Molecular and Cellular Biochemistry 117: 23-33, 1992. © 1992 Kluwer Academic Publishers. Printed in the Netherlands.

Isolation and characterization of the ecto-5'-nucleotidase from a rat glioblastoma cell line Javier Turnay, Nieves Olmo, J. Mafia Navarro, Jos6 G. Gavilanes and M. Antonia Lizarbe Departamento de Bioqu[mica y Biologta Molecular. Facultad de Ciencias Qu[micas. Universidad Complutense. 28040-Madrid. Spain Received 13 January 1992; accepted 6 June 1992

Summary 5'-Nucleotidase has been purified from rat glioblastoma cells (Rugli cells). The enzyme has been solubilized from plasma membranes by using Triton X-100 and CHAPS. Two affinity chromatographies on concanavalin A and 5'-AMP-Sepharose render the purified enzyme with a high specific activity (76.36/zmol AMP.min-l.mgl). The purified enzyme gives a single polypepfide band on SDS-PAGE with an apparent molecular mass of 74 kDa. Active forms with an apparent molecular mass of 135 kDa and 268 kDa are observed when the purified enzyme is analyzed by gel filtration in the presence of either 0.6% sodium deoxycholate or 0.1% Triton X-100, respectively. The purified 5'-nucleotidase presents optimum activity at pH 7.8-8.1 either in the presence or in the absence of Mg2+. A linear Arrhenius plot is observed in the 25--46° C temperature range and an activation energy of 33.7 KJ/mol is calculated. The enzyme is inhibited by EDTA; the activity is partially restored by different divalent cations as Zn 2+, Mn 2÷, and Co 2÷. The hydrolysis of nucleosides 5'-monophosphate shows Michaelis kinetic. The enzyme is inhibited by nucleosides di- and triphosphate. 5'-Nucleotidase is a glycoprotein, being its activity inhibited at different extent by various lectins. (Mol Cell Biochem 117: 23--33, 1992).

Key words." 5'-Nucleotidase, Rugli cells

Introduction 5'-Nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) catalyzes the hydrolysis of nucleosides 5'monophosphate to nucleosides and inorganic phosphate. This enzyme is a membrane-bound glycoprotein found in a large variety of eukaryotic cells as well as in microorganisms, and has been purified from different tissues [1-8]. It is primarily located in the plasma mem-

brane being frequently used as a marker enzyme of the cell surface (ectoenzyme) [9]. Nevertheless, fractionation studies have revealed the presence of this enzyme activity in lysosomes, in the Golgi apparatus, in endocytotic recycling pool, and in cytosolic cell fractions [2, 10--13]. The topography of the membrane bound enzyme remains to be determined. This ectoenzyme has

Address for offprints: M.A. Lizarbe, Departamento de Bioqufmica y Biolog~a Molecular, Facultad de Ciencias Qutmicas, Universidad Cornplutense, 28040-Madrid, Spain

24 been described as either a transmembrane protein [1415] or a short-stalked integral membrane protein without cytoplasmic domain [1]. More recently, the ectoenzymes from several cell types and tissues have been reported to be anchored to the plasma membrane via a phosphatidylinositol-glycan [16-19]. The physiological function of the ectoenzyme is not completely understood [20], but it may be involved in the cellular uptake of nucleoside derivatives from nucleosides 5'-monophosphate [2, 21]. Studies about this enzyme have been stimulated by the growing evidence on the important role that adenosine plays in intercellular communication in several tissues, including brain [22, 23]. In the central nervous system, 5'-nucleotidase is predominantly localised at the glia cells, particularly in astroglial plasma membranes [24]. Moreover, 5'nucleotidase activity appears increased in malignant human anaplastic astrocytomas and glioblastomas in comparison to benign astrocytomas or normal tissue [25]. We have previously studied the 5'-nucleotidase activity from rat glioblastoma (Rugli) cultured cells [3]. We have recently reported the effect of extracellular matrix proteins on the 5'-nucleotidase activity of intact Rugli cells [26]. In the present paper we describe the purification and biochemical characterization of this enzyme from such an established cell line.

Experimentalprocedures

(NO3) 2 and 2 mM 5'-AMP in the Tris-maleate buffer. Controls included both cells incubated with buffer and cells incubated with lead nitrate, under identical conditions. After incubation, the cells were washed with Tris-maleate buffer, post-fixed with 1% OsO4 for 30 min, dehydrated in acetone, and embedded in an EponAraldite mixture. Thin sections were stained with 3% uranyl acetate in 50% ethanol for 15 min and with 80 mM lead citrate for 7 min. The electron micrographs were taken with a JEOL 2000 FX electron microscope at 80-100 kV.

Enzyme assays Enzymatic activity was determined by measuring the hydrolysis rate for different nucleoside 5'-monophosphates. The routinary assay was performed by using radioactive 5'-AMP as substrate as previously described [3]. One unit equals the hydrolysis of 1 ~mol of nucleoside 5'-monophosphate/min. Kinetic studies for different nucleoside monophosphate substrates were performed by spectrophotometrical measurement of the Pi liberated after the addition of the corresponding nucleotide [27]. Inhibition kinetics were measured at different inhibitor concentrations: ADP and ATP, at 10, 25, 75, and 150/zM; Con A and L. culinaris lectin, at 25, and 50 nM.

Mater&&

Enzyme purification

Concanavalin A-Sepharose, AMP-Sepharose, and Sephacryl S-300 were obtained from Pharmacia. Nucleotides, lectins, a-methyl mannopyranoside, deoxycholate, proteins for gel chromatography calibration, and the calibration kits for SDS-polyacrylamide-gel electrophoresis were purchased from Sigma. Triton X-100 and 3-[(3-cholamidopropyl)dimethylammonio]-l-propane sulfate (CHAPS) were obtained from Boehringer Mannheim; [2-3H]AMP (20 Ci/mmol) was from Amersham. All other reagents were of analytical grade.

5'-Nucleotidase was purified from cultures of rat glioblastoma cells (Rugli cells) by a modification of the methods of Dieckhoff et al. [7] and Grondal & Zimmermann [8]. All operations were carried out in a cold room at 0-4 ° C. Cells were cultured in DMEM medium supplemented with 10% fetal bovine serum, glutamine (300/zg/ml), penicillin (50IU/ml), and streptomycin (50/zg/ml). Cells were harvested from 20-30 culture flasks (150 cm2) and were collected by centrifugation after scraping with a rubber policeman. The cell pellet was then homogenized in 200 ml of buffer A [40 mM Tris, pH 7.4, containing 100 mM NaCl, 1 mM CaC12, 1 mM MgC12, 1 mM MnC12, 1 mM NAN3, and 1 mM phenylmethylsulfonyl fluoride (PMSF)] in a Dounce homogenizer. The crude homogenate was immediately centrifuged at 200,000 g (rav= 81.2 ram) for 30 rain. The pellet was resuspended in 100 ml of buffer A, containing 1% Triton X-100, for

Cytochemical localization of 5'-nucleotidase Cell monolayers were fixed in 3% glutaraldehyde in 50mM Tris-maleate buffer, pH 7.0, for 10 min at 4°C. After exhaustive washing with cold buffer, cells were incubated for l h at 37°C in the presence of 2 mM Pb

25

Fig. 1. Cytochemicallocalizationof 5'-nucleotidaseactivityin Rugli cells. Cellswere incubated in the presence of lead nitrate without (A) and with 5'-AMP (B, C, D). Electron micrographs show a heavy precipitate of lead phosphate only in the plasma membranes of the cells incubated in the presence of 5'-AMP. Magnifications: (A, B)× 4,400, bar = 1.7/zm; (C) × 3,000, bar = 2.5 izm; (D) × 12,000, bar-~ 0.61zm. 30 min. After centrifugation at 200,000 g for 30 min, the pellet was resuspended in 50 ml of buffer A, containing 5% C H A P S , and it was again homogenized as above described. The homogenate was overnight maintained under continuous stirring and further clarified by centrifugation at 200,000 g for 30 min. The supernatant was applied to a Con A-Sepharose column (7 ml bed volume) equilibrated in buffer B (40mM Tris, p H 7.4, containing 0.1% Triton X-100, 100raM NaC1, l m M CaCI2, 1 m M MgCI:, 1 m M MnC12, and 0.5 mM NAN3). The column was washed with 50 ml of buffer B and the enzyme activity was eluted with 50 ml of buffer B, containing 0.4 M a-methyl mannopyranoside. AMPase activity containing fractions were pooled and stored over-

night at - 2 0 ° C. After thawing and dialysis against buffer B, the pool was applied to an AMP-Sepharose column (3 ml bed volume) equilibrated in buffer B. The column was washed with 30 ml of the same buffer and the enzyme activity was eluted with 50 ml of buffer B, containing 10raM AMP. The fractions containing 5'nucleotidase activity were pooled and dialysed against buffer B. The enzyme was stored at - 20 ° C.

Other analytical procedures Protein concentration was determined according to the method of Bradford [28] by using bovine serum albumin

26 a/. [30]. Molecular weight determination of active 5'nucleotidase was carried out on a column (1.6 × 70.0cm) of Sephacryl S-300 equilibrated in either 40 mM Tris, pH 7.4, containing 0.1% Triton X-100, and 100raM NaC1, or 40mM Tris, pH 8.3, containing 0.6% deoxycholate, and 100 mM NaC1. The elution profile of the ecto-5'-nucleotidase was obtained by routinary enzyme activity assay above described. Independent chromatographies for tyroglobulin, catalase, alkaline phosphatase, bovine serum albumin, ovalbumin, pepsin, and myoglobin were used for column calibration.

Results Ioslation of 5'-nucleotidase We have previously reported the hydrolysis of extracellular 5'-AMP by intact glioblastoma (Rugh) cells in monolayer culture (5.06 _+ 1.05nmol AMP/celFmin) [3]. However, mechanically disrupted Rugli cells hydrolyze 6.40 _+ 1.13 nmol AMP/ceU/min. These values suggest an outer surface cell membrane localiTation for 79 % of the total 5'-nucleotidase cellular activity. Moreover, the cytochemical and electron microscopic studies (Fig. 1) show the predominant ectoenzyme character of the 5'-nucleotidase from Rugli cells. These facts have determined the enzyme purification procedure employed, which is summarized in Table 1. The process includes three extraction steps and two affinity chromatographies on Con A- and AMP-Sepharose. The critical step is the treatment with 1% Triton X-100 which removes 70% of the contaminant proteins. Increased detergent concentrations produce 30-40% loss of the enzyme activity whereas lower Triton X-100 concentrations do not result in significant 5'-nucleotidase purification. It is also essential the freezing of the

Fig. 2. Analysis of purified Rugli 5'-nucleotidase by silver-staining PAGE-SDS. Samples were reduced with 5% 13-mercaptoethanol and the electrophoresis was performed on 10% polyacrylamide gels. (A) High molecular weight standard proteins (molecular weight units in kDa). (B) Purified 5'-nucleotidase.

dissolved in buffer A or buffer B as standard. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl suffate (SDS-PAGE) was carried out according to Laemrnli [29] on 10% (w/v) polyaerylamide gels. Silver staining was performed as described by Oaldey et Table 1. Summary of the purification of 5'-nucleotidase from Rugli cells. Fraction

Total protein (mg)

Total activity (U)

Specific activity (U/rag) protein)

Yield (%)

Purification (-fold)

Cell homogenate 200,000 g pellet 1% Triton X-100 extraction (P) 5% CHAPS extraction (S) Con A-Sepharose chromatography AMP-Sepharose chromatography

487 340 90 53 0.560 0.022

29.37 23.83 20.22 17.66 6.35 1.68

0.06 0.07 0.23 0.33 11.34 76.36

100 81 69 60 22 6

1.0 1.2 3.8 5.6 189.0 1272.7

AMPase activity was determined by using the radioisotope assay (3). One unit (U) equals the hydrolysis of 1 ~mol of AMP/min at 37° C. (P) pellet; (S) supernatant.

27

•~

120

B

A

e--,\

,/' "\ I'\

,o

40

1.6 I

3.2

3.3 /

1/T .10 a (K-l)

30

40 50 60 Temperature

(°C)

8

10 pH

Fig 3. Effect of temperature and pH on the 5'-nucleotidase activity. (A) Purified enzyme was incubated for 15 rain in the presence of 100/~M AMP at the indicated temperature. The pH was maintained at 7.4 for all the temperatures tested. Results are expressed as U/rag of protein (I unit equals the hydrolysis of i/xmol of AMP/rain). Data are the average of four different experimental determinations with duplicate samples. Inset: Arrhenius plot for the 5'-nucleotidase activity. (B) Purified enzyme was incubated for 15 rain in the presence of 100/zM AMP at 3T C in 50 mM Tris (pH 6.5 to 9.0; - 0 - - 0 - 0 ) , MES/Tris (pH 5.0 to 7.5; - J - l g l ) , or glycine eJNaOH (8.0 to 10.0; - A - A - & ) buffers, contzining 0.12 M NaC1. Identical results were obtained in the presence of 2 mM Mg z+. Results are expressed as U/rag protein. Data are the average of four different experimental determinations with duplicate samples.

enzyme fractions eluted from the Con A-Sepharose chromatography, prior to its application on the AMPSepharose cohimn. The omission of this step results in a tight-binding of the protein to the AMP-Sepharose and a very poor recovery. The 5'-nucleotidase activity elutes in a broad peak from both chromatographies. Only the central parts of such peaks have been considered, which explains the apparent low recovery after these two steps. A 1300-fold purified enzyme is obtained throughout this isolation procedure with a very high specific activity (76.36 U/rag of protein). Assays performed in the presence of 25/zM adenosine 5'-(cc, [3-methylene) diphosphate (AOPCP) show total inhibition of the enzyme activity. This indicates that the measured enzyme activity corresponds to 5'-nucleotidase activity [3, 9]. The purified enzyme gives a single band when analyzed by electrophoresis in SDS-polyacrylamide gels and silver staining (Fig. 2). Regarding the enzyme stability, the purified 5'-nucleotidase can be stored at 4°C for at least two weeks in 40raM Tris buffer, pH 7.4, containing 0.1M NaC1 and 0.1% Triton X-100, without significant loss of activity (less than 10%). At - 20° C, the enzyme can be stored for months.

Molecular weight of the purified 5'-nucleotidase Analysis of the purified 5'-nucleotidase ectoenzyme from Rugli cells by SDS-PAGE gives a single band of 74 kDa apparent molecular weight (Fig. 2). Determination of the molecular weight of the active enzyme by gel filtration yields different molecular weights. Thus, gel filtration on Sephacryl S-300 in the presence of 0.6% sodium deoxycholate renders 135 kDa, whereas the value obtained in the presence of 0.1% Triton X-100 is 268 kDa.

Effects of temperature and pH on the enzyme activity The influence of the temperature on the AMPase activity of the purified 5' nucleotidase is shown in Fig. 3A. The enzyme activity is stable up to about 46 ° C. An Arrhenius plot gives a straight line between 25 and 46°C (Fig. 3A, inset) showing an activation energy of 33.7 KJ/mol for the catalyzed reaction. 5' Nucleotidase isolated from Rugli cells shows optimum activity in the 7.8--8.1 pH range, as observed in Fig. 3B.

28

100 so

g

60

~

M g 2*

.K

B

\

C a 2*

~

-Q

Mn 2 .

20

60 10(] n2*] (~uM)

g. ~

C O 2+

20

Ni 2 . Z n 2+

5

lO

2o

5

[ X2+] (mM)

lb

2o Ix2+] (mM)

Fig. 4. Effect of divalent metal cations on the 5'-nucleotidase activity. Purified enzyme was assayed after exhaustive dialysis against 40 mM Tris, pH 7.4, containing 0.1% Triton X-100 and 100mM NaCI. (A) Dialyzed enzyme was incubated at 370C for 15 min with 100 p.M AMP in 50mM Tris buffer, pH 7.4, containing 0.12 M NaC1 and different concentrations of divalent metal cations. (B) Dialyzed enzyme was first incubated at 37° C for 15 rain with 100/zM EDTA in 50 mM Tris buffer, pH 7.4, containing 0.12 M NaCI. Afterwards, different divalent metal cation concentrations were added. After 15 min of incubation, the reaction was started by addition of 100/zM AMP. Results are expressed as percentage of the control activity in the absence of both EDTA and divalent metal cation. Inset: AMPase activity recovery after EDTA inactivation by Zn 2÷ addition. (Q) Mg 2÷, ( 0 ) Ca2+; (~7) Mn2+; ( V ) C02+; ( ~ ) Ni:+; (1B) Zn2+.

Effects of divalent cations Fig. 4A shows the effect of increasing concentrations of different divalent cations on the AMPase activity of the purified enzyme from Rugli cells. These divalent cations are inhibitors of the AMPase activity with the exception of Mg 2÷ which has no detectable effect, and Ca 2+ which exerts only a very slight inhibition. Z n 2+ , Ni 2+, Co 2+, and Mn 2+ are strong inhibitors of the 5'nucleotidase activity in the miilimolar range. The most potent inhibitor is Zn 2+ which inhibits 95% of the AMPase activity at 0.1 mM concentration. The enzymatic activity of purified 5'-nucleotidase is reduced up to about 25% in the presence of 100/~M EDTA, and is completely inhibited at 1raM EDTA (Table 2). This result suggests that the ecto-5'-nucleotidase from Rugli cells is a metalloenzyme. AMPase inactivation by 100tzM EDTA is largely reverted (80-90%) by exhaustive dialysis against free EDTA buffer; this result suggests that the endogenous cation is not removed at this EDTA concentration. However, higher EDTA concentrations (e.g. 1 mM) completely inhibit the enzyme activity which is not recovered after either dialysis or addition of exogenous cations. This may be explained based on an enzyme

denaturation after the complete removal of the endogenous cation. The AMPase activity is partially restored by the addition of divalent metal ions after inactivation by 100/.tM EDTA. The highest effect is observed for Z n 2+ ions; 10 and 20/~M Zn 2+ restore the AMPase activity at about 50% and 90%, respectively (Fig. 4B; inset). But Zn 2+ reactivation disappears at higher concentrations. This biphasic behaviour is also observed at higher concentraTable 2. Inhibition of purified Rugli 5'-nucleotidase activity by EDTA. [EDTA] (raM)

AMPase activity (%)

0 0.05 0.10 0.25 O.50 0.75 1.00

100 37 ± 2.1 24 5:2.5 18 ± 0.5 12 ± 0.4 7 ± 0.3 3 ± 0.4

Purified enzyme was incubated at 37° C for 15 min with the indicated EDTA concentrations in 50mM Tris buffer, pH 7.4, containing 0.12M NaC1. The reaction was started by the addition of 100/.tM AMP. Results are expressed as percentage of the control activity without EDTA. Data are the average of three different experimental determinations with duplicate samples.

.0ot

29

,_,1oo[ A I

I

_

~

A

T

P

3 ,oi f/ 20 I

I

100

200

300

[S] (juM)

1

2

3

4

5

Vq/[S] (U.mg protein'l.pM -1)

Fig. 5. AMPase activity inhibition kinetics of 5'-nucleotidase purified from Rugli cells by ADP and ATP. (A) Plots of v o vs. AMP concentration. (B) Eadie-Hofstee plots. Without inhibitors ( ~ ) , pins 75/zM ATP (A), and plus 25/xM ADP (A). Data are the average of three different experimental determinations with duplicate samples.

tions for other divalent metal ions: Co 2+ > Mn 2+ > Ni 2+ > Ca 2+ (Fig. 4B). M g 2+ does not present such a biphasic response and it slowly restores the AMPase activity after EDTA inactivation; about 75% of the original activity is recovered at 15 mM Mg 2+. Complete reactivation is not achieved in any case. This could result from the above suggested denaturation of the enzyme due to removal of the endogenous metal ion by the EDTA treatment.

Substrate specificity Hydrolysis of 5'-AMP by the ecto-5'-nucleotidase exhibits a Michaelis-Menten kinetic (KM = 19.6+ Table 3. Summary of kinetic parameters of 5'-nucleotidase purified from Rugli cells. SUBSTRATE

V ~ , (U/mg)

KM (/zM)

V~/KM

Y-AMP 5'-GMP 5'-UMP 5'-IMP 5'-CMP

98.38 + 64.67599.77 + 46.09 + 99.21+

19.64 + 38.15+ 30.51 513.53 + 69.86+

5.01 1.70 3.27 3.41 1.42

1.50 1.68 1.98 1.02 2.15

1.09 3.11 2.21 1.43 4.09

The kinetic parameters have been obtained from the computer analyses of the plots of Vo vs. substrate concentration (_+ SD). Each plot was obtained from three experimental determinations with duplicate samples for each substrate concentration (at least 12 concentration ranging from 4 to 300 ~M).

1.1/xM; Vm,~= 98.4 + 1.5 U/mg of protein) (Table 3). This KMvalue of the purified enzyme is twice lower than the one observed for the 5'-nucleotidase in intact cells (KM = 36.2+ 2.7/zM). The activity of 5'-nucleotidase towards a number of nucleotides monophosphate has been determined by measuring P~ release. The results are also shown in Table 3. The enzyme exhibits higher affinity for IMP and AMP than for UMP, GMP, and C MP. However, the hydrolysis rate of AMP is slightly lower than that observed for other substrates. Substrate specificity measurement, expressed by V ~ j K M(Table 3), reveals highest specificity for AMP; for IMP and LIMP the values obtained were 1.5-times lower and 3-times lower for GMP and CMP. These results may be interpreted in terms of AMP as physiological substrate for the enzyme. No activity is observed toward 2'-AMP, 3'-AMP or p-nitro-phenylphosphate.

Inhibition by nucleotides 5'-Nucleotidase is strongly inhibited by micromolar concentrations of nucleosides di- and tri-phosphate. In this context, the inhibition of 5'-nucleotidase by ATP and ADP has been studied by using AMP as substrate (Fig. 5). ATP shows a partial competitive inhibition type. The Ki obtained at 75/zM ATP is 76.4 _+ 1.5/xM. In contrast with ATP, ADP exhibits complete compet-

30

Lentil Con A

7

I

100

I

200

i"! ".,. . . .

r

1 2 3 4 5 Vo/IS] (Uxmg protein'l~ jui -1 )

300

Is]

Fig. 6. AMPase activity inhibition kinetics of 5'-nucleotidase purified from Rugli cells by lectins. (A) Plots of Vo vs. AMP concentration. (B) Eadie-Hofstee plots. Without inhibitors i l l ) , pins 50 nM Lens cu//nar/s agglutinin ( • ) , and plus 50 nM Con A (A). Data are the mean of three different experimental determinations with duplicate samples.

itive inhibition of 5'-nucleotidase. The Ki for ADP is 7.4+ 0.7 tzM.

Effect of lectins The effect of different lecfins on the 5'-nucleotidase activity was studied for both intact cells and the purified enzyme (Table 4). Phytolacca americana agglutinin

does not exert any effect in the activity of 5'-nucleotidase from Rugli cells. Con A and Lens culinaris agglutinin exhibit an inhibitory effect on both AMPase activities. Wheat germ agglutinin strongly inhibits AMPase activity from intact cells, but has almost no effect on the purified ectoenzyme. The kinetic studies show that the inhibition of the purified enzyme observed with Con A and Lens culinar/s is non-competitive (Fig. 6). These results confirm the

Tab/e 4. Rugli AMPase activity inhibition on intact cells and purified enzyme by different lectins. Lectin

Residue specificity

Concanavalm A

ct-D-Glucosyl and a-D-marmosyl

Lens cu//nar/s

a-D-Glucosyl and a-D-mannosyl

Wheat germ

N-Acetyl-~D-glucosaminyl and sialic acid

Phytolacca americana

N-Acetyl-[5-D-glycosaminyl oligomers

[Lectin] (raM)

50 200 1000 50 200 1000

50 200 1000 50 200 1000

Activity (%) Intact cells

Purified enzyme

54.1 + 13.2 + 3.4 + 87.3 + 54.8+ 31.5 + 91.7 + 72.0 + 31.3 + 101.6 + 102.5+ 100,8 +

50.8 + 39.9 + 36.0 + 61.8 + 51.0+ 44.3 + 99.8 + 98.7 + 96.5 + 100.1 + 99.8+ 100.3 +

2.4 1.1 0.8 5.3 2.2 0.8 4.7 3.1 2.7 1.5 2.4 2.3

4.1 1.2 2.0 1.7 2.1 0.9 5.3 2.7 2.5 6.2 4.1 2.5

The effect of lectins on the 5'-Nucleotidase of intact Rugli cells was performed in suspension (2.5 × 1@ cells) as described previously (3). Activity is expressed as percentage of control values in the absence of lectins (100% activity on intact cells = 2.13 nmol AMP hydrolyzed/celFmin; 100% activity for the purified enzyme = 76.4 U/rag protein), and are the mean of three different experimental determinations with duplicate samples (+ SD).

31 higher affinity of the purified enzyme for Con A, showhag a Ki of 34.9 riM, similar to those reported for other nucleotidases.

Discussion 5'-Nucleotidase (ectoenzyme) from glioblastoma cultured cells (Rugli) has been purified to homogeneity based on the results obtained by SDS-PAGE after silver-staining. The single polypeptide band obtained by this analysis has an apparent molecular weight of 74kDa. This is in agreement with the apparent Mr reported for the 5'-nucleotidase from several other different tissue sources, which is generally around 70,000 by SDS-PAGE [5, 7, 31, 32]. Active forms of 135 kDa and 268 kDa have been observed by gel filtration in the presence of detergents. Gel filtration experiments in the presence of Triton X-100 and deoxycholate have been performed in order to analyze the molecular weight of active forms of the enzyme. In the presence of deoxycholate the activity of 5'-nucleotidase exhibits an apparent molecular weight of 135 kDa whereas the value obtained in the presence of Triton X-100 is 268 kDa. These values actually correspond to the apparent molecular weight of the enzyme-detergent micelle complexes, since the detergent concentrations employed are above the corresponding critical micellar concentrations. Nevertheless, the values obtained and the reported average molecular weights for the micelles of the detergents employed [33, 34] suggest the existence of at least dimeric active forms of the 5'-nucleotidase in the enzyme-detergent complexes. Different molecular weights have also been described for 5'-nucleotidase depending on the detergent used for the enzyme solubilization [7, 8]. But, no active forms have been detected at about 70 kDa molecular weight, which has been interpreted in terms of a dimer structure for the native 5'-nucleotidase [5--8, 32, 35]. The purified enzyme exhibits high stability at neutral pH in the presence of 0.1% Triton X-100. This kind of 5'-nucleotidase preparation has been used for the enzyme characterization. The obtained Arrhenius plot is linear in the 25-46 ° C temperature range. Linear Arrhenius plots have been described for purified 5'-nucleotidase from different sources [4, 6], although a biphasic Arrhenius plot has also been reported for this enzyme [36]. The membrane-bound enzyme may show both linear [4] and biphasic plots [6]. All of this suggests an important role of the lipid or detergent environment on

the thermodynamical behaviour of the AMPase activity. The optimum pH of the purified 5'-nucleotidase from the Rugli cells is around 8.0. This has been analyzed in the 5,0-10.0 pH range by using different buffers. It has been described that certain buffers, such as glycine/ NaOH or carbonate/bicarbonate, may modify the AMPase activity [5]. However, we have not found such an effect when using the glycine/NaOH buffer. The pH/activity curve is not modified in the presence of 2 mM MgCI2 (Fig. 3B). A second AMPase activity peak at high pH values in the presence of Mg 2+ has been previously reported [5, 9]. This Mg 2+ effect, which is not observed for the purified Rugh 5'-nucleotidase, may be due to alkaline phosphatase contamination, since alkaline phosphatase activity is strongly dependent on divalent metal ions [4]. The absence of phosphatases on the purified Rugli cells 5'-nucleotidase is corroborated by the absence of any activity against 2'AMP, 3'-AMP, and p-nitrophenylphosphate. The purified enzyme from Rugli cells is suggested to be a metalloenzyme since it is inhibited by EDTA treatment. Moreover, the enzyme activity is not completely restored after addition of Zn z+, Co >, Mn 2+, Ni >, Ca 2+, or Mg >. Concerning the effect of divalent cations, it has been reported that the enzymes from bull seminal plasma, chicken gizzard, and snake venom contains Zn 2+ as endogenous cation [20] which can be removed by treatment with I mM nitrilotriacetic acid, being the activity restored after 2 h incubation in the presence of Zn 2+, Cu 2+ or Co2+. However, the activity of the purified enzyme from Rugli ceils cannot be recovered after complete inhibition by EDTA, as also described for the enzyme from lymphocyte membranes [4]. The KM of the purified enzyme from rat ghoblastoma cells is 19.6 + 1.1 #M. Similar KM values have been also reported for other purified 5'-nucleotidase enzymes [5, 7, 8, 37]. The enzyme exhibits highest specificity for AMP, as it has been also reported for the enzyme from other sources [6, 8]. However, some 5'-nucleofidases present higher specificity for pyrimidine-ribose nucleotides such as IMP [35, 38]. The enzyme is inhibited by ATP according to a partial competitive pattern. This type of ATP partial competitive inhibition of AMPase activity has been also found in 5'-nucleotidase purified from small intestine smooth muscle [39]. The abnormal inhibitor effect of ATP could be explained by the existence of a mixed inhibition; ATP should act as a normal competitive inhibitor but

32 also as a chelator of the enzyme-bound metal cation [37, 39]. Lectins are also inhibitors of the purified enzyme from Rugli cells. The effects of different lectins on Rugli 5'-nucleotidase would suggest that this enzyme is of the high-mannose type, similar to the 5'-nucleotidase from bovine lymphocyte-plasma membrane [37] and 5'-nucleotidase from Torpedo marmorata electric organ [8]. However, the very low or lack of inhibition obtained by treatment with Phytolacca americana and wheat germ agglutinins suggest the absence of [3-N-acetylglucosaminyl or sialic acid residues, in contrast with 5'-nucleotidase from bovine brain [6] and bovine synaptosomal plasma membrane 5'-nucleotidase [40], where a heterogeneity of the glycosidic moiety of the enzyme has been reported. All of this indicates a glycoprotein character for the 5'-nucleotidase from rat glioblastoma cells. Finally, a high inhibition of the 5'-nucleotidase from intact cells is observed in the presence of wheat germ agglutinin but no effect is observed in the purified enzyme. Con A and Lens culinaris lectins inhibit both purified enzyme and enzyme present in intact cells although in a different extent. The inhibition produced in the purified enzyme may be interpreted in terms of the specific binding of lectins to the carbohydrate residues near the active site. However the inhibition produced by wheat germ agglutinin, which only is observed for the membrane-bound enzyme should be interpreted in terms of structural rearrangements of the membrane induced by binding of the lectin to other membrane glycoconjugates.

Acknowledgements Authors are indebted to Dr. Carlos Barba and Mr. Agustin Fermindez, from the Servicio de Microscopia Electr6nica (UCM), for their skilful assistance on the electron microscopy studies. This work has been supported by grant No. PB88--0129 from the DGICYT (Spain).

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