ARCHIVES
OF BIOCHEMISTRY
AND BIOPHYSICS
Vol. 296, No. 1, July, pp. 161-169, 1992
A Novel Human Phosphotransferase Highly Specific for Adenosine Edward
P. Garvey’
and Thomas
Wellcome Research Laboratories,
A. Krenitsky
Research Triangle Park, North Carolina 27709
Received December 9, 1991
A novel nucleoside phosphotransferase, referred to as adenosine phosphotransferase (Ado Ptase), was partially purified 1230-fold from human placenta. This enzyme differed from other known nucleoside phosphotransferases in its substrate specificity. Using AMP as the phosphate donor, it readily phosphorylated Ado. Changes in the sugar moiety were tolerated. dAdo and ddAdo were phosphate acceptors and dAMP was a donor. No other nucleotide or nucleoside common in nature displayed appreciable activity as donor or acceptor substrate, respectively. In the absence of nucleoside, the enzyme catalyzed the hydrolysis of AMP, typical of other nucleoside phosphotransferases. However, in the presence of Ado, little, if any, hydrolysis occurred. Ado Ptase had an absolute requirement for a metal cation, with Mg2+ and, to a lesser extent, Mn*+ fulfilling this requisite. The apparent K,,, for Ado was 0.2 mM. However, the donor AMP displayed cooperativity in both transfer and hydrolytic reactions. This cooperativity was eliminated by nucleotides, 2,3-diphosphoglycerate, and inorganic phosphate. ADP and 2,3-diphosphoglycerate were especially potent. In the presence of these effecters, the apparent K,,, for AMP was 3.0 mM in the transfer reaction and 4.0 mM in the hydrolytic reaction. Kinetic data suggest that there are two nucleotide binding sites on Ado Ptase, one for the donor, the other for an effector. AMP appeared to bind to both sites. Although this novel enzyme might play a role in the anabolism of nucleoside analogues, the normal physiological role of this nucleoside phosphotranso 1992 Academic P~RW, IIIC. ferase is not understood.
The enzymes that phosphorylate nucleosides fall into two groups on the basis of the phosphate donor and the reaction mechanism. One group, the nucleoside kinases, utilizes nucleoside triphosphates as phosphate donors in direct displacement reactions. The other group catalyzes 1 To whom correspondence
should be addressed. Fax: (919) 248-8747.
0003s9861/92 $5.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form
the phosphorylation reaction with nucleoside monophosphates as donors. This second class, the nucleoside phosphotransferases, catalyzes reactions that proceed through a phosphorylated enzyme intermediate. The overall reaction consists of the conversion of acceptor nucleoside to its monophosphate and the accompanied conversion of the donor nucleoside monophosphate to its unphosphorylated nucleoside. Nucleoside phosphotransferases seem to be a part of a family of enzymes that convert nucleoside monophosphates to nucleosides. This family includes another class of enzymes, the nucleoside monophosphate hydrolases (i.e., nucleotidases). The distinction between phosphotransferases and nucleotidases is sometimes not sharp. It is based on the relative rates of phosphoryl transfer from the donor nucleoside monophosphate to acceptor nucleoside vs the rate of transfer to water. With nucleoside phosphotransferases, appropriate acceptor nucleosides compete very effectively with water as the phosphate acceptor. Although these enzymes display nucleotidase activity in the absence of acceptor nucleoside, very little hydrolysis of the donor nucleoside monophosphate occurs in the presence of appropriate nucleoside. In contrast, with true nucleotidases, the presence of nucleoside does not appreciably alter the rate of the hydrolytic reaction and little, if any, transfer of phosphate to an acceptor nucleoside occurs. On the other hand, at least one 5’-nucleotidase has considerable phosphotransferase activity. The soluble 5’-nucleotidase that catalyzes nucleoside exchange at high concentrations of acceptor nucleoside (1) represents an activity that falls somewhere between the two extremes just outlined. Enzymes that phosphorylate nucleosides have attracted much attention because of their importance in the activation of nucleoside analogues that have antiviral or antitumor activity. Of particular relevance to this paper are the studies of enzymes that phosphorylate purine dideoxynucleosides that have activity against the human immune deficiency virus (HIV). The soluble 5’-nucleotidase that catalyzes nucleoside exchange (1) also phosphorylates 161
Inc. reserved.
162
GARVEY
AND
ddIno and ddGuo using IMP as the phosphate donor (2). This enzyme does not phosphorylate ddAdo directly. Dideoxyadenosine is initially converted by adenosine deaminase to ddIno prior to phosphorylation (3,4). We describe in this report a novel nucleoside phosphotransferase which we refer to as adenosine phosphotransferase (Ado Ptase).’ It exhibited marked specificity for adenosine as the acceptor nucleoside and also was capable of phosphorylating adenosine analogues such as ddAdo. MATERIALS
AND
METHODS
KRENITSKY Nucleoside monophosphate hydrolysis to nucleoside was assayed by the same technique. Assay buffer for AMP hydrolysis (i.e., [8-“C]AMP being converted to [&“C]Ado) was the same as above, except that no nucleoside was present and radioactive AMP was at 0.1 mM. The “high K,,,” 5’-nucleotidase (5) was assayed in either the hydrolytic or the phosphotransfer direction in 50 mM imidazole-HCl, pH 6.5, 20 mM MgC&, 0.5 M NaCl, 0.5 mg of BSA/ml, 5 mM IMP, and 5 mM ATP. Radioactive Ino was at 0.1 mM, where the phosphotransfer reaction was assayed. The following activities were assayed using published procedures: nonspecific phosphatases withp-nitrophenylphosphate as substrate (6); purine nucleoside phosphorylase, adenosine deaminase, AMP deaminase (7); and adenosine kinase, AMP kinase (8). None of these activities were detected in the final preparation of Ado Ptase.
Chemicals and Radiochemicak Highly pure imidazole, AMP and ADP affinity resins, 2,3-DPG, PRib-PP, and all nucleosides and nucleotides (except those noted below) were purchased from Sigma (St. Louis, MO). The purities of AMP, ADP, and ATP were examined by ion-exchange HPLC. AMP purity was greater than 99%, with less than 0.1% ADP or ATP impurity. ADP purity was 94%, with a 6% AMP contamination. ATP was 99% pure, with a 1% ADP contamination. ATP (“special quality”) was purchased from Boehringer Mannheim (Indianapolis, IN). Orange A resin was a product of Amicon (Danvers, MA). DEAE-Sepharose Fast Flow, butylSepharose 4B, agarose-hexane-ATP (type IV), Mono Q HR 5/5, Superose 12 HR 10/30, and concanavalin A-Sepharose were from Pharmacia LKB Biotechnology (Piscataway, NJ). IS-“C]Ado, [8-i4C]Ino, [8“C]Guo, [8-3H]dGuo, [S-“C]dAdo, and [2’,3’-3H]ddAdo were purchased from Moravek Biochemicals (Brea, CA); [U-“C]Cyd and [5’-3H]Thd were from Amersham (Arlington Heights, IL); [8-i4C]AMP and [814C]IMP were from ICN Radiochemicals (Costa Mesa, CA); and [U14C]ATP, [U-“C]Urd, and [2-“C]dCyd from DuPont New England Nuclear Research Products (Wilmington, DE).
Enzyme Assays The standard reaction mixture used to assay nucleoside phosphory(pH 7.2), 20 mM MgClz, 0.5 mg lation contained 50 mM Hepes-NaOH of BSA/ml, 5 mM AMP (as donor), 5 mM ATP (as activator), and 0.1 mM radioactive nucleoside. During the early steps of purification and when labeled Ado was used as substrate, the adenosine deaminase inhibitor EHNA was included at 10 pM. The reaction was initiated by enzyme. Typical volumes were 20 ML. Reaction mixtures were incubated at 37°C and 2-~1 aliquots were spotted onto polyethyleneimine, thinlayer chromatography plates, prespotted with 10 nmol of unlabeled nucleoside. Plates were developed in either 50% MeOH or 0.5 M LiC1/0.5 M acetic acid. Product and substrate were quantitated either by scanning individual lanes on a Bioscan System 200 Imaging Scanner (used to follow activity through purifications), or visualizing and cutting out uv spots and counting the radioactivity by liquid scintillation spectrophotometry with ScintiLene (Fisher). The enzyme activity was proportional to the amount of enzyme used. Unless stated otherwise, all rates were calculated from data collected during the initial 15% conversion of substrate. Single time points were taken during purification of Ado Ptase. For the determination of kinetic parameters, three time points were taken, and the rate was calculated by a linear least square fit to the data (the r square value for these fits was consistently greater than 0.99). One unit of enzyme is the amount that converts one nanomole of labeled substrate to product per minute.
’ Abbreviations used: Ado Ptase, adenosine phosphotransferase; ddAdo, dideoxyadenosine; EHNA, 9-(erythro-2-hydroxy-l-nonyl)adenine; 2,3-DPG, 2,3diphosphoglycerate; Ap3A, P1,Pa-di(adenosine5’) triphosphate; Ap,A, P’,P-di(adenosine-5’) tetraphosphate; APS, adenosine 5’-phosphosulfate; AMP-C-P, cY&methylene ADP, ADP-CP, &y-methylene ATP, pNPP, p-nitrophenylphosphate.
Purification of Ado-Specific AMP-Dependent Phosphotransferase (Ado Ptase) from Human Placenta All of the following steps were performed at 4’C. Preparation of extract. Amnion and chorion were removed from fresh placenta, which was then rinsed with 0.9% NaCl (3 X 500 ml). The tissue (500-700 g) was then homogenized in a Waring blender (4 X 2 min, with 2-min periods in between for cooling), in 3-4 vol of Buffer A (20 mM imidazole-HCl (pH 7.0), 20 mM MgClr) with the addition of the following protease inhibitors: 0.5 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 0.5 mM phenanthrolene, 0.5 pg/ml leupeptin, and 10 fig/ ml soybean trypsin inhibitor. The homogenate was filtered through a single layer of cheesecloth and then centrifuged in a Sorvall RCdB centrifuge twice at 27,500g for 30 min. Supernatant was taken as extract (Table I), which could be stored at -70°C without a significant loss in activity over several months. Purifications were typically performed with 500 ml of extract. Ammonium sulfate fractionation. Ado Ptase precipitated between 30 and 55% (NHI)&SOI saturation; solid (NHI)$OI (85 g,. followed by an additional 84 g) was added to give the percentage saturations indicated. Precipitated protein was pelleted at 25,000g for I5 min and redissolved in 100-200 ml Buffer A (plus protease inhibitors described above). This material was dialyzed against three changes (4 h each) of 2 liters Buffer A (plus protease inhibitors). The dialysate was applied to a 2.5 X 17-cm column DEAE-Sephurose. of DEAE-Sepharose Fast Flow that had been equilibrated in Buffer A. Flow rate was 1 ml/min and 15-min fractions were collected. The resin was then washed with Buffer A until absorbance at 280 nm decreased to below 0.2. Ado Ptase was eluted in a large volume by raising the imidazole concentration of Buffer A to 100 mM. Active fractions were pooled (typically 200-300 ml) and protein was precipitated with the addition of solid (NH&SO4 to 60% saturation. Pelleted protein was dissolved in a minimum volume of Buffer A (2 ml) and then dialyzed against 2 liters of Buffer A overnight. 30 mg of protein in the dialyzed pool FPLC Mono Q. Approximately from DEAE-Sepharose was applied to Mono Q HR 5/5 (5 X 50 mm) column, which had been equilibrated with Buffer A at a flow of 1 ml/ min. One-milliliter fractions were collected. After washing with 5 ml of Buffer A, the column was developed with a linear gradient of 0 to 300 mM NaCl (in Buffer A) in a volume of 30 ml. Fractions containing activity were pooled. Ado Ptase consistently eluted at about 140 mM NaCl. Butyl Sepharose 4B. The Mono Q pool was made 1 M (NH&SO4 by addition of solid (NH4)$S01. This solution was applied at a flow of 0.3 ml/min to a 1 X 3-cm column of butyl-Sepharose that had been equilibrated with Buffer B (Buffer A plus 1 M (NH&SOI). Two-milliliter fractions were collected. After the resin was washed with Buffer B until absorbance at 280 nm decreased below 0.1, Ado Ptase was eluted with a linear gradient of 1 to 0 M (NH,)zSOI (in Buffer A) in a total volume of 20 ml. Ado Ptase eluted at about 0.5 M (NH4)$04. Active fractions were pooled and dialyzed against 1 liter of Buffer A overnight.
A NOVEL
Application
NUCLEOSIDE
PHOSPHOTRANSFERASE Buffer A plus 0.1 M NaCl
Buffer A 0.1 M lmidazole
FROM
HUMAN
PLACENTA
Buffer A plus 0.3 M NaCl
H
4
8
12
16
50
54
58 62
66
70 74
a6
90
163
94
98102
%
lioli4ila
Fractions
FIG. 1. DEAE-Sepharose chromatography of Ado Ptase. 150 ml of dialysate (from the previous (NH&SO, step) that contained -500 units of Ado Ptase was applied to DEAE-Sepharose as described under Materials and Methods. After elution of Ado Ptase, the resin was first washed with 0.1 M NaCl in Buffer A and then 0.3 M NaCl in Buffer B. Note that the “high K,,,” 5’-nucleotidase was assayed in the phosphotransfer direction (i.e., the phosphorylation of [‘*C]Ino in the presence of IMP), while the “low K” 5’-nucleotidase was assayed in the hydrolytic direction (i.e., the cleavage of [i4C]AMP to Ado).
ATP Sepharose. The dialyzed sample was loaded at a flow rate of 0.5 ml/min onto ATP-Sepharose (1 X 2.5 cm). The resin was washed with Buffer A until absorbance at 280 nm decreased below 0.1. Then enzyme activity was eluted with 0.25 M NaCl in Buffer A. Active fractions were pooled and the volume was reduced to 0.4 ml by Amicon ultrafiltration (Centricon 10). FPLC Superose 12. A volume of 0.2 ml of the ATP-Sepharose pool (typically, 0.1-0.2 mg protein) was applied to the FPLC Superose 12size exclusion column, which had been equilibrated in Buffer C (Buffer was performed at 0.3 ml/min, and A plus 0.1 M NaCl). Chromatography 0.3-ml fractions were collected. Protein standards had the following retention volumes: catalase (10.9 ml), aldolase (11.4 ml), BSA (12.2 ml), ovalbumin (12.9 ml); void volume was 7.8 ml. Ado Ptase eluted at 10.9 ml. Active fractions were pooled, concentrated by Amicon ultrafiltration to approximately 0.3 ml, and stored at 4°C. Under these conditions, Ado Ptase activity lost -25% activity over a 2-week period. Protein determination. Protein concentration was determined by the Bradford method (9) with bovine serum albumin as a standard. Treatment to remoue MgC1,. Ado Ptase (200 ~1) was dialyzed in a microdialyzer against two 80-ml changes (5 h each) of 20 mM imidazoleHCl, pH 7.0 (made with Chelex-treated distilled water), in the presence of 8 g Chelex. EDTA was then added to enzyme to a concentration of 0.5 mM. Enzyme was dialyzed as described above for 12 h. Trace amounts of EDTA-Mgz+ were finally removed by FPLC Superose 12 chromatography in Buffer C minus MgClz, made with Chelex-treated distilled water.
RESULTS Purification
of Ado Ptase
An enzyme activity was detected in crude extracts of human placenta that phosphorylated ddAdo and other nucleoside analogues in the presence of AMP, albeit at a
very low rate (-0.5 pmol product/min/mg protein). We also observed this phosphorylating activity in a T lymphoblastoid cell line (CCRF-CEM) (data not shown). Because of the availability of material, we purified this activity from human placenta. In addition, we used Ado rather than ddAdo as the substrate to monitor purification because of the significant increase in activity (>lOO-fold). To assay crude extracts, the adenosine deaminase inhibitor EHNA was included in the assay mixture. With every purification technique used, the Ado-phosphorylating activity copurified with the ddAdo-phosphorylating activity (data not shown). Ado Ptase was separated from adenosine kinase, which is known to phosphorylate purine nucleosides and purine nucleoside analogues (10). Most of the Ado kinase activity was removed from the preparation during (NH&SO4 fractionation, as Ado kinase precipitated above 60% (NHJ2S04. Residual Ado kinase did not bind to DEAESepharose, and the final preparation did not catalyze the phosphorylation of Ado using ATP as the phosphate donor. Ado Ptase was separated by DEAE-Sepharose chromatography (Fig. 1) from the two soluble 5’-nucleotidases that have been isolated from human placenta (5,11,12). The hydrolytic enzyme that prefers AMP to IMP, termed “low-K,” 5’-nucleotidase (11,12), did not bind to the column. In contrast, the enzyme that prefers IMP to AMP, labeled “high K,,,” 5’-nucleotidase (5, 12), binds more tightly to DEAE-Sepharose than Ado Ptase and did not
164
GARVEY
AND TABLE
Purification
Step
Volume (ml)
Protein bvd
Extract (NHJsSOI DEAE-Sepharose FPLC Mono Q ATP-Sepharose FPLC Superose 12
500 210 110 15 2 0.9
8670 714 55 7 0.7 0.2
KRENITSKY I
of Ado Ptase from Human Placenta Units (nmol/min) 1750 932 1100 465 100 44
Specific activity (units/mg) 0.2 1.3 20 66 152 246
Recovery (%) 53 63 27 6 3
Fold purification 6.5 100 330 760 1230
elute with Ado Ptase. In addition, both of the 5’-nucleo- preparation still contained five significant protein species tidase activities were separable from Ado Ptase using as judged by SDS-PAGE analysis (data not shown). Thus, other chromatographic techniques not shown in Table I. the most successful preparation is at best lo-20% pure. AMP-Sepharose and concanavalin A-Sepharose sepa- However, no activities that would interfere with the assay rated low K,,, 5’-nucleotidase from Ado Ptase, and AMP- for Ado Ptase were detected in the final preparation. All Sepharose and Orange A resin separated the high K,,, 5’- subsequent studies were performed with this enzyme. nucleotidase from Ado Ptase. It is noteworthy that, whereas the high K,,, 5’-nucleo- Rates of Phosphate Donation vs. Acceptance tidase transfers a phosphoryl group from a nucleoside In the absence of an acceptor nucleoside, Ado Ptase monophosphate to a nucleoside (1) or to a nucleoside ancatalyzed the hydrolysis of AMP. A preparation that had alogue (2,13), the low K,,, 5’-nucleotidase apparently does a specific activity of 200 units/mg in the phosphotransfer not. In our laboratory, the low Km 5’-nucleotidase from direction had a specific activity of 140 units/mg when human placenta was purified by AMP-Sepharose chromatography (11). Even though this preparation was able assayed for the hydrolysis of [14C]AMP. We determined to hydrolyze AMP with a specific activity of 1.0 pmol/ whether Ado Ptase was a phosphotransferase or a 5’-nucleotidase by measuring the rate of phosphorylation of min/mg, it was not able to phosphorylate either Ado or Ino (
OF BIOCHEMISTRY
AND BIOPHYSICS
Vol. 296, No. 1, July, pp. 161-169, 1992
A Novel Human Phosphotransferase Highly Specific for Adenosine Edward
P. Garvey’
and Thomas
Wellcome Research Laboratories,
A. Krenitsky
Research Triangle Park, North Carolina 27709
Received December 9, 1991
A novel nucleoside phosphotransferase, referred to as adenosine phosphotransferase (Ado Ptase), was partially purified 1230-fold from human placenta. This enzyme differed from other known nucleoside phosphotransferases in its substrate specificity. Using AMP as the phosphate donor, it readily phosphorylated Ado. Changes in the sugar moiety were tolerated. dAdo and ddAdo were phosphate acceptors and dAMP was a donor. No other nucleotide or nucleoside common in nature displayed appreciable activity as donor or acceptor substrate, respectively. In the absence of nucleoside, the enzyme catalyzed the hydrolysis of AMP, typical of other nucleoside phosphotransferases. However, in the presence of Ado, little, if any, hydrolysis occurred. Ado Ptase had an absolute requirement for a metal cation, with Mg2+ and, to a lesser extent, Mn*+ fulfilling this requisite. The apparent K,,, for Ado was 0.2 mM. However, the donor AMP displayed cooperativity in both transfer and hydrolytic reactions. This cooperativity was eliminated by nucleotides, 2,3-diphosphoglycerate, and inorganic phosphate. ADP and 2,3-diphosphoglycerate were especially potent. In the presence of these effecters, the apparent K,,, for AMP was 3.0 mM in the transfer reaction and 4.0 mM in the hydrolytic reaction. Kinetic data suggest that there are two nucleotide binding sites on Ado Ptase, one for the donor, the other for an effector. AMP appeared to bind to both sites. Although this novel enzyme might play a role in the anabolism of nucleoside analogues, the normal physiological role of this nucleoside phosphotranso 1992 Academic P~RW, IIIC. ferase is not understood.
The enzymes that phosphorylate nucleosides fall into two groups on the basis of the phosphate donor and the reaction mechanism. One group, the nucleoside kinases, utilizes nucleoside triphosphates as phosphate donors in direct displacement reactions. The other group catalyzes 1 To whom correspondence
should be addressed. Fax: (919) 248-8747.
0003s9861/92 $5.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form
the phosphorylation reaction with nucleoside monophosphates as donors. This second class, the nucleoside phosphotransferases, catalyzes reactions that proceed through a phosphorylated enzyme intermediate. The overall reaction consists of the conversion of acceptor nucleoside to its monophosphate and the accompanied conversion of the donor nucleoside monophosphate to its unphosphorylated nucleoside. Nucleoside phosphotransferases seem to be a part of a family of enzymes that convert nucleoside monophosphates to nucleosides. This family includes another class of enzymes, the nucleoside monophosphate hydrolases (i.e., nucleotidases). The distinction between phosphotransferases and nucleotidases is sometimes not sharp. It is based on the relative rates of phosphoryl transfer from the donor nucleoside monophosphate to acceptor nucleoside vs the rate of transfer to water. With nucleoside phosphotransferases, appropriate acceptor nucleosides compete very effectively with water as the phosphate acceptor. Although these enzymes display nucleotidase activity in the absence of acceptor nucleoside, very little hydrolysis of the donor nucleoside monophosphate occurs in the presence of appropriate nucleoside. In contrast, with true nucleotidases, the presence of nucleoside does not appreciably alter the rate of the hydrolytic reaction and little, if any, transfer of phosphate to an acceptor nucleoside occurs. On the other hand, at least one 5’-nucleotidase has considerable phosphotransferase activity. The soluble 5’-nucleotidase that catalyzes nucleoside exchange at high concentrations of acceptor nucleoside (1) represents an activity that falls somewhere between the two extremes just outlined. Enzymes that phosphorylate nucleosides have attracted much attention because of their importance in the activation of nucleoside analogues that have antiviral or antitumor activity. Of particular relevance to this paper are the studies of enzymes that phosphorylate purine dideoxynucleosides that have activity against the human immune deficiency virus (HIV). The soluble 5’-nucleotidase that catalyzes nucleoside exchange (1) also phosphorylates 161
Inc. reserved.
162
GARVEY
AND
ddIno and ddGuo using IMP as the phosphate donor (2). This enzyme does not phosphorylate ddAdo directly. Dideoxyadenosine is initially converted by adenosine deaminase to ddIno prior to phosphorylation (3,4). We describe in this report a novel nucleoside phosphotransferase which we refer to as adenosine phosphotransferase (Ado Ptase).’ It exhibited marked specificity for adenosine as the acceptor nucleoside and also was capable of phosphorylating adenosine analogues such as ddAdo. MATERIALS
AND
METHODS
KRENITSKY Nucleoside monophosphate hydrolysis to nucleoside was assayed by the same technique. Assay buffer for AMP hydrolysis (i.e., [8-“C]AMP being converted to [&“C]Ado) was the same as above, except that no nucleoside was present and radioactive AMP was at 0.1 mM. The “high K,,,” 5’-nucleotidase (5) was assayed in either the hydrolytic or the phosphotransfer direction in 50 mM imidazole-HCl, pH 6.5, 20 mM MgC&, 0.5 M NaCl, 0.5 mg of BSA/ml, 5 mM IMP, and 5 mM ATP. Radioactive Ino was at 0.1 mM, where the phosphotransfer reaction was assayed. The following activities were assayed using published procedures: nonspecific phosphatases withp-nitrophenylphosphate as substrate (6); purine nucleoside phosphorylase, adenosine deaminase, AMP deaminase (7); and adenosine kinase, AMP kinase (8). None of these activities were detected in the final preparation of Ado Ptase.
Chemicals and Radiochemicak Highly pure imidazole, AMP and ADP affinity resins, 2,3-DPG, PRib-PP, and all nucleosides and nucleotides (except those noted below) were purchased from Sigma (St. Louis, MO). The purities of AMP, ADP, and ATP were examined by ion-exchange HPLC. AMP purity was greater than 99%, with less than 0.1% ADP or ATP impurity. ADP purity was 94%, with a 6% AMP contamination. ATP was 99% pure, with a 1% ADP contamination. ATP (“special quality”) was purchased from Boehringer Mannheim (Indianapolis, IN). Orange A resin was a product of Amicon (Danvers, MA). DEAE-Sepharose Fast Flow, butylSepharose 4B, agarose-hexane-ATP (type IV), Mono Q HR 5/5, Superose 12 HR 10/30, and concanavalin A-Sepharose were from Pharmacia LKB Biotechnology (Piscataway, NJ). IS-“C]Ado, [8-i4C]Ino, [8“C]Guo, [8-3H]dGuo, [S-“C]dAdo, and [2’,3’-3H]ddAdo were purchased from Moravek Biochemicals (Brea, CA); [U-“C]Cyd and [5’-3H]Thd were from Amersham (Arlington Heights, IL); [8-i4C]AMP and [814C]IMP were from ICN Radiochemicals (Costa Mesa, CA); and [U14C]ATP, [U-“C]Urd, and [2-“C]dCyd from DuPont New England Nuclear Research Products (Wilmington, DE).
Enzyme Assays The standard reaction mixture used to assay nucleoside phosphory(pH 7.2), 20 mM MgClz, 0.5 mg lation contained 50 mM Hepes-NaOH of BSA/ml, 5 mM AMP (as donor), 5 mM ATP (as activator), and 0.1 mM radioactive nucleoside. During the early steps of purification and when labeled Ado was used as substrate, the adenosine deaminase inhibitor EHNA was included at 10 pM. The reaction was initiated by enzyme. Typical volumes were 20 ML. Reaction mixtures were incubated at 37°C and 2-~1 aliquots were spotted onto polyethyleneimine, thinlayer chromatography plates, prespotted with 10 nmol of unlabeled nucleoside. Plates were developed in either 50% MeOH or 0.5 M LiC1/0.5 M acetic acid. Product and substrate were quantitated either by scanning individual lanes on a Bioscan System 200 Imaging Scanner (used to follow activity through purifications), or visualizing and cutting out uv spots and counting the radioactivity by liquid scintillation spectrophotometry with ScintiLene (Fisher). The enzyme activity was proportional to the amount of enzyme used. Unless stated otherwise, all rates were calculated from data collected during the initial 15% conversion of substrate. Single time points were taken during purification of Ado Ptase. For the determination of kinetic parameters, three time points were taken, and the rate was calculated by a linear least square fit to the data (the r square value for these fits was consistently greater than 0.99). One unit of enzyme is the amount that converts one nanomole of labeled substrate to product per minute.
’ Abbreviations used: Ado Ptase, adenosine phosphotransferase; ddAdo, dideoxyadenosine; EHNA, 9-(erythro-2-hydroxy-l-nonyl)adenine; 2,3-DPG, 2,3diphosphoglycerate; Ap3A, P1,Pa-di(adenosine5’) triphosphate; Ap,A, P’,P-di(adenosine-5’) tetraphosphate; APS, adenosine 5’-phosphosulfate; AMP-C-P, cY&methylene ADP, ADP-CP, &y-methylene ATP, pNPP, p-nitrophenylphosphate.
Purification of Ado-Specific AMP-Dependent Phosphotransferase (Ado Ptase) from Human Placenta All of the following steps were performed at 4’C. Preparation of extract. Amnion and chorion were removed from fresh placenta, which was then rinsed with 0.9% NaCl (3 X 500 ml). The tissue (500-700 g) was then homogenized in a Waring blender (4 X 2 min, with 2-min periods in between for cooling), in 3-4 vol of Buffer A (20 mM imidazole-HCl (pH 7.0), 20 mM MgClr) with the addition of the following protease inhibitors: 0.5 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 0.5 mM phenanthrolene, 0.5 pg/ml leupeptin, and 10 fig/ ml soybean trypsin inhibitor. The homogenate was filtered through a single layer of cheesecloth and then centrifuged in a Sorvall RCdB centrifuge twice at 27,500g for 30 min. Supernatant was taken as extract (Table I), which could be stored at -70°C without a significant loss in activity over several months. Purifications were typically performed with 500 ml of extract. Ammonium sulfate fractionation. Ado Ptase precipitated between 30 and 55% (NHI)&SOI saturation; solid (NHI)$OI (85 g,. followed by an additional 84 g) was added to give the percentage saturations indicated. Precipitated protein was pelleted at 25,000g for I5 min and redissolved in 100-200 ml Buffer A (plus protease inhibitors described above). This material was dialyzed against three changes (4 h each) of 2 liters Buffer A (plus protease inhibitors). The dialysate was applied to a 2.5 X 17-cm column DEAE-Sephurose. of DEAE-Sepharose Fast Flow that had been equilibrated in Buffer A. Flow rate was 1 ml/min and 15-min fractions were collected. The resin was then washed with Buffer A until absorbance at 280 nm decreased to below 0.2. Ado Ptase was eluted in a large volume by raising the imidazole concentration of Buffer A to 100 mM. Active fractions were pooled (typically 200-300 ml) and protein was precipitated with the addition of solid (NH&SO4 to 60% saturation. Pelleted protein was dissolved in a minimum volume of Buffer A (2 ml) and then dialyzed against 2 liters of Buffer A overnight. 30 mg of protein in the dialyzed pool FPLC Mono Q. Approximately from DEAE-Sepharose was applied to Mono Q HR 5/5 (5 X 50 mm) column, which had been equilibrated with Buffer A at a flow of 1 ml/ min. One-milliliter fractions were collected. After washing with 5 ml of Buffer A, the column was developed with a linear gradient of 0 to 300 mM NaCl (in Buffer A) in a volume of 30 ml. Fractions containing activity were pooled. Ado Ptase consistently eluted at about 140 mM NaCl. Butyl Sepharose 4B. The Mono Q pool was made 1 M (NH&SO4 by addition of solid (NH4)$S01. This solution was applied at a flow of 0.3 ml/min to a 1 X 3-cm column of butyl-Sepharose that had been equilibrated with Buffer B (Buffer A plus 1 M (NH&SOI). Two-milliliter fractions were collected. After the resin was washed with Buffer B until absorbance at 280 nm decreased below 0.1, Ado Ptase was eluted with a linear gradient of 1 to 0 M (NH,)zSOI (in Buffer A) in a total volume of 20 ml. Ado Ptase eluted at about 0.5 M (NH4)$04. Active fractions were pooled and dialyzed against 1 liter of Buffer A overnight.
A NOVEL
Application
NUCLEOSIDE
PHOSPHOTRANSFERASE Buffer A plus 0.1 M NaCl
Buffer A 0.1 M lmidazole
FROM
HUMAN
PLACENTA
Buffer A plus 0.3 M NaCl
H
4
8
12
16
50
54
58 62
66
70 74
a6
90
163
94
98102
%
lioli4ila
Fractions
FIG. 1. DEAE-Sepharose chromatography of Ado Ptase. 150 ml of dialysate (from the previous (NH&SO, step) that contained -500 units of Ado Ptase was applied to DEAE-Sepharose as described under Materials and Methods. After elution of Ado Ptase, the resin was first washed with 0.1 M NaCl in Buffer A and then 0.3 M NaCl in Buffer B. Note that the “high K,,,” 5’-nucleotidase was assayed in the phosphotransfer direction (i.e., the phosphorylation of [‘*C]Ino in the presence of IMP), while the “low K” 5’-nucleotidase was assayed in the hydrolytic direction (i.e., the cleavage of [i4C]AMP to Ado).
ATP Sepharose. The dialyzed sample was loaded at a flow rate of 0.5 ml/min onto ATP-Sepharose (1 X 2.5 cm). The resin was washed with Buffer A until absorbance at 280 nm decreased below 0.1. Then enzyme activity was eluted with 0.25 M NaCl in Buffer A. Active fractions were pooled and the volume was reduced to 0.4 ml by Amicon ultrafiltration (Centricon 10). FPLC Superose 12. A volume of 0.2 ml of the ATP-Sepharose pool (typically, 0.1-0.2 mg protein) was applied to the FPLC Superose 12size exclusion column, which had been equilibrated in Buffer C (Buffer was performed at 0.3 ml/min, and A plus 0.1 M NaCl). Chromatography 0.3-ml fractions were collected. Protein standards had the following retention volumes: catalase (10.9 ml), aldolase (11.4 ml), BSA (12.2 ml), ovalbumin (12.9 ml); void volume was 7.8 ml. Ado Ptase eluted at 10.9 ml. Active fractions were pooled, concentrated by Amicon ultrafiltration to approximately 0.3 ml, and stored at 4°C. Under these conditions, Ado Ptase activity lost -25% activity over a 2-week period. Protein determination. Protein concentration was determined by the Bradford method (9) with bovine serum albumin as a standard. Treatment to remoue MgC1,. Ado Ptase (200 ~1) was dialyzed in a microdialyzer against two 80-ml changes (5 h each) of 20 mM imidazoleHCl, pH 7.0 (made with Chelex-treated distilled water), in the presence of 8 g Chelex. EDTA was then added to enzyme to a concentration of 0.5 mM. Enzyme was dialyzed as described above for 12 h. Trace amounts of EDTA-Mgz+ were finally removed by FPLC Superose 12 chromatography in Buffer C minus MgClz, made with Chelex-treated distilled water.
RESULTS Purification
of Ado Ptase
An enzyme activity was detected in crude extracts of human placenta that phosphorylated ddAdo and other nucleoside analogues in the presence of AMP, albeit at a
very low rate (-0.5 pmol product/min/mg protein). We also observed this phosphorylating activity in a T lymphoblastoid cell line (CCRF-CEM) (data not shown). Because of the availability of material, we purified this activity from human placenta. In addition, we used Ado rather than ddAdo as the substrate to monitor purification because of the significant increase in activity (>lOO-fold). To assay crude extracts, the adenosine deaminase inhibitor EHNA was included in the assay mixture. With every purification technique used, the Ado-phosphorylating activity copurified with the ddAdo-phosphorylating activity (data not shown). Ado Ptase was separated from adenosine kinase, which is known to phosphorylate purine nucleosides and purine nucleoside analogues (10). Most of the Ado kinase activity was removed from the preparation during (NH&SO4 fractionation, as Ado kinase precipitated above 60% (NHJ2S04. Residual Ado kinase did not bind to DEAESepharose, and the final preparation did not catalyze the phosphorylation of Ado using ATP as the phosphate donor. Ado Ptase was separated by DEAE-Sepharose chromatography (Fig. 1) from the two soluble 5’-nucleotidases that have been isolated from human placenta (5,11,12). The hydrolytic enzyme that prefers AMP to IMP, termed “low-K,” 5’-nucleotidase (11,12), did not bind to the column. In contrast, the enzyme that prefers IMP to AMP, labeled “high K,,,” 5’-nucleotidase (5, 12), binds more tightly to DEAE-Sepharose than Ado Ptase and did not
164
GARVEY
AND TABLE
Purification
Step
Volume (ml)
Protein bvd
Extract (NHJsSOI DEAE-Sepharose FPLC Mono Q ATP-Sepharose FPLC Superose 12
500 210 110 15 2 0.9
8670 714 55 7 0.7 0.2
KRENITSKY I
of Ado Ptase from Human Placenta Units (nmol/min) 1750 932 1100 465 100 44
Specific activity (units/mg) 0.2 1.3 20 66 152 246
Recovery (%) 53 63 27 6 3
Fold purification 6.5 100 330 760 1230
elute with Ado Ptase. In addition, both of the 5’-nucleo- preparation still contained five significant protein species tidase activities were separable from Ado Ptase using as judged by SDS-PAGE analysis (data not shown). Thus, other chromatographic techniques not shown in Table I. the most successful preparation is at best lo-20% pure. AMP-Sepharose and concanavalin A-Sepharose sepa- However, no activities that would interfere with the assay rated low K,,, 5’-nucleotidase from Ado Ptase, and AMP- for Ado Ptase were detected in the final preparation. All Sepharose and Orange A resin separated the high K,,, 5’- subsequent studies were performed with this enzyme. nucleotidase from Ado Ptase. It is noteworthy that, whereas the high K,,, 5’-nucleo- Rates of Phosphate Donation vs. Acceptance tidase transfers a phosphoryl group from a nucleoside In the absence of an acceptor nucleoside, Ado Ptase monophosphate to a nucleoside (1) or to a nucleoside ancatalyzed the hydrolysis of AMP. A preparation that had alogue (2,13), the low K,,, 5’-nucleotidase apparently does a specific activity of 200 units/mg in the phosphotransfer not. In our laboratory, the low Km 5’-nucleotidase from direction had a specific activity of 140 units/mg when human placenta was purified by AMP-Sepharose chromatography (11). Even though this preparation was able assayed for the hydrolysis of [14C]AMP. We determined to hydrolyze AMP with a specific activity of 1.0 pmol/ whether Ado Ptase was a phosphotransferase or a 5’-nucleotidase by measuring the rate of phosphorylation of min/mg, it was not able to phosphorylate either Ado or Ino (
