Cytosol 5′-nucleotidase from Artemia embryos. Purification and properties

Comp. Biochem. PhysioL Vol. 86B, No. 1, pp. 49-53, 1987 Printed in Great Britain

0305-0491/87 $3.00 + 0.00 Pergamon Journals Ltd

CYTOSOL 5'-NUCLEOTIDASE FROM ARTEMIA EMBRYOS. PURIFICATION A N D PROPERTIES ROSA M. PINTO, Josl~ CANALES, ANGELESFARALDO, ANTONIOSILLEROa n d MARIA A. Gf3NTHER SILLERO Instituto de Investigaciones Biom6dicas del C.S.I.C., Departamento de Bioquimica, Facultad de Medicina de la Universidad de Extremadura, Badajoz, Spain

(Received 18 March 1986) Abstract--1. Cytosol 5'-nucleotidase (EC 3.1.3.5) has been purified near homogeneity from Artemia embryos. 2. The enzyme cleaves preferentially IMP and GMP, and to a lesser extent other 5'-mononucleotides. The substrate-velocity plot was hyperbolic with GMP and sigrnoidal with AMP. 3. The hydrolysis of GMP is stimulated both by ATP and fl,~,-methyleneadenosine 5'-triphosphate with the same activation constant of around 0.6 raM. Both nucleotides decreased So.5 without affecting V. 4. The molecular mass of the native purified enzyme was 165 kDa, and one major band of 42 kDa was detected after sodium dodecyl sulphate polyacrylamide gel electrophoresis.

INTRODUCTION W e have previously c o m m u n i c a t e d the occurrence in Artemia cysts o f a nucleotidase activated by A T P (Pinto et al., 1985). The results presented here show t h a t this activity c o r r e s p o n d s to the cytosol 5'-nucleotidase previously described in rat liver (Fritzson, 1969; V a n den Berghe et al., 1977; Itoh, 1981a), chicken liver (Itoh et al., 1978) a n d rat p o l y m o r p h o n u c l e a r leucocytes ( W o r k u a n d Ncwby, 1983). T h e physiological role o f this nucleotidase is still u n k n o w n . As part o f a general project o n m e t a b olism o f purine nucleotides in Artemia carried o u t in o u r l a b o r a t o r y in the last years ( R e n a r t et al., 1976; Pinto et al., 1983; Canales et al., 1985), it seemed to us o f interest to further characterize this enzyme. In this paper, the purification o f Artemia cytosolic 5'-nucleotidase a n d its m a i n characteristics are reported. MATERIALS AND METHODS

Materials Artemia eggs were from Bio-Marine Research, Hawthorne, CA. Sephacryl S-300 (superfine) and Sepharose 4B were from Pharmacia Fine Chemicals. DEAE-SH-cellulose (0.85 rthq/g) was obtained from Serva and Cibacron Blue F3G-A from Ciba AG, Basel. Nucleotides were purchased from Sigma Chemical Co. and Boehringer Mannheim. [UJ4C]GMP (specific radioactivity 520 mCi/mmol) was obtained from the Radiochemical Centre, Amersham. Proteins used as molecular mass markers in sucrose gradient centrifugation were from Boehringer Mannheim. Plastic-backed (20 x 20cm) sheets of PEI-impregnated cellulose (MNPolygram, CEL 300 PEI/UV2~,), were from MachereyNagel & Company, Diiren, FRG. Sample concentrators Minicon B15 were purchased from Amicon Corp.

Abbreviations used: AMP-PCP, B,?-methyleneadenosine 5"-triphosphate; Pi, inorganic phosphate; TLC, thin layer chromatography. C.B.P. 86/IB--D

49

Methods Preparation of the extracts. All operations were conducted at 0-4°C. Before use, the cysts were resuspended (20 ml/g) in glass distilled water. The cysts that sedimented were collected and washed twice with glass distilled water followed by treatment with 1% cold NaC10 for 10 min. This chemical was removed by successive washes in cold distilled water. A last wash was performed with homogenization buffer, and the cysts were filtered through a piece of.cloth. The wet cysts were disrupted in a cold mortar with 10 vol of 50mM Tris-HCl buffer, pH 7.3, 1 mM EDTA and 10 mM 2-mercaptoethanol (buffer A). The homogenate was filtered through glass wool and centrifuged at 27,000g for 10 min. The resulting supernatant was filtered again through glass wool and centrifuged for 80 min at 105,000 g. 5'-Nucleotidase activity: assay 1. This activity was followed through evaluation of the free inorganic phosphate (Pi) liberated from the corresponding substrate. The reaction mixture contained, in a final volume of 0.2 ml, the following components: 100 mM imidazole-HC1 buffer, pH 7.0, 7.5mM MgC12, 0.1% bovine serum albumin, 0.2M NaC1, substrate as indicated, and enzyme. After incubation at 37°C, the reaction was stopped by addition of 1.45 ml of a solution prepared shortly before use by mixing 6 vol of 3.4mM ammonium molybdate in 0.5M HESO4, 1 vol of 10% (wt/vol) ascorbic acid, 0.47 vol of 8% (wt/vol) sodium dodecyl sulphate and 0.55 vol of water. The samples were incubated for 20 min at 45°C and Aaz0 was determined after cooling at room temperature. The amount of Pi formed was calculated from standard curves. In our conditions, 1 nmol of Pi rendered a net As20 reading of 0.015-0.020, which was stable for at least 2 hr.. 5'Nucleotidase activity: assay 2. The reaction mixture contained in a final volume of 50#1 the following components: 50mM Tris-HC1 buffer, pH 7.5, 6 m M MgC1v 0.24 mM [U-14C]GMP, 0.1 M NaCI, nucleoside 5'-triphosphate when indicated, and enzyme. After incubation at 37°C the reaction was terminated by immersing the reaction vessels in a boiling water bath for 1 min. Subsequently they were cooled in ice and centrifuged for 1.5 min at about 8500# in a Beckman Microfuge B centrifuge. Aliquots of 5#1 were subjected to thin layer chromatography (TLC) on PEI-cellulose, using 0.5 M LiCI as ascendent eluant. After chromatography, the plate was

ROSA M. PINTO et al.

50

monitored on a TLC linear analyzer (Model LB 283 from Labor Berthold), with the entrance window (250 mm x 15 mm) covered with thin foil 1.5 mg/cm2 polyethylene. The count pulses were collected and stored in a multichannel analyzer. Total radioactivity of individual peaks was calculated by integrating selected regions of interest of the radioactive profile displayed at the screen of the display unit. The percentage contribution of each region to the total measured radioactivity was obtained after background subtraction, and was used as a measure of the enzymatic activity. Controls with labelled GMP and guanosine were carried out in parallel. All measurements were performed with a gain of 3. 5'-Nucleotidase activity: assay 3. The components of the reaction mixture were the same as in assay 1, except that NaC1 was lowered to 0.12 M in order to decrease the ionic strength of the final sample to be analyzed by high pressure liquid chromatography. Final concentration of nucleotides in the reaction mixture were as indicated. The reaction was stopped by heating the assay mixtures for 1 min in a boiling water bath, diluted 10-fold with glass distilled water and filtered through a Millipore filter. Aliquots of 5 #1 were injected into a Nova Pack Cl8 column (3.9 x 150 mm) from Waters Assoc. Nucleotides were eluted with a linear gradient of sodium phosphate (5-100 raM), pH 7.0 in I0 mM tetrabutylammonium and 20% methanol. Chromatography was performed in an HP 1090 chromatograph commanded by an HP-85B computer and equipped with an HP 3390 A integrator. A filter of 250 nm was used to detect the peaks. In assays 1,2 and 3, one unit (U) of activity corresponds to the transformation of 1/~mol of substrate per min in the above conditions. Activity on p-nitrophenylphosphate. The reaction mixture (0.2 ml) contained 50 mM Tris-HC1 buffer, pH 8.0, 6 mM MgC12, 3 mM p-nitrophenylphosphate and enzyme. The mixture was incubated at 37°C, the reaction stopped by the addition of 1.3 ml of 0.2 N NaOH, and the absorbance measured at 405 nm. Catalase (EC 1.11.1.6) and alcohol dehydrogenase (EC 1.1.1.1) were assayed as described by Martin and Ames (1961). Protein was determined by the methods of Lowry et al., (1951) or Bradford (1976) with bovine serum albumin as the standard. Sucrose density gradient centrifugation. Molecular mass was determined by sucrose density gradient centrifugation according to the method of Martin and Ames (1961). The linear gradient (10 ml) used was 5-20% (wt/vol) sucrose in 50raM Tris-HC1 buffer, pH 7.3, 1 mM EDTA and 10mM 2-mercaptoethanol. Centrifugation was performed at 4°C for 17hr at 35,000rpm utilizing the SW-41 rotor from Beckman. After centrifugation fractions of nine drops were collected from the bottom. Gel electrophoresis. Discontinuous gel electrophoresis in the presence of sodium dodecyl sulphate was performed according to Laemmli (1970), as described in Pinto et al., (1983). Staining of proteins was performed by the sensitive method of Morrissey (1981). Coupling of Cibacron Blue F3G-A to Sepharose 4B was performed as described in Pinto et al. (1983).

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Fig. 1. Chromatography of 5'-nucleotidase on Sephacryl S-300. Details as described in the text (step 3 of purification). 5"-Nucleotidase (@ @) was measured by assay 1 in the presence of 2.5 mM GMP as substrate. Activity on p-nitrophenylphosphate (O ©) was assayed as described in Methods. 105,000 g supernatant contained in a final volume of 147 ml, 1 unit of enzyme activity. Step 2. Ammonium sulphate fractionation. To the solution from the previous step, 14.4g of solid amm o n i u m sulphate/100 ml (0.25 saturation) were added. After stirring for 30 min, the suspension was centrifuged at 27,000 g for 20 min and the precipitate was discarded. The supernatant was filtered through glass wool and brought to 0.45 saturation with a m m o n i u m sulphate by the addition of 12.5 g/100 ml and treated as above. The precipitate was resuspended in buffer A supplemented with 0.2 M NaC1, up to a volume of 8 ml. Step 3. Chromatography on Sephacryl S-300. 6 ml of the solution from the previous step were applied to a column of Sephacryl S-300 (2 × 100cm) equilibrated with 0.2 M NaC1 in buffer A. The material was eluted with the same buffer at a flow rate of 22 ml/hr. Fractions of 1.9 ml were collected. The major portion 15

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RESULTS

Purification o f the enzyme The experimental conditions were chosen to favor m a x i m u m recovery o f the enzyme, which was achieved after supplementing all buffers with 2-mercaptoethanol at a final concentration of 10 raM. All operations were carried out at 0-4°C. Step 1, 105,000 g supernatant. 15 g of A rtemia cysts were treated as described in Methods. The resulting

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Artemia cytosol 5'-nucleotidase

of the fractions was 2.9 ml. Fractions 56-90 were pooled.

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Fig. 3. Chromatography of 5'-nucleotidase on Cibacron blue F3G-A Sepharose 4B. Details as described in the text (step 5 of purification). Arrow 1, elution with 0.15 M NaCI in buffer A; arrow 2, start of the gradient. Nucleotidase activity was assayed as in Fig. 2.

of the 5'-nucleotidase activity was in fractions 62-88, which were pooled and processed as follows. Activity on p-nitrophenylphosphate was also measured in the eluted fractions. Step 4. Chromatography on DEAE-cellulose. To 47 mg of protein from the previous step, in a volume of 47 ml, enough buffer A was added to bring the NaC1 concentration to 0.1 M. The solution was applied to a DEAE-cellulose column (2 x 6.5 cm) equilibrated with 0.1 M NaCI in buffer A. The column was then washed with the same buffer until the absorbance at 280 nm of the effluent was near zero. Nucleotidase activity was eluted with 140ml of a linear gradient (0.1-O.5 M) NaC1 in buffer A. The volume

the above pool were brought to 233 ml with buffer A and applied to a Cibacron blue-Sepharose column (2 x 5.7 cm) equilibrated in buffer A supplemented with 0.15 M NaC1. The column was then washed with the same buffer (60 ml). Nucleotidase activity was eluted with 140 ml of a linear gradient of NaC1 (0.15-1.0 M) in buffer A, collecting 3 ml fractions at a flow rate of 32 ml/hr. Fractions 66-92 comprising the major portion of the enzymatic activity were pooled. A summary of a typical purification run is presented in Table 1. A purification of 500-fold with a recovery of 38% was obtained with that procedure. The purified enzyme from step 5, free of activity on p-nitrophenylphosphate, was used to explore the following properties of the enzyme. When more active enzyme solutions were required (experiments in Fig. 4, Table 2, and determination of the molecular mass by sucrose density gradient centrifugation, see below), an aliquot of the enzyme from step 5, previously supplemented with 0.01% bovine serum albumin, was concentrated several-fold by means of a Minicon B 15 membrane.

Substrate specificity Results obtained with partially purified enzyme had shown that G M P and X M P were substrates of the nucleotidase (Pinto et al., 1985). With purified enzyme it was also possible to measure the stochiometry of the reaction: 1 mole of guanosine was formed per mole of G M P hydrolyzed. The activity of the enzyme on several compounds with terminal phosphate was also tested (Table 2). I M P and G M P

Table 1. Purification of cytosol 5'-nucleotidase from Artemia embryos Specific Volume Protein Activity* activity Step (ml) (mg) (U) (mU/mg)

Yield %

1. 105,000g supernatant 147 485 1.05 2.2 100 2. (NH4)2 SOa fractionation 8 124 1.07 8.6 102 3. Sephacryl S-300 chromatography 68 68 1.14 16.7 108 4. DEAE-cellulose chromatography 147 9.1 0.81 89.2 77 5. Cibacron blue-Sepharose chromatography 122 0.37 0.40 1100 38 *5'-Nucleotidase activity was determined with 2.5 mM GMP as substrate under the conditions described for assay 1. Table 2. Substrate specificityof Artemia 5'-nucleotidase 10 mM 2.5 mM Activityratio Substrate (mU/ml) (mU/ml) 10 mM/2.5 mM IMP GMP XMP AMP CMP 3' A M P 3' G M P Glucose 6-phosphate L-Glycerol 3-phosphate

12.8 12.3 6.6 6.3 0.4 <0.05 <0.05 <0.05 <0.05

6.4 5.1 1.4 0.6 0.2 -----

2.0 2.4 4.7 10.5 2.0 -----

The activity was determined by assay 1, using enzyme from step 5 concentrated by a Minicon B 15 membrane. Concentrations of nucleotides were as indicated in the table.

52

ROSA M. PINTOet al. 3O

GTP (3-fold). UTP and CTP were not activators of the nucleotidase.

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Molecular mass o f the native enzyme and subunit composition

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The molecular mass of the native enzyme was determined by employing sucrose density gradient centrifugation as described by Martin and Ames (1961). A molecular mass of around 165 kDa was determined for the purified enzyme from step 5 after performing five different runs and using alcohol dehydrogenase or catalase as reference proteins. Polyacrylamide gel electrophoresis of the purified 5-nucleotidase in the presence of sodium dodecyl sulphate revealed a major band and a faint minor protein staining band. Using appropriate standards, the molecular mass estimated for the major band was 42 kDa, suggesting that the structure of the native enzyme is a tetramer as it is the case for the liver enzyme (Itoh, 1982). Other properties o f the enzyme

The nucleotidase has a pH optimum around 7.0 when measured by assay 1 with either G M P or AMP as substrates at a concentration of 2.5 and 20 mM, respectively. The enzyme also requires Mg 2÷ for activity. In the presence of 2.5 mM G M P maximum activity was obtained in the presence of 5 mM MgC12.

DISCUSSION

were the best substrates followed by XMP, A M P and CMP. Nucleoside Y-phosphates (AMP, GMP), glucose 6-phosphate and glycerol 3-phosphate were not hydrolyzed by the enzyme to an appreciable extent. In our experimental conditions, the saturation curve was hyperbolic (nil = 1.0) using G M P as substrate, and an apparent Km value of 2-3 mM was obtained from a Lineweaver-Burk plot. With A M P as substrate, the kinetics were slightly sigmoidal (n u = 1.3-1.5) and a So.5 value around 60 mM was obtained. The ratio of the activity of the enzyme at two different substrate concentrations, 10 and 2.5 mM, can be taken as an approximate index of cooperativity of the nucleotidase. The enzyme shows maximum cooperativity towards A M P (Table 2).

We detected accidentally, the presence of this enzyme while exploring the occurrence of G M P synthetase (EC 6.3.4. I) in A rtemia extracts. The reaction mixture for the assay of the synthetase activity contained, among other reactants, XMP and ATP. In the presence, but not in the absence of ATP, XMP was preferentially cleaved to xanthosine. The pursuit of this activity led us to the finding of a nucleotidase in Artemia cysts extracts. In the course of the purification of this activity we became aware that it could correspond to the cytosolic 5'-nucleotidase previously described in other sources. The hydrolysis of nucleoside Y-phosphate is carried out by two different enzymes, one bound to membranes (Drummond and Yamamoto, 1971; Bailyes et al., 1982) and the other located in the cytosolic cell fraction. Both Influence o f A T P on enzyme activity have the same entry in the Enzyme Commission (EC The hydrolysis of G M P by an enzyme preparation 3.1.3.5). The cytosolic enzyme was firstly described in from step 2 was stimulated by ATP with an activation chicken (Itoh et al., 1967; Naito and Tsushima, 1976) constant value of 2.5 mM (Pinto et al., 1985). With and rat (Fritzson, 1969) livers. In those papers the the purified enzyme this value decreased to 0.6 mM preferential cleavage of IMP and G M P by the en[Fig. 4(a)]. ATP activates the hydrolysis of G M P by zyme was described, at the time that its specificity decreasing the apparent Km value (0.12 mM) without towards nucleoside 5'-monophosphates was clearly affecting the maximum velocity (result not shown). stated. Nucleoside 2' or 3'-phosphates or ribose We also explored whether fl,y-methyleneadenosine 5'-phosphate were not hydrolyzed by the enzyme. In 5'-triphosphate (AMP-PCP) also stimulated the 1977, Van den Berghe et al., reported the activation cleavage of GMP. An activation constant of 0.7 mM of partially purified nucleotidase from rat liver by was obtained in this case thus showing that the ATP. Later the same effect was confirmed with more hydrolysis of ATP to A D P was not requisite for the purified enzyme from both chicken (Itoh et al., 1978) activation of the enzyme by the nucleotide [Fig. 4(b)]. and rat liver (Itoh, 1981a,b). The kinetics of the Other nucleoside 5'-triphosphates (all at 3 mM final enzyme with A M P as substrate is sigrnoidal. ATP concentration) were also tested as activators using the decreases the sigmoidicity of the enzyme (Van den assay 2 and G M P as substrate: higher activation was Berghe, 1977; Itoh et al., 1978). The molecular mass obtained by ATP (8-fold) and to a lesser extent by described for the rat liver enzyme is around 200 kDa

Artemia cytosol 5'-nucleotidase

and it is composed of four identical subunits (Itoh, 1982). The properties of the purified cytosol 5'-nucleotidase from Artemia are rather similar to those mentioned above. In addition we present here conclusive evidence that the hydrolysis of ATP to ADP is not a requisite for the activation of the nucleotidase, as the ATP analog, fl, ~-methyleneadenosine 5'-triphosphate, has the same activation constant as ATP (around 0.7 mM). We have observed that the addition of sulphydryl groups in the extraction buffers is critical for the purification of the enzyme. We spent time pursuing the possible existence of different molecular forms, due to the elution profiles obtained after chromatography on Sephacryl S-300, in the absence of 2-mercaptoethanol. The addition of that component to the elution buffer changed the chromatographic profile of the enzyme rendering a more definite peak of nucleotidase activity (Fig. 1). Concerning the physiological role of this enzyme, several hypothesis have been forwarded. Bontemps et al. (1983) favor the view that the cytosol 5'-nucleotidase and AMP deaminase contribute to form a futile cycle between AMP and adenosine that could help to adapt the rate of adenine nucleotide catabolism to an increased rate of synthesis. Itoh (1981 b) points to the possibility that the enzyme both eliminates excess of IMP generated by AMP deaminase and protects the cell against sudden utilization of ATP by removal of AMP. In this sense, Worku and Newby (1983) point out that cytosol 5'-nucleotidase is responsible for adenosine production in rat polymorphonuclear leucocytes. Finally Tjernshaugen and Fritzson (1984), from experiments with regenerating rat liver, conclude that the main metabolic action of the enzyme is the production of inosine to be exported from the liver to other tissues. We are at present investigating the role of this nucleotidase in Artemia. T h e enzyme does not seem to decrease significantly during development from encysted gastrula to free swimming larva, although the induction of an alkaline phosphatase activity after hatching interferes with a precise determination of the cytosol 5'-nucleotidase.

53

nosine in isolated hepatocytes. Proc. natl Acad. Sci. USA 80, 2829-2833.

Bradford M. M. (1976) A rapid and sensitivemethod for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72, 248-254. Canales J., Fernfindez A., Faraldo A., Pinto R. M., Sillero A. and Sillero M. A. G. (1985) IMP dehydrogenase from Artemia embryos: Molecular forms, purification and properties. Comp. Biochem. Physiol. 81B, 837-844. Drummond G. I. and Yamamoto M. (1971) Nucleotide phosphomonoesterases. In The Enzymes (Edited by Boyer P. D.), 3rd edn, Vol. 4, pp. 337-354. Academic Press, New York. Fritzson P. (1969) Nucleotidase activities in the soluble fraction of rat liver homogenate. Partial purification and properties of a 5'-nucleotidase with pH optimum 6.3. Biochim. biophys. Acta 178, 534-541. Itoh R. (1981a) Purification and some properties of cytosol 5'-nucleotidase from rat liver. Biochem. biophys. Acta 657, 402-410. Itoh R. (1981b) Regulation of cytosol 5'-nucleotidase by adenylate energy charge. Biochim. biophys. Acta 659, 31-37. Itoh R. (1982) Studies on some molecular properties of cytosol 5-nucleotidase from rat liver. Biochim. biophys. Aeta 716, 110-113. Itoh R., Mitsui A. and Tsushima K. (1967) 5'-Nucleotidase of chicken liver. Biochim. biophys. Acta 146, 151-159. Itoh R., Usami C., Nishino T. and Tsushima K. (1978) Kinetic properties ofcytosol 5'-nucleotidase from chicken liver. Biochim. biophys. Acta 526, 154-162. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680485. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Martin R. G. and Ames B. N. (1961) A method for determining the sedimentation behavior of enzymes: application to protein mixtures. J. biol. Chem. 236, 137~1379. Naito Y. and Tsushima K. (1976) Cytosol 5'-nucleotidase from chicken liver. Purification and some properties. Biochim. biophys. Acta 438, 159-168. Pinto R. M., Faraldo A., Fern~.ndezA., Canales J., Sillero A. and Sillero M. A. G. (1983) Adenylosuccinate lyase from Artemia embryos. Purification and properties. J. biol. Chem. 258, 12513-12519. Pinto R. M., Faraldo A., Canales J., Fernfindez A., Sillero A. and Sillero M. A. G. (1985) Occurrence of a nucleotidase activated by ATP in Artemiacysts extracts. 2nd Int. Symp. on the brine shrimp Artemia. Antwerpen, Belgium. Acknowledgement--This investigation was supported by the Renart M. F., Renart J., Sillero M. A. G. and Sillero A. Comisirn Asesora de Investigacibn Cientifica y Trcnica (1976) Guanosine monophosphate reductase from Arte(Grant No. 993/81). mia salina: Inhibition by xanthosine monophosphate and activation by diguanosine tetraphosphate. Biochemistry 15, 4962-4966. Tjernshaugen H. and Fritzson P. (1984) Activity of cytosolic REFERENCES 5'-nucleotidase in regenerating rat liver after partial hepatectomy. Int. J. Biochem. 16, 607413. Bailyes E. M., Newby A. C., Siddle K. and Luzio J. P. (1982) Solubilization and purification of rat liver Van den Berghe G., Van Pottelsberghe C. and Hers H.-G. (1977) A kinetic study of the soluble 5'-nucleotidase of rat 5'-nu¢leotidase by use of a zwitterionic detergent and a liver. Biochem. J. 162, 611416. monoclonal-antibody immunoadsorbent. Biochem. J. Worku Y. and Newby A. C. (1983) The mechanism of 203, 245-251. adenosine production in rat polymorphonuclear leucoBontemps F., Van den Berghe G. and Hers H.-G. (1983) cytes. Biochem. J. 214, 325-330. Evidence for a substrate cycle between AMP and ade-