 30 cm) of 10 g of DEAE-cellulose, type 40, that had been equilibrated with 0.005 M Tris.HCl of pH 7.2. After the column was washed with about 200 ml of the same buffer, the enzyme was eluted with 0.06 M sodium acetate, pH 5.8. All tubes containing enzyme activity were combined, constituting step 3. One-third of the step 3 eluate was applied directly to a column of 3 ml of AGAMP type 3 resin that had been washed with 0.06 M sodium acetate, pH 5.8. The remainder was stored at -20°. The column was washed with the same buffer until no more protein was recovered by slow elution with water, fractions of 2 ml being collected every 15 min. This eluate, which could be stored in the frozen state, represents step 4. For regeneration, the AGAMP column was washed first with 1 M NaCl, then with 1 M NaOH, and finally with 0.06 M sodium acetate, pH 5.8. RESULTS AND CONCLUDING REMARKS Purification by Affinity Chromatography. The course of isolation of the nucleotide phosphotransferase is shown in Table 1. The extraction of the enzyme with the aid of lysozyme is much more efficient than the procedure applied previously (2); it yields nearly nine times as much enzyme per g of cells in step 1 with an almost threefold specific activity. Equally impressive is the increase in the recovery of enzyme when the last steps are compared: 42 units/mg of original cells (step 4 in Table 1) as compared with 5 or 7 units in Table 1 of ref. 2. Also, the specific activity of the final eluate from the affinity column is considerably higher than the final activities recorded before. The phosphotransferase isolated with the aid of lysozyme was not as readily adsorbed to the DEAE-cellulose column (step 3) as were the extracts examined previously (2). This difficulty was overcome by applying the enzyme at pH 7.2. The activity was then easily eluted, with removal of the bulk of contaminating phosphatases, by lowering the pH and raising the salt concentration (Fig. 1). A series of affinity resins was tested. The phosphotransferase was adsorbed readily to both AGNAD, type 4, and AGAMP, type 4, but was difficult to recover. The first set of experiments with AGNAD, type 1, gave promising results, since much contaminating protein was removed and enzyme activity could be recovered by means of dilute nucleoside solutions. The reproducibility was, however, poor. With some of the enzyme preparations, the release of a substance having a spectrum similar to AMP was observed; this suggested an occasional contamination with a pyrophosphatase that hydrolyzed the
0
2
.16 01-j.0.J. 14 18 22 26 30 10
0.
34 38 42 46 50
Fraction number
FIG. 1. Purification of nucleotide phosphotransferase on DEAE-cellulose (step 3 in Table 1). The activity is expressed as ,mol/ml of thymidine 3',5'-diphosphate (as a measure of transferase) and of p-nitrophenol (as a measure of phosphatase and phosphotransferase) formed by 0.5 ml of enzyme in 30 min at 37°. O, Absorbance at 280 nm; 0, phosphotransferase; 0, p-nitrophenol released.
NAD moiety of the affinity resin. Attempts to inhibit this activity with pyrophosphate failed. AGAMP, both types 2 and 3, adsorbed the phosphotransferase. Since the activity was more easily recovered from type 3, this medium was adopted for the final procedure. A typical elution pattern is shown in Fig. 2. The procedure described here not only provides enzyme at higher specific activity than reported previously but is also a convenient method for concentrating the protein. Electropherograms of preparations corresponding to step 4 showed two major bands at pH 4.3; no bands were seen at pH 8.9.
.!
0
4C0
3c
0.6
-
._
._-
20
10
01LA v.F
i
I
1
_~I~
Ikc.
Eluates (H20)
0
0.4
0-.2
Jo J
o
FIG. 2. Purification of nucleotide phosphotransferase on AGAMP, type 3. Symbols and expression of activity are as in the legend of Fig. 1, except that protein is represented as ,ug/ml.
3228
Proc. Natl. Acad. Sci. USA 74 (1977)
Biochemistry: Brunngraber and Chargaff
Table 2. Action of purified nucleotide phosphotransferase on ribonucleosides, nucleotides, and nucleosidonucleotides*
Exp.
Substrate
Product, % of substrate 1440 mint 60 mint 30 mint
95% (2') -28
(2') -67
') -59
(3)-33
-
100 100 79 98 95 72 45 39 39 76 75 44 28 26 16 18 19 11 70 68 52 ApA 24 63 41 CpA 38 35 23 ApC * Assay mixture (total volume, 0.4 ml): 0.1 M sodium acetate (pH 5.2), 100 mM p-nitrophenylphosphate, 16 jtg of step 4 enzyme, 15 mM acceptor. Incubation at 37°. Portions of 10 or 20 ul of the mixtures were developed on paper in solvent 1. t Incubation time. 1 2 3 4 5 6 7 8 9
C A 5'-CMP 5'-AMP 3':5'-cAMP CpC
When solutions other than water were tested as eluents from the AGAMP, type 3, column, no activity was eluted by 0.1 M salt solutions or by 5 mM dTMP or AMP, whereas 1 mM thymidine, deoxyguanosine, or NAD did release the phosphotransferase, sometimes with a higher specific activity (8001000) than in the experiment shown in Table 1. It is not unlikely that these fluctuations are attributable to variations between different lots of AGAMP rather than to the nature of the eluent used. Phosphate Transfer. The enzyme preparations secured by affinity chromatography were tested with a large number of the acceptors that had been used before (1, 2), namely, A, dA, AMP, dAMP; dG, dGMP; C, dC, CMP, dCMP; dT, dTMP. As phosphate donors, p-nitrophenylphosphate, 2'-AMP, 2'-GMP, and 3'-TMP were used. The results previously recorded (1, 2) with regard to the particular hydroxyls phosphorylated enzymically were duplicated completely, nor were any differences observed when enzyme preparations eluted from the affinity columns with either dT, dG, or NAD were compared. No indications of the existence of more than one phosphotransferase were obtained. A selection of experiments, showing the total yields of phosphorylation products during different periods of incubation, is assembled in Table 2. It includes four ribonucleosidonucleotides that could not be tested before owing to the contamination of our previous enzyme preparations with a ribophosphodiesterase (unpublished experiments with S.-H. Chang). In the present procedure most, if not all, of the interfering activity was removed. This made possible the study of ApC, CpC, CpA, and ApA as phosphate acceptors. Table 2 summarizes the total yields of enzymic phosphorylation products, regardless of the distribution of isomers. With all nucleosidonucleotides, a new UV-absorbing component appeared on the chromatograms that exhibited a spectrum identical with that of the respective precursor. The maximum amount of product was formed after incubation for 60 min, with generally only a slight further increase when the incubation was extended to 24 hr. CpA formed an exception; on very long incubation a partial decomposition of the CpAp formed was observed. With the nucleosides, the conversion to 2'(3')-nucleotides was virtually complete. In our previous study (2), when initial velocities were compared, the amounts of Cp formed from C and of pCp formed from pC were the same, in contrast to the present findings with an excess of enzyme (Table 2). A detailed
C
A
(5')
u-
(5')
13
C
A
(2')
(2') 75% (3)
(2') (3')
0
()
(2') (3')
C
A
28 71
(2') (3')
71 29
0
(2) -29 (3') -71
35%
68% (5')
39% -~(3'
A
A
0
.5
(5')
I
FIG. 3. Positional discrimination in enzymic phosphate transfer to ribonucleosides and their derivatives. In all cases one phosphate moiety was transferred to a hydroxyl, with the indicated relative frequencies (as % of total product). The yields of total phosphorylation products (as % of acceptor) are given on the left of each structure.
study showed that with limiting amounts of enzyme both acceptors are phosphorylated at the same rate during the first 2 hr, but that subsequently the formation of pCp falls off whereas that of the monophosphate Cp continues to completion. Positional Discrimination in Phosphate Transfer. The nucleosides C and A offer three hydroxyls to the transferase, the 5'-nucleotides pC and pA afford two, and the nucleosidonucleotides ApC and ApA afford four hydroxyls. Since in all cases only one phosphate group appears to be transferred enzymically, the selections made by the enzyme are of interest. In a mixed dimer like ApC, in which the phosphate bridge goes from the 3' position of A to the 5' position of C, alkaline hydrolysis of the phosphorylation product yields by itself sufficient data for a decision. The product obtained by the enzyme action on ApC was isolated on paper and treated with alkali. Since hydrolysis was complete, no phosphorylation of the 2' hydroxyl of adenosine could have occurred; the isolation of less than 1% of pAp and C similarly showed that phosphorylation of the 5' hydroxyl of adenosine was minimal. The principal hydrolysis products were AMP and CMP, which latter was shown by liquid chromatography to consist of 29% 2'-CMP and 71% 3'CMP. With two identical bases, as in ApA, it is necessary to resort to enzymic hydrolysis for the analysis of the product. Spleen phosphodiesterase II, which produces 3'-nucleotides, was used. The enzymic hydrolysis of the phosphorylation product of ApA was nearly complete (98%). Liquid chromatography showed the hydrolysate to consist of 14% 2'-AMP and 86% 3'-AMP. The same proportions were found when the AMP mixture was first isolated by paper chromatography and then analyzed by liquid
Biochemistry: Brunngraber and Chargaff chromatography. From these values it can be calculated that the transferase produced 28% ApAp(2') and 72%6 ApAp(3'). The small amount not hydrolyzed by the spleen enzyme (about 2%) must have been all (5')pApA, since the product was completely hydrolyzed with alkali. This would not have been the case if some (2')pApA had also been formed. The various findings are summarized in Fig. 3. The values for the nucleosides and 5'-nucleotides were redetermined with enzyme preparations purified as described here; the results completely duplicated the findings reported before (1, 2). Concluding Remarks. Several biological systems on which an enzyme of the type described here could have a regulatory effect have been discussed before (1, 2). One possible mode of interference by the nucleotide phosphotransferase in the biosynthesis of the nucleic acid could be the inactivation of the primer, in polymerase catalysis requiring such a compound, via the phosphorylation of the terminal 3' hydroxyl indispensable for chain growth. Another possible effect could be on the terminal 3' OH group of a transfer RNA. From the exper-
Proc. Natl. Acad. Sci. USA 74 (1977)
3229
iments with nucleosidonucleotides presented here it may be co.ided that it will be exclusively the 3' OH end that will be phosphorylated. The results summarized in Fig. 3 reveal a surprising shift in positional selectivity, especially when the cytosine-containing compounds are compared. It is also noteworthy that the nucleoside carrying the 3' -- 5'-phosphate bridge is practically not attacked by the enzyme. This work was aided by research grants from the National Institutes of Health, U.S. Public Health Service, and the National Science Foundation. 1. Brunngraber, E. F. & Chargaff, E. (1970) Proc. Natl. Acad. Sci. USA 67,107-112. 2. Brunngraber, E. F. & Chargaff, E. (1973) Biochwmistry 12, 3005-312. 3. Brunngraber, E. F. & Chargaff, E. (1973) Biochemistry 12, 3012-016. 4. Razzeli, W. E. (1963) in Methods in Enzymology, eds. Colowick, S. P. & Kaplan, N. 0. (Academic Press, New York and London), Vol. 6, pp. 236-258.
Vol. 74, No. 8, pp.,3226-3229, August 1977 Biochemistry
Nucleotide phosphotransferase of Escherichia coli: Purification by affinity chromatography (affinity resins/phosphorylation of ribonucleosidonucleotides/positionsal specificities regarding hydroxyls)
ELINOR F. BRUNNGRABER AND ERWIN CHARGAFF* Cell Chemistry Laboratory, The Roosevelt Hospital, New York, New York 10019
Contributed by Erwin Chargaff, May 19,1977
ABSTRACT Improved extraction and purification procedures permit the isolation from Eschenchia coli W cells of much larger quantities and of more highly purified preparations of nucleotide phosphotransferase. Of various affinity resins tested for efficiency of purification, columns of agarose/5'-AMP (AGAMP), type 3, proved the best. In this way a 300- to 450-fold purification of the enzyme was achieved in a few steps. The enzyme, which, as reported before, transfers organically bound phosphate to the 2' or 3' hydroxyls of nucleosides and nucleotides, was tested in its behavior toward a series of ribonucleosidonucleotides, namely, CpC, ApA, CpA, and ApC. All were phosphate acceptors, but a detailed comparative study of adenosine and cytidine, 5'-AMP and 5'-CMP, and ApA and ApC revealed peculiar specificities in the relative distribution of the phosphorylated products.
We have reported previously the discovery (1) and partial purification (2) of a nucleotide phosphotransferase capable of transferring phosphoric acid from an organic phosphate donor to the 2' or 3' hydroxyls of nucleosides and nucleotides. The enzyme preparations then available were used in a search for the possible biological functions of this interesting enzyme. It was found that deoxyribooligothymidylic acid preparations (2) and NAD (3) could also serve as phosphate acceptors, but it proved impossible to test oligoribonucleotides as acceptors, since the enzyme specimens were contaminated with a ribonuclease or ribophosphodiesterase. The present paper explores the use of affinity chromatography in order to overcome this difficulty. The resin agarose/adenylic acid (AGAMP) proved especially valuable for the purification of the transferase. This procedure is presented here, together with the application of preparations thus purified to the phosphorylation of ribonucleosidonucleotides. MATERIALS AND METHODS Escherichia coli W, grown on a high peptone medium and harvested in the early logarithmic phase, served as the enzyme source. Frozen preparations of these organisms were supplied by General Biochemicals, Chagrin Falls, OH, and by Grain Processing Corp., Muscatine, IA. The enzymes used in the isolation of the phosphotransferase and the investigation of the enzymic products, pancreatic deoxyribonuclease I (EC 3.1.4.5), spleen phosphodiesterase II (EC 3.1.4. 18), and lysozyme (EC 3.2.1.17), were obtained commercially, as was the phosphate donor p-nitrophenylphosphate. The various nucleosides, nucleotides, and nucleosidonucleotides tested as acceptors came from P-L Biochemicals, Inc., Milwaukee, WI. DEAE-cellulose Type 40 (Schleicher and Schuell) had a capacity of 0.93 mequiv/g. The affinity resins, agarose/adenylic The costs of publication of this article were defrayed in part by the payment of page charges from funds made available to support the research which is the subject of the article. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
acid (AGAMP) and agarose/nicotinamide adenine dinucleotide (AGNAD), were also supplied by P-L Biochemicals. Procedures described previously (1, 2) were followed consistently for the assay of the activity of nucleotide phosphotransferase and the study of acceptor specificity. The release of p-nitrophenol was estimated through the reading of the absorbance at 410 nm. Gel electrophoresis was performed with 7.5% gels at pH 4.3 and at pH 8.9. For the analysis of the products of the enzymic phosphate transfer, the incubation mixture was streaked on Whatman no. 1 paper and developed in one of two solvents: solvent 1, n-propyl alcohol/concentrated ammonia/water/, 11:2:7, vol/vol/vol; solvent 2, n-propyl alcohol/concentrated ammonia/water/, 11:7:2, vol/vol/vol. When nucleosidonucleotides served as acceptors, the dinucleotides produced by phosphorylation were eluted from the paper with water and the extracts were concentrated in a vacuum; this was followed by hydrolysis, either with 0.3 M KOH (300, 18 hr) or with spleen phosphodiesterase (p. 245 of ref. 4). The hydrolysis products were separated on paper (solvent 2) and the appropriate zone was then analyzed by liquid chromatography in the Varian Aerograph LCS 1000 under the conditions described before (1). ISOLATION OF ENZYME In a typical run, 50 g of E. coli W cells were suspended at room temperature in 100 ml of water with the aid of a Sorvall Omnimixer at low speed. The mixture was poured into 100 ml of 0.02 M Tris.HCl, pH 7.5, containing 0.01 M EDTA and 150 mg of lysozyme.t After 10 min the mixture was adjusted to 0.015 M MgCl2 by the addition of an 1 M solution, and 10 ml of a 0.1% solution of deoxyribonuclease was added. The mixture was stirred in the Omnimixer for 30 sec, kept for 10 min, and centrifuged (10,000 X g, 1 hr, 40). All of the following procedures were performed in the cold. The supernatant solution represents step 1 in Table 1. This solution was brought to 25% saturation by the addition of solid ammonium sulfate. A small precipitate was removed (20,000 X g, 20 min) and the supernatant was adjusted to 65% saturation with respect to ammonium sulfate. After 1 hr the
Abbreviations: AGAMP resin, agarose/hexane/adenosine 5'-phosphate resin; AGNAD resin, agarose/hexane/nicotinamide adenine dinucleotide resin. * To whom correspondence should be addressed. t We are grateful to J. G. Stavrianopoulos for suggesting this procedure. Occasionally batches of bacteria were encountered on which lysozyme appeared to act less effectively, as was shown by the small change in viscosity when the cell suspension was added to the lysozyme. This was especially apparent when the volume of homogenizing medium used per gram of cells was smaller than specified here. By avoiding the use of too dense a cell suspension or by washing the cells with the Tris-HCl buffer before exposing them to lysis, this difficulty could be overcome.
3226
Biochemistry: Brunngraber and Chargaff
Proc. Nati. Acad. Sci. USA 74 (1977)
3227
Table 1. Purification of nucleotide phosphotransferase of E. coli by affinity chromatography* Vol., Protein, ml S.A.t Procedure % Step mg/ml 1 2 3 4
Lysis (NH4)2SO4 DEAE-cellulose AGAMP type 3
130 51 122 10
17.4 35.2 0.85 0.32
2.2 2.2 37.6 660
100 79 78 42
U~~~~~~~~~~~~
10
* In this experiment 50 g of E. coli W cells were processed. (S.A.) was determined with 5'-thymidylic acid as acceptor (2); it is expressed as gmol of thymidine 3',5'-diphosphate produced in 1 hr by 1 mg of protein in 1 ml of assay mixture.
-O
t The specific activity
precipitate was spun down (20,000 X g, 20 min) and resuspended in about 50 ml of water, and the solution was dialyzed overnight, first against 1 liter of water and then against the same volume of 0.005 M Tris-HCI, pH 7.2. This represents step 2. The transferase activity then was adsorbed on a column (2.5 X 30 cm) of 10 g of DEAE-cellulose, type 40, that had been equilibrated with 0.005 M Tris.HCl of pH 7.2. After the column was washed with about 200 ml of the same buffer, the enzyme was eluted with 0.06 M sodium acetate, pH 5.8. All tubes containing enzyme activity were combined, constituting step 3. One-third of the step 3 eluate was applied directly to a column of 3 ml of AGAMP type 3 resin that had been washed with 0.06 M sodium acetate, pH 5.8. The remainder was stored at -20°. The column was washed with the same buffer until no more protein was recovered by slow elution with water, fractions of 2 ml being collected every 15 min. This eluate, which could be stored in the frozen state, represents step 4. For regeneration, the AGAMP column was washed first with 1 M NaCl, then with 1 M NaOH, and finally with 0.06 M sodium acetate, pH 5.8. RESULTS AND CONCLUDING REMARKS Purification by Affinity Chromatography. The course of isolation of the nucleotide phosphotransferase is shown in Table 1. The extraction of the enzyme with the aid of lysozyme is much more efficient than the procedure applied previously (2); it yields nearly nine times as much enzyme per g of cells in step 1 with an almost threefold specific activity. Equally impressive is the increase in the recovery of enzyme when the last steps are compared: 42 units/mg of original cells (step 4 in Table 1) as compared with 5 or 7 units in Table 1 of ref. 2. Also, the specific activity of the final eluate from the affinity column is considerably higher than the final activities recorded before. The phosphotransferase isolated with the aid of lysozyme was not as readily adsorbed to the DEAE-cellulose column (step 3) as were the extracts examined previously (2). This difficulty was overcome by applying the enzyme at pH 7.2. The activity was then easily eluted, with removal of the bulk of contaminating phosphatases, by lowering the pH and raising the salt concentration (Fig. 1). A series of affinity resins was tested. The phosphotransferase was adsorbed readily to both AGNAD, type 4, and AGAMP, type 4, but was difficult to recover. The first set of experiments with AGNAD, type 1, gave promising results, since much contaminating protein was removed and enzyme activity could be recovered by means of dilute nucleoside solutions. The reproducibility was, however, poor. With some of the enzyme preparations, the release of a substance having a spectrum similar to AMP was observed; this suggested an occasional contamination with a pyrophosphatase that hydrolyzed the
0
2
.16 01-j.0.J. 14 18 22 26 30 10
0.
34 38 42 46 50
Fraction number
FIG. 1. Purification of nucleotide phosphotransferase on DEAE-cellulose (step 3 in Table 1). The activity is expressed as ,mol/ml of thymidine 3',5'-diphosphate (as a measure of transferase) and of p-nitrophenol (as a measure of phosphatase and phosphotransferase) formed by 0.5 ml of enzyme in 30 min at 37°. O, Absorbance at 280 nm; 0, phosphotransferase; 0, p-nitrophenol released.
NAD moiety of the affinity resin. Attempts to inhibit this activity with pyrophosphate failed. AGAMP, both types 2 and 3, adsorbed the phosphotransferase. Since the activity was more easily recovered from type 3, this medium was adopted for the final procedure. A typical elution pattern is shown in Fig. 2. The procedure described here not only provides enzyme at higher specific activity than reported previously but is also a convenient method for concentrating the protein. Electropherograms of preparations corresponding to step 4 showed two major bands at pH 4.3; no bands were seen at pH 8.9.
.!
0
4C0
3c
0.6
-
._
._-
20
10
01LA v.F
i
I
1
_~I~
Ikc.
Eluates (H20)
0
0.4
0-.2
Jo J
o
FIG. 2. Purification of nucleotide phosphotransferase on AGAMP, type 3. Symbols and expression of activity are as in the legend of Fig. 1, except that protein is represented as ,ug/ml.
3228
Proc. Natl. Acad. Sci. USA 74 (1977)
Biochemistry: Brunngraber and Chargaff
Table 2. Action of purified nucleotide phosphotransferase on ribonucleosides, nucleotides, and nucleosidonucleotides*
Exp.
Substrate
Product, % of substrate 1440 mint 60 mint 30 mint
95% (2') -28
(2') -67
') -59
(3)-33
-
100 100 79 98 95 72 45 39 39 76 75 44 28 26 16 18 19 11 70 68 52 ApA 24 63 41 CpA 38 35 23 ApC * Assay mixture (total volume, 0.4 ml): 0.1 M sodium acetate (pH 5.2), 100 mM p-nitrophenylphosphate, 16 jtg of step 4 enzyme, 15 mM acceptor. Incubation at 37°. Portions of 10 or 20 ul of the mixtures were developed on paper in solvent 1. t Incubation time. 1 2 3 4 5 6 7 8 9
C A 5'-CMP 5'-AMP 3':5'-cAMP CpC
When solutions other than water were tested as eluents from the AGAMP, type 3, column, no activity was eluted by 0.1 M salt solutions or by 5 mM dTMP or AMP, whereas 1 mM thymidine, deoxyguanosine, or NAD did release the phosphotransferase, sometimes with a higher specific activity (8001000) than in the experiment shown in Table 1. It is not unlikely that these fluctuations are attributable to variations between different lots of AGAMP rather than to the nature of the eluent used. Phosphate Transfer. The enzyme preparations secured by affinity chromatography were tested with a large number of the acceptors that had been used before (1, 2), namely, A, dA, AMP, dAMP; dG, dGMP; C, dC, CMP, dCMP; dT, dTMP. As phosphate donors, p-nitrophenylphosphate, 2'-AMP, 2'-GMP, and 3'-TMP were used. The results previously recorded (1, 2) with regard to the particular hydroxyls phosphorylated enzymically were duplicated completely, nor were any differences observed when enzyme preparations eluted from the affinity columns with either dT, dG, or NAD were compared. No indications of the existence of more than one phosphotransferase were obtained. A selection of experiments, showing the total yields of phosphorylation products during different periods of incubation, is assembled in Table 2. It includes four ribonucleosidonucleotides that could not be tested before owing to the contamination of our previous enzyme preparations with a ribophosphodiesterase (unpublished experiments with S.-H. Chang). In the present procedure most, if not all, of the interfering activity was removed. This made possible the study of ApC, CpC, CpA, and ApA as phosphate acceptors. Table 2 summarizes the total yields of enzymic phosphorylation products, regardless of the distribution of isomers. With all nucleosidonucleotides, a new UV-absorbing component appeared on the chromatograms that exhibited a spectrum identical with that of the respective precursor. The maximum amount of product was formed after incubation for 60 min, with generally only a slight further increase when the incubation was extended to 24 hr. CpA formed an exception; on very long incubation a partial decomposition of the CpAp formed was observed. With the nucleosides, the conversion to 2'(3')-nucleotides was virtually complete. In our previous study (2), when initial velocities were compared, the amounts of Cp formed from C and of pCp formed from pC were the same, in contrast to the present findings with an excess of enzyme (Table 2). A detailed
C
A
(5')
u-
(5')
13
C
A
(2')
(2') 75% (3)
(2') (3')
0
()
(2') (3')
C
A
28 71
(2') (3')
71 29
0
(2) -29 (3') -71
35%
68% (5')
39% -~(3'
A
A
0
.5
(5')
I
FIG. 3. Positional discrimination in enzymic phosphate transfer to ribonucleosides and their derivatives. In all cases one phosphate moiety was transferred to a hydroxyl, with the indicated relative frequencies (as % of total product). The yields of total phosphorylation products (as % of acceptor) are given on the left of each structure.
study showed that with limiting amounts of enzyme both acceptors are phosphorylated at the same rate during the first 2 hr, but that subsequently the formation of pCp falls off whereas that of the monophosphate Cp continues to completion. Positional Discrimination in Phosphate Transfer. The nucleosides C and A offer three hydroxyls to the transferase, the 5'-nucleotides pC and pA afford two, and the nucleosidonucleotides ApC and ApA afford four hydroxyls. Since in all cases only one phosphate group appears to be transferred enzymically, the selections made by the enzyme are of interest. In a mixed dimer like ApC, in which the phosphate bridge goes from the 3' position of A to the 5' position of C, alkaline hydrolysis of the phosphorylation product yields by itself sufficient data for a decision. The product obtained by the enzyme action on ApC was isolated on paper and treated with alkali. Since hydrolysis was complete, no phosphorylation of the 2' hydroxyl of adenosine could have occurred; the isolation of less than 1% of pAp and C similarly showed that phosphorylation of the 5' hydroxyl of adenosine was minimal. The principal hydrolysis products were AMP and CMP, which latter was shown by liquid chromatography to consist of 29% 2'-CMP and 71% 3'CMP. With two identical bases, as in ApA, it is necessary to resort to enzymic hydrolysis for the analysis of the product. Spleen phosphodiesterase II, which produces 3'-nucleotides, was used. The enzymic hydrolysis of the phosphorylation product of ApA was nearly complete (98%). Liquid chromatography showed the hydrolysate to consist of 14% 2'-AMP and 86% 3'-AMP. The same proportions were found when the AMP mixture was first isolated by paper chromatography and then analyzed by liquid
Biochemistry: Brunngraber and Chargaff chromatography. From these values it can be calculated that the transferase produced 28% ApAp(2') and 72%6 ApAp(3'). The small amount not hydrolyzed by the spleen enzyme (about 2%) must have been all (5')pApA, since the product was completely hydrolyzed with alkali. This would not have been the case if some (2')pApA had also been formed. The various findings are summarized in Fig. 3. The values for the nucleosides and 5'-nucleotides were redetermined with enzyme preparations purified as described here; the results completely duplicated the findings reported before (1, 2). Concluding Remarks. Several biological systems on which an enzyme of the type described here could have a regulatory effect have been discussed before (1, 2). One possible mode of interference by the nucleotide phosphotransferase in the biosynthesis of the nucleic acid could be the inactivation of the primer, in polymerase catalysis requiring such a compound, via the phosphorylation of the terminal 3' hydroxyl indispensable for chain growth. Another possible effect could be on the terminal 3' OH group of a transfer RNA. From the exper-
Proc. Natl. Acad. Sci. USA 74 (1977)
3229
iments with nucleosidonucleotides presented here it may be co.ided that it will be exclusively the 3' OH end that will be phosphorylated. The results summarized in Fig. 3 reveal a surprising shift in positional selectivity, especially when the cytosine-containing compounds are compared. It is also noteworthy that the nucleoside carrying the 3' -- 5'-phosphate bridge is practically not attacked by the enzyme. This work was aided by research grants from the National Institutes of Health, U.S. Public Health Service, and the National Science Foundation. 1. Brunngraber, E. F. & Chargaff, E. (1970) Proc. Natl. Acad. Sci. USA 67,107-112. 2. Brunngraber, E. F. & Chargaff, E. (1973) Biochwmistry 12, 3005-312. 3. Brunngraber, E. F. & Chargaff, E. (1973) Biochemistry 12, 3012-016. 4. Razzeli, W. E. (1963) in Methods in Enzymology, eds. Colowick, S. P. & Kaplan, N. 0. (Academic Press, New York and London), Vol. 6, pp. 236-258.
