Isolation and characterization of diadenosine tetraphosphate (Ap4A) hydrolase from Schizosaccharomyces pombe

Biochimica et Biophysica Acta, 1161 (1993) 139-148 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4838/93/$06.00

139

BBAPRO 34405

Isolation and characterization of diadenosine tetraphosphate (Ap 4 A ) hydrolase from Schizosaccharomycespombe Angela K. Robinson, Carlos E. de la Pefia and Larry D. Barnes Department of Biochemistry, University of Texas Health Science Center, San Antonio, TX (USA) (Received 1 June 1992) (Revised manuscript received 31 August 1992)

Key words: Diadenosine tetraphosphate; Ap4A; Diadenosine tetraphosphate hydrolase; Fission yeast; (S. pombe)

An enzyme that catalyzes the asymmetric hydrolysis of mP4A has been partiall$ purified from the fission yeast, Schizosaccharomyces pombe. The crude supernatant fraction from log-phase cells was fractionated by (NH4)2SO4 precipitation followed by chromatography on DEAE-cellulose, Red A dye-ligand and QAE-Sepharose resins. Two peaks of AP4A hydrolase activity, designated major and minor, were separated on the Red A dye-ligand resin. Both the major and minor AP4A hydrolase have an apparent molecular mass of 49 kDa based on gel filtration chromatography. On a SDS polyacrylamidegel, a protein of 22 kDa exhibited AP4A hydrolase activity. Both forms of the enzyme have a K m value in the range of 22 to 36/~M for AP4A. Both forms of the enzyme asymmetricallyhydrolyze Ap4A to AMP and ATP as determined by HPLC. AP4A is the optimal substate among several nucleotides and dinucleoside polyphosphates tested at 10 #M. A divalent metal cation is required for activity. Concentrations of Pi below 30 mM stimulate AP4A hydrolase while higher concentrations inhibit the activity.PI is not a substrate for this Ap4A-degradative enzyme. Fluoride, from 50/zM to 20 mM, has no significant effect on Ap4A hydrolase activity.

Introduction Diadenosine 5', 5"-P1, p 4.tetraphosphat e (mP4A) is present in prokaryotes and eukaryotes at basal levels of 10 -8 to 10 -6 M [1]. Exceptions are platelets and adrenal chromaffin cells in which mpaA is present at 10 -4 M in secretory granules [1-3]. AP4A has been proposed to be a nucleotide coupling protein synthesis to other processes of proliferation, such as DNA replication [4], and as a signal nucleotide involved in the adaptive response to oxidative stress [5,6]. Ap4A also

Correspondence to: L.D. Barnes, Department of Biochemistry, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78284-7760, USA. Abbreviations: Ap 4, adenosine tetraphosphate; AP4A, diadenosine 5', 5"-P 1, p4-tetraphosphate; AP4N, adenosine-5'-tetraphospho-5'nucleoside, where N is guanosine, cytosine, or uridine; ApnA, diadenosine 5', 5"-P 1, pn-polyphosphate, n = 2-6; GPnG, diguanosine 5', 5"-P l, pn-polyphosphate, n = 3-5; Hepes, 4-(2-hydroxyethyl)-lpiperazine-N-ethanesulfonic acid; HPLC, high-performance liquid chromatography; m7GP3Am, 7-methylguanosine 5', 5"-P 1, p3-triphospho-2'-O-methyladenosine; mTGpaCm, 7-methylguanosine 5', 5"-P 1, p3-triphospho-2'-O-methylcytidine; mTGoaG, 7-methylguanosine 5', 5"-P ~, p3-triphospho-guanosine; 2'-O-mAMP, 2'-0methyl adenosine 5'-monophosphate; 2'-O-mCMP, 2'-O-methyl cytidine 5'-monophosphate; PMSF, phenylmethylsulfonyl fluoride.

modulates the activities of the vascular and adrenal neurosecretory systems in higher eukaryotes [7-9]. Potential functions of AP4A in different cellular processes have been reviewed [10-12]. Three types of catabolic enzymes specific for Ap4A and other dinucleoside polyphosphates have been isolated. One type of enzyme, AP4A asymmetric hydrolase, catalyzes the hydrolysis of Ap4A to AMP and ATP and is stimulated by Mg 2÷ or Mn 2÷. This hydrolase has been isolated from several eukaryotic organisms, including human cells [13-18]. A second type of mpam hydrolase catalyzes the symmetrical hydrolysis of Ap4A to ADP. This hydrolase was initially discovered in Physarum polycephalum [19,20], and it is present in Escherichia coli [21,22]. The E. coli gene encoding Ap4A hydrolase has been cloned and sequenced [23,24]. The third type of enzyme, Ap4A phosphorylase, catalyzes the phosphorolysis of Ap4A in the presence of Pi and a divalent cation to yield ADP and ATP [25]. This enzyme is present in the budding yeast, Saccharomyces cerevisiae, [25] and in the protozoan, Euglena gracilis, [26]. Saccharomyces cerevisiae contains a second Ap4A phosphorylase whose protein sequence has a 60% identity with that of the first Ap4A phosphorylase [27]. The yeast APA1 and APA2 genes, encoding AP4A phosphorylase I and Ap4A phosphorylase II,

140 respectively, have been cloned and sequenced [27-29].

APA1 has been physically mapped on chromosome III of Saccharomyces cereuisiae [30]. Here, we describe the partial purification and characterization of an asymmetric ApaA hydrolase from the fission yeast, Schizosaccharomyces pombe. AP4m hydrolase from S. pombe is more similar to the APaA asymmetric hydrolase present in higher eukaryotes, including humans, than to the Ap4A phosphorylase from the budding yeast, Saccharomyces cerevisiae, or the ApnA symmetric hydrolases from P. polycephalum and E. coli. A preliminary account of part of this work has been reported [31]. Materials and Methods

Materials AP4A was custom-labeled with tritium by Amersham. The [3H]Ap4A was purified by chromatography on a boronate resin [32] and analyzed for purity by HPLC as described [33]. Unlabeled adenine and guanine nucleotides were purchased from Sigma, except for ApaG , mPaU , APaC and the mRNA cap nucleotide analogues. APnG was kindly donated by M. Morozumi, A. Kuninaka, Y. Furuichi and A. Shatkin. Ap4U and ApaC were synthesized from ATP and the appropriate pyrimidine monophosphate by reaction with 1-ethyl-3(3 dimethylaminopropyl) carbodiimide in the presence of MgC12 and Hepes buffer (pH 6.9), as described by Ng and Orgel [34]. ApnU and Ap4C were purified by chromatography on DEAE-cellulose by gradient elution with ammonium acetate (pH 6.0). Fractions containing APnU and ApaC were lyophilized and analyzed for purity by HPLC [33]. ApaU and ApaC were characterized by UV spectroscopy and HPLC analysis of enzyme-catalyzed reaction products in comparison with nucleotide standards. The mRNA cap nucleotide analogues, mTGp3Am, mTGP3Cm, and mTGp3G, were from Pharmacia LKB. DEAE-cellulose (microgranular DE52) and the Partisphere 5 SAX HPLC column were purchased from Whatman. Matrex Gel Red A dyeligand resin was from Amicon. The QAE-Sepharose and Sephadex G-75 resins and the Mono-Q H R 5 / 5 HPLC column were manufactured by Pharmacia LKB. Proteins used as molecular weight standards for gel filtration chromatography and polyacrylamide gel electrophoresis, thymidine 5'-monophosphate p-nitrophenyl ester, p-nitrophenyl phenylphosphonate, phenylmethylsulfonyl fluoride, leupeptin and pepstatin A were purchased from Sigma. Crotalus adamanteus phosphodiesterase I was from Worthington.

2.2" l0 s cells/ml. Yeast were cultured in 120 1 batches at 30°C with an aeration of 170 1/min and an agitation of 250 rpm in a New Brunswick model IF-250 basic fermentor equipped with ML-4100 microprocessor instrumentation.

Assay of ZP4A hydrolase activity AP4A hydrolase from S. pombe was assayed by formation of [3H]AMP and [3H]ATP from [3H]AP4A. Routinely, activity was measured in 50 mM HepesNaOH (pH 7.5), 100 /zM [3H]Ap4A and 0.2 mM MnC12 at 37°C for 10 min in a volume of 100 /xl. In some experiments, the concentrations of Ap4A and MnC12 were varied as noted in the figure legends. MnCI 2 was replaced with MgCI2, COC12, or CaC12 in some experiments as noted in the figure legends. The reaction products were separated from residual substrate by column chromatography on a boronate-derivatized resin as described in detail [32]. Enzymic activity was expressed as nmol of AMP + ATP formed/min per mg protein. Activity was a linear function of the mass of protein and the time of incubation under these assay conditions. A non-radioisotopic assay based on HPLC analysis was used to determine the substrate specificity of ApnA hydrolase and to identify reaction products. ApaA and other potential substrates were incubated in the absence or presence of ApaA hydrolase in 10 mM Hepes-NaOH (pH 7.5) and 0.2 mM MnC12 at 37°C for 20 min in a volume of 100 /zl. The reaction was stopped by quick freezing the assay solution on solid CO 2 or by immediately injecting the entire assay solution onto the HPLC column. Solutions were analyzed on a Whatman Partisphere 5 SAX (strong anion-exchange) column eluted isocratically with 0.20 M ammonium phosphate (pH 5.5). Some assay solutions were eluted from the column using a gradient from 50 mM ammonium phosphate (pH 5.2) to 450 mM ammonium phosphate (pH 5.7). The stated concentrations refer to the concentration of phosphate. The column was eluted at 1.0 or 1.5 ml/min at about 24°C using a Beckman model 110 pump. Nucleotides were detected with a Beckman model 160 detector and the absorbance was recorded with an Altex model C-R1A integrator-recorder. Routinely, 1 nmol of each nucleotide, AMP, ADP, ATP, AP4A and other potential nucleotide substrates, was detected at 254 nm with a full scale absorbance range of 0.05. Nucleotides were identified by retention time and peak areas were integrated for quantitative analysis. The percent hydrolysis of a substrate was calculated by dividing the peak areas of the substrate in the presence and absence of enzyme [20].

Culture of Schizosaccharomyces pombe S. pombe 972 h-, kindly provided by P. Russell, was

Assay of phosphodiesterase activity

cultured in 1% yeast extract (Marcor), 2% peptone (Difco) and 3% glucose, and harvested at 1.4.10 s to

AP4A hydrolase was assayed for 5'-phosphodiesterase activity using thymidine 5'-monophosphate

141 p-nitrophenyl ester [35] and p-nitrophenyl phenylphosphonate [36] as potential substrates. AP4A hydrolase was incubated with 1 mM of each substrate in 50 mM Hepes-NaOH (pH 7.5) at about 25°C and the formation of any p-nitrophenolate was monitored spectrophotometrically at 405 nm in a continuous assay. Snake venom phosphodiesterase I was assayed with these substrates under the same conditions as a positive control.

Determination of the apparent molecular mass of Ap4A hydrolase The major and minor forms of AP4A hydrolase eluted from the Red A dye-ligand column were subjected to gel filtration chromatography to determine their apparent molecular masses. A Sephadex G-75 column (1.5 x 28 era) was equilibrated in 50 mM Hepes-NaOH (pH 7.5), 10% glycerol. The chromatography was also done in buffer containing 10 mM 2-mercaptoethanol. Approx. 0.42 ml fractions were collected. The actual volume of each fraction was calculated from the mass of each fraction. About 1 mg each of bovine serum albumin, ovalbumin, carbonic anhydrase and myoglobin were used as molecular mass standards. Values of Kay were calculated from the elution volumes of the proteins and the void volume of the column as determined by the elution volume of thyroglobulin.

Gel electrophoresis Protein samples were analyzed for purity by electrophoresis on 12.5% (w/v) polyacrylamide gels under denatured protein conditions. Proteins were denatured by dissociation in 10 mM sodium phosphate (pH 7.2) 1% (w/v) SDS and 1% (v/v) 2-mercaptoethanol at 65°C for 15 min. Dissociated proteins were subjected to electrophoresis with the discontinuous buffer system for slab gels as described by Laemmli [37] and modified by Studier [38]. Gels were stained in silver nitrate and destained as described [39]. Bovine serum albumin (66 kDa), ovalbumin (45 kDa), rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (36 kDa), bovine erythrocyte carbonic anhydrase (29 kDa), bovine pancreas trypsinogen (24 kDa) and soybean trypsin inhibitor (20.1 kDa) served as molecular mass standards. In some experiments, electrophoresis on SDS gels was modified to test for association of AP4A hydrolase activity with a particular protein band. The major and minor AP4A hydrolase Mono-Q fractions and standard proteins were dissociated at 37°C for 10 min in the phosphate buffer described above. In some experiments, proteins were dissociated in the absence of 2-mercaptoethanol, and the gel was run under non-reducing, denaturing conditions. The gel was pre-electrophoresed for 30 min prior to electrophoresis of proteins. After electrophoresis, lanes of the gel containing

the enzyme were washed for 45 min in 50 mM HepesNaOH (pH 7.5), 1 mM 2-mercaptoethanol at 25°C with gentle agitation to elute the SDS. The washing was repeated twice in buffer without 2-mercaptoethanol. The washed gel was sliced into 1.1 mm segments on a set of razor blades. Each gel segment was incubated in 150 /zl of 50 mM Hepes-NaOH (pH 7.5), 0.2 mM MnC12 and 100 /.~M [3H]AP4A at 25°C for 90 min, about 16 h at 4°C and 20 min at 37°C to assay for activity. Other lanes, from the same SDS gel, containing Ap4A hydrolase and standard proteins were stained for protein with a silver stain [39]. Stained gels were scanned densitometrically and the data were analyzed using the public domain software Image v. 1.44 program (written by W. Rasband, NIH) with instrumentation similar to that described previously [40]. In preliminary experiments, we determined that there was no significant difference in the electrophoretic patterns of standard proteins dissociated at 65°C for 15 min or dissociated at 37°C for 10 min. Also, extensive washing of the gel after electrophoresis to elute the SDS did not elute standard proteins to any significant extent based on the intensity of staining. This procedure to measure enzymic activity after SDSPAGE is a modification of the procedure described by Lacks and Springhorn [41].

Miscellaneous Phenylmethylsulfonyl fluoride was prepared as a 100 mM stock solution in 100% isopropanol [42]. It was diluted into buffers at 4°C about 30-60 rain before using. Leupeptin and pepstatin A were added to buffers just prior to using. Conductivity of column eluates was measured at 4°C with a Radiometer CDM2 conductivity meter and CDC 114 conductivity cell. Protein mass was measured according to Lowry et al. [43] after diluting samples into 1% (w/v) SDS. Absorbance values of samples were corrected for absorbance due to the Hepes buffer. Bovine serum albumin was used as the protein standard. Results

Isolation of Ap4A hydrolase About 1 kg wet wt of S. pombe was processed at a time for partial purification of Ap4A hydrolase through the Red A dye-ligand chromatography. Red A dyeligand fractions originating from 4 kg of yeast were combined for further purification. Yeast were broken in 50 mM Hepes-NaOH (pH 7.5), 10% (v/v) glycerol, 0.5 mM PMSF, 0.5 /.~g leupeptin/ml and 0.5 /~g pepstatin A / m l by two passes through a chilled Manton-Gaulin homogenizer at a pressure of 700-800 kg/cm2. The homogenate (3 ml of buffer per gm wet wt of yeast) was centrifuged at 28 000 X g for 30 min to obtain the crude supernatant fraction.

142 1B

0.30

-080

060

4-

12-

Minor Ap4A I Hyarolase ~

I

~

~0.20

,~

9-

"

~

6-

-o.10

I°.4°I =o 020

0 " 220

240

260

280

300

320

340

000 360

test for possible interconversion between the two forms. The elution procedure was the same as used in the initial separation of the two forms. The major mpaA hydrolase fraction re-chromatographed as a single peak of activity that eluted with a similar concentration of KC1 as originally. The minor ApaA hydrolase fraction also re-chromatographed as a single peak of activity that eluted with a similar concentration of KCI as originally. Major Ap4A hydrolase fractions isolated from four 1-kg batches of S. pombe were combined at this stage in the isolation and further purified. Similarly, minor m P a A hydrolase fractions were combined and further purified. The major mp4A hydrolase fraction was dialyzed in 20 mM Tris-HC1 (pH 7.5), 10% glycerol and applied to a 1.5 × 28 cm QAE-Sepharose column equilibrated in the same buffer. The column was eluted with buffer followed by buffer containing 50 mM KCI and a 250 ml linear gradient of 50-200 mM KC1 in buffer. A single peak of Ap4A hydrolase activity was eluted with 50-65 mM KC1 in buffer. Fractions containing activity were pooled and concentrated in an Amicon diaflo unit as described above. The major Ap4A hydrolase was further purified on a Mono-Q HPLC column equilibrated in 20 mM Tris-HC1 (pH 7.5), 10% glycerol. Both the Mono-Q column and the sample injector loop were packed in ice. After the enzyme was injected onto the column, the column was eluted with buffer for 2 min, buffer containing 30 mM KCI for 3 min and a linear gradient of 30-90 mM KC1 in buffer over a 40 rain period. The flow rate was 1 mi/min. The column eluate was monitored at 280 nm at a full scale absorbance of 0.15. 1-ml fractions were collected on ice. A single peak of ApaA hydrolase eluted with 50-60 mM KCI in buffer. Fractions containing activity were pooled, then concentrated and exchanged into 50 mM Hepes-NaOH (pH 7.5), 10% glycerol buffer in an Amicon diaflo unit with a YM-5 membrane. The minor Ap4A hydrolase fraction from the Red A dye-ligand column was subjected to chromatography on QAE-Sepharose in the same size column and under the same conditions as used with the major AP4A hydrolase. A single peak of AP4A hydrolase activity was eluted 80-100 mM KCI in buffer. Fractions containing activity were pooled and concentrated in an Amicon diaflo unit. The minor AP4A hydrolase after QAE-Sepharose chromatography was subjected to HPLC on the Mono-Q column under the same conditions described for the major Ap4A hydrolase, except the column was eluted with 50 mM KCI in buffer, then a linear gradient of 50 mM to 120 mM KC1 in buffer. The minor m P a m hydrolase eluted from the Mono-Q column with 65-75 mM KCI in buffer, and fractions containing activity was processed in the same manner as described with the major ApaA hydrolase Mono-Q fractions. Both the major and minor APaA hydrolase

-0 O0

Fraction Number

Fig. 1. Elution profile of the AP4A hydrolase DEAE-cellulose fraction subjected to chromatography on a Red A dye-ligand matrix column equilibrated in 50 mM Hepes-NaOH (pH 7.5), 10% glycerol. The 2.5×63 cm column was eluted with buffer followed by elution with a 1200 ml linear gradient of 0-0.4 M KCI in buffer, and 4.3 ml fractions were collected. The AP4A hydrolase activity (A), absorbance at 280 nm ([]), and the KCI concentration (©) were measured for each of the indicated fractions.

The crude supernatant was subjected to (NH4)2SO 4 fractionation and Ap4A hydrolase precipitated between 45 and 65% saturation. The pellet containing APaA hydrolase was dissolved in 20 mM Hepes-NaOH (pH 7.5), 10% glycerol, 0.5 mM PMSF, 0.5 /zg leupeptin/ml and 0.5 tzg pepstatin A/ml, and was extensively dialyzed in 20 mM Hepes-NaOH (pH 7.5), 10% glycerol and 0.5 mM PMSF. The dialyzed (NH4)2SO 4 fraction was applied to a 5 × 49 cm DEAE-cellulose column equilibrated in 20 mM Hepes-NaOH (pH 7.5), 10% glycerol. The column was eluted initially with buffer at 100 m l / h and subsequently was eluted with a 2 liter, linear gradient of 0-0.4 M KCI in buffer. APaA hydrolase was eluted with 10-50 mM KC1 in buffer. Endogenous 5'phosphodiesterase activity was eluted from the column in the initial buffer wash. Fractions containing Ap4 A hydrolase activity were pooled and applied directly to a 2.5 × 63 cm column of Red A dye-ligand resin equilibrated with 50 mM Hepes-NaOH (pH 7.5), 10% glycerol. The column was eluted initially with buffer at 30 m l / h and subsequently was eluted with a 1200 ml, linear gradient of 0-0.4 M KCI in buffer. Two peaks of APaA hydrolase activity were eluted (Fig. 1). The first peak of activity, designated as the minor AP4A hydrolase based on relative activity, was eluted with 90-130 mM KCI in buffer. The second peak of activity, designated as the major ApaA hydrolase, was eluted with 140-180 mM KC1 in buffer. Pooled fractions from each peak of ApaA hydrolase activity were concentrated under N z pressure in Amicon diaflo units containing PM-10 membranes. The major ApaA hydrolase and minor Ap4A hydrolase fractions were each re-chromatographed on individual Red A dye-ligand columns to

143 preparations were stored at -80°C. Results of the purification of AP4A hydrolase are summarized in Table I. Both the major and minor AP4A hydrolase preparations are heterogeneous after HPLC on the Mono-Q column as determined electrophoretically on SDS polyacrylamide gels (see Fig. 2 for a SDS gel of the major Ap4A hydrolase). Although the specific activity of the major hydrolase preparation did not increase after chromatography on the Mono-Q column, the enzyme preparation was less heterogeneous than the corresponding QAE-Sepharose fraction as determined electrophoretically.

A P 4 A hydrolase Mono-Q fraction and standard pro-

Determination of apparent molecular mass The major and minor A P 4 A hydrolase activities eluted from the Red A dye-ligand column were subjected to gel filtration chromatography on a calibrated Sephadex G-75 column as described in Materials and Methods. The apparent molecular mass of both forms of the enzyme was 49 + 1 kDa based on elution of m P a A hydrolase activity relative to elution of standard proteins. The major Red A - A P a A hydrolase fraction was also subjected to gel filtration chromatography in buffer containing 10 mM 2-mercaptoethanol. The value of the apparent molecular mass differed by less than 7% from the value obtained under non-reducing conditions, and there was no detectable activity associated with a protein of lower molecular mass. Although the major AP4A hydrolase Mono-Q fraction was heterogeneous, a specific protein band on SDS-PAGE exhibited enzymic activity. The major

Requirement of a divalent metal cation In preliminary experiments with dialyzed crude supernatant fraction, it was determined that a divalent metal cation is required for maximal A P 4 A hydrolase activity. Activity was assayed as a function of the concentration of Mn 2+, M g 2+, C o 2+ and Ca 2÷, each with C1- as the anion, for the major Red A dye-ligand A P 4 A hydrolase fraction, and the result is shown in Fig. 3. Each of the cations stimulated the activity in a characteristic concentration-dependent manner. Mn 2+ from 0.2 to 0.5 mM maximally stimulated the activity among the metal cations tested. The minor Red A dye-ligand AP4A hydrolase fraction exhibited a similar pattern of dependence of activity upon the concentration of the same four cations, except that the stimulation by M g 2+ was weaker relative to Mn 2+ and was similar to the stimulation by Co 2÷. Activities of the major and minor A P 4 A hydrolase fractions in the

teins were subjected to SDS-PAGE modified as described in Materials and Methods to facilitate detection of enzymic activity. The results are shown in Fig. 2. After electrophoresis under reducing conditions, AP4A hydrolase activity was associated with a protein with an apparent molecular mass of 22 + 1 kDa. We were unable to detect enzymic activity when the gels were run under non-reducing conditions in the absence of 2-mercaptoethanol. In a preliminary experiment, the minor m P a A hydrolase Mono-Q fraction also yielded a 22 kDa protein band on a SDS gel that exhibited enzymic activity.

TABLE I

Purification of ZP4h hydrolasefrom Schizosaccharomycespombe Mass of cells for isolation was 4 kg of S. pombe 972 h Fraction

Protein (mg)

Total activity a (/~mol AXP/min)

Crude supernatant

217219

438

2.0

45-65% saturated (NH4)2SO 4

100138

255

2.6

58.3

7 508

115

14.3

26.3

DEAE cellulose Red A dye ligand Major Peak Minor Peak QAE Sepharose from Major Red A from Minor Red A Mono-Q HPLC Major Minor

145 112 1.5 12.7 0.13 b 0.05 b

Specific activity a (nmol AXP/min per mg)

Yield (%) 100

44.8 13.3

388 133

10.2 3.0

9.6 2.5

6 200 194

2.2 0.6

0.44 0.03

3 387 708

0.1 0.01

a Activity was assayed with 100/zM [3H]Ap4A in the presence of 0.2 mM MnC! 2. AXP equals AMP+ATP. b Protein mass calculated from absorbance at 280 nm assuming 1 absorbance unit equals 1.1 mg/ml. Protein mass for all other fractions was determined by the method of Lowry et aL [43].

144 presence of 0.05 to 5 mM ZnCI 2 were less than 2% of the activities assayed in the presence of 0.2 mM MnC12. Neither form of ApnA hydrolase had detectable activity when assayed in the presence of 0.5 mM to 10 mM EDTA without the addition of a divalent metal cation.

Substrate specificity and identification of reaction products The products of the hydrolysis of mpaA by the major Ap4A hydrolase from the Mono-Q HPLC column were identified as AMP and ATP by coelution with nucleotide standards on a strong anion-exchange HPLC column (Fig. 4). The reaction products, AMP and ATP, were unchanged by the addition of Pi in the assay (Fig. 4C and D). The relative activity of the major mPam hydrolase from the Mono-Q column with various mono-and di-nucleotides as potential substrates is shown in Table II. The specific activity of the major ApaA hydrolase with p-nitrophenyl phenylphosphonate and thymidine A

a

b

c

d

e

f

500

•

,

•

,

•

,

•

,

.

,

Mg2 + P.

i,= :>

100

~J <

C a

Ol 0

1

2

2+

i 3

i 4

i 5

6

[DIVALENTCATION], mM Fig. 3. Effect of divalent metal cations on the activity of Ap4A hydrolase. The major Ap4A hydrolase from the Red A dye-ligand column was assayed in 50 mM Hepes-NaOH (pH 7.5), with 100/zM [3H]Ap4A at 37°C for 10 min with different concentrations of the indicated divalent cations, each present as the chloride salt. Mn 2+ (e), Mg 2+ ([3), Co 2+ (O), Ca 2+ (11).

100

5'-monophosphate p-nitrophenyl ester as substrates was compared to the specific activity of snake venom phosphodiesterase I as a measure of non-specific 5'phosphodiesterase activity. The specific activity of the major ApaA hydrolase from the Red A dye-ligand column with these substrates was 1-5% of the corresponding specific activity of the snake venom phosphodiesterase I. Hydrolysis of Ap4m by the minor Ap4m hydrolase from the Mono Q column also yielded AMP and ATP (data not shown). The specific activity of the minor ApaA hydrolase from the Red A dye-ligand column with the 5'-phosphodiesterase substrates was 1-5% of the corresponding specific activity of the snake venom phosphodiesterase I.

Fig. 2. Electrophoretic pattern of the Mono-Q HPLC major AP4A hydrolase fraction on a SDS polyacrylamide gel. The enzyme and standard proteins were subjected to electrophoresis under the conditions described in Materials and Methods. Panel A: lane 1: standard proteins stained with silver nitrate; (a), bovine serum albumin; (b), ovalbumin; (c), glyceraldehyde-3-phosphate dehydrogenase; (d), carbonic anhydrase; (e), trypsinogen; (f), trypsin inhibitor. Lane 2: Ap4A hydrolase stained for protein with silver nitrate. Panel B: the stained gel lane (2) containing AP4A hydrolase was digitized and analyzed using the Image software. Another lane of the gel containing mP4A hydrolase was sliced into 1.1 mm segments immediately after electrophoresis, and each gel segment was assayed for Ap4A hydrolase activity. The length of the gel sliced for assay of enzymic activity was normalized to the length (mm) of the stained gel. The activity of Ap4A hydrolase in the gels segments was superimposed on the densitometric plot of the digitized gel. The densitometric plot is represented by the continuous, solid line. Activity of Ap4A hydrolase is expressed as nmol AXP which represents nmol of [3H]AMP + [ 3H]ATP formed.

Kinetics with h p 4 A as substrate The activity of the major Ap4A hydrolase from the QAE-Sepharose column was measured as a function of the concentration of ApaA from 5 to 300 /~M in the presence of 0.2 mM MnCI z. The enzyme exhibited Michaelis-Menten kinetics as indicated by linear Eadie-Hofstee plots. The apparent value of the g m for Ap4A was 22 __+3/xM (mean _ S.D., n --- 3). Pi is a substrate, along with A P 4 A , for A P 4 A phosphorylase from Saccharornyces cerevisiae [25]. Although Ap4A hydrolase from S. pombe is active in the absence of added Pi, we measured the effect of 1 to 200 mM Pi on the activity of the major Ap4A hydrolase from the QAE-Sepharose column in the presence of 100 ~M Ap4A and 0.2 mM MnCI 2. At concentrations from 1 mM to 30 mM, Pi stimulated the activity in a

~ii~

B

i~;i ¸

4

3

c2_ x <(

II

i

1

2

t~ i

i 1

o

i

r

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40

60

80

mm

145

A

B

D

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Fig. 4. HPLC identification of the products, AMP and ATP, of AP4A hydrolysis catalyzed by AP4A hydrolase. Nucleotide standards and assay solutions were subjected to chromatography on a Whatman Partisphere 5 SAX column eluted with a gradient from 50 mM ammonium phosphate (pH 5.2) to 450 mM ammonium phosphate (pH 5.7), at 1.5 ml/min. Nucleotides were detected spectrophotometrically at 254 nm at a full scale absorbance of 0.05. (A), elution profile of 1 nmol each of AMP, ADP, AP4A and ATP as standards; (B), assay solution of 10 mM Hepes-NaOH (pH 7.5), 0.2 mM MnCI 2 and 1 nmol of AP4A incubated 10 min at 37°C in the absence of enzyme; (C), same assay solution as in (B), except incubated in the presence of 0.04/zg of AP4A hydrolase (major Mono-Q HPLC fraction) and (D), same assay solution as in (C), except 5 mM Pi was also present. The number associated with each peak is the elution time in rain.

variable, concentration-dependent manner with a maximum stimulation of two-fold. At concentrations greater than 30 mM, Pi was increasingly inhibitory, such that at 100 mM Pi the activity was 95% inhibited. The effects of NaF and thiol compounds on the activity of the major Ap4A hydrolase were measured in the presence of 100 /xM m P 4 A and 0.2 mM MnCI 2. The activity changed by 10% or less in the presence of 0.05 mM to 20 mM NaF. Dithiothreitol and 2-mercaptoethanol, at concentrations from 0.1 mM to 20 mM, maximally stimulated the activity by 30% at a concentration of 1 mM of either thiol compound. The minor A P 4 A hydrolase from the the QAE-Sepharose column also exhibited Michaelis-Menten kinetics. The apparent value of the K m for A P 4 A w a s 36 ± 7 /zM (mean ± S.D., n -- 3). The effects of Pi, NaF, dithiothreitol and 2-mercaptoethanol on the activity of the minor A P 4 A hydrolase were similar to the effects of these compounds on the activity of the major Ap4A hydrolase as described above. Discussion

AP4A hydrolase from the fission yeast S. pombe exists in two forms which we have designated as major and minor, based on their relative activities. Three different protease inhibitors were included in buffers in the early stages of purification to decrease the possibility of proteolysis and the generation of spurious forms of the enzyme. The separation of the major and minor forms of Ap4A hydrolase was reproducible in four different preparations subjected to chromato-

graphy on the Red A dye-ligand resin. Elution of both the major and minor A P a A hydrolases as single peaks upon re-chromatography on Red A dye-ligand resin indicates that the two forms are not interconvertible by a simple equilibrium process. The two forms have the same reaction products for AP4A, and they have the same apparent molecular weights. The effects of different divalent metal cations, Pi, fluoride and thiol compounds on their activities are also similar. The difference in the apparent K m values of the two forms for m P a A may not be significant. The major and minor forms of m P a m hydrolase differ in their charge based on their differential elution from the Red A dye-ligand and QAE-Sepharose resins. The basis for this difference in charge is presently unknown. We do not know if the two forms have the same primary sequence with the charge difference due to a posttranslational modification or if they have different primary sequences with a difference in the number of charged amino-acid residues. The two forms also differ in their apparent values of Vm~,, but this difference is presently not meaningful, since both forms are heterogeneous. Since most properties of the two forms of AP4A hydrolase are the same, the following discussion and conclusions are applicable to both forms unless noted otherwise. Ap4A hydrolase from S. pombe catalyzes the asymmetric hydrolysis of A P 4 A to yield AMP and ATP. Such asymmetric hydrolysis of A P 4 A places this enzyme in the same group as the corresponding enzyme present in several other eukaryotic species [13-18,44,45] and distinguishes it from the symmetric Ap4A hydrolases present in P. polycephalum [19,20] and E. coli

146 TABLE II

Substrate specificity of Schizosaccharomyces pombe diadenosine tetraphosphate hydrolase Diadenosine tetraphosphate hydrolase (major Mono-Q fraction) was incubated in 10 mM Hepes-NaOH (pH 7.5) and 0.2 mM MnCI z with the indicated nucleotides for 20 min at 37°C. The mass of enzyme used ranged from 0.04 to 0.4/zg. Residual substrate and products in the assay solution were analyzed quantitatively by HPLC as described in Materials and Methods. Data are representative values of a minimum of two assays with an experimental difference of 10% or less. Substrate (10/zM)

Relative hydrolysis a

Reaction products

(%) AMP, ADP, ATP, ApzA, AP6A, GP6G Ap 4

< 0.5 4

ATP + Pi AMP + ADP AMP + ATP AMP + ATP (from AP4)

AP3A AP4A AP5A

16 100 2

AP4G

39

ATP + GMP (60%) b GTP + AMP (40%)

AP4U

27

ATP + UMP (56%) UTP + AMP (44%)

AP4C

9

ATP + CMP (64%) CTP + AMP (36%)

Gp3G Gp4G GPsG

2 12 1

m7GP3 G m7GP3Cm m7GP3Am

5 6 6

GMP + GDP GMP + GTP GMP + GTP (from Gp 4) c m7GDP c + GMP m7GDP + 2'-O-mCMP c m7GDP + 2'-O-mAMP c

The percent of substrate hydrolyzed was calculated from the peak areas of the substrates in the absence and presence of the enzyme. The percent hydrolysis of each substrate per /~g of enzyme was expressed relative to the hydrolysis of AP4A. b Percentage values represent the distribution of the indicated nucleotides as products. c Presumed products because nucleotide standards for GP4, mTGDP, 2'-O-mAMP and 2'-O-mCMP were not available. a

[21,22] and the Ap4A phosphorylases present in Saccharomyces cerevisiae [25] and E. gracilis [26]. Activity of the S. pombe Ap4A hydrolase in the absence of Pi and the absence of ADP as a reaction product when the enzyme is assayed in the presence of Pi further distinguish this enzyme from the AP4A phosphorylases. Also, polyclonal antibody against Saccharomyces cerevisiae h P 4 A phosphorylase I [46] does not cross-react with the major Mono Q HPLC fraction of S. pombe h P 4 A hydrolase on a Western blot (Robinson, A.K. and Barnes, L.D., unpublished data). S. pombe Ap4A hydrolase is similar to other asymmetric Ap4A hydrolases in exhibiting negligible activity with AMP, ADP, ATP and Ap2A as potential substrates [17,44,45]. Activity of the S. pombe Ap4A lay-

drolase with Ap3A as substrate is about 16% of the activity with AP4A and the activity with ApsA and AP6A is very low. In contrast, asymmetric Ap4A hydrolases from Anemia [17], lupin [45], human cells [15], rat liver [16] and mouse Ehrlich ascites tumor cells [44] exhibit negligible activity with Ap3A as substrate, while exhibiting significant activity with Ap5A and AP6A when the latter dinucleotides have been tested [17,45]. Some of the asymmetric Ap4A hydrolases [13,16,17,44] have greater activity with Gp4G than with Ap4A as a substrate, but the S. pombe Ap4A hydrolase has eight times greater activity with AP4A than with GP4G. The S. pombe Ap4A hydrolase is similiar to Ap4A phosphorylase II from Saccharomyces cerevisiae [27] in terms of relative activities with Ap4A and GP4G as substrates. S. pombe Ap4A hydrolase catalyzes the asymmetric hydrolysis of AP4G, AP4U, and Ap4C always yielding ATP and the respective nucleotide monophosphate as the preferential pair of products. Lupin AP4A hydrolase also cleaves these three dinucleoside tetraphosphates to yield four products with each substrate, but the distribution of each pair of products for each substrate was not specified [45]. Anemia Ap4A hydrolase catalyzes the hydrolysis of Ap4G to yield GTP and AMP as the predominant pair of products [17]. The differences in activities among the asymmetric Ap4A hydrolases with different dinucleoside polyphosphates as substrates may be more apparent than real. Most of the experiments on substrate specificity, including the present one, are based on relative activities at one concentration of substrate which may not be saturating for all potential substrates. Prescott et al. [17] showed that GP4G was a better substrate than Ap4A for Anemia Ap4A hydrolase based on determination of the specifity constant, kcat/K m. S. pombe AP4A hydrolase exhibits low, but detectable activity with three different mRNA 5' terminal cap nucleotide analogues. Such analogues have not been assayed as potential substrates for asymmetric Ap4A hydrolases from other organisms. The symmetric Ap4A hydrolase from P. polycephalum exhibited very low activity when assayed with mVGp3Cm and m7GP3Am [20]. We do not know if S. pombe Ap4A hydrolase will hydrolyze the 5' terminal cap nucleotide structure on intact mRNA. Such information may be critical in determining in vivo functions of Ap4A catabolic enzymes. However, the different ApnA catabolic enzymes are clearly distinct from the specific mRNA decapping enzymes from HeLa cells [47], Saccharomyces cerevisiae [48] and rat liver [49]. The asymmetric Ap4A hydrolases from different sources [13-18,44,45] have apparent K m values for A P 4 g ranging from 1 to 40/xM, and the corresponding values for the two forms of S. pombe Ap4A hydrolase are within this range. All asymmetric AP4A hydrolases require a divalent metal cation for activity, although

147 there are variations among these enzymes with respect to specific divalent metal cation requirements. S. pombe APaA hydrolase activity is optimal with Mn 2+, while the corresponding enzymes from rat liver [16], brine shrimp [17], human leukemia cells [15], mouse Ehrlich ascites ceils [44] and lupin seeds [45] have optimal activity with Mg 2+. S. pombe mP4m hydrolase is active in the presence of Co 2+ and Ca 2+, but the enzymes from rat liver [16], brine shrimp [17] and lupin seeds [45] are inactive in the presence of Ca 2+, and the enzyme from lupin seeds [45] is also inactive with CO 2+. Z n 2+ has no effect on the activity of the A P 4 A hydrolases from S. pombe, brine shrimp [17] and lupin [45], while it inhibits the enzymes from rat liver [50] and sea urchin [51]. The general pattern of activity of S. pombe Ap4A hydrolase with the different divalent metal cations is more similar to that of Saccharomyces cerevisiae A P 4 A phosphorylase [25] than to that of other asymmetric Ap4A hydrolases. Optimal activity of the symmetric Ap4A hydrolase from E. coli occurs with Co 2+ [21,22]. Guranowski reported that fluoride was a strong, noncompetitive inhibitor of four different asymmetric A P 4 A hydrolases, but that fluoride, at concentrations up to 25 mM, had no effect on the E. coli symmetric AP4A hydrolase or on the Saccharomyces cerevisiae ApgA phosphorylase [52]. The asymmetric Ap4A hydrolase from S. pombe appears to be an exception to his observation that asymmetric AP4A hydrolases are distinguished by their response to fluoride. The determinations of the apparent molecular mass of 49 kDa for AP4A hydrolase by gel filtration chromatography and of 22 kDa on a SDS polyacrylamide gel were both based on detection of enzymic activity. These data indicate that S. pombe Ap4A hydrolase is a dimer or a non-spheroidal monomer or a monomer complexed with another protein during gel filtration chromatography. If the enzyme is a dimer in its native state, then the monomer is active. Also, if the enzyme is a dimer, the monomers are not covalently linked by disulfide bonds based on the results of the gel filtration chromatography. All of the asymmetric AP4A hydrolases that have been examined are single polypeptide chains with molecular masses between 17 and 22 kDa [13,15-18,45], with the exception of mouse liver AP4A hydrolase which has a reported molecular mass of 64 kDa [14]. In comparison, the symmetric AP4A hydrolases [19-22] and Ap4A phosphorylases [26,28,29] are single polypeptide chains with molecular masses ranging from 26 to 36 kDa. We conclude that S. pombe AP4A hydrolase is either a non-spheroidal, single polypeptide chain with a molecular mass of 22 kDa or a homodimer. The significance of the two forms of S. pombe A P 4 A hydrolase is unknown. Prescott et al. [17] detected two forms of Artemia asymmetric Ap4A hydrolase that

have the same apparent molecular mass and that differ in charge. Saccharomyces cerevisiae contains two forms of AP4A phosphorylase that differ by about 400 Da in molecular mass and that have a 60% sequence identity [27-29]. The physiological function and regulation of two forms of a particular Ap4 A catabolic enzyme in different organisms remain to be determined.

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