Eur. J. Biochem. 76, 331 - 337 (1977)
Dinucleosidetriphosphatase from Rat Liver Purification and Properties Maria A. G. SILLERO, Rita VILLALBA, Alfredo MORENO, Miguel QUINTANILLA, Carmen D . LOBATON, and Antonio SILLERO Instituto de Enzimologia del Consejo Superior de Investigaciones Cientificas, Departamento de Fisiologia y Bioquimica Facultad de Medicina, Universidad de Valladolid (Received December 28, 1976)
An enzyme is here described in rat liver cytoplasmic extracts which hydrolyzes dinucleoside triphosphates. Compounds of this kind are present in Arterniu sulinu and Duphniu mugnu extracts and capping the 5’ end of RNAs of diverse origin and nature. Diadenosine, diguanosine and diuridine triphosphates were substrates of the reaction with similar I/ and K, values (7, 2 and 25 pM respectively). The products of the reaction were the corresponding nucleoside di and monophosphates. Dixanthosine triphosphate was the unique dinucleoside triphosphate tested which was not substrate of the enzyme. None of the following compounds were substrates of the reaction: dinucleoside tetraphosphates (diadenosine, and diguanosine tetraphosphates), dinucleoside diphosphates (diadenosine and diguanosine diphosphates, NAD , NADP’), nucleoside 5’-phosphates (AMP, ADP, ATP, p4A), bis-p-nitrophenyl phosphate and glucose 6-phosphate. The enzyme requires Mg2+ or Mn2+,is maximally active at a pH value of approximately 7.5, and has a molecular weight of 29 800 as estimated by filtration on Sephadex G-75. AMP, ADP and ATP were competitive inhibitors of the reaction with Ki values in the 100 pM range. Based on its substrate specificity the name of dinucleosidetriphosphatase or dinucleoside-triphosphate nucleotidehydrolase has been adopted for the enzyme through this paper. This enzyme is different from dinucleosidetetraphosphatase (EC 3.6.1.17) and from other enzymes recently described which liberate m7pG or m’GDP from the dinucleoside triphosphate caps of certain RNAs. +
Abbreviations. Ap4A, PtP4-bis(5‘-adenosyl) tetraphosphate; Ap3A, PtP3-bis(5‘-adenosyl) triphosphate; ApzA, PtP2-bis(5’adenosyl) diphosphate; cyclic AMP, adenosine 3’: 5’-monophosphate; cyclic GMP, guanosine 3’: 5’-monophosphate; Gp4G, PtP4bis(5’-guanosyl) tetraphosphate; Gp3G, PtP3-bis(5’-guanosyl) triphosphate; GpzG, P~P2-bis(5’-guanosyl)diphosphate; m’pG, 7methylguanosine 5‘-phosphate; m7Gp3N, P’,P3-[5’,5’-(7-methylguanosyl)nucleosyl] triphosphate; p4A, guanosine 5’-tetraphosphate; Up4U, PtP4-bis(5‘-uridyl) tetraphosphate; UpsU, P t P 3 bis(5’-uridyl) triphosphate; Xp4X, PtP4-bis(5‘-xanthosy1) tetraphosphate; Xp3X, PtP3-bis(5’-xanthosy1) triphosphate. Enzymes. Adenosine deaminase or adenosine aminohydrolase (EC 3.5.4.4); adenylate kinase or ATP: AMP phosphotransferase (EC 2.7.4.3) ; alkaline phosphatase or orthophosphoric monoester phosphohydrolase (alkaline optimum) (EC 3.1.3.1); bis(S-guanosy1)tetraphosphatase or diguanosine-tetraphosphate guanylohydrolase (EC 3.6.1.17); glucose-6-phosphate dehydrogenase or Dglucose-6-phosphate: NADP’ I-oxidoreductase (EC 1.1.1.49); glyceraldehyde-phosphate dehydrogenase or ~-glyceraldehyde-3phosphate: NAD’ oxidoreductase (phosphorylating) (EC 1.2.1.12); hexokinase or ATP : D-hexose 6-phosphotransferase (EC 2.7.1.1); lactate dehydrogenase or L-lactate: NAD’ oxidoreductase (EC 1.1.1.27); lysyl-tRNA synthetase or L-lysine: tRNALyS ligase (AMPforming (EC 6.1.1.6); phosphodiesterase I or oligonucleate 5’nucleotidohydrolase (EC 3.1.4.1); phosphoglycerate kinase or ATP: 3-phospho-~-glycerateI-phosphotransferase (EC 2.7.2.3) and pyruvate kinase or ATP : pyruvate 2-0-phosphotransferase (EC 2.7.1.40).
A splitting activity on diadenosine triphosphate (Ap3A) has been recently described in rat liver extracts [l]. The synthesis of this nucleotide from biological material was described by Zamecnik and collaborators as a by-product of the reaction in vitro of activation of lysine by the lysyl-tRNA synthetase from Escherichiu coli [2]. To our knowledge the presence of this nucleotide in liver extracts has not been so far described. A similar nucleotide, diguanosine triphosphate (Gp3G), is present in Arterniu dins extracts at concentrations of around 1 mM [3]. Recently a methylated derivative of GpJG has been described capping the 5’ end of certain RNAs [4- 61. The function of dinucleoside triphosphates is largely unknown. The object of this report is to describe the purification of a hydrolytic activity on Ap3A present in rat liver extracts. The results presented here support the idea that this activity corresponds to a specific enzyme for which the name of dinucleosidetriphosphatase or dinucleoside-triphosphate nucleotidehydrolase will be used throughout this paper. This enzyme is different from other phosphodiesterases recently described acting on methyl derivatives of dinucleoside
Rat Liver Dinucleosidetriphosphatase
332
triphosphates, present at the 5' end of certain RNAs [7 - 111; it also differs from dinucleosidetetraphosphatase [12], which hydrolyzes the family of dinucleoside tetraphosphates to the corresponding nucleoside tri and monophosphates. Comparative properties between this last enzyme and the one reported here will be also presented in this report. MATERIALS AND METHODS Preparations of Substrates Some of the substrates used for the characterization of this enzyme Ap3A, Gp3G, diuridine triphosphate (Up3U), dixanthosine triphosphate (Xp3X), diadenosine tetraphosphate (Ap4A), diguanosine tetraphosphate (Gp4G) and diguanosine diphosphate (Gp2G) are not commercially available and have been obtained by chemical synthesis or from Artemia cysts extracts. The synthesis and characterization of Ap3A has been previously published [l]. Up3U was obtained as a by-product during the synthesis of diuridine tetraphosphate (Up4U) previously described [12]. Uridine 5'-phosphomorpholidate (0.54 mmol) was allowed to react with the triethylamine salt of pyrophosphate (0.35 mmol) in a medium of anhydrous pyridine (10ml). After standing 5 days at 30 "C, pyridine was eliminated from the reaction mixture by flask evaporation, and the resulting material was resuspended in 8 ml of glass-distilled water, applied to a DEAE-cellulose column (37.5 x 2.6 cm) and fractionated with 3.2 1 of a linear gradient (0.06-0.25 M) of ammonium bicarbonate, pH 8.6. Up3U eluted between 0.15 - 0.16 M ammonium bicarbonate. Gp3G was obtained from Artemia cysts [13] or by chemical synthesis [14]. Dixanthosine triphosphate (Xp3X) was obtained by deamination of diguanosine triphosphate. 2.2 pmol Gp3G was dissolved in 1 ml of 1.7 M HC1, and 0.25ml of 4 M NaN02 was added slowly with stirring. The mixture, after standing for 10 min at room temperature, was applied to a Sephadex G-10 column (12 x 1.7 cm) previously equilibrated with glass-distilled water. Fractions of 1 ml were collected. An ultraviolet-absorbing peak, eluted in the fractions 10-17, clearly separated from the rest of the compounds of the reaction mixture. This peak was characterized as Xp3X by the following criteria. (a) Ultraviolet absorption spectrum (Amax at 248 and 276 nm at pH 7; A,, at 236 and 260 nm at pH 1 ; no peaks were observed at 233 and 343 nm at pH 7, and at 430 nm at pH 11, indicating that the 2-nitropurine or 8-nitropurine derivatives were not synthesized in significant amounts in the course of the reaction [15,16]. (b) This nucleotide was subjected to extensive digestion with phosphodiesterase I and the products of the reaction analyzed by thin-layer chromatography on Cel 300 poly(ethy1eneimine) (PEI)/UV cellulose
(from Macherey-Nagel). Elution was performed with water followed by 1.2 M LiCl. A unique spot with the same mobility as a marker Qf XMP, run in parallel, was detected by ultraviolet light. That spot and that of the marker were eluted from the PEI-cellulose plate with 3 M LiCl and the following spectral characteristics were found. Ratios for the spot eluate at pH 7: A2jo/A260 = 1.32; A280/A260 = 1.02; A290/ A 2 6 ~= 0.51. Ratios for the XMP marker eluate at pH 7: A2jo/A260 = 1.29; Azso/&o = 1.08; A290/ A260 = 0.54. The characterization of Ap3A, Gp3G and Up3U was made based on the following criteria: insensivity to alkaline phosphatase, ratio phosphorus to base, analysis of the products of hydrolysis after treatment with phosphodiesterase I, and spectral characteristics. Dinucleoside polyphosphates, Ap4A, Gp4G and Gp2G, were obtained and characterized as previously described [12,14]. Other nucleotides used were purchased from Boehringer, and diadenosine diphosphate (Ap2A) from Sigma. Enzymatic Assays Enzymatic assays, and the corresponding controls without either enzyme or substrate, were carried out at 37 "C in a volume of 1 ml. Variation in absorbance was measured in a Varian Techtron Model 635 spectrophotometer. Details of some of the methods used to measure the hydrolysis of potential substrates of the phosphatase have been published elsewhere [l, 12,141 and only new experimental conditions will be specified. All the enzymes used were from Boehringer. Coupled Assays When the products of the reaction were (or presumed to be) nucleoside diphosphates, ADP, GDP or UDP, the reaction was coupled to the pyruvate kinase/lactate dehydrogenase system [ 141. ATP and GTP were evaluated with phosphoglycerate kinase and glyceraldehyde-3-phosphatedehydrogenase [ 141; ATP was also measured with hexokinase and glucose6-phosphate dehydrogenase [14]. AMP was evaluated with alkaline phosphatase/adenosine deaminase [ 11. Hyperchromicity Assay In this method we took advantage of the fact that the hydrolysis of 1 pmol Ap3A is associated with an increase of 5.0 A259 unit [l]. Other Methods NAD', NADP', Pi and p-nitrophenol were measured as previously described [14]. In every case, 1 unit (U) is the amount of enzyme able to transform 1 pmol substrate/min at 37 "C.
M. A. G. Sillero, R . Villalba, A. Moreno, M. Quintanilla, C. D. L o b a t h , and A. Sillero
Determination of the Molar Absorption Coefficient for Ap3A Two methods were followed for this determination. In one of them, 0.873 A259 unit of Ap3A was incubated with 50 mM Tris-HC1 buffer, pH 7.0, 5 mM MgC12 and phosphodiesterase I in a volume of 1 ml. An increase of absorbance of 0.174 A259 unit was observed. As the molar absorption coefficient of both AMP and ADP is 1 5 . 4 lo3 ~ M-l cm-' when measured at 259 nm and pH 7.0, the corresponding coefficient for Ap3A is 0.873 x 2 x 15.4 x 103/1.047 = 25.75 x lo3 M-l cm-l. This method is similar to that previously described by Randerath et al. [17] for the determination of Ap4A molar absorption coefficient. In the other method 47.8 A259 unit Ap3A was incinerated and from its content in inorganic phosphate (5.56 pmol), a molar absorption coefficient of 25.8 x lo3 M-l cm-' was calculated, in good agreement with the value found above. RESULTS
Purfication of Enzyme Female white rats were used through these experiments. All operations were carried out at nearly 4 "C. The chromatographic behaviour of dinucleosidetriphosphatase was studied in parallel with that of dinucleosidetetraphosphatase. As shown below, the chromatographic properties of both enzymes are different. Step 1. 150000 x g Supernatant. The livers from 7 rats (35.8 g) were homogenized with two volumes of
333
50 mM Tris-HC1 buffer, pH 7.5, 0.5 mM EDTA (buffer T) in a Waring blendor. The homogenate was centrifuged at 27000 x g for 15 min, and the supernatant was further centrifuged at 150000 x g for 60 min. Step 2. Ammonium Sulfate Fractionation. To the solution from the previous step, 16.4 g of solid ammonium sulfate/100 ml (0.3 saturation) were added. After stirring for 30 min, the suspension was centrifuged at 27 000 x g during 15 min and the precipitate was discarded. The supernatant was brought to 0.6 saturation with ammonium sulfate by the addition of 18.1 g/lOO ml and treated as above. The precipitate was resuspended in 15.2 ml of buffer T. Step 3. Chromatography on Sephadex G-100. The solution from the previous step was applied to a Sephadex G-100 column (2.5 x 100 cm) equilibrated with buffer T and eluted with the same buffer. Fractions of 8.3 ml were collected. Fractions 45 - 53 containing the major portion of dinucleosidetriphosphatase were pooled. Dinucleosidetetraphosphatase eluted between fractions 48 - 59 (results not shown). Step 4. Chromatography on DEAE-SH-Cellulose. The pooled fractions from the previous step were applied to a column (1.5 x 22 cm) of DEAE-SH-cellulose previously equilibrated with buffer T. The column was then washed with the same buffer until the absorbance at 280nm of the effluent was near zero (Fig. 1). The volume of the fractions 1-42 was 8.2 ml. Dinucleosidetriphosphatase was eluted with 280 ml of a linear gradient (0.025- 0.4 M) of KCl in buffer T. Fractions of 4.1 ml were collected. Fractions 57 - 64, containing the major portion of dinucleosidetriphos-
Fraction number
Fig. 1. Chromatography ojdinucleoside tri and tetraphosphatase on DEAE-SH-cellulose. Details as described in the text (step 4). (0)Absorbance at 280 nm; (0)activity on Ap3A; (A) activity onGp4G
334
Rat Liver Dinucleosidetriphosphatase
phatase, were pooled. Dinucleosidetetraphosphatase eluted between fractions 66- 75 (Fig. 1). Step 5. Chromatography on DEAE-Sephadex. The pooled fractions from the previous step were dialyzed for 3 h against 2 1 buffer T, which was changed twice during this period. The dialyzed solution (about 29 ml) was applied to a column of DEAE-Sephadex A-50 medium (1.6 x 2.5 cm) previously equilibrated with 0.025 M KCI in buffer T, and the column washed with this buffer until the absorbance at 280nm of the effluent was negligible (Fig. 2). The volume of the fractions 1- 23 was of 4.1 ml. The phosphatase activity was eluted with 60 ml of a linear gradient (0.0250.4 M) of KC1 in buffer T. Fractions of 1.1 ml were collected. Fractions 37 - 62 containing the major portion of dinucleosidetriphosphatase were pooled. Step 6. Chromatography on DEAE-Neocell. 16 ml from the previous step was applied to a DEAENeocell (Serva) column (1.3 x 2.2 cm) previously equilibrated with buffer T. The column was then washed with buffer T containing 0.05 M KCl, until the absorbance at 280 nm of the effluent was negligible.
Fraction
number
Fig. 2. Chromatography of dinucleosidetriphosphatase on DEAESephadex. Step 5 of purification as described in the text. (0)Absorbance at 280 nm; (0)enzyme activity
The enzyme was eluted with 25 ml of a linear gradient (0.05- 0.125 M) of KC1 in buffer T, and 1.O-ml fractions were collected. The enzyme eluted between fractions 8-23. This step was performed twice using 16 ml from step 5 each time, and is critical for the removal of the contaminant adenylate kinase, which is eliminated in great extent in the 0.05 M KC1 wash. A summary of a typical purification run is given in Table 1. A purification of 150-fold with a recovery of 7 % was obtained. At this stage the material is still not homogeneous as judged by dodecylsulfate/polyacrylamide gel electrophoresis. Substrate Specificity The following compounds were tested as substrates of the phosphatase : (a) dinucleoside polyphosphates, A p A , Gp3G, U P ~ U ,X P ~ X ,A p d , Gp4G, A p A , Gp2G, NAD+ and NADP+ ; (b) nucleoside 5'-phosphates, p4A, ATP, ADP, AMP; (c) a substrate of phosphodiesterase, bis-p-nitrophenyl phosphate and (d) glucose 6-phosphate. In every case the hydrolysis of the potential substrates was coupled to evaluation of different products of reaction as specified in Table 2. NAD' and NADP' were assayed as substrates through evaluation of the residual pyridine nucleotides after incubation with dinucleosidetriphosphatase. For each substrate, the concentration at which it was tested, the percentage velocity of hydrolysis by dinucleosidetriphosphatase and, when pertinent, the K , value found are also included in Table 2. The results presented indicate that the substrates of the enzyme are Ap3A, Gp3G and Up3U. The maximum velocity and K, values for these nucleotides are rather similar (Table 2). Dixanthosine triphosphate (Xp3X) does not seem to be a substrate of the reaction, as the velocity at 200 pM Xp3X concentration was less than 6 % of that obtained with 50 pM Ap3A. The presence of Xp3X did not inhibit the hydrolysis of Ap3A by the phosphatase in neither of the two following experimental conditions : (a) 13 pM Ap3A, 46 pM Xp3X and using the pyruvate kinase/lactate dehydrogenase coupled assay; (b) 130 pM Ap3A, 200 pM Xp3X and using the alkaline phosphatase coupled assay. These results show that in our experi-
Table 1. Purification of rat liver dinucleosidetriphosphatase of Ap3A to alkaline phosphatase and adenosine deaminase 35.8 g rat liver were used. The activity was followed coupling the h y d-0lysis ~ Step
150000 x g supernatant (N&)zS04 fraction Sephadex G-100 chromatography DEAE-cellulose chromatography DEAE-Sephadex chromatography DEAE-Neocell chromatography
Activity
Specific activity
mg
U
mU/mg
%
2339 851 137 17.9 6.0 1.0
2.3 1.2 1.4 0.8 0.54 0.15
1.o 1.4 10.2 44.6 90.0 150.0
100 51 61 35 23
Volume
Protein
ml 69 15.2 71 31 36 35
Recovery
I
M. A. G. Sillero, R. Villalba, A. Moreno, M. Quintanilka, C. D. Lobaton, and A. Sillero
335
Table 2. Substrate specificity of rat liver dinucleosidetriphosphatase Velocities were calculated as pmol substrate transformed/min for 1 ml enzyme preparation (DEAE-Neocell) at 37 "C. Assay methods as described in Materials and Methods Substrate
Concentration
Product evaluated
Relative velocity
Ap3A GP~G UP3U XP3X
FM 50 30 200 200
ADP GDP UDP Pi
100 46 37
Dinucleosidetriphosphatase from Rat Liver Purification and Properties Maria A. G. SILLERO, Rita VILLALBA, Alfredo MORENO, Miguel QUINTANILLA, Carmen D . LOBATON, and Antonio SILLERO Instituto de Enzimologia del Consejo Superior de Investigaciones Cientificas, Departamento de Fisiologia y Bioquimica Facultad de Medicina, Universidad de Valladolid (Received December 28, 1976)
An enzyme is here described in rat liver cytoplasmic extracts which hydrolyzes dinucleoside triphosphates. Compounds of this kind are present in Arterniu sulinu and Duphniu mugnu extracts and capping the 5’ end of RNAs of diverse origin and nature. Diadenosine, diguanosine and diuridine triphosphates were substrates of the reaction with similar I/ and K, values (7, 2 and 25 pM respectively). The products of the reaction were the corresponding nucleoside di and monophosphates. Dixanthosine triphosphate was the unique dinucleoside triphosphate tested which was not substrate of the enzyme. None of the following compounds were substrates of the reaction: dinucleoside tetraphosphates (diadenosine, and diguanosine tetraphosphates), dinucleoside diphosphates (diadenosine and diguanosine diphosphates, NAD , NADP’), nucleoside 5’-phosphates (AMP, ADP, ATP, p4A), bis-p-nitrophenyl phosphate and glucose 6-phosphate. The enzyme requires Mg2+ or Mn2+,is maximally active at a pH value of approximately 7.5, and has a molecular weight of 29 800 as estimated by filtration on Sephadex G-75. AMP, ADP and ATP were competitive inhibitors of the reaction with Ki values in the 100 pM range. Based on its substrate specificity the name of dinucleosidetriphosphatase or dinucleoside-triphosphate nucleotidehydrolase has been adopted for the enzyme through this paper. This enzyme is different from dinucleosidetetraphosphatase (EC 3.6.1.17) and from other enzymes recently described which liberate m7pG or m’GDP from the dinucleoside triphosphate caps of certain RNAs. +
Abbreviations. Ap4A, PtP4-bis(5‘-adenosyl) tetraphosphate; Ap3A, PtP3-bis(5‘-adenosyl) triphosphate; ApzA, PtP2-bis(5’adenosyl) diphosphate; cyclic AMP, adenosine 3’: 5’-monophosphate; cyclic GMP, guanosine 3’: 5’-monophosphate; Gp4G, PtP4bis(5’-guanosyl) tetraphosphate; Gp3G, PtP3-bis(5’-guanosyl) triphosphate; GpzG, P~P2-bis(5’-guanosyl)diphosphate; m’pG, 7methylguanosine 5‘-phosphate; m7Gp3N, P’,P3-[5’,5’-(7-methylguanosyl)nucleosyl] triphosphate; p4A, guanosine 5’-tetraphosphate; Up4U, PtP4-bis(5‘-uridyl) tetraphosphate; UpsU, P t P 3 bis(5’-uridyl) triphosphate; Xp4X, PtP4-bis(5‘-xanthosy1) tetraphosphate; Xp3X, PtP3-bis(5’-xanthosy1) triphosphate. Enzymes. Adenosine deaminase or adenosine aminohydrolase (EC 3.5.4.4); adenylate kinase or ATP: AMP phosphotransferase (EC 2.7.4.3) ; alkaline phosphatase or orthophosphoric monoester phosphohydrolase (alkaline optimum) (EC 3.1.3.1); bis(S-guanosy1)tetraphosphatase or diguanosine-tetraphosphate guanylohydrolase (EC 3.6.1.17); glucose-6-phosphate dehydrogenase or Dglucose-6-phosphate: NADP’ I-oxidoreductase (EC 1.1.1.49); glyceraldehyde-phosphate dehydrogenase or ~-glyceraldehyde-3phosphate: NAD’ oxidoreductase (phosphorylating) (EC 1.2.1.12); hexokinase or ATP : D-hexose 6-phosphotransferase (EC 2.7.1.1); lactate dehydrogenase or L-lactate: NAD’ oxidoreductase (EC 1.1.1.27); lysyl-tRNA synthetase or L-lysine: tRNALyS ligase (AMPforming (EC 6.1.1.6); phosphodiesterase I or oligonucleate 5’nucleotidohydrolase (EC 3.1.4.1); phosphoglycerate kinase or ATP: 3-phospho-~-glycerateI-phosphotransferase (EC 2.7.2.3) and pyruvate kinase or ATP : pyruvate 2-0-phosphotransferase (EC 2.7.1.40).
A splitting activity on diadenosine triphosphate (Ap3A) has been recently described in rat liver extracts [l]. The synthesis of this nucleotide from biological material was described by Zamecnik and collaborators as a by-product of the reaction in vitro of activation of lysine by the lysyl-tRNA synthetase from Escherichiu coli [2]. To our knowledge the presence of this nucleotide in liver extracts has not been so far described. A similar nucleotide, diguanosine triphosphate (Gp3G), is present in Arterniu dins extracts at concentrations of around 1 mM [3]. Recently a methylated derivative of GpJG has been described capping the 5’ end of certain RNAs [4- 61. The function of dinucleoside triphosphates is largely unknown. The object of this report is to describe the purification of a hydrolytic activity on Ap3A present in rat liver extracts. The results presented here support the idea that this activity corresponds to a specific enzyme for which the name of dinucleosidetriphosphatase or dinucleoside-triphosphate nucleotidehydrolase will be used throughout this paper. This enzyme is different from other phosphodiesterases recently described acting on methyl derivatives of dinucleoside
Rat Liver Dinucleosidetriphosphatase
332
triphosphates, present at the 5' end of certain RNAs [7 - 111; it also differs from dinucleosidetetraphosphatase [12], which hydrolyzes the family of dinucleoside tetraphosphates to the corresponding nucleoside tri and monophosphates. Comparative properties between this last enzyme and the one reported here will be also presented in this report. MATERIALS AND METHODS Preparations of Substrates Some of the substrates used for the characterization of this enzyme Ap3A, Gp3G, diuridine triphosphate (Up3U), dixanthosine triphosphate (Xp3X), diadenosine tetraphosphate (Ap4A), diguanosine tetraphosphate (Gp4G) and diguanosine diphosphate (Gp2G) are not commercially available and have been obtained by chemical synthesis or from Artemia cysts extracts. The synthesis and characterization of Ap3A has been previously published [l]. Up3U was obtained as a by-product during the synthesis of diuridine tetraphosphate (Up4U) previously described [12]. Uridine 5'-phosphomorpholidate (0.54 mmol) was allowed to react with the triethylamine salt of pyrophosphate (0.35 mmol) in a medium of anhydrous pyridine (10ml). After standing 5 days at 30 "C, pyridine was eliminated from the reaction mixture by flask evaporation, and the resulting material was resuspended in 8 ml of glass-distilled water, applied to a DEAE-cellulose column (37.5 x 2.6 cm) and fractionated with 3.2 1 of a linear gradient (0.06-0.25 M) of ammonium bicarbonate, pH 8.6. Up3U eluted between 0.15 - 0.16 M ammonium bicarbonate. Gp3G was obtained from Artemia cysts [13] or by chemical synthesis [14]. Dixanthosine triphosphate (Xp3X) was obtained by deamination of diguanosine triphosphate. 2.2 pmol Gp3G was dissolved in 1 ml of 1.7 M HC1, and 0.25ml of 4 M NaN02 was added slowly with stirring. The mixture, after standing for 10 min at room temperature, was applied to a Sephadex G-10 column (12 x 1.7 cm) previously equilibrated with glass-distilled water. Fractions of 1 ml were collected. An ultraviolet-absorbing peak, eluted in the fractions 10-17, clearly separated from the rest of the compounds of the reaction mixture. This peak was characterized as Xp3X by the following criteria. (a) Ultraviolet absorption spectrum (Amax at 248 and 276 nm at pH 7; A,, at 236 and 260 nm at pH 1 ; no peaks were observed at 233 and 343 nm at pH 7, and at 430 nm at pH 11, indicating that the 2-nitropurine or 8-nitropurine derivatives were not synthesized in significant amounts in the course of the reaction [15,16]. (b) This nucleotide was subjected to extensive digestion with phosphodiesterase I and the products of the reaction analyzed by thin-layer chromatography on Cel 300 poly(ethy1eneimine) (PEI)/UV cellulose
(from Macherey-Nagel). Elution was performed with water followed by 1.2 M LiCl. A unique spot with the same mobility as a marker Qf XMP, run in parallel, was detected by ultraviolet light. That spot and that of the marker were eluted from the PEI-cellulose plate with 3 M LiCl and the following spectral characteristics were found. Ratios for the spot eluate at pH 7: A2jo/A260 = 1.32; A280/A260 = 1.02; A290/ A 2 6 ~= 0.51. Ratios for the XMP marker eluate at pH 7: A2jo/A260 = 1.29; Azso/&o = 1.08; A290/ A260 = 0.54. The characterization of Ap3A, Gp3G and Up3U was made based on the following criteria: insensivity to alkaline phosphatase, ratio phosphorus to base, analysis of the products of hydrolysis after treatment with phosphodiesterase I, and spectral characteristics. Dinucleoside polyphosphates, Ap4A, Gp4G and Gp2G, were obtained and characterized as previously described [12,14]. Other nucleotides used were purchased from Boehringer, and diadenosine diphosphate (Ap2A) from Sigma. Enzymatic Assays Enzymatic assays, and the corresponding controls without either enzyme or substrate, were carried out at 37 "C in a volume of 1 ml. Variation in absorbance was measured in a Varian Techtron Model 635 spectrophotometer. Details of some of the methods used to measure the hydrolysis of potential substrates of the phosphatase have been published elsewhere [l, 12,141 and only new experimental conditions will be specified. All the enzymes used were from Boehringer. Coupled Assays When the products of the reaction were (or presumed to be) nucleoside diphosphates, ADP, GDP or UDP, the reaction was coupled to the pyruvate kinase/lactate dehydrogenase system [ 141. ATP and GTP were evaluated with phosphoglycerate kinase and glyceraldehyde-3-phosphatedehydrogenase [ 141; ATP was also measured with hexokinase and glucose6-phosphate dehydrogenase [14]. AMP was evaluated with alkaline phosphatase/adenosine deaminase [ 11. Hyperchromicity Assay In this method we took advantage of the fact that the hydrolysis of 1 pmol Ap3A is associated with an increase of 5.0 A259 unit [l]. Other Methods NAD', NADP', Pi and p-nitrophenol were measured as previously described [14]. In every case, 1 unit (U) is the amount of enzyme able to transform 1 pmol substrate/min at 37 "C.
M. A. G. Sillero, R . Villalba, A. Moreno, M. Quintanilla, C. D. L o b a t h , and A. Sillero
Determination of the Molar Absorption Coefficient for Ap3A Two methods were followed for this determination. In one of them, 0.873 A259 unit of Ap3A was incubated with 50 mM Tris-HC1 buffer, pH 7.0, 5 mM MgC12 and phosphodiesterase I in a volume of 1 ml. An increase of absorbance of 0.174 A259 unit was observed. As the molar absorption coefficient of both AMP and ADP is 1 5 . 4 lo3 ~ M-l cm-' when measured at 259 nm and pH 7.0, the corresponding coefficient for Ap3A is 0.873 x 2 x 15.4 x 103/1.047 = 25.75 x lo3 M-l cm-l. This method is similar to that previously described by Randerath et al. [17] for the determination of Ap4A molar absorption coefficient. In the other method 47.8 A259 unit Ap3A was incinerated and from its content in inorganic phosphate (5.56 pmol), a molar absorption coefficient of 25.8 x lo3 M-l cm-' was calculated, in good agreement with the value found above. RESULTS
Purfication of Enzyme Female white rats were used through these experiments. All operations were carried out at nearly 4 "C. The chromatographic behaviour of dinucleosidetriphosphatase was studied in parallel with that of dinucleosidetetraphosphatase. As shown below, the chromatographic properties of both enzymes are different. Step 1. 150000 x g Supernatant. The livers from 7 rats (35.8 g) were homogenized with two volumes of
333
50 mM Tris-HC1 buffer, pH 7.5, 0.5 mM EDTA (buffer T) in a Waring blendor. The homogenate was centrifuged at 27000 x g for 15 min, and the supernatant was further centrifuged at 150000 x g for 60 min. Step 2. Ammonium Sulfate Fractionation. To the solution from the previous step, 16.4 g of solid ammonium sulfate/100 ml (0.3 saturation) were added. After stirring for 30 min, the suspension was centrifuged at 27 000 x g during 15 min and the precipitate was discarded. The supernatant was brought to 0.6 saturation with ammonium sulfate by the addition of 18.1 g/lOO ml and treated as above. The precipitate was resuspended in 15.2 ml of buffer T. Step 3. Chromatography on Sephadex G-100. The solution from the previous step was applied to a Sephadex G-100 column (2.5 x 100 cm) equilibrated with buffer T and eluted with the same buffer. Fractions of 8.3 ml were collected. Fractions 45 - 53 containing the major portion of dinucleosidetriphosphatase were pooled. Dinucleosidetetraphosphatase eluted between fractions 48 - 59 (results not shown). Step 4. Chromatography on DEAE-SH-Cellulose. The pooled fractions from the previous step were applied to a column (1.5 x 22 cm) of DEAE-SH-cellulose previously equilibrated with buffer T. The column was then washed with the same buffer until the absorbance at 280nm of the effluent was near zero (Fig. 1). The volume of the fractions 1-42 was 8.2 ml. Dinucleosidetriphosphatase was eluted with 280 ml of a linear gradient (0.025- 0.4 M) of KCl in buffer T. Fractions of 4.1 ml were collected. Fractions 57 - 64, containing the major portion of dinucleosidetriphos-
Fraction number
Fig. 1. Chromatography ojdinucleoside tri and tetraphosphatase on DEAE-SH-cellulose. Details as described in the text (step 4). (0)Absorbance at 280 nm; (0)activity on Ap3A; (A) activity onGp4G
334
Rat Liver Dinucleosidetriphosphatase
phatase, were pooled. Dinucleosidetetraphosphatase eluted between fractions 66- 75 (Fig. 1). Step 5. Chromatography on DEAE-Sephadex. The pooled fractions from the previous step were dialyzed for 3 h against 2 1 buffer T, which was changed twice during this period. The dialyzed solution (about 29 ml) was applied to a column of DEAE-Sephadex A-50 medium (1.6 x 2.5 cm) previously equilibrated with 0.025 M KCI in buffer T, and the column washed with this buffer until the absorbance at 280nm of the effluent was negligible (Fig. 2). The volume of the fractions 1- 23 was of 4.1 ml. The phosphatase activity was eluted with 60 ml of a linear gradient (0.0250.4 M) of KC1 in buffer T. Fractions of 1.1 ml were collected. Fractions 37 - 62 containing the major portion of dinucleosidetriphosphatase were pooled. Step 6. Chromatography on DEAE-Neocell. 16 ml from the previous step was applied to a DEAENeocell (Serva) column (1.3 x 2.2 cm) previously equilibrated with buffer T. The column was then washed with buffer T containing 0.05 M KCl, until the absorbance at 280 nm of the effluent was negligible.
Fraction
number
Fig. 2. Chromatography of dinucleosidetriphosphatase on DEAESephadex. Step 5 of purification as described in the text. (0)Absorbance at 280 nm; (0)enzyme activity
The enzyme was eluted with 25 ml of a linear gradient (0.05- 0.125 M) of KC1 in buffer T, and 1.O-ml fractions were collected. The enzyme eluted between fractions 8-23. This step was performed twice using 16 ml from step 5 each time, and is critical for the removal of the contaminant adenylate kinase, which is eliminated in great extent in the 0.05 M KC1 wash. A summary of a typical purification run is given in Table 1. A purification of 150-fold with a recovery of 7 % was obtained. At this stage the material is still not homogeneous as judged by dodecylsulfate/polyacrylamide gel electrophoresis. Substrate Specificity The following compounds were tested as substrates of the phosphatase : (a) dinucleoside polyphosphates, A p A , Gp3G, U P ~ U ,X P ~ X ,A p d , Gp4G, A p A , Gp2G, NAD+ and NADP+ ; (b) nucleoside 5'-phosphates, p4A, ATP, ADP, AMP; (c) a substrate of phosphodiesterase, bis-p-nitrophenyl phosphate and (d) glucose 6-phosphate. In every case the hydrolysis of the potential substrates was coupled to evaluation of different products of reaction as specified in Table 2. NAD' and NADP' were assayed as substrates through evaluation of the residual pyridine nucleotides after incubation with dinucleosidetriphosphatase. For each substrate, the concentration at which it was tested, the percentage velocity of hydrolysis by dinucleosidetriphosphatase and, when pertinent, the K , value found are also included in Table 2. The results presented indicate that the substrates of the enzyme are Ap3A, Gp3G and Up3U. The maximum velocity and K, values for these nucleotides are rather similar (Table 2). Dixanthosine triphosphate (Xp3X) does not seem to be a substrate of the reaction, as the velocity at 200 pM Xp3X concentration was less than 6 % of that obtained with 50 pM Ap3A. The presence of Xp3X did not inhibit the hydrolysis of Ap3A by the phosphatase in neither of the two following experimental conditions : (a) 13 pM Ap3A, 46 pM Xp3X and using the pyruvate kinase/lactate dehydrogenase coupled assay; (b) 130 pM Ap3A, 200 pM Xp3X and using the alkaline phosphatase coupled assay. These results show that in our experi-
Table 1. Purification of rat liver dinucleosidetriphosphatase of Ap3A to alkaline phosphatase and adenosine deaminase 35.8 g rat liver were used. The activity was followed coupling the h y d-0lysis ~ Step
150000 x g supernatant (N&)zS04 fraction Sephadex G-100 chromatography DEAE-cellulose chromatography DEAE-Sephadex chromatography DEAE-Neocell chromatography
Activity
Specific activity
mg
U
mU/mg
%
2339 851 137 17.9 6.0 1.0
2.3 1.2 1.4 0.8 0.54 0.15
1.o 1.4 10.2 44.6 90.0 150.0
100 51 61 35 23
Volume
Protein
ml 69 15.2 71 31 36 35
Recovery
I
M. A. G. Sillero, R. Villalba, A. Moreno, M. Quintanilka, C. D. Lobaton, and A. Sillero
335
Table 2. Substrate specificity of rat liver dinucleosidetriphosphatase Velocities were calculated as pmol substrate transformed/min for 1 ml enzyme preparation (DEAE-Neocell) at 37 "C. Assay methods as described in Materials and Methods Substrate
Concentration
Product evaluated
Relative velocity
Ap3A GP~G UP3U XP3X
FM 50 30 200 200
ADP GDP UDP Pi
100 46 37
