Enzymatic properties of the liver membrane ecto-ATPDase are similar to those of the chicken oviduct ecto-ATPDase that we have previously purified and cloned.. HeLa cells transfected with
Trang 1Purification, characterization, cloning, and expression of the chicken liver ecto-ATP-diphosphohydrolase
Aileen F Knowles1, Agnes K Nagy2, Randy S Strobel3and Mae Wu-Weis1
1
Department of Chemistry, San Diego State University, San Diego, CA, USA;2West Los Angeles Veterans Affairs Medical Center, Los Angeles, CA, USA;3Department of Natural Sciences, Metropolitan State University, St Paul, MN, USA
We previously demonstrated that the major ecto-nucleoside
triphosphate phosphohydrolase in the chicken liver
membranes is an ecto-ATP-diphosphohydrolase
(ecto-ATPDase) [Caldwell, C., Davis, M.D & Knowles, A.F
(1999) Arch Biochem Biophys 362, 46–58] Enzymatic
properties of the liver membrane ecto-ATPDase are similar
to those of the chicken oviduct ecto-ATPDase that we have
previously purified and cloned Using antibody developed
against the latter, we have purified the chicken liver
ecto-ATPDase to homogeneity The purified enzyme is a
glycoprotein with a molecular mass of 85 kDa and a specific
activity of 1000 UÆmg protein)1 Although slightly larger
than the 80-kDa oviduct enzyme, the two ecto-ATPDases
are nearly identical with respect to their enzymatic properties
and mass of the deglycosylated proteins The primary
sequence of the liver ecto-ATPDase deduced from its cDNA
obtained by RT-PCR cloning also shows only minor
differences from that of the oviduct ecto-ATPDase
Immunochemical staining demonstrates the distribution of
the ecto-ATPDase in the bile canaliculi of the chicken liver
HeLa cells transfected with the chicken liver ecto-ATPDase cDNA express an ecto-nucleotidase activity with character-istics similar to the enzyme in its native membranes, most significant of these is stimulation of the ATPDase activity by detergents, which inhibits other members of the ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) family The stimulation of the expressed liver ecto-ATPDase
by detergents indicates that this property is intrinsic to the enzyme protein, and cannot be attributed to the lipid environment of the native membranes The molecular identification and expression of a liver ecto-ATPDase, reported here for the first time, will facilitate future investigations into the differences between structure and function of the different E-NTPDases, existence of liver ecto-ATPDase isoforms in different species, its alteration in pathogenic conditions, and its physiological function Keywords: ecto-ATP-diphosphohydrolase; chicken liver; E-NTPDase; expression; immunoaffinity purification
E(cto)-ATPases (E-ATPases; also known as E-NTPDases)
(EC 3.6.1.5) are ubiquitous cell surface glycoproteins that
hydrolyze nucleoside triphosphates Some will also
hydro-lyze nucleoside diphosphates Their physiological substrates
are probably the ligands of purinergic receptors, e.g
extracellular ATP, ADP, and UTP [1] They may also play
a role in regulating substrate concentration of ecto-protein
kinases [2] A substantial literature on the characterization
of the E-ATPases in intact cells and plasma membrane
preparations has accumulated since the 1970s (reviewed in
[3]) Because of their low abundance and the lability of some
E-ATPases to detergents, only the E-ATPases of rabbit muscle transverse tubules [4], chicken gizzard [5], human placenta [6], and chicken oviduct [7] have been purified to homogeneity On the other hand, the cDNA sequences of more than two dozen related E-ATPases and soluble E-type ATPases have been reported, establishing an E-ATPase gene family [8] The cDNAs of membrane-bound E-ATPases encode proteins of 500 amino acids The bulk of the E-ATPase protein is extracellular with two transmembranous domains near the N- and C-termini Variable numbers of potential N-glycosylation sites and protein kinase consensus motifs occur in the sequences More importantly, all contain five highly conserved apyrase consensus regions [9,10] and 10 conserved cysteine residues, the latter are probably involved in disulfide bond formation The E-ATPases can be divided into two groups based
on their substrate selectivity and inhibition by azide The ecto-ATP-diphosphohydrolases (ecto-ATPDases or ecto-apyrases) hydrolyze NDPs as well as NTPs and are inhibited by high concentrations of azide, whereas the ecto-ATPases show little activity toward NDPs and are not inhibited by azide The ecto-ATPDases are comprised of different isoforms The majority of the ecto-ATPDases that have been cloned are closely related to CD39, a cell surface antigen that is expressed on activated lymphocytes [11,12] CD39s from several species have 60–90% identity
in their primary sequences [12–15] Biochemical and
Correspondence to A F Knowles, Department of Chemistry,
San Diego State University, San Diego, CA 92182-1030, USA.
Fax: + 1619 594 4634, Tel.: + 1619 594 2065,
E-mail: aknowles@chemistry.sdsu.edu
Abbreviations: DMEM, Dulbecco’s modified Eagle’s medium.
Definitions: E-ATPases are a family of cell surface (ecto) ATPases that
hydrolyze extracellular ATP; they are also known as the E-NTPDases.
Ecto-ATP-diphosphohydrolase (ecto-ATPDases) and ecto-ATPases
are two different subfamilies of the E-ATPases E-type ATPases are
ATPases that have similar enzymatic characteristics and sequence
homology to the E-ATPases, however, they are not membrane
proteins.
(Received 17 December 2001, revised 18 March 2002,
accepted 22 March 2002)
Trang 2immunolocalization studies indicated that CD39s are
vas-cular ecto-ATPDases [16–20] Two other ecto-ATPDases,
cloned from chicken oviduct [21] and human brain [22], can
be distinguished from the CD39/ecto-ATPDases because of
significant sequence divergence from the latter
Interest-ingly, these two ecto-ATPDases have only 44% identity in
their primary sequences This cannot be entirely accounted
for by species differences because the chicken and human
ecto-ATPases have 57% sequence identity Thus, the true
relationship of the chicken and human ecto-ATPDases
remains to be established
We previously reported the immunoaffinity purification
of the chicken oviduct ecto-ATPDase to homogeneity [7]
and its molecular cloning [21] An ecto-ATPDase similar to
the chicken oviduct enzyme is present in the chicken liver
membranes [23] We report here the complete purification of
the chicken liver ecto-ATPDase, its enzymatic properties
and localization, cloning of the full-length cDNA and its
expression
E X P E R I M E N T A L P R O C E D U R E S
Materials
The production and characterization of monoclonal
anti-bodies against the chicken oviduct ecto-ATPDase were
described previously [7] Of the six monoclonal antibodies
generated, MC18 was most suitable for Western blot
analysis because of its strong and specific binding to the
chicken ecto-ATPDase [7,23] MC22 was employed to
prepare the immunoaffinity column using hydrazide
acti-vated Affi-gel [7] and MC27 was used for
immunolocaliza-tion Goat anti-(mouse IgG) IgG conjugated to alkaline
phosphatase was purchased from Promega Chicken liver
polyA+RNA, MarathonTMcDNA amplification kit and
Advantage 2 PCR enzyme system were purchased from
Clontech Pfu Turbo DNA polymerase was purchased from
Strategene Dulbecco’s modified Eagle’s media (DMEM),
OptiMEM, fetal bovine serum, Lipofectamine, and
genet-icin were purchased from Life Technologies Inc
N-Glyco-sidase F and restriction enzymes were purchased from New
England Biolabs ATP, ADP, and all other biochemical
reagents were purchased from Sigma Chemical Co
Oligo-nucleotides used as primers for PCR and sequencing were
synthesized at the San Diego State University
Microchemi-cal Core Facility
Purification of chicken liver ecto-ATPDase
Liver from freshly killed chickens was purchased at a local
poultry farm Membranes were prepared by homogenizing
1 lb of livers in 500 mL of isolation buffer (50 mM Tris/
HCl, pH 7.4, 0.25M sucrose, and 1 mM EGTA) in a
Waring blender for 1 min After filtering the homogenate
through cheesecloth, the filtrate was homogenized again in a
Dounce homogenizer and then centrifuged at 5000 r.p.m in
an SS34 rotor in a Sorvall centrifuge for 10 min The
supernatant was centrifuged again at 16 000 r.p.m for
20 min to precipitate the membranes The membrane pellet
was washed three times by repeated resuspension in a buffer
containing 50 mMTris/HCl, pH 7.4 and 1 mMEGTA and
centrifugation To extract the ecto-ATPDase, the
mem-branes were solubilized in 50 mM Tris/HCl, pH 7.4
containing 5% NP-40 at 2 mg proteinÆmL)1and stirring
at 4C overnight After centrifugation at 16 000 r.p.m for
20 min, the supernatant was filtered through Whatman
no 1 filter paper
The filtrate containing the extracted membrane proteins were applied to a DEAE Biogel A column (2.5· 43 cm) pre-equilibrated with 50 mMTris/HCl, pH 7.4 containing 0.1% NP-40 (chromatography buffer) After washing the column to elute the unbound proteins, the column was developed with a NaCl gradient consisting of 375 mL of chromatography buffer and 375 mL of chromatography buffer containing 1M NaCl The ATPase activity was eluted as a broad peak while more than 90% of the solubilized proteins remained bound to the column Total recovery of activity was nearly 100%, partly attributable to the activating effect of NP-40 of liver membrane ecto-ATPDase [23]
The proteins eluted from the DEAE Biogel A column
(1.5· 10 cm) pre-equilibrated with chromatography buffer After washing off the unbound proteins, the chicken liver ecto-ATPDase and other bound glycoproteins were eluted with 200 mL of chromatography buffer containing 1% a-methylmannoside Fractions containing ATPase activity were pooled and applied to a second DEAE Biogel A column (1.5· 10 cm) as a means of concentrating the proteins The bound enzyme was eluted in a small volume of chromatography buffer containing 1M NaCl Fractions containing ATPase activity were pooled and desalted on Sephadex G-25 column (1.5· 48 cm) The desalted and partially purified chicken liver ecto-ATPDase fraction ( 20 mL) was added to 3 mL of MC22-hydrazide resin [7], and equilibrated overnight by rocking at 4C The slurry was poured into a small column, and washed sequentially with 50 mL each of chromatogra-phy buffer, buffer containing 0.5MNaCl, and buffer again The enzyme was eluted with 50 mM glycine, pH 2.5 containing 0.1% NP-40 Fractions of 1 mL were collected into tubes containing 0.1 mL of 1MTris/HCl, pH 8.0 plus 0.1% NP-40 The emergence of an 85-kDa protein coincided with elution of the ATPase activity (not shown)
ATPase assays During the purification procedure, ATPase activities of the chicken liver ecto-ATPDase were assayed in 0.5 mL reac-tion mixture containing 50 mM Tris/HCl, pH 7.4, 0.1% NP-40, 4 mMMgCl2and 4 mMATP at 37C for 5–30 min After terminating the reactions by the addition of 0.1 mL 10% trichloroacetic acid, the denatured proteins were removed by centrifugation Aliquots of the supernatant were used for determination of Pi released by the AAM reagent (10 mMammonium molybdate/5N H2SO4/acetone,
1 : 1 : 2, v/v/v) [7] Absorbance of the phosphomolybdate complex was read at 355 nm
For characterizing the enzymatic properties of the purified chicken liver ecto-ATPDase, enzyme assays were carried out in 0.25-mL reaction mixtures using the buffer systems and substrate concentrations indicated in the legends Phosphate released was determined by the mala-chite green reagent as described previously [7]
ATP and ADP hydrolysis activities of intact COS or HeLa cells transfected by chicken liver ecto-ATPDase
Trang 3cDNA were determined using either attached cells in
six-well plates or cell suspension obtained after trypsinization
For the six-well plates, cells were washed twice with 1 mL of
buffered isotonic solution (0.1M NaCl, 0.01M KCl, and
25 mM Tris/HCl, pH 7.4) after aspiration of the culture
media The cells were then overlaid with 1 mL buffered
isotonic solution containing 5 mMMgCl2and 5 mMATP
or ADP After incubation at 37C for 15–30 min, the
reaction mixture was collected by Pasteur pipettes and
added to 0.1 mL 10% trichloroacetic acid Aliquots (0.1–
0.4 mL) of the solution were used for determination of
phosphate released using the AAM reagent Alternatively,
cells grown in 10-cm plates were trypsinized, suspended
in culture media, and collected by centrifugation After
washing with buffered isotonic solution, the cells were
resuspended in the same solution at 1–3 mg proteinÆmL)1
Aliquots of cell suspension (50–100 lg cell protein) were
used for enzyme assays in 0.5 mL isotonic reaction mixture
with substrates as described above The reaction was carried
out for 10–30 min at 37C and stopped by the addition of
0.1 mL 10% trichloroacetic acid The suspension was
centrifuged to remove denatured proteins Aliquots of the
supernatant solution were used for Pidetermination by the
AAM reagent
RT-PCR cloning
Chicken liver polyA-RNA (1 lg) was reverse transcribed
using avian myoblastosis virus reverse transcriptase and
oligo(dT) as the primer (MarathonTMcDNA amplification
kit, Clontech) Double-stranded cDNA was prepared
according to the manufacturer’s instruction and served as
template in the PCR Oligonucleotides corresponding to the
5¢ and 3¢ ends of the chicken oviduct cDNA (GenBank
accession no AF041355) were used: forward primer,
reverse primer, 5¢-TTGGATTTCCAGAAACACTGGA-3¢
PCR was carried out in a 50-lL reaction mixture containing
0.5 lL (from a total of 10 lL) cDNA template, 0.5 lM
primers, 0.1 mM dNTP, and 1.25 U Pfu Turbo DNA
polymerase (Strategene) Thermal cycling on a PTC-200
Peltier thermal cycler (MJ Research, Waltham, MA, USA)
began with 4.5 min at 94C followed by 35 cycles of 94 C
for 45 s, 55C for 1 min and 72 C for 3 min and ending
with 10 min at 72C A PCR product of 1.5 kb that
corresponded to the length of the coding region of the
chicken ecto-ATPDase was obtained An aliquot (2 lL) of
the reaction mixture was used for further amplification with
the same primers and thermal cycling conditions but with
Advantage 2 Taq DNA polymerase (Clontech) for
subse-quent TA cloning The 1.5-kb PCR product was gel purified
and ligated to pCR2.1 (Invitrogen) and an aliquot of the
ligation mixture was used to transform INVaF Escherichia
coli cells (Invitrogen) White colonies that grew on agar
containing ampicillin were selected and the presence of
1.5-kb insert was verified by digestion with EcoRI DNA
from one recombinant plasmid was isolated and digested
with EcoRI The resultant fragment was ligated with the
mammalian expression vector pcDNA3 (Invitrogen)
linear-ized with EcoRI and treated with bovine pancreatic alkaline
phosphatase An aliquot of the ligation mixture was used to
transform DH5a E coli cells Orientation of the insert in the
recombinant pcDNA3 was determined with appropriate
restriction enzymes One clone (pcDNA3-CL8) with the correct orientation was propagated for preparation of DNA for sequencing and transfection DNA sequencing was provided by the San Diego State University Microchemical Core Facility
Transient and stable transfection COS-7 and HeLa cells were grown in DMEM containing 10% fetal bovine serum, penicillin (100 UÆmL)1) and streptomycin (100 lgÆmL)1) Cells were plated either in six-well plates or 10-cm plates and were used for transfection after reaching 50–70% confluence In the six-well plates, the cells were washed twice with OptiMEM and then layered with 1 mL OptiMEM containing DNA (1 lg per well) and Lipofectamine (5 lL per well) which had been premixed and incubated according to the manufacturer’s instruction After 5 h, 1 mL of DMEM containing 20% fetal bovine serum was added to the wells Twenty-four hours after transfection, the medium was replaced by fresh DMEM/ 10% fetal bovine serum ATPase and ADPase activities were determined 48–72 h after transfection When transfec-tion was carried out in 10-cm plates, the cells were overlaid with 6.4 mL OptiMEM containing premixed DNA (5 lg) and Lipofectamine (30 lL) After 5 h, DMEM and serum were added to bring the volume of the media to 10 mL and
a final serum concentration to 10% After 24 h, the media were replaced by fresh DMEM containing 10% serum After another 24–48 h, the cells were harvested by trypsi-nization Enzyme activities were determined using 25–50 lL cell suspension (50–100 lg cell protein)
For stable transfection, HeLa cells were first transfected
in 10-cm plates with the chicken liver cDNA (in pcDNA3) Two days after transfection, the cells were harvested and divided into two T-25 flasks The cells were allowed to attach overnight and geneticin was added at 400 lgÆmL)1 Medium was replaced every three days The established geneticin-resistant clones were propagated for activity determination and characterization
Deglycosylation Purified chicken liver ecto-ATPDase (2 lg) was precipitated
by the addition of 9 vol of ice-cold acetone After centrifugation for 30 min, the protein pellet was dissolved
in 0.5% SDS/2% 2-mercaptoethanol and heated for 10 min
at 100C Phosphate buffer (pH 7.5) and NP-40 were added to a final concentration of 50 mMand 1%, respect-ively The protein solution was incubated with 1000 U of N-glycosidase F at 37C for 16 h Aliquots of the protein solution were used for Western blot analysis
Gel electrophoresis and Western blot analysis SDS/PAGE was carried out on a mini-gel apparatus (Bio-Rad) in slab gels of 7.5% acrylamide according to Laemmli [24] The gel was stained with silver nitrate [7] For Western blot analysis, protein samples were mixed with sample buffer containing SDS but without reducing agents since the epitope recognized by MC18 is destroyed by reduction of disulfide bonds Separated proteins were transferred to poly(vinylidene difluoride) membranes The membranes were first blocked with NaCl/Tris (0.5 NaCl and 20 m
Trang 4Tris/HCl, pH 7.4) with 2% BSA and then incubated for 2 h
with the monoclonal antibody MC18 diluted in NaCl/Tris/
2% BSA After washing four times with NaCl/Tris
containing 0.1% Tween 20, the membranes were incubated
in a solution containing goat anti-(mouse IgG) Ig
conju-gated to alkaline phosphatase for 1.5 h After three washes
with NaCl/Tris containing 0.1% Tween-20 and a final wash
with NaCl/Tris, the immunoreative bands were detected by
treating the membranes with the alkaline phosphatase
substrates (Bio-Rad)
Immunolocalization
Paraffin-embedded sections of chicken liver fixed in 10%
buffered formalin were used Immunoperoxidase
localiza-tion of ecto-ATPDase in these seclocaliza-tions were performed with
purified monoclonal antibody, MC27, using a standard
immunohistochemical protocol [25]
R E S U L T S
Purification of chicken liver ecto-ATPDase
Chicken liver ecto-ATPDase, an integral membrane
pro-tein, was solubilized from liver membranes by NP-40 and
purified by ion-exchange, lectin-affinity, and immunoaffinity
chromatographic separations as described in Experimental
procedures The purification scheme is summarized in
Table 1 The most effective purification was obtained by
immunoaffinity chromatography with a 50-fold
purifica-tion in one single step The significant loss of total activity
probably resulted from irreversible binding of the majority
of the liver ecto-ATPDase to MC22 Total purification
was 2000-fold The final preparation had a specific
ATPase activity of 1200 lmolÆmin)1Æmg)1protein, similar
to that of the purified chicken oviduct ecto-ATPDase
( 800 lmolÆmin)1Æmg)1protein) [7] The purified enzyme
is stable indefinitely at 4C in buffer containing 0.1%
NP-40, but suffers significant loss of activity upon freezing
The purified chicken liver ecto-ATPDase contains a
single protein band (Fig 1A, lane 2) with an apparent
molecular mass of 85 kDa, which is slightly higher than the
molecular mass of the purified chicken oviduct
ecto-ATPDase, 80 kDa (Fig 1A, lane 1) In Western blot
analysis with MC 18, an 85-kDa protein was also detected
in both the chicken liver membranes (Fig 1B, lane 3) and
the purified ecto-ATPDase preparation (Fig 1B, lane 1)
Because the epitope detected by MC18 was sensitive to
disulfide reduction, the samples used for Western blot
analysis were not treated with 2-mercaptoethanol Under
these circumstances, a higher molecular mass band of
180 kDa was often detected in the protein sample (Fig 1B, lane 1) This result suggests that the enzyme is able to form dimers When the chicken liver ecto-ATPDase was treated with N-glycosidase F which removes N-linked oligosaccharides, Western blot analysis with MC18 revealed the presence of two protein bands with molecular masses of
55 and 100 kDa (Fig 1B, lane 2) The molecular masses of the deglycosylated chicken liver ecto-ATPDase monomer is the same as the deglycosylated chicken oviduct ecto-ATPDase that we previously reported [21] Thus, the slightly higher molecular mass of the native liver ecto-ATPDase indicates that its glycosylation is more extensive than that of the oviduct enzyme
Table 1 Purification of ecto-ATPDase from chicken liver ND, not determined.
Fraction
Volume (mL)
Total protein (mg)
Total activity (lmolÆmin)1)
Specific activity
( lmolÆmin)1Æmg)1)
Yield (%)
Purification (fold) Liver membranes 525 10200 6608 0.638 100 1
M22-hydrazide gel 6.6 0.017 21.3 1242 0.32 1947
Fig 1 Molecular masses of native and deglycosylated purified chicken liver ecto-ATPDase (A) Purified chicken liver oviduct ecto-ATPDase (0.3 lg) and liver ecto-ATPDase (0.1 lg) were dissolved in SDS gel sample buffer containing 2-mercaptoethanol and applied to a 7.5% polyacrylamide gel After electrophoresis, the gel was silver stained Lane 1, chicken oviduct ectoATPDase; lane 2, chicken liver ATPDase (B) Western blot analysis of purified chicken liver ecto-ATPDase before and after deglycosylation Purified chicken liver ecto-ATPDase (2 lg) was treated with N-glycosidase F as described in Experimental procedures An aliquot of the protein solution was treated with SDS gel sample buffer without 2-mercaptoethanol After electrophoresis on 7.5% polyacrylamide gel and transfer to poly(vinylidene difluoride) membrane, the membrane was probed with monoclonal antibody MC18 Lane 1, purified chicken liver ecto-ATPDase; lane 2, deglycosylated chicken liver ecto-ecto-ATPDase; lane 3, chicken liver plasma membranes.
Trang 5Enzymatic characteristics of the purified chicken liver
ecto-ATPDase
Most E-ATPases have broad substrate specificity with
respect to nucleoside triphosphates Ecto-ATPDases and
CD39 also hydrolyze nucleoside diphosphates The Km
values obtained for ATP of the purified chicken liver
ecto-ATPDase is 0.51 mM and that for ADP is 5.3 mMin the
presence of 5 mMMgCl2at pH 7.4 Lower Kmvalues for
ATP and ADP, 0.13 mMand 0.72 mM, respectively, were
obtained at pH 6.4 Unlike some other E-ATPases that
exhibit similar or higher ATP hydrolysis activity in the
presence of Ca2+[26], the CaATPase activity of the purified
chicken liver ecto-ATPDase is 30% of the MgATPase
activity at a divalent ion-ATP concentration of 5 mM at
pH 7.4 On the other hand, the CaADPase activity is
80% of the MgADPase activity at a divalent ion-ADP concentration of 5 mM(data not shown)
The ATPase and ADPase activities of the purified chicken liver ecto-ATPDase were affected differently by
pH Figure 2 shows that the pH–activity curves of ATP and ADP hydrolysis do not coincide While maximal ATP hydrolysis activity was obtained in the pH range of 7.5–8.5, the pH optima for ADP was lower at 6.0–6.5 Thus, the ADPase/ATPase ratios vary significantly at different pH values For example, a higher ADPase/ATPase ratio was obtained at pH 6.4 ( 0.5) than at pH 8.0 ( 0.1) Because
of the lower Km for ADP and higher ADPase activity observed at pH 6–6.5, ADPase activities were determined at
pH 6.4 in several of the experiments reported below
We showed previously that, in contrast to the chicken smooth muscle ecto-ATPase, which is inactivated by most detergents, the chicken liver plasma membrane ecto-ATPDase activity is increased by Triton X-100 and NP-40 [23,27] As described above, the enzyme was extracted from the membranes by 5% NP-40, and all solutions used in its purification contained 0.1% NP-40 Table 2 shows that the purified chicken liver ecto-ATPDase was not affected by ConA or suramin, the former activates while the latter inhibits the chicken smooth muscle ecto-ATPase [23,28] On the other hand, it was inhibited by high concentrations
of azide, an inhibitor of CD39 and most ecto-ATPDases [29–31], and high concentrations of fluoride, vanadate, and pyrophosphate, inhibitors of the purified chicken oviduct ecto-ATPDase [7,31] Like the other ecto-ATPDases, the ADPase activity of the purified chicken liver ecto-ATPDase was more sensitive to azide inhibition than its ATPase activity at either pH 7.4 or pH 6.4 (Fig 3) At pH 7.4,
5 mM azide inhibited ADP hydrolysis by 70% whereas ATP hydrolysis was inhibited by only 10% Inhibition of ADP and ATP hydrolysis by azide was greater when the pH
of assay solutions was 6.4 Azide inhibition was diminished
if Ca2+ was used in place of Mg2+ (data not shown) Inhibition of the enzyme by fluoride and vanadate was also more pronounced with ADP as the substrate and was greater at lower pH values In contrast to azide, fluoride, and vanadate, 5 mMpyrophosphate inhibited ATP hydro-lysis significantly ( 50%), and the extent of inhibition was insensitive to pH This difference could be the result of different modes of inhibition by these compounds Pyro-phosphate was previously shown to be a competitive inhibitor of the oviduct ADPase activity [32], whereas
Fig 2 Effect of pH on ATP and ADP hydrolysis by the purified chicken
liver ecto-ATPDase Enzyme assays were carried out in 0.25-mL
reaction mixtures using a wide-range buffer system (piperazine
dihydrochloride/glycylglycine/NaOH [31]), covering the pH range of
4.6–9.5 The assay solutions contained 50 m M buffer, 0.1% NP-40,
5 m M MgATP (d) or 10 m M MgADP (m) with 0.2–0.4 lg chicken
liver ecto-ATPDase The reaction was carried out for 5 min at 37 C.
Table 2 Effect of modulators on ATP and ADP hydrolysis of the purified chicken liver ecto-ATPDase Purified chicken liver ecto-ATPDase (0.2– 0.4 lg) was preincubated with the indicated concentrations of modulators in a 0.25-mL reaction mixture at 37 C for 5 min before initiating the reaction by the addition of ATP or ADP Buffers were 50 m M Tris/HCl for pH 7.4 and 50 m M Mops for pH 6.4 Reaction time was 5 min Duplicate samples were run for each condition Results presented were the average (± SD) of three experiments Values are given as percent activity.
Addition
ATP hydrolysis ADP hydrolysis
ConA (50 lgÆmL)1) 102 ± 2.9 92 ± 1.6 110 ± 13.3 91 ± 2.9 Suramin (0.1 m M ) 106 ± 7.9 103 ± 3.6 112 ± 1.6 105 ± 8.2 Azide (10 m M ) 79 ± 3.6 57 ± 2.6 15 ± 0.4 8 ± 0.8 Fluoride (10 m M ) 98 ± 2.9 88 ± 1.2 9 ± 2.2 3 ± 0.8 Vanadate (1 m M ) 93 ± 2.3 78 ± 1.1 46 ± 3.4 12 ± 2.1 Pyrophosphate (5 m M ) 49 ± 1.9 56 ± 3.3 3 ± 0.4 7 ± 2.5
Trang 6inhibition by azide was of the mixed and uncompetitive type
[31] The mechanism of inhibition of fluoride and vanadate
has not been investigated, but they are unlikely to be
competitive inhibitors
Immunolocalization of the chicken liver ecto-ATPDase
When thin sections of chicken liver were stained with the
chicken ecto-ATPDase monoclonal antibody, MC27, the
protein could be seen to be distributed at the bile canaliculi
(Fig 4) This localization of the ecto-ATPDase agrees with
previous finding of distribution of cell surface ATPase
activity in rat liver as determined by cytochemical staining
[33–36] Besides oviduct and liver, the chicken
ecto-ATPDase is also present in the apical membranes of the
oxyntic-peptic cells [37] The distribution of the
ecto-ATPDase on these epithelial cells is distinctly different from
the other ATPDase in the E-ATPase family, the CD39s
[13,17,19]
Molecular cloning of chicken liver ecto-ATPDase
The results described above indicate that: (a) the enzymatic
properties of the chicken liver ecto-ATPDase are similar to
that of the oviduct ATPDase; (b) the chicken liver
ecto-ATPDase binds strongly to the monoclonal antibodies of
the oviduct ecto-ATPDase, as well as an antibody [21]
developed against the N-terminus of the chicken oviduct
ecto-ATPDase (data not shown); and (c) the deglycosylated
chicken liver and oviduct ecto-ATPDases have the same
molecular mass, i.e. 55 kDa [21] It seems likely that the
two enzymes may have similar primary sequences despite
the different molecular masses of the native enzymes We decided to obtain the cDNA of the chicken liver ecto-ATPDase using RT-PCR starting with chicken liver polyA+ RNA Under the appropriate PCR conditions described in Experimental procedures, a 1.5-kb PCR product was obtained, which was introduced into the cloning vector, pCR2.1, by TA cloning Three separate clones were sequenced using T7 promotor, M13, and gene specific primers, all yielding the same nucleotide sequence (Fig 5, GenBank accession no AF426405) The deduced primary sequence of the chicken liver ecto-ATPDase (Fig 5) is nearly identical to that of the oviduct ecto-ATPDase differing in seven amino acids out of 493 amino acids It has the same two transmembranous domains, five apyrase conserved regions (ACRs), 10 conserved cysteine residues, and 12 potential N-glycosylation sites as the oviduct ecto-ATPDase [21] Northern blot analysis using total chicken liver RNA revealed a major transcript of
2 kb (data not shown)
Expression of the chicken liver ecto-ATPDase cDNA
To verify that the cDNA we obtained encodes an ecto-ATPDase, we carried out transient transfection in both COS-7 and HeLa cells Both host cells express low ectonucleotidase activities, i.e 5–10 nmolÆmin)1Æmg)1 pro-tein with either MgATP or MgADP as the substrates Cells transfected with the chicken liver ecto-ATPDase cDNA displayed 10- to 30-fold higher ATP hydrolysis activities Because the expression level was higher in HeLa cells, which also have a more rapid growth rate than COS cells, we chose HeLa cells for stable transfection in order to characterize the expressed enzyme with respect to azide inhibition and the effect of NP-40 on ATP and ADP hydrolysis Table 3 shows that: (a) in the absence of NP-40, the ATP hydrolysis activities were similar at pH 7.4 and 6.4, whereas ADP hydrolysis activity at pH 6.4 was threefold greater than at
pH 7.4; (b) NP-40 (0.1%) increased both ATP and ADP hydrolysis activities by threefold to fivefold at both pH values; (c) ADPase/ATPase ratios varied between 0.2 and 0.64 depending on the pH and the presence of NP-40; (d)
Fig 3 Inhibition of ATP and ADP hydrolysis activities of purified
chicken liver ecto-ATPDase by azide ATP and ADP hydrolysis
reac-tions were carried out in 0.25-mL reaction mixtures containing 50 m M
Tris/HCl, pH 7.4 or 50 m M Mops, pH 6.4 with 5 m M MgCl 2 , 5 m M
ATP or 5 m M (ADP) in the absence and presence of the indicated
concentrations of sodium azide.MgATP hydrolysis at pH 7.4 (s) and
6.4 (d); MgADP hydrolysis at pH 7.4 (h) and 6.4 (j).
Fig 4 Immunolocalization of ecto-ATPDase in chicken liver Thin sections of chicken liver were stained with monoclonal antibody MC27
as described in Experimental procedures Stained bile canaliculi are indicated by arrowheads Bar ¼ 10 lm.
Trang 7both ATPase and ADPase activities were inhibited by
10 mM azide, but ADPase activity was more sensitive to
azide inhibition than ATP hydrolysis and inhibition in
general was greater at pH 6.4 than at pH 7.4 These characteristics are similar to those displayed by the enzyme
in its native membranes [23] and the purified enzyme described above Figure 6 shows that MC18 detected protein bands of molecular masses of 80–85 kDa in HeLa cells expressing the chicken ecto-ATPDase but not in HeLa cells transfected with the pcDNA3 vector alone
D I S C U S S I O N
The existence of a cell surface ATPase in the bile canaliculi
of liver was first demonstrated by ATPase activity staining
in the 1950s [33,34] Interestingly, this activity was both increased in magnitude and altered in cellular location in rat hepatomas induced by carcinogens [33,35] Early biochemi-cal characterization of an ATPase activity in rat liver microsomes showed that it had the unusual properties of hydrolyzing also UTP and UDP and that hydrolyses of these nucleotides were inhibited by 5 mM azide [38] We found that the extent of azide inhibition of ATPase activity
in different plasma membrane preparations correlated with the ability of the membranes to hydrolyze ADP, and concluded that azide inhibition is a characteristic of a membrane-bound ATP diphosphohydrolase (ATPDase) [30] We also showed that this activity is abundant in liver
Fig 5 Nucleotide sequence of the chicken liver ecto-ATPDase and the
deduced primary sequence Nucleotide numbers are on the left side and
amino-acid residue numbers are on the right side of the figure The
transmembranous domains of the protein at the N- and C-terminus are
shaded The five apyrase conserved regions (ACRs) are underlined and
in bold The 10 cysteine residues conserved in all E-ATPases are
indicated by a bold C with shading Asparagine residues involved in
potential N-glycosylation are indicated by bold italic N The
amino-acid residues that differ between chicken liver and oviduct
ecto-ATPDases are at number 278–280, 316, 357, 461, 462 The different
amino-acid residues in the chicken oviduct ecto-ATPDase are shown
in parentheses following the corresponding amino-acid residue in the
liver ecto-ATPDase.
Table 3 Effect of NP-40 and azide on the ecto-ATPDase activities of HeLa Cells stably transfected by chicken liver ecto-ATPDase cDNA HeLa cells were stably transfected with the chicken liver ecto-ATPDase cDNA as described under Experimental procedures Cells were grown in DMEM containing 10% fetal bovine serum and 400 lgÆmL)1geneticin For ectonucleotidase assays, cells collected after trypsinization were used Enzyme assays were carried out in a 0.5-mL reaction mixture containing either 50 m M Tris/HCl (pH 7.4) or 50 m M Mops (pH 6.4) with 5 m M MgATP or
5 m M MgADP and 1 m M ouabain (inhibitor of the Na + /K + -ATPase) and 0.1 m M sodium azide (inhibitor of the mitochondrial ATPase) with
25 lL of trypsinized cells (50–75 lg cell protein) Reaction was carried out for 30 min at 37 C Duplicate samples were run for each condition Results presented were average (± SD) of three experiments Values are given as lmol P i Æmin)1Æmg protein)1.
Addition
None 0.26 ± 0.04 0.05 ± 0.01 0.24 ± 0.05 0.15 ± 0.05 0.1% NP-40 0.84 ± 0.28 0.28 ± 0.04 0.75 ± 0.22 0.48 ± 0.17
10 m M azide 0.19 ± 0.04 0.01 ± 0.01 0.14 ± 0.03 0.03 ± 0.05 0.1% NP-40 + 10 m M azide 0.59 ± 0.16 0.10 ± 0.02 0.40 ± 0.11 0.06 ± 0.05
Fig 6 Expression of the chicken liver ecto-ATPDase in transiently and stably transfected HeLa cells HeLa cells transfected with vector alone
or chicken liver ecto-ATPDase cDNA were frozen to lyse the cells Cell lysates (25 lg protein) were treated by SDS gel sample buffer without reducing agent, and subjected to SDS/PAGE on 7.5% acrylamide gel Separated proteins were transferred to a poly(vinylidene difluoride) membrane, which was probed with monoclonal antibody MC18 Lane 1, chicken liver plasma membrane; lane 2, lysates of HeLa cells transfected with pcDNA vector; lane 3, lysates of HeLa cells trans-fected with chicken liver ecto-ATPDase cDNA.
Trang 8and several other tissues [30] Subsequently, an ATPase
possessing NTPase and NDPase activities was partially
purified from rat liver [39], and was shown to be responsible
for the E-ATPase activity of intact rat hepatocytes [40]
That the ecto-ATPDase is the major E-ATPase in livers
was supported by our recent study of the E-ATPase activity
in the chicken liver plasma membranes We showed that the
chicken liver E-ATPase differs from the chicken smooth
muscle ecto-ATPase with respect to substrate utilization,
divalent ion requirement, thermal stability, azide inhibition,
and response to a panel of modulators [23] On the other
hand, its enzymatic properties are similar to the chicken
oviduct ecto-ATPDase that we have previously purified and
cloned [7,21] Utilizing antibodies developed for the chicken
oviduct ecto-ATPDase, we achieved the first complete
purification of a liver ecto-ATPDase
The purified chicken liver ecto-ATPDase is an 85-kDa
membrane glycoprotein Although slightly larger than the
chicken oviduct ecto-ATPDase, it is nearly identical to the
chicken oviduct ecto-ATPDase with respect to enzymatic
properties The similar properties of the two chicken
ecto-ATPDases and their cross-reactivities with the same
antibodies suggest that they may have similar primary
sequences in spite of their different molecular masses This
proved to be the case when we obtained the cDNA of the
chicken liver ecto-ATPDase by RT-PCR cloning The two
chicken ecto-ATPDases differ in only seven amino acids out
of 493 amino-acid residues, all located in the C-terminal half
of the molecules that are less conserved in the E-ATPases
[21] There is an additional N-glycosylation site in the liver
ecto-ATPDase These substitutions appear to have
negli-gible effect on the enzymatic properties of the liver
ecto-ATPDase Upon transfection of HeLa cells with the chicken
liver ecto-ATPDase cDNA, expression of the cDNA was
demonstrated both by Western blotting and enzyme
activity The expressed protein still binds the chicken
ecto-ATPDase monoclonal antibody MC18 but there is some
evidence of heterogeneity in glycosylation in HeLa cells
(Fig 6) The expressed enzyme was able to hydrolyze ATP
and ADP, was inhibited by 10 mMazide, and was markedly
stimulated by the detergent NP-40 (Table 3) The response
to detergents constitutes the most striking difference
between the chicken ecto-ATPDases (E-NTPDase III)
and chicken ATPase (E-NDPDase II) The
ecto-ATPases of chicken gizzard, brain, and transverse tubules
are inactivated by most detergents, except digitonin
[4,5,23,27,41,42] In contrast, the chicken ecto-ATPDases
are activated by the same detergents and are extracted from
the membranes by high concentrations of NP-40 [7,23] (and
this study) This study shows unambiguously that the
activity of the chicken liver ecto-ATPDase is increased by
NP-40 even when the protein is expressed in nonliver host
cells, leading to the conclusion that this property is intrinsic
to the chicken liver ecto-ATPDase protein and cannot be
attributed to any specific lipid environment of the liver cell
membranes
The differential effects of detergents on the chicken
ecto-ATPase and ecto-ATPDase are most likely related to the
different regulatory mechanisms of the two enzymes
Compounds that promote oligomer formation, such as
Con A and chemical cross-linking agents increase the
activity of chicken ecto-ATPase [5,23,43], whereas
deter-gents and other amphiphilic molecules, which prevent
oligomer formation of membrane proteins, decrease its activity [27,28] In contrast, the chicken liver ecto-ATPDase activity is not affected by ConA [23] while the chicken stomach ecto-ATPDase is actually markedly inhibited by a chemical cross-linking agent [44] We and others have proposed that ecto-ATPase requires oligomerization for high activity [23,28,43,45], but the chicken ecto-ATPDase does not [23] Interestingly, the CD39s, although also ecto-ATPDases, are adversely affected by detergents [20,46] Studies from Guidotti’s laboratory showed that while the full-length membrane-bound rat CD39/ecto-ATPDase was inhibited by detergents, recombinant forms that lack either
or both transmembranous domains had lower activities and were not inhibited by detergents [46,47] It has been proposed that these domains are involved in the formation
of a tetramer of the enzyme [46]
Our finding that the chicken liver ecto-ATPDase, whether expressed in the native or host cell membrane, is activated
by detergents while unaffected by ConA, indicates that its activity is not dependent on its oligomerization status While
it is likely that the enzyme is fully active as monomers, the possibility that the enzyme has a more detergent-resistant quaternary structure cannot be ruled out at present In either case, it will be of interest to determine if this unusual characteristic can be attributed to its transmembranous domains that have different sequences than those of the chicken smooth muscle ecto-ATPase and rat CD39/ecto-ATPDase However, activation by detergents may be a unique property of the chicken ecto-ATPDases because the ecto-ATPDase activity of the rat liver plasma membranes
is inhibited by NP-40 (A F Knowles, unpublished data) Nevertheless, like the chicken ecto-ATPDases, the rat liver ecto-ATPDase is also inhibited by azide (A F Knowles &
A K Nagy, unpublished data) In contrast, the ecto-ATPDase activity of porcine liver, shown also to be distributed in the bile canaliculi, was reported to be less sensitive to azide inhibition [48,49] Furthermore, analysis of the N-terminal amino-acid sequence of porcine liver ATP-Dase revealed identity to the rat liver lysosomal ATPase [49] suggesting that it may not be a member of the E-ATPase family The possibility that more than one enzyme protein contribute to the overall liver ATPDase activity in different species will require further investigation
The function of the liver ecto-ATPDase is not established While its localization in the bile canaliculi is suggestive of its involvement in bile acid secretion, experimental evidence is tenuous [50] The proposal that it may play a role in purine salvage is more likely because 5¢-nucleotidase, which hydrolyzes AMP to adenosine, is also present in the bile canaliculi [50] The liver ecto-ATPDase may also be important in regulating signaling by extracellular nucleo-tides because they elicit a variety of responses in liver [51] It
is expected then that the protein may be colocalized with a purinergic receptor The molecular identification of a liver ecto-ATPDase should facilitate future investigation of its physiological function
A C K N O W L E D G E M E N T S
This work was supported by the California Metabolic Research Foundation We thank Dr Jacques Perrault for providing the HeLa cells, and Drs Charles Caldwell and Rita Lim for helpful discussions.
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