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Diadenosine 5′,5‴-P1,P4-tetraphosphate hydrolase is present in human erythrocytes, leukocytes and platelets
Pergamon
hr. ./. Binchem.Cell Bid. Vol. 27, No. 2, pp. 201-206, 1995 Copyright Q 1995Elsevier ScienceLtd Printed in Great Britain. All rights reserved 0020-711X(94)00074-X 1357-2725/95$9.50 + 0.00
5’, 5”-P’, P4-Tetra t in Human Erythrwytes,
is
L
ocytes
STEVEN HANKIN, N. MATTHEW, H. THORNE, ALEXANDER G. McLENNAN* Cellular and Metabolic Regulation Group, Department P.O. Box 147, Liverpool L69 3BX, U.K.
of Biochemistry,
University of Liverpool,
hydrohue (Ap,A--+ An asymmetrically-cleaving diadenosine 5’,5’“-P’,F% ATP + AMP) is present in all higher eukaryotes and co intracellular level of the alarmone nucleotide diademWe 5',5"'-P',P4-tetr Thii myme has previously heen isolated from unfractiomtted huawr blood edls. The aim oft@ as part of our study report is to determine the contribution made by of the roles of Ap,A as an intra- and extracell partially purified from isolated human erythrocytes and by probiq bngel permeation chromatography and characterized manobbts witb an antibody raised agabts cleariy present in all tbree ceI1types. Each The erytiyte and platelet Km for tbe leukocyte enzyme inbWion above 10 c M Ap,A. The spec cells, &fold lower than that in leukocytes and platelets. However, the erytbroayte accounted for 97% of tbe total activity of unfwtionati blood eetls (336 U out of 346 U/ml blood). The study shows that leukocytes, platelets amI erytbrocytes all contain Ap,A bydr&aae activity. The last observation is of particular interest given tbe reported absenceof Ap,A from enucleated erytbrocytes. Two possible iaterpretations are that the enzyme is retain& from the reticulocyte stage, or that erytbrocytes retain a capacity, previously unobse~ed, for dim&o&de polypbospbate synthesis. Keywords: Ap,A hydrolase Blood Erythrocytes ht. .I. Biochem.
Leukocytes Platelets
Cell Biol. (1995) 27, 201-206
INTRODUCTION
Diadenosine polyphosphates, such as diadenosine 5’S”-P’,P3-triphosphate (Ap,A), *To whom correspondence should be addressed. Received 2 June 1994; accepted 7 November 1994. Abbreviations: Ap,A, diadenosine 5’,5”‘-P’,P’-triphosphate; Ap.,A, diadenosine 5’5”-P’,P4-tetraphosphate; Ap,A, diadenosine 5’,5”‘-P’,P5-pentaphosphate; ApA, diadenosine 5’,5”‘-P’,P6-hexaphosphate; Ap,A hydrolase, diadenosine 5’,5”-P’,P“-tetraphosphate pyrophosphohydrolase (EC 3.6.1.17); TBST, 10 mM Tris-HCI pH 7.4, 0.15 M NaCl, 0.1% Tween-20.
diadenosine 5’,5”‘-P’,P4-tetraphosphate (AphA), diadenosine 5’,5”‘-P ‘,P5-pentaphospbate (Ap,A) and diadenosine 5’,5”‘-P’,P6-hexaphosphate (Ap,A) have been shown to be present in and secreted from certain neurosecretory granules and the dense granules of bIood platelets. These compounds have neurotransmitter and vasomotor activity, affect platelet aggregation and may be novel regulators of blood pressure with properties distinct from ATP (Flodgaard and Klenow, 1982;’ Ogilvie, 1992; Pintor and MirasPortugal, 1993; Pohl et al., 1991; S&titer et al., 1994). In addition, virtually all cells contain 201
202
Steven
Hankin
submicromolar to micromolar cytosolic levels of Ap,A and Ap,A, with the latter compound having been implicated in a number of intracellular events, including DNA replication and cellular responses to metabolic stress (Yakovenko and Formazyuk, 1993). Mammalian cells typically contain a specific Ap,A hydrolase (EC 3.6.1.17) that hydrolyses Ap,A in an asymmetric fashion to yield AMP and ATP. This enzyme also hydrolyses higher homologues (e.g. Ap,A and ApeA) and is presumed to be involved in the regulation of the intracellular level of such nucleotides (Guranowski and Sillero, 1992). The human enzyme has been purified from leukaemia cells (Ogilvie and Antl, 1983), placenta (Lazewska et al., 1993) and unfractionated blood cells (Pintor et al., 1991). In the last case, it is important to know the relative contributions made by different blood cell fractions, since (i) the substrate AplA is reportedly absent from human erythrocytes (Flodgaard and Klenow, 1982; Ogilvie, 1992), although it has been shown to modulate O,-binding to haemoglobin (Bonaventura et al., 1992); (ii) the metabolic inertness of the stored Ap4A pool in platelets has been ascribed in part to an apparent lack of hydrolase activity in these cells (Flodgaard and Klenow, 1982); (iii) there may be tissue-specific isoforms of Ap,A hydrolases differing in size and K, value (Cameselle et al., 1982). Here, we report the presence of Ap*A hydrolase in all three major human blood cell fractions: erythrocytes, leukocytes and platelets. MATERIALS
AND
METHODS
Materials
All blood cell fractions were obtained from Merseyside Regional Blood Transfusion Service. ATP Monitoring Reagent (contains firefly luciferase and luciferin) was from BioOrbit. Dextran T-500 was obtained from Pharmacia. All other reagents were from Sigma. Homogeneous human placental Ap,A hydrolase (spec. act., 4 U/mg protein; kcat, 1.3 s-l) was prepared as described (Lazewska et al., 1993). Lysis buffer contained 30 mM Hepes-KOH, pH 8.0, 5 mM EDTA, 1 mM phenylmethylsulphonyl fluoride, 1 mM benzamidine, 5 p M tram -epoxysuccinylL-leucylamido-(4-guanidino)butane (E-64). Ap, A hydrolase assay
The rate of Ap,A degradation was determined using a luciferase-based bioluminescence
et u/
assay to detect the ATP product (Prescott et al., 1989). The assay contained 35 mM HepesKOH, pH 7.8, 5 mM Mg acetate, 20 PM Ap,A, ATP Monitoring Reagent (20,ul) and enzyme fraction in a total volume of 100 ~1 and was incubated at 25°C. The initial rate of increase in luminescence was measured using a LKB Wallac 1250 luminometer and converted into units of enzyme activity (pm01 Ap,A degraded per min) using a calibration curve. Partial purification platelets
of Ap,A
hydrolase from
Platelet-enriched plasma (250 ml) was centrifuged at 200g for 5 min (25”C), the supernatant removed and centrifuged again at 16,OOOg, 10 min, 25°C. The resulting pellet was washed twice by resuspension and centrifugation (16,000 g, 10 min, 25°C) in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 20 mM EDTA. The platelet pellet (8-10 g) was resuspended in lysis buffer (1 g ‘platelets per 2 ml lysis buffer). The cell suspension was freeze/thawed 3 times in liquid nitrogen and then homogenized using a motor-driven Teflon-tipped pestle. The homogenate was centrifuged at 20,OOOg for 20 min, the supernatant brought to 30% saturation by the addition of solid ammonium sulphate, then centrifuged at 20,OOOg for 20 min at 4°C. The supernatant was removed, brought to 70% saturation with ammonium sulphate and centrifuged as before. The cream coloured pellet was redissolved in Superdex buffer (30mM HepesKOH pH 7.8, 50 mM KCl, 0.1 mM EDTA, 1 mM 2-mercaptoethanol) and filtered (0.2 pm filter). A sample (0.5 ml, 15.6mg protein/ml) was applied to a high performance gel filtration column (HiLoad 16/60 Superdex 75) and then eluted at 1 ml/min with Superdex buffer. Fractions (1 ml) were collected and assayed for Ap,A hydrolase activity. Active fractions (8-10 ml) were pooled and concentrated to a volume of 250-300 ~1 with a Centriconconcentrator (Amicon). Loss of activity was less than 10%. Partial purification erythrocytes
of Ap,A
hydrolase from
Lysis buffer (40 ml) was added to 20 ml erythrocyte concentrate. The cell suspension was then left at 4°C for 1 hr to lyse the cells. A 30-70% ammonium sulphate fraction was prepared as described above and the red protein pellet redissolved in Superdex buffer. A sample (0.5 ml, 132 mg protein/ml) was applied to the Superdex column, The column was eluted and
203
Ap,A hydrolase in human blood cells
fractions collected, assayed and concentrated as above. Partial pu$cation leukocytes
of Ap,A
hydrolase from
Dextran (6% in PBS) was added to 30 ml leukocyte-enriched blood in a ratio of 1:9. The sample was left to sediment at room temperature for 20 min. The top, straw-coloured layer was removed and centrifuged at 15Og for 5 min. The supernatant was discarded and the pellet (2-3 g) resuspended in water to lyse any contaminating erythrocytes. After centrifugation at 15Og for 5 min, the cell pellet was resuspended in water and the lysis step repeated until all the remaining erythrocytes were removed. The final leukocyte pellet was resuspended in lysis buffer (0.5 g cells/ml buffer) and left on ice for 1 hr. The sample was freeze-thawed three times, homogenized, and a 3O-70% ammonium sulphate fraction prepared as described above. This was redissolved in Superdex buffer and a 0.5 ml sample (4.3 mg protein/ml) applied to the Superdex column. The column was eluted and fractions collected, assayed and concentrated as above. Production of a polyclonal Ap,A hydrolase
antibody to human
Pure placental Ap*A hydrolase (20 pg) in Freund’s complete adjuvant was injected subcutaneously at two sites into the breast of a chicken. After 30 days, a boost comprising the decapeptide LSHEHQAYRC conjugated to keyhole limpet haemocyanin (4 mg peptide, sequence data derived from tryptic digest of placental enzyme) was given. Eggs were collected and an immunoglobulin fraction prepared from the yolk (Gassman et al., 1990). The anti-Ap,A hydrolase antibody was affinity-purified on a 0.5 ml column of Affi-Gel 102 (BioRad) on which was immobilized 15 mg of the above peptide. After washing with PBS, the antibody was eluted with 100 mM glycine, pH 2.5, 100 mM NaCl, 10% ethylene glycol (Smith and Fisher, 1984). Electrophoresis and immunoblotting
Samples of the partially purified, concentrated Ap,A hydrolase fractions were subjected to SDS-PAGE in a 17.5% gel and electroblotted on to nitrocellulose in 10 mM 3-(cyclohexylamino)- I-propane-sulphonic acid (CAPS), pH 11, 10% methanol according to standard procedures. After blocking in 10 mM Tris-HCl
pH 7.4, 0.15 M NaCl, 0.1% Tween-20 (TBST) containing 1% milk protein, the blot was incubated overnight at room temperature with a I:2000 dilution of the anti-hydrolase antibody in TBST. After washing, the second antibody (alkaline phosphatase-conjugated rabbit antichicken IgG, 1: 10,000 dilution) was added for 3 hr at room temperature. The blot was finally developed in TBST, pH 9.5, containing 0.33 mg/ ml nitro blue tetrazolium and 0.165 mg/ml 5-bromo-4-chloro-3-indolyl phosphate. Protein assay
Protein was determined by the Coomassie blue dye binding method using bovine-y -globulin as standard (Peterson, 1982). RESULTS
Specific Ap4A hydrolase activity was readily detectable in extracts of all three major blood cell fractions: erythrocytes, leukocytes and platelets. Extracts were partially purified by ammonium sulphate fractionation and high performance gel filtration. The Superdex elution profile for the erythrocyte fraction is shown in Fig. 1. Calibration of the Superdex column with standard proteins showed that all three enzymes had an apparent M, of 19,200 (Table 1) while
EluConVolume (ml) Fig. 1. Chromatography of 0.5 ml of a 30-70% ammonium sulphate fraction of human erythrocytes on a HiLoad 16J60 Superdex 75 high performance gel filtration column. Conditions were as described in Material and Methods. The column was calibrated with the following molecular weight standards: 1, conalbumin (77 kDa); 2, bovine serum albumin (67 kDa); 3, ovalbumin (45 kDa); 4, fi-lactoglobulin (36.8 kDa); 5, carbonic anhydrase (29 kDa); 6, chymotrypsinogen A (25 kDa); 7, soybean trypsin inhibitor (20.1 kDa); 8, myoglobin (17 kDa) and 9, cytochrome c (12.4 kDa). Ap.,A hydrolase activity (a); elution positions of protein standards (m); A,, (-). Apart from the A, profile, the elution of the Ap,A hydrolases from similar fractions prepared from leukocytes and platelets was identical.
Steven Hankin e/ a/.
204
Table 1. Comparison between Ap4A hydrolases from different blood cell fractions
M by
Cell type Erythrocytes Leukocytes Platelets
(n = 3)
Specific activity (mU/mg protein)
Specific activity (mu/IO6 cells)h
Specific activity (mu/ml blood)’
19,200
0.70 & 0.05
0.009
0.067
336.0
19,200 19,200
1.50 + 0.20 0.70 * 0.05
0.126 0.133
0.647 0.017
Mr by
gel filtration
SDS-PAGE
19,200
19,200 19,200
Kt, (PM)
5.2 5.0
BIn order to eliminate any possible contribution from non-specific phosphodiesterases and interference from phosphatases, activity was determined from the sum of the activities of the Superdex fractions. The protein concentration of the applied sample was taken to represent the total cellular soluble protein as the bulk of this appears in the 30-70% ammonium sulphate fraction. bCell numbers in the purified suspensions were determined by counting in a haemocytometer before lysis. Estimates of purity were >99.9% in each case. ‘I ml of whole blood was taken to contain 5 x IO9 erythrocytes, 8 x IO6 leukocytes and 3 x IO8 platelets.
SDS-PAGE and immunoblotting with an antibody raised against the human placental hydrolase (Table 1 and Fig. 2) also showed them to be identical in size to each other and to the placental enzyme, which has previously been sized electrophoretically at M, = 19,200 (Lazewska et al., 1993; Thorne, N.M.H. unpublished). Hence, no differences in size were observed among the blood cell enzymes as has been suggested for the hydrolases from, different rat tissues (Cameselle et al., 1982). Crossreaction with the anti-placental enzyme antibody also shows that the enzymes under examination are indeed the specific, asymmetrically cleaving Ap,A hydrolases. All three enzymes displayed MichaelisMenten kinetics up to 10 PM ApJA; however, higher concentrations caused inhibition (Fig. 3). This is not due to (i) product inhibition, since initial rates were calculated from the first few seconds of the progress curves; (ii) chelation of Mg2+, since this is in clear excess over Ap,A at 5 mM; (iii) the presence of an unidentified uncompetitive or non-competitive inhibitor in the substrate, since Ap,A repurified by ionexchange chromatography (MonoQ) and gel
A
B
C
D
Fig. 2. lmmunoblot of human blood cell Ap,A hydrolases. Samples were electrophoresed, blotted on to nitrocellulose and the blot probed with an antibody to the placental enzyme as described in Materials and Methods. (A) 1 pg homogeneous placental hydrolase; (B) 66 ,ug erythrocyte fraction; (C) 82 pg leukocyte fraction; (D) 21 fig platelet fraction.
filtration (BioGel P2) yielded the same result; the only known contaminants of Ap,A are AMP, ADP, ATP and p,A, all of which are competitive inhibitors (Vallejo et al., 1976) and would thus be expected simply to reduce both K, and V,,,, if present as contaminants; (iv) some peculiarity of the assay system, since no inhibition of the Ap,A hydrolase from the green alga Scenedesmus obliquus was observed even up to 400 PM Ap,A using the same assay (McLennan et al., 1994) while an identical inhibition was observed with the human placental Ap,A hydrolase using an entirely different (hyperchromicity) assay system (Hankin S. and McLennan A. G., unpublished observation). Hence, the reason appears to be substrate inhibition. This has not previously been reported for an Ap,A hydrolase and is probably due to substrate crowding, i.e. the binding of two adenosine moieties from two different Ap,A
0'
0
I
I
1
100
200
300
[A~44 (PM) Fig. 3. Dependence of initial reaction velocity on substrate concentration for the leukocyte Ap,A hydrolase. Assays were performed as described in Materials and Methods at different Ap,A concentrations. Similar profiles were obtained with the erythrocyte and platelet enzymes.
Ap.,A hydrolase in human blood cells
205
cytes, they could only account for 10% of the erythrocyte activity. Also, the enzyme from leukocytes and platelets cannot be due to contaminating erythrocytes because of the 15-fold higher specific activities measured for these cells. Thus, all three cell types contain Ap,A hydrolase. The high erythrocyte concentration in whole blood means that red cells are by far the most important source (97%) of the blood cell enzyme. The presence of a significant level of Ap,A hydrolase in enucleated red cells is particularly interesting, since they do not synthesize proteins or Ap,A and presumably have low levels of aminoacyl-tRNA synthetases, the enzymes responsible for Ap,A synthesis (Goerlich et al., 1982). There are several possible reasons for the presence of hydrolase in erythrocytes. First, it may be a relic of the reticulocyte stage with no true function in erythrocytes. Second, red cells may retain an unobserved capacity for a low level of Ap,A synthesis. This may be catalysed by residual aminoacyl-tRNA synthetases, or may be the result of the chemical condensation of two ATP molecules, as previously suggested (McLennan and Zamecnik, 1992). In either case, Ap,A hydrolase activity would be required to prevent the undesirable accumulation of this nucleotide. Finally, it is possible that other dinucleoside polyphosphates, e.g. Ap,A, Ap,A or Gp,G (Coste et al., 1987) are the true substrates for the hydrolase in red cells. The presence of a ‘normal’ concentration of hydrolase activity in platelets contrasts with an earlier report ascribing the metabolic stability of the stored Ap,A pool to the apparent absence of this enzyme (Flodgaard and Klenow, 1982). Hydrolytic activity against Ap,A has been detected in detergent-solubilized DISCUSSION platelet lysates (Liithje and Ogilvie, 1983); howBased on previously published results, the ever, as the sole product was AMP, the activity presence of Ap,A hydrolase activity in all three could have been due to phosphodiesterase acmajor blood cell fractions was unexpected. It tivity. Presumably, compartmentalization of is important to note that the presence of the enzyme in the cytosol and its substrate(s) in the enzyme in erythrocytes cannot be due to the dense granules is sufficient to account for the contamination by the other blood cells since, stability of the stored AplA. Nevertheless, this according to the data in Table 1, this would finding must be considered in future studies of require either a white cell: red cell ratio of 1: 10 the synthesis, storage, turnover and secretion of or a platelet: red cell ratio of 4: 1 in the purified the physiologically active platelet dinucleotides cell fractions, neither of which is remotely poss- Ap,A, Ap,A and Ap,A (Schliiter et ui., 1994). ible, considering the normal ratios in whole blood. Part of the erythrocyte activity may be Acknow~ledgement-The work was supported by grants due to reticulocytes, which normally amount to from the Science and Engineering Research Council and the I % of the red cell population. Even so, assum- Royal Society. S.H. is the holder of an SERC Research ing that they had an activity similar to leukoStudentship.
molecules to the sites normally occupied by both adenosine moieties of a single Ap,A. The erythrocyte and platelet enzymes both had K, values of 0.70 PM, determined from Eadie-Hofstee plots, while that of the leukocyte enzyme was slightly higher at 1.50 PM. The leukocyte pellet prepared by dextran sedimentation consists of about 70% neutrophils, the remainder being mainly lymphocytes, but whether the higher K, is in some way related to the heterogeneity of the leukocyte fraction is unknown at present. The K,,, for the unfractionated blood cell enzyme was also reported to be 0.7 PM (Pintor et al., 1991) while those for the leukaemic cell and placental enzymes were 0.5 PM (Ogilvie and Antl, 1983) and 1OpM (Lazewska et al., 1993) respectively. Interestingly, our own measurements for the placental enzyme gave a K,,, value of 0.8 PM. Hence, the human enzyme may exist in two kinetically distinguishable forms, differing in K,,, by about IO-fold, as previously reported for the enzyme from Artemia embryos (Prescott et al., 1989) and for the enzymes from different rat tissues (Cameselle et al., 1982). Comparison of the specific activities of the different blood cell hydrolases (Table 1) shows that, in terms of protein, both the platelet and leukocyte enzymes have similar values. On the other hand, erythrocytes have a 15-fold lower specific activity. In terms of enzyme activity per cell, leukocytes clearly have the highest activity. The 50-fold lower activity in platelets can be accounted for by their approx. 50-fold lower cell volume; erythrocytes and neutrophils (the major leukocyte) have similar cell volumes.
206
Steven H ankin et al REFERENCES
Bonaventura C., Cashon R., Colacino J. M. and Hilderman R. H. (1992) Alteration of hemoglobin function by diadenosine 5’,5”‘-Pi ,P4-tetraphosphate and other alarmones. J. biol. Chem. 267, 46524657. Cameselle J. C., Costas M. J., Sillero M. A. G. and Sillero A. (1982) Bis-(5’-guanosyl)tetraphosphatases in rat tissues. Biochem.
J. 201, 405409.
Coste H., Brevet A., Plateau P. and Blanquet S. (1987) Nonadenylated bis(5’-nucleosidyl)tetraphosphates occur in Saccharomyces cerevisiae and Escherichia coli and accumulate upon temperature shift or exposure to cadmium. J. biol. Chem. 262, 12096-12100. Flodgaard H. and Klenow H. (1982) Abundant amounts of diadenosine 5’,5”‘-P’,P4-tetraphosphate are present and releasable, but metabolically inactive, in human platelets. Biochem.
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737-742.
Gassmann M., Thommes P., Weiser T. and Hiibscher LJ. (1990) Efficient production of chicken egg-yolk antibodies against a conserved mammalian protein. FASEB J. 4, 2528-2532. Goerlich O., Foeckler R. and Holler E. (1982) Mechanism of synthesis of adenosine (S’)tetraphospho(5’)adenosine (AppppA) by aminoacyl-tRNA synthetases. Eur. J. Biothem. 124, 135-140. Guranowski A. and Sillero A. (1992) Enzymes cleaving dinucleoside polyphosphates. In .4p& and Other Dinucleoside Polyphosphates (Edited by McLennan A. G.), pp. 81-133. CRC Press, Boca Raton, Florida. Lazewska D., Starzynska E. and Guranowski A. (1993) Human placental (asymmetrical) diadenosine 5’,S”‘P’,P4-tetraphosphate hydrolase: purification to homogeneity and some properties. Protein Expr. Purif: 4, 45-51. Liithje J. and Ogilvie A. (1983) The presence of diadenosine 5’,5”‘-P’,P3-triphosphate (Ap,A) in human platelets. Biothem.
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115, 253-260.
McLennan A. G. and Zamecnik P. C. (1992) Dinucleoside polyphosphates-an introduction. In Apd and Other Dinucleoside Polyphosphates (Edited by McLennan A. G.), pp. l-7. CRC Press, Boca Raton, Florida. McLennan A. G., Mayers M., Hankin S., Thorne N. M. H., Prescott M. and Powls P. (1994) The green alga Scenedesmus obliquus contains both diadenosine 5’,5”-P’,P4tetraphosphate (asymmetrical) pyrophosphohydrolase and phosphorylase activities. Biochem. J. 300, 183-189.
Ogilvie A. (1992) Extracellular functions for Ap,A. In Apg and Other Dinucleoside Polyphosphates (Edited by McLennan A. G.), pp. 229-273. CRC Press, Boca Raton, Florida. Ogilvie A. and Ant1 W. (1983) Diadenosine tetraphosphatase from human leukemia cells. Purification to homogeneity and partial characterization. J. biol. Chem. 258,4105-4109. Peterson G. L. (1982) Determination of total protein. Merhs Enzymol. 91, 95-l 19. Pintor R. M., Costas M. J., FernLndez A., Canales J., Garcia-Agundez J. A. and Cameselle J. C. (1991) Dinucleoside tetraphosphatase from human blood cells. Purification and characterization as a high specific activity enzyme recognized by an anti-rat tetraphosphatase antibody. FEBS Left. 287, 85-88. Pinlor J. and Miras-Portugal M. T. (1993) Diadenosine polyphosphates (ApXA) as new neurotransmitters. Drug. Dev.
Res. 28, 259-262.
Pohl U., Ogilvie A., Lamontagne D. and Busse R. (1991) Potent effects of Ap,A and Ap,A on coronary resistance and autacoid release of intact rabbit hearts. Amer. J. Physiol. 260, H1692-H 1697. Prescott M., Milne A. D. and McLennan A. G. (1989) Characterization of the bis(S-nucleosidyl)tetraphosphate pyrophosphohydrolase from encysted embryos of the brine shrimp Artemia. Biochem. J. 259, 831-838. Schliiter H., Offers E., Briiggeman G., van der Giet M., Tepel M., Nordhoff E., Karas M., Spieker C., Witzel H. and Zidek W. (1994) Diadenosine phosphates and ;he physiological control of blood pressure. Nature 367, 186-188. Smith D. E. and Fisher P. A. (1984) Identification, developmental regulation and response to heat-shock of two antigenically related forms of a major nuclear-envelope protein in Drosophila embryos-application of an improved method for affinity purification of antibodies using polypeptides immobilized on nitrocellulose blots. J. cell. Biol.
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Vallejo G.,G., Lobaton C. D., Quintanilla M., Sillero A. and Sillero M. A. G. (1976) Dinucleoside tetraphosphatase in rat liver and Artemia. Biochim. Biophys. Acta 438, 304-309.
Yakovenko I. N. and Formazyuk V. E. (1993) Diadenosine oligophosphates: metabolic pathways and role in regulation of the functional activity of cells. Biokhimiya (Engl. trans.) 58, l-15.
hr. ./. Binchem.Cell Bid. Vol. 27, No. 2, pp. 201-206, 1995 Copyright Q 1995Elsevier ScienceLtd Printed in Great Britain. All rights reserved 0020-711X(94)00074-X 1357-2725/95$9.50 + 0.00
5’, 5”-P’, P4-Tetra t in Human Erythrwytes,
is
L
ocytes
STEVEN HANKIN, N. MATTHEW, H. THORNE, ALEXANDER G. McLENNAN* Cellular and Metabolic Regulation Group, Department P.O. Box 147, Liverpool L69 3BX, U.K.
of Biochemistry,
University of Liverpool,
hydrohue (Ap,A--+ An asymmetrically-cleaving diadenosine 5’,5’“-P’,F% ATP + AMP) is present in all higher eukaryotes and co intracellular level of the alarmone nucleotide diademWe 5',5"'-P',P4-tetr Thii myme has previously heen isolated from unfractiomtted huawr blood edls. The aim oft@ as part of our study report is to determine the contribution made by of the roles of Ap,A as an intra- and extracell partially purified from isolated human erythrocytes and by probiq bngel permeation chromatography and characterized manobbts witb an antibody raised agabts cleariy present in all tbree ceI1types. Each The erytiyte and platelet Km for tbe leukocyte enzyme inbWion above 10 c M Ap,A. The spec cells, &fold lower than that in leukocytes and platelets. However, the erytbroayte accounted for 97% of tbe total activity of unfwtionati blood eetls (336 U out of 346 U/ml blood). The study shows that leukocytes, platelets amI erytbrocytes all contain Ap,A bydr&aae activity. The last observation is of particular interest given tbe reported absenceof Ap,A from enucleated erytbrocytes. Two possible iaterpretations are that the enzyme is retain& from the reticulocyte stage, or that erytbrocytes retain a capacity, previously unobse~ed, for dim&o&de polypbospbate synthesis. Keywords: Ap,A hydrolase Blood Erythrocytes ht. .I. Biochem.
Leukocytes Platelets
Cell Biol. (1995) 27, 201-206
INTRODUCTION
Diadenosine polyphosphates, such as diadenosine 5’S”-P’,P3-triphosphate (Ap,A), *To whom correspondence should be addressed. Received 2 June 1994; accepted 7 November 1994. Abbreviations: Ap,A, diadenosine 5’,5”‘-P’,P’-triphosphate; Ap.,A, diadenosine 5’5”-P’,P4-tetraphosphate; Ap,A, diadenosine 5’,5”‘-P’,P5-pentaphosphate; ApA, diadenosine 5’,5”‘-P’,P6-hexaphosphate; Ap,A hydrolase, diadenosine 5’,5”-P’,P“-tetraphosphate pyrophosphohydrolase (EC 3.6.1.17); TBST, 10 mM Tris-HCI pH 7.4, 0.15 M NaCl, 0.1% Tween-20.
diadenosine 5’,5”‘-P’,P4-tetraphosphate (AphA), diadenosine 5’,5”‘-P ‘,P5-pentaphospbate (Ap,A) and diadenosine 5’,5”‘-P’,P6-hexaphosphate (Ap,A) have been shown to be present in and secreted from certain neurosecretory granules and the dense granules of bIood platelets. These compounds have neurotransmitter and vasomotor activity, affect platelet aggregation and may be novel regulators of blood pressure with properties distinct from ATP (Flodgaard and Klenow, 1982;’ Ogilvie, 1992; Pintor and MirasPortugal, 1993; Pohl et al., 1991; S&titer et al., 1994). In addition, virtually all cells contain 201
202
Steven
Hankin
submicromolar to micromolar cytosolic levels of Ap,A and Ap,A, with the latter compound having been implicated in a number of intracellular events, including DNA replication and cellular responses to metabolic stress (Yakovenko and Formazyuk, 1993). Mammalian cells typically contain a specific Ap,A hydrolase (EC 3.6.1.17) that hydrolyses Ap,A in an asymmetric fashion to yield AMP and ATP. This enzyme also hydrolyses higher homologues (e.g. Ap,A and ApeA) and is presumed to be involved in the regulation of the intracellular level of such nucleotides (Guranowski and Sillero, 1992). The human enzyme has been purified from leukaemia cells (Ogilvie and Antl, 1983), placenta (Lazewska et al., 1993) and unfractionated blood cells (Pintor et al., 1991). In the last case, it is important to know the relative contributions made by different blood cell fractions, since (i) the substrate AplA is reportedly absent from human erythrocytes (Flodgaard and Klenow, 1982; Ogilvie, 1992), although it has been shown to modulate O,-binding to haemoglobin (Bonaventura et al., 1992); (ii) the metabolic inertness of the stored Ap4A pool in platelets has been ascribed in part to an apparent lack of hydrolase activity in these cells (Flodgaard and Klenow, 1982); (iii) there may be tissue-specific isoforms of Ap,A hydrolases differing in size and K, value (Cameselle et al., 1982). Here, we report the presence of Ap*A hydrolase in all three major human blood cell fractions: erythrocytes, leukocytes and platelets. MATERIALS
AND
METHODS
Materials
All blood cell fractions were obtained from Merseyside Regional Blood Transfusion Service. ATP Monitoring Reagent (contains firefly luciferase and luciferin) was from BioOrbit. Dextran T-500 was obtained from Pharmacia. All other reagents were from Sigma. Homogeneous human placental Ap,A hydrolase (spec. act., 4 U/mg protein; kcat, 1.3 s-l) was prepared as described (Lazewska et al., 1993). Lysis buffer contained 30 mM Hepes-KOH, pH 8.0, 5 mM EDTA, 1 mM phenylmethylsulphonyl fluoride, 1 mM benzamidine, 5 p M tram -epoxysuccinylL-leucylamido-(4-guanidino)butane (E-64). Ap, A hydrolase assay
The rate of Ap,A degradation was determined using a luciferase-based bioluminescence
et u/
assay to detect the ATP product (Prescott et al., 1989). The assay contained 35 mM HepesKOH, pH 7.8, 5 mM Mg acetate, 20 PM Ap,A, ATP Monitoring Reagent (20,ul) and enzyme fraction in a total volume of 100 ~1 and was incubated at 25°C. The initial rate of increase in luminescence was measured using a LKB Wallac 1250 luminometer and converted into units of enzyme activity (pm01 Ap,A degraded per min) using a calibration curve. Partial purification platelets
of Ap,A
hydrolase from
Platelet-enriched plasma (250 ml) was centrifuged at 200g for 5 min (25”C), the supernatant removed and centrifuged again at 16,OOOg, 10 min, 25°C. The resulting pellet was washed twice by resuspension and centrifugation (16,000 g, 10 min, 25°C) in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 20 mM EDTA. The platelet pellet (8-10 g) was resuspended in lysis buffer (1 g ‘platelets per 2 ml lysis buffer). The cell suspension was freeze/thawed 3 times in liquid nitrogen and then homogenized using a motor-driven Teflon-tipped pestle. The homogenate was centrifuged at 20,OOOg for 20 min, the supernatant brought to 30% saturation by the addition of solid ammonium sulphate, then centrifuged at 20,OOOg for 20 min at 4°C. The supernatant was removed, brought to 70% saturation with ammonium sulphate and centrifuged as before. The cream coloured pellet was redissolved in Superdex buffer (30mM HepesKOH pH 7.8, 50 mM KCl, 0.1 mM EDTA, 1 mM 2-mercaptoethanol) and filtered (0.2 pm filter). A sample (0.5 ml, 15.6mg protein/ml) was applied to a high performance gel filtration column (HiLoad 16/60 Superdex 75) and then eluted at 1 ml/min with Superdex buffer. Fractions (1 ml) were collected and assayed for Ap,A hydrolase activity. Active fractions (8-10 ml) were pooled and concentrated to a volume of 250-300 ~1 with a Centriconconcentrator (Amicon). Loss of activity was less than 10%. Partial purification erythrocytes
of Ap,A
hydrolase from
Lysis buffer (40 ml) was added to 20 ml erythrocyte concentrate. The cell suspension was then left at 4°C for 1 hr to lyse the cells. A 30-70% ammonium sulphate fraction was prepared as described above and the red protein pellet redissolved in Superdex buffer. A sample (0.5 ml, 132 mg protein/ml) was applied to the Superdex column, The column was eluted and
203
Ap,A hydrolase in human blood cells
fractions collected, assayed and concentrated as above. Partial pu$cation leukocytes
of Ap,A
hydrolase from
Dextran (6% in PBS) was added to 30 ml leukocyte-enriched blood in a ratio of 1:9. The sample was left to sediment at room temperature for 20 min. The top, straw-coloured layer was removed and centrifuged at 15Og for 5 min. The supernatant was discarded and the pellet (2-3 g) resuspended in water to lyse any contaminating erythrocytes. After centrifugation at 15Og for 5 min, the cell pellet was resuspended in water and the lysis step repeated until all the remaining erythrocytes were removed. The final leukocyte pellet was resuspended in lysis buffer (0.5 g cells/ml buffer) and left on ice for 1 hr. The sample was freeze-thawed three times, homogenized, and a 3O-70% ammonium sulphate fraction prepared as described above. This was redissolved in Superdex buffer and a 0.5 ml sample (4.3 mg protein/ml) applied to the Superdex column. The column was eluted and fractions collected, assayed and concentrated as above. Production of a polyclonal Ap,A hydrolase
antibody to human
Pure placental Ap*A hydrolase (20 pg) in Freund’s complete adjuvant was injected subcutaneously at two sites into the breast of a chicken. After 30 days, a boost comprising the decapeptide LSHEHQAYRC conjugated to keyhole limpet haemocyanin (4 mg peptide, sequence data derived from tryptic digest of placental enzyme) was given. Eggs were collected and an immunoglobulin fraction prepared from the yolk (Gassman et al., 1990). The anti-Ap,A hydrolase antibody was affinity-purified on a 0.5 ml column of Affi-Gel 102 (BioRad) on which was immobilized 15 mg of the above peptide. After washing with PBS, the antibody was eluted with 100 mM glycine, pH 2.5, 100 mM NaCl, 10% ethylene glycol (Smith and Fisher, 1984). Electrophoresis and immunoblotting
Samples of the partially purified, concentrated Ap,A hydrolase fractions were subjected to SDS-PAGE in a 17.5% gel and electroblotted on to nitrocellulose in 10 mM 3-(cyclohexylamino)- I-propane-sulphonic acid (CAPS), pH 11, 10% methanol according to standard procedures. After blocking in 10 mM Tris-HCl
pH 7.4, 0.15 M NaCl, 0.1% Tween-20 (TBST) containing 1% milk protein, the blot was incubated overnight at room temperature with a I:2000 dilution of the anti-hydrolase antibody in TBST. After washing, the second antibody (alkaline phosphatase-conjugated rabbit antichicken IgG, 1: 10,000 dilution) was added for 3 hr at room temperature. The blot was finally developed in TBST, pH 9.5, containing 0.33 mg/ ml nitro blue tetrazolium and 0.165 mg/ml 5-bromo-4-chloro-3-indolyl phosphate. Protein assay
Protein was determined by the Coomassie blue dye binding method using bovine-y -globulin as standard (Peterson, 1982). RESULTS
Specific Ap4A hydrolase activity was readily detectable in extracts of all three major blood cell fractions: erythrocytes, leukocytes and platelets. Extracts were partially purified by ammonium sulphate fractionation and high performance gel filtration. The Superdex elution profile for the erythrocyte fraction is shown in Fig. 1. Calibration of the Superdex column with standard proteins showed that all three enzymes had an apparent M, of 19,200 (Table 1) while
EluConVolume (ml) Fig. 1. Chromatography of 0.5 ml of a 30-70% ammonium sulphate fraction of human erythrocytes on a HiLoad 16J60 Superdex 75 high performance gel filtration column. Conditions were as described in Material and Methods. The column was calibrated with the following molecular weight standards: 1, conalbumin (77 kDa); 2, bovine serum albumin (67 kDa); 3, ovalbumin (45 kDa); 4, fi-lactoglobulin (36.8 kDa); 5, carbonic anhydrase (29 kDa); 6, chymotrypsinogen A (25 kDa); 7, soybean trypsin inhibitor (20.1 kDa); 8, myoglobin (17 kDa) and 9, cytochrome c (12.4 kDa). Ap.,A hydrolase activity (a); elution positions of protein standards (m); A,, (-). Apart from the A, profile, the elution of the Ap,A hydrolases from similar fractions prepared from leukocytes and platelets was identical.
Steven Hankin e/ a/.
204
Table 1. Comparison between Ap4A hydrolases from different blood cell fractions
M by
Cell type Erythrocytes Leukocytes Platelets
(n = 3)
Specific activity (mU/mg protein)
Specific activity (mu/IO6 cells)h
Specific activity (mu/ml blood)’
19,200
0.70 & 0.05
0.009
0.067
336.0
19,200 19,200
1.50 + 0.20 0.70 * 0.05
0.126 0.133
0.647 0.017
Mr by
gel filtration
SDS-PAGE
19,200
19,200 19,200
Kt, (PM)
5.2 5.0
BIn order to eliminate any possible contribution from non-specific phosphodiesterases and interference from phosphatases, activity was determined from the sum of the activities of the Superdex fractions. The protein concentration of the applied sample was taken to represent the total cellular soluble protein as the bulk of this appears in the 30-70% ammonium sulphate fraction. bCell numbers in the purified suspensions were determined by counting in a haemocytometer before lysis. Estimates of purity were >99.9% in each case. ‘I ml of whole blood was taken to contain 5 x IO9 erythrocytes, 8 x IO6 leukocytes and 3 x IO8 platelets.
SDS-PAGE and immunoblotting with an antibody raised against the human placental hydrolase (Table 1 and Fig. 2) also showed them to be identical in size to each other and to the placental enzyme, which has previously been sized electrophoretically at M, = 19,200 (Lazewska et al., 1993; Thorne, N.M.H. unpublished). Hence, no differences in size were observed among the blood cell enzymes as has been suggested for the hydrolases from, different rat tissues (Cameselle et al., 1982). Crossreaction with the anti-placental enzyme antibody also shows that the enzymes under examination are indeed the specific, asymmetrically cleaving Ap,A hydrolases. All three enzymes displayed MichaelisMenten kinetics up to 10 PM ApJA; however, higher concentrations caused inhibition (Fig. 3). This is not due to (i) product inhibition, since initial rates were calculated from the first few seconds of the progress curves; (ii) chelation of Mg2+, since this is in clear excess over Ap,A at 5 mM; (iii) the presence of an unidentified uncompetitive or non-competitive inhibitor in the substrate, since Ap,A repurified by ionexchange chromatography (MonoQ) and gel
A
B
C
D
Fig. 2. lmmunoblot of human blood cell Ap,A hydrolases. Samples were electrophoresed, blotted on to nitrocellulose and the blot probed with an antibody to the placental enzyme as described in Materials and Methods. (A) 1 pg homogeneous placental hydrolase; (B) 66 ,ug erythrocyte fraction; (C) 82 pg leukocyte fraction; (D) 21 fig platelet fraction.
filtration (BioGel P2) yielded the same result; the only known contaminants of Ap,A are AMP, ADP, ATP and p,A, all of which are competitive inhibitors (Vallejo et al., 1976) and would thus be expected simply to reduce both K, and V,,,, if present as contaminants; (iv) some peculiarity of the assay system, since no inhibition of the Ap,A hydrolase from the green alga Scenedesmus obliquus was observed even up to 400 PM Ap,A using the same assay (McLennan et al., 1994) while an identical inhibition was observed with the human placental Ap,A hydrolase using an entirely different (hyperchromicity) assay system (Hankin S. and McLennan A. G., unpublished observation). Hence, the reason appears to be substrate inhibition. This has not previously been reported for an Ap,A hydrolase and is probably due to substrate crowding, i.e. the binding of two adenosine moieties from two different Ap,A
0'
0
I
I
1
100
200
300
[A~44 (PM) Fig. 3. Dependence of initial reaction velocity on substrate concentration for the leukocyte Ap,A hydrolase. Assays were performed as described in Materials and Methods at different Ap,A concentrations. Similar profiles were obtained with the erythrocyte and platelet enzymes.
Ap.,A hydrolase in human blood cells
205
cytes, they could only account for 10% of the erythrocyte activity. Also, the enzyme from leukocytes and platelets cannot be due to contaminating erythrocytes because of the 15-fold higher specific activities measured for these cells. Thus, all three cell types contain Ap,A hydrolase. The high erythrocyte concentration in whole blood means that red cells are by far the most important source (97%) of the blood cell enzyme. The presence of a significant level of Ap,A hydrolase in enucleated red cells is particularly interesting, since they do not synthesize proteins or Ap,A and presumably have low levels of aminoacyl-tRNA synthetases, the enzymes responsible for Ap,A synthesis (Goerlich et al., 1982). There are several possible reasons for the presence of hydrolase in erythrocytes. First, it may be a relic of the reticulocyte stage with no true function in erythrocytes. Second, red cells may retain an unobserved capacity for a low level of Ap,A synthesis. This may be catalysed by residual aminoacyl-tRNA synthetases, or may be the result of the chemical condensation of two ATP molecules, as previously suggested (McLennan and Zamecnik, 1992). In either case, Ap,A hydrolase activity would be required to prevent the undesirable accumulation of this nucleotide. Finally, it is possible that other dinucleoside polyphosphates, e.g. Ap,A, Ap,A or Gp,G (Coste et al., 1987) are the true substrates for the hydrolase in red cells. The presence of a ‘normal’ concentration of hydrolase activity in platelets contrasts with an earlier report ascribing the metabolic stability of the stored Ap,A pool to the apparent absence of this enzyme (Flodgaard and Klenow, 1982). Hydrolytic activity against Ap,A has been detected in detergent-solubilized DISCUSSION platelet lysates (Liithje and Ogilvie, 1983); howBased on previously published results, the ever, as the sole product was AMP, the activity presence of Ap,A hydrolase activity in all three could have been due to phosphodiesterase acmajor blood cell fractions was unexpected. It tivity. Presumably, compartmentalization of is important to note that the presence of the enzyme in the cytosol and its substrate(s) in the enzyme in erythrocytes cannot be due to the dense granules is sufficient to account for the contamination by the other blood cells since, stability of the stored AplA. Nevertheless, this according to the data in Table 1, this would finding must be considered in future studies of require either a white cell: red cell ratio of 1: 10 the synthesis, storage, turnover and secretion of or a platelet: red cell ratio of 4: 1 in the purified the physiologically active platelet dinucleotides cell fractions, neither of which is remotely poss- Ap,A, Ap,A and Ap,A (Schliiter et ui., 1994). ible, considering the normal ratios in whole blood. Part of the erythrocyte activity may be Acknow~ledgement-The work was supported by grants due to reticulocytes, which normally amount to from the Science and Engineering Research Council and the I % of the red cell population. Even so, assum- Royal Society. S.H. is the holder of an SERC Research ing that they had an activity similar to leukoStudentship.
molecules to the sites normally occupied by both adenosine moieties of a single Ap,A. The erythrocyte and platelet enzymes both had K, values of 0.70 PM, determined from Eadie-Hofstee plots, while that of the leukocyte enzyme was slightly higher at 1.50 PM. The leukocyte pellet prepared by dextran sedimentation consists of about 70% neutrophils, the remainder being mainly lymphocytes, but whether the higher K, is in some way related to the heterogeneity of the leukocyte fraction is unknown at present. The K,,, for the unfractionated blood cell enzyme was also reported to be 0.7 PM (Pintor et al., 1991) while those for the leukaemic cell and placental enzymes were 0.5 PM (Ogilvie and Antl, 1983) and 1OpM (Lazewska et al., 1993) respectively. Interestingly, our own measurements for the placental enzyme gave a K,,, value of 0.8 PM. Hence, the human enzyme may exist in two kinetically distinguishable forms, differing in K,,, by about IO-fold, as previously reported for the enzyme from Artemia embryos (Prescott et al., 1989) and for the enzymes from different rat tissues (Cameselle et al., 1982). Comparison of the specific activities of the different blood cell hydrolases (Table 1) shows that, in terms of protein, both the platelet and leukocyte enzymes have similar values. On the other hand, erythrocytes have a 15-fold lower specific activity. In terms of enzyme activity per cell, leukocytes clearly have the highest activity. The 50-fold lower activity in platelets can be accounted for by their approx. 50-fold lower cell volume; erythrocytes and neutrophils (the major leukocyte) have similar cell volumes.
206
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