Molecular and Cellular Biochemistry 213: 11–16, 2000.
© 2000 Kluwer Academic Publishers. Printed in the Netherlands.
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Identification of brain ecto-apyrase as a
phosphoprotein
Márcia R. Wink,1 Guido Lenz,2 Richard Rodnight,1 João J.F. Sarkis1
and Ana M.O. Battastini1
1
Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde; 2Departamento de Biofísica,
Instituto de Biociências, UFRGS, Porto Alegre, Brasil
Received 21 September 1999; accepted 12 May 2000
Abstract
Ecto-apyrase is a transmembrane glycoprotein that hydrolyzes extracellular nucleoside tri- or diphosphates. Apyrase activity
is affected by several physiological and pathological conditions indicating the existence of regulatory mechanisms. Considering that apyrase presents consensus phosphorylation sites, we studied the phosphorylation of this enzyme. We found an overlay of the immunoblotting and phosphorylated bands in three different preparations from rat brain: (a) hippocampal slices, (b)
synaptic plasma membrane fragments and (c) cultured astrocytes. In addition, two-dimensional electrophoresis separations
with human astrocytoma cells were done to identify unequivocally the coincidence between the immunodetected and phosphorylated protein. These observations indicate that apyrase can be detected as a phosphoprotein, with obvious implications in
the regulation of this enzyme. (Mol Cell Biochem 213: 11–16, 2000)
Key words: ecto-apyrase, ecto-ATPdiphosphohydrolase, ecto-ATPase, apyrase phosphorylation
Introduction
Extracellular ATP and its hydrolysis products mediate a wide
variety of physiological responses in the central and peripheral nervous systems mainly through binding to purinergic
receptors [1]. The ATP released by synapses is hydrolyzed
by ectonucleotidases which constitute a highly sophisticated
pathway designed to control the rate, amount and timing of
ATP degradation and adenosine formation. The hydrolysis of
ATP to AMP is catalyzed either by ecto-ATPase or ecto-apyrase [2–7] and the AMP formed is catabolized to adenosine
by the action of an ecto-5′-nucleotidase [8]. This degradation
cascade plays important roles in the signaling mediated by
purines, since ATP and ADP act on purinoceptors of type 2
(P2X and P2Y), while adenosine and AMP act on purinoceptors of type 1, with different, even completely antagonistic
effects [9, 10]. So, the consequences experienced by the cell
will be modulated by the activity of these ecto-enzymes, indicating the importance of their regulation.
Apyrase presents remarkable changes in its activity in different physiological and pathological situations. For example, we have reported a fast learning-specific decrease in
synaptosomal apyrase activity from rat brain [11, 12], and an
increase in the apyrase and 5′-nucleotidase activities in the
hippocampus of rats submitted to different ischaemic episodes. Interestingly, one of the biochemical consequences of
ischaemia is an increase in the extracellular levels of adenosine derived from ATP and this increase may be related to
survival of neural cells [13].
Phosphorylation of proteins is one of the most widespread
mechanisms of cellular regulation. The importance of this
regulation can be assessed by the prognosis that there are
probably as many as 2000 protein kinase genes and at least
1000 phosphatase genes which phosphorylate and dephosphorylate around one third of all known proteins [14]. The
importance of phosphorylation is even more strengthened by
the fact that kinases and phosphatases are the major effectors of the main signal transduction pathways [14].
Address for offprints: A.M.O. Battastini, Departamento de Bioquímica - ICBS - UFRGS, Rua Ramiro Barcelos, 2600-anexo, CEP 90035-003, Porto Alegre,
RS, Brasil
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The cDNA of ecto-apyrase has been isolated from rat and
human brain and chicken oviduct [6, 7, 15]. Analysis of the
amino acid sequences from different sources has shown several potential phosphorylation sites [5–7, 15], indicating that
ecto-apyrase could be regulated by phosphorylation. Here we
show that the ecto-apyrase present in different brain tissue
preparations and cell culture can be detected as a phosphorylated protein, with implications for the regulation of the
activity of this enzyme.
Materials and methods
Materials
[γ32P]ATP was obtained from Amersham International, and
[32P]Na2HPO4 from CNEN, São Paulo. All electrophoresis
chemicals, material and media for cell culture and other reagents were from Sigma Chemical Co. (St. Louis, MO, USA).
Secondary antibodies (rabbit and mouse Ig, horseradish peroxidase-linked whole antibody) and the ECL luminol kit were
purchased from Amersham International. A polyclonal antiserum against apyrase was obtained by immunizing rabbits with
potato apyrase grade VII from Sigma. This antiserum has been
used to detect ecto-apyrase from synaptic plasma membrane
from rat brain [16] and tegumental membrane of Schistosoma
mansoni [17]. Monoclonal anti-CD39 (apyrase) was obtained
from Zymed Laboratories Inc. (San Francisco, CA, USA).
[32P]orthophosphate for 1 h at 30°C. The reaction was stopped
with 1 ml of 10% TCA. After a minimum of 10 min on ice,
slices were washed twice with 4% TCA to remove excess of
radioactivity, briefly washed with water to remove acid and
then immediately dissolved in SDS-PAGE sample buffer.
Labeling of astrocyte cultures with [32P]phosphate
Primary astrocyte cultures (a generous gift from Dr. C. Gottfried, UFRGS) were prepared from neonatal hippocampus as
described previously [19]. After 20 days in vitro culture medium was withdrawn and the cells (60 mm dishes) were
washed twice with 300 µl of the same HEPES-buffered incubation medium as used for slice incubation. Medium (500 µl)
was added and the cells were pre-incubated for 30 min at
30°C. The pre-incubation medium was then withdrawn and
250 µl of medium containing 30 µCi of [32P]phosphate was
added. After 1 h at 30°C the radioactive medium was removed
and the cells solubilized with the aid of a mini-spatula in
50 µl of lysis solution containing 137 mM NaCl, 20 mM Tris,
pH 8.0, 0.5 mM EDTA, 10% glycerol, 1% Triton X-100 and
the following protease inhibitors: 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) 0.1 mg/ml, aprotinin 50 µg/ml,
pepstatin 2 µM and leupeptin 2 µM. The cells were centri-
Isolation and labeling of synaptic plasma membranes
Synaptic plasma membrane fragments (SPM) were prepared
according to Jones and Matus [18]. Samples (80 µg) were
incubated at 30°C in a basic medium containing as final concentration: 10 mM Tris-HCl (pH 7.5), 1 mM MgCl2 and 10 µM
ATP plus 6 µCi [γ32P]ATP. The reaction was started by addition of [γ32P]ATP and stopped after 2 min with 0.5 ml of 10%
trichloroacetic acid (TCA). Precipitated proteins were washed
with 4% TCA and finally with 80% ethanol. Pellets were
dissolved in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer.
Preparation and labelling of slices
Rats were killed by decapitation and hippocampi dissected
on ice. Transverse slices (0.4 mm thick) were cut with a
McIlwain chopper and labeled with 32P. Briefly, two adjacents
slices were pre-incubated for 30 min in 500 µl of medium containing (mM): NaHEPES, 26, pH 7.4; NaCl, 124; KCl, 3.85;
MgSO4, 1.23; glucose, 12; CaCl2, 1; gassed with 95% O2 and
then incubated with 100 µl of medium containing 60 µCi of
Fig. 1. Representative immunoblot and phosphorylation pattern of rat hippocampal slices. Hippocampal slices were incubated with 60 µCi of [32P]orthophosphate for 60 min as detailed and 40 µg of protein was immediately
separated by SDS-PAGE and electroblotted onto nitrocellulose. Blots were
submitted to immunodetection with polyclonal potato tuber apyrase antisera (1:1000) by the luminol method and then exposed to X-ray film at –70°C
with intensifying screens. Lane 1 represents the immunoblot and lane 2
shows the corresponding autoradiograph of radiolabeled proteins. Arrow
points to the phosphorylated bands which immunoreacts with apyrase antibody. Positions of molecular size markers are shown in kDa. Data are representative examples of at least four experiments.
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buffer containing: 2% SDS, 100 mM Tris-HCl, pH 6.8, 1 mM
EDTA, 10% glycerol, 5% β-mercaptoethanol and 0.0025%
bromophenol blue, vortexed, boiled for 5 min and separated
on 8% acrylamide-SDS gel. For two-dimensional electrophoresis, U87 astrocytomas were lysed in 2D sample buffer
containing 9 M urea, 12.5 mM lysine, 4% Nonidet-P-40 and
2% (v/v) mercaptoethanol, and the samples were separated
according to net charge by non-equilibrated pH gel electrophoresis (NEPHGE) for the first dimension and then by
SDS-PAGE slab gel for the second dimension as described
previously [20]. The acrylamide concentration was 3% for
NEPHGE and 8% for SDS-PAGE. Gels were dried and exposed or electroblotted onto nitrocellulose membranes using
a semi-dry transfer cell (Trans-blot RD, BioRad) for 1 h at
15 V.
Immunodetection of apyrase
Fig. 2. Representative immunoblot and phosphorylation pattern of synaptic
plasma membrane (SPM) and astrocytes culture. Panel A: the SPM were
incubated with 6 µCi [γ-32P]ATP. Panel B: the astrocytes were incubated with
30 µCi of [32P]-orthophosphate as detailed in Materials and methods. In both
cases the samples (50 µg) were separated by SDS-PAGE and electroblotted
onto nitrocelullose. Blots were submitted to immunodetection with polyclonal
potato tuber apyrase antisera (1:1000) by the luminol method and then exposed to X-ray film at –70°C with intensifying screens. Lane 1 represents the
immunoblot and lane 2 shows the corresponding autoradiograph of radiolabeled proteins. Arrow points to the phosphorylated bands which immunoreacts with apyrase antibody. Positions of molecular size markers are shown
in kDa. Data are representative of at least 4 experiments.
fuged in a bench centrifuge for 5 min at 4oC. A concentrated
SDS-PAGE sample buffer was added to the supernatant.
Labelling of human U87 astrocytoma cells with
[32P]phosphate
Additional studies were also performed with U87 astrocytoma cells (a generous gift from Dr. A.B. da Rocha, SOAD/
HCPA). The protocol used was essentially the same as described above, with exception that 75 cm 2 flasks were used
and the cells were washed and pre-incubated with 10 ml of
HEPES-buffered medium and incubated with 3 ml of medium
containing 400 µCi of [32P]phosphate. The cells were dissolved in sample buffer for SDS-PAGE or two-dimensional
electrophoresis (2D).
Electrophoresis and blotting
For SDS-PAGE, the different labelled samples (slices, SPM,
astrocyte and U87 cells) were diluted in SDS-PAGE sample
Nitrocellulose membranes were blocked with 5% nonfat dry
milk in Tris-buffered saline (TBS), pH 7.5 overnight, at 4°C.
The blocking buffer was removed and washed with TBS containing 0.05% Tween 20 (TTBS). The nitrocellulose blots
were incubated at room temperature with polyclonal potato
tuber apyrase antisera (1:1000) for 2 h or anti-CD39 (1:100)
for 4 h in 5% milk buffer. After washing with TTBS and incubating for 1 h with anti-rabbit or anti-mouse horseradish peroxidase conjugated secondary antibody (in blocking buffer),
immunoreactivity was detected by ECL luminol kit from
Amersham International, using Kodak X-Omat X-ray film.
In all experiments two parallel gels were run, both were
transferred and one was immunodetected, before exposing, taking care to allow the decay of the light from ECL (membranes
were left overnight before exposing). The other membrane was
first exposed to X-ray film and then immunodetected. In both
cases the phosphorylation pattern was compared with the
immunoreactive bands. Great care was taken to ensure correct matching of the phosphorylation pattern on the autoradiographs and the ECL prints of the immunoreactive bands.
Results
Apyrase immunodetected with polyclonal or monoclonal antibodies overlayed to a phosphoprotein in several preparations
in unidimensional as well as two-dimensional electrophoresis.
The phosphorylation patterns and the immunoreactive bands
identified by a polyclonal potato tuber apyrase anti-sera of
three preparations are shown in Figs 1 and 2. When hippocampal slices were 32P-labeled and analysed by electrophoresis, two main bands appeared, with apparent Mr’s of ~ 120 and
~ 80 kDa which overlapped with immunodetected bands on
the autoradigraphs of the nitrocellulose membranes (Fig. 1).
14
When the samples were frozen after labeling, and analyzed
later, it was observed a different immunodetection pattern
with the appearance of lower Mr bands, probably due to proteolysis during freeze-thawing process. It is interesting to note
that the intensity of the immunodetected bands changed in
the frozen tissue, and these changes were accompanied by
the same intensity changes in the phosphorylated bands (data
not shown). In the SPM fraction labeled with [γ-32P]ATP, a
broad immunoreactive band which corresponded to a phosphorylated band in the autoradiograph was observed (Fig.
2A). In astrocyte cultures the polyclonal antibody recognized
a doublet of ~ 62 and ~ 58 kDa, with the corresponding bands
in the phosphorylation pattern (Fig. 2B).
Considering the limited resolution of one dimensional SDS
PAGE, U87 human astrocytoma cells were labeled with [32P]phosphate, run on two-dimensional gel electrophoresis and
the phosphorylation pattern was compared to the immunoreactive spots. An overview of the two-dimensional electrophoretic mobilities of the phosphorylated proteins is shown
in Fig. 3B, where the position of the phosphoprotein identified with the monoclonal antibody is indicated. As shown in
Fig. 3C, two main spots with Mr’s ~ 55 kDa were immunodetected with an anti-human CD39 monoclonal antibody. The
strongest spot was located at the front of the NEPHGE gel
and the other spot migrated to a position which is between pI
5–7. It can be observed an overlay of these immunodetected
spots with phosphorylated proteins in the autoradiograph
(Fig. 3D). The anti-apyrase immunoreactive band can also
be seen in the unidimensional separation that was run in parallel with the two-dimensional gel (Fig. 3A).
Discussion
The apyrase activity presents remarkable changes in different physiological and pathological situations [11–13]. Considering that the fast kinetics of some of these changes can
not be attributed to changes in the expression or degradation
of the enzyme, another possible explanation for these alterations should be investigated. Apyrase is a transmembrane
protein, which is anchored by the N- and C-terminal, with
intracellular sequences of around 20 amino acids. If intracellular regulation is considered, as is the case for several
receptors and ion channels [21], phosphorylation is one of
the best candidate mechanisms, even more if it is considered
that the intracellular sequences has at least two consensus
phosphorylation sites [6, 7].
In the present work we investigated if apyrase can be detected as a phosphoprotein by using the overlay of the immunodetected apyrase with phospho-labeled bands or spots. This
was done with four different preparations, using both unidimensional as well as two-dimensional electrophoresis. The
results presented here show that in rat hippocampal slices at
least two bands, with apparent Mr of ~ 120 and ~ 80 kDa were
immunodetected with anti-apyrase polyclonal antibody, which
corresponded to phosphorylated bands. It was also possible
to observe a much fainter band of lower molecular mass,
around 62 kDa which could be a non-glycosylated form of
the protein. In contrast, SDS-PAGE of the SPM, a fraction
enriched in membrane proteins, where ecto-apyrase has been
characterized [16], revealed a relatively stronger immunoreactive band which overlayed with a well separated phosphorylated band at ~ 64 kDa (Fig. 2A). The use of this membrane
fraction eliminated a reasonable amount of the phosphoproteins of the cells (compare Fig. 1 with Fig. 2A), increasing the chance that the immunodetected apyrase corresponds
to the phosphorylated protein. This observation suggests that
either apyrase in synaptic membranes was deglycosylated
during subcellular fractionation or the deglycosylated form
was concentrated during this process. The detection of an
immunoreactive band with the same Mr in astrocyte cultures
adds to the evidence shown above. The doublet of around
62 kDa observed in samples prepared from astrocytes, probably represents different glycosylation variations considering the Mr and the sharpness of the bands [22, 23]. The
apparent inconsistency in the number of immunodetected and
labeled bands observed when the results of slices are compared with SPM and astrocytes culture, could be explained
by the presence of a great variety of cells and proteins in
slices. A possible cross-immunoreactivity between the polyclonal antisera and other proteins related to ecto-apyrase
should also be considered. Anyway, the close correspondence
between the migration of the immunoreactive bands in all
preparations analyzed by SDS-PAGE and the phosphorylated
bands makes the results obtained with one-dimensional electrophoresis a strong argument in favor to the existence of a
phosphorylated form of apyrase.
To confirm the identification of apyrase as a phosphoprotein we used a two-dimensional separation of 32P-labeled
proteins obtained from U87 astrocytoma cell line which was
immunodetected with a specific monoclonal antibody. Although apyrase, perhaps due to glycosylation and/or low
solubility in the two-dimensional sample buffer, was only
partially separated in NEPHGE, with the majority of protein
remaining on the front of the gel, it was possible to observe
overlay of the phosphorylated and immunodetected spots
(Figs 3C and 3D). The overlay of the spot that remained on
the front of the NEPHGE is total, whereas the overlay of the
fraction that migrated in the NEPHGE is partial, with the
phosphorylated spot shifted to a slightly acidic position in relation to the immunodetected one, which agrees with the
increased negative charge of the phosphorylated when compared to the unphosphorylated protein. The absence of an
immunodetected spot at the exact position of the phosphoprotein might be due to the presence of a low fraction of
phosphorylated protein which could not be detected by West-
15
overlay seen in the two-dimensional gels can also be seen in
the SDS-PAGE (Fig. 3A). Moreover, the apparent Mr is in
agreement with the calculated Mr’s for the human apyrase
which is 59 kDa [3]. Finally, our findings show, for the first
time, the immunodetection of apyrase in a two-dimensional
gel electrophoresis separation where it can be identified as a
phosphoprotein.
In conclusion, the existence of putative phosphorylation
sites together with the detection of immunoreactive bands in
four different preparations from brain which coincide with
radiolabeled bands in the autoradiograph patterns of the
phosphorylated proteins, make the results shown here a very
strong argument in favor of the existence of a phosphorylated
form of apyrase. Experiments are in progress to determine
the relation between the phosphorylation and changes in
apyrase activity.
Acknowledgements
We thank Dr. A. Masuda from Departamento de Biotecnologia,
UFRGS, for assistance in antisera preparation, to Dr. J.M.
Ribeiro from NIH-USA for the help with the analysis of the
amino acid sequence and D. Oppelt for help with two-dimensional electrophoresis procedures. M.R. Wink was the recipient of a CNPq-fellowship. This work was supported by grants
from FINEP-Brazil and PRONEX.
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Fig. 3. Representative immunoblot and phosphorylation pattern of human
U87 astrocytoma cell line. The astrocytoma cells were labelled with 400
µCi of [32P]-orthophosphate and 100 µg of protein was separated in SDSPAGE (Panel A) or two-dimensional electrophoresis (Panels B, C and D).
The gels were dried and exposed (Panel B) or electroblotted onto nitrocellulose membranes (Panels A, C and D). Proteins were immunodetected with
anti-CD39 antibody (1:100) and then the nitrocellulose membranes were
exposed to X-ray films as detailed in Materials and methods. Panel A:
lane 1 represents the immunoblot and lane 2 shows the corresponding autoradiograph of the radiolabeled proteins. Arrow points to the phosphorylated
band which immunoreacts with anti-CD39 antibody. Positions of molecular size markers are shown in kDa. Panel B: the two dimensional slab gel
were dried and exposed to X-ray films at –70°C with intensifying screens
in order to show the overview of the phosphoproteins in astrocytoma cells.
The position of the phosphoprotein identified with the monoclonal antibody
is indicated with an arrow. Panel C shows the immunoblot and Panel D
shows the corresponding autoradiograph of radiolabeled proteins. Arrow
points to the phosphorylated spots which immunoreacts with apyrase antibody. Positions of molecular size markers are shown in kDa. Data are representative of at least 3 experiments.
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