Báo cáo khoa học: Chick embryo anchored alkaline phosphatase and mineralization process in vitro Influence of Ca 2+ and nature of substrates doc

Chick embryo anchored alkaline phosphatase and mineralization
process
in vitro
Influence of Ca
2+
and nature of substrates
Eva Hamade, Ge
´
rard Azzar, Jacqueline Radisson, Rene
´
Buchet and Bernard Roux
Laboratoire de Physico-Chimie Biologique, UMR CNRS 5013, Universite
´
Claude Bernard, Lyon I, Villeurbanne, France
Bone alkaline phosphatase with glycolipid anchor (GPI-
bALP) from chick embryo femurs in a medium without
exogenous inorganic phosphate, but containing calcium
and GPI-bALP substrates, served as in vitro model of min-
eral formation. The mineralization process was initiated by
the formation of inorganic phosphate, arising from the
hydrolysis of a substrate by GPI-bALP. Several minerali-
zation media containing different substrates were analysed
after an incubation time ranging from 1.5 h to 144 h. The
measurements of Ca/P
i
ratio and infrared spectra permitted
us to follow the presence of amorphous and noncrystalline
structures, while the analysis of X-ray diffraction data
allowed us to obtain the stoichiometry of crystals. The
hydrolysis of phosphocreatine, glucose 1-phosphate, glucose
6-phosphate, glucose 1,6-bisphosphate by GPI-bALP pro-

duced hydroxyapatite in a manner similar to that of
b-glycerophosphate. Several distinct steps in the mineral
formation were observed. Amorphous calcium phosphate
was present at the onset of the mineral formation, then
poorly formed hydroxyapatite crystalline structures were
observed, followed by the presence of hydroxyapatite crys-
tals after 6–12 h incubation time. However, the hydrolysis of
either ATP or ADP, catalysed by GPI-bALP in calcium-
containing medium, did not lead to the formation of any
hydroxyapatite crystals, even after 144 h incubation time,
when hydrolysis of both nucleotides was completed. In
contrast, the hydrolysis of AMP by GPI-bALP led to the
appearance of hydroxyapatite crystals after 12 h incubation
time. The hydroxyapatite formation depends not only on the
ability of GPI-bALP to hydrolyze the organic phosphate but
also on the nature of substrates affecting the nucleation
process or producing inhibitors of the mineralization.
Keywords: anchored bone alkaline phosphatase; minerali-
zation; Ca
2+
; hydroxyapatite.
Alkaline phosphatase (bALP) (EC 3.1.3.1) is one of the
most frequently used biochemical markers of osteoblast
activity [1–4]. The role of bALP in mineral formation was
evidenced in the case of hypophosphatasia, an inheritable
disorder leading to a defective bone formation and a
deficiency of bALP [5]. Mice deficient in the tissue
nonspecific bALP gene mimic a severe form of hypophos-
phatasia, indicating the importance of bALP in initiating
mineral formation [6]. It has been suggested that bALP

could be involved in the mineralization process by hydro-
lyzing organic phosphates to release free inorganic phos-
phate at sites of mineralization [7]. However, it is still not
clear which organic phosphates are hydrolyzed by bALP.
Osseous bALP, localized in the matrix vesicles, exist as
a phosphatidylinositol-glycolipid (GPI) anchored protein
[8,9]. Mineralization is initiated within and at the surface of
extracellular matrix vesicles derived from osteoblasts [10].
Bone is constantly destroyed or resorbed by the osteoclasts
and then replaced by the osteoblasts [11]. Poorly crystal-
line hydroxyapatite [HA/Ca
10
(PO
4
)
6
(OH)
2
;Ca/P
i
molar
ratio ¼ 1.67] is the major component of bone and other
calcified tissues [12]. The maturation of the initial deposits
of a solid phase of calcium phosphate in bone and other
changes that occur with time, were investigated by using
in vitro models, matrix vesicles or osteoblastic cell-cultures
[13–34,34–40]. The most common precursors of HA are
amorphous calcium phosphate [ACP/Ca
3
(PO

4
)
2
, XH
2
O;
Ca/P
i
molar ratio ¼ 1.5] [13–15], brushite [(CaHPO
4
)
3
Æ
2H
2
O; Ca/P
i
molar ratio ¼ 1] [16] and octacalcium phos-
phate [OCP/Ca
8
(PO
4
)
6
H
2
;Ca/P
i
molar ratio ¼ 1.33] [17],
although the presence of OCP, brushite [18,19] was not

reproducible and appeared most likely due to preparative
artefacts [20]. Despite the remaining difficulties in charac-
terizing the composition of mineral samples that initiate
mineralization within osteoblast cell-cultures [25–27,32,33,
35,38,40] and matrix vesicles [28–31,34], these models
permitted a better delineation of the roles of various
proteins such as type III sodium-dependent phosphate
transporter [26], type I collagen [28], type II and X collagen
Correspondence to G. Azzar, Laboratoire de Physico-Chimie
Biologique, UMR CNRS 5013, Universite
´
Claude Bernard,
Lyon 1, 6 rue Victor Grignard, Baˆ timent Euge
`
ne Chevreul,
69622 Villeurbanne Cedex, France.
Fax: + 33 4 72 43 15 43, Tel.: + 33 4 72 44 83 24,
E-mail:
Abbreviations: ACP, amorphous calcium phosphate; bALP, bone
alkaline phosphatase; GPI, phosphatidylinositol-glycolipid;
GPI-bALP, bone alkaline phophatase with glycolipid anchor;
HA, hydroxyapatite; OCP, octacalcium phosphate; pNPP, para-
nitrophenylphosphate; b-GP, b-glycerophosphate.
Enzyme: Alkaline phosphatase (EC 3.1.3.1).
(Received 4 February 2003, accepted 19 March 2003)
Eur. J. Biochem. 270, 2082–2090 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03585.x
[30,32] bALP [29], annexin II [30], annexin V [28,30] and
annexin VI [30]. Very often an organic phosphate com-
pound, b-glycerophosphate (b-GP) was added in the
medium (in vitro models, matrix vesicles and cell cultures)

to stimulate the mineralization process [25,31–34,34–40].
This exogenous organic phosphate compound is a substrate
for bALP, producing an increase in phosphate content in
the medium. To our knowledge, no other organic phosphate
than b-GP was supplemented in the mineralization medium,
with the exception of ATP [41].
The aim of this work was to examine whether other
organic phosphate compounds, present in the cells at
much higher concentrations than b-GP, could be the
substrates for phosphatidylinositol-glycolipid anchored
alkaline phosphatase (GPI-bALP), producing inorganic
phosphate and initiating the mineralization process. Our
results demonstrate that crystal formation obtained in the
presence of GPI-bALP (extracted from chick embryo
femurs), without supplementing exogenous P
i
, depended
on the type of substrates and incubation conditions.
Determination of Ca/P
i
molar ratio, IR spectroscopy and
X-ray analysis revealed that the hydrolysis of substrates,
such as AMP, b-GP, phosphocreatine, glucose 1-phos-
phate, glucose 6-phosphate and glucose 1,6-bisphosphate,
by GPI-bALP, produced inorganic phosphate and initi-
ated the formation of HA. In contrast to these results, the
hydrolysis of other substrates such as ADP and ATP by
GPI-bALP, was not accompanied by the formation of
HA. The nature of the substrates hydrolyzed by GPI-
bALP, under in vitro conditions, can influence the onset

and the formation of HA. Whether this property or age-
related change of organic phosphate content could
influence the first steps of matrix vesicle-induced miner-
alization under in vivo conditions remains to be investi-
gated.
Materials and methods
Materials
Chick embryo femurs were isolated from 17-day-old-eggs
obtained from a local producer. HA, N-octyl b-
D
-gluco-
pyranoside, para-nitrophenylphosphate (pNPP), phospho-
creatine, glucose 6-phosphate, glucose 1-phosphate, glucose
1,6-bisphosphate, nucleotides, protease inhibitors (leupep-
tin, pepstatin, phenylmethylsulfonyl fluoride, benzamidin),
were purchased from Sigma Chemical Co., St. Louis, MO,
USA. AcA 202 and Octyl Sepharose were obtained from
Pharmacia San Diego, CA, USA. All others reagents were
of the highest purity commercially available.
Preparation and purification of femur chick embryo
GPI anchored alkaline phosphatase
Membrane bound femur chick embryo alkaline phospha-
tase (GPI-bALP) was prepared according to the procedure
described by Radisson et al. [42]. Briefly, femurs were
removed immediately, rinsed in phosphate buffer saline
containing 8 m
M
Na
2
HPO

4
, 1.5 m
M
KH
2
PO
4
pH 7.4,
120 m
M
NaCl and 1.35 m
M
KCl. They were homogenized
in 0.1
M
Tris/HCl, pH 8.5, with 1 m
M
MgCl
2
,1l
M
leupeptin, 0.72 l
M
pepstatin, 1 m
M
benzamidin and
0.25 m
M
phenylmethanesulfonyl fluoride, using a Virtis
homogenizer for 5 min at 4 °C. The homogenate obtained

was centrifuged at 90 000 g, for 180 min at 4 °C. GPI-
bALP was extracted from the pellet, with 60 m
M
N-octyl
b-
D
-glucopyranoside for 60 min at 4 °Cin0.1
M
Tris/HCl,
pH 8.5, 1 m
M
MgCl
2
,1l
M
leupeptin, 0.72 l
M
pepstatin
and 1 m
M
benzamidin. After centrifugation (90 000 g,
30 min), in the first step the surfactant-solubilized enzyme
was further purified by gel filtration chromatography (ACA
202) equilibrated in 0.1
M
Tris/HCl pH 8.5, 0.1
M
NaCl and
1m
M

MgCl
2
. In the second step, GPI-bALP was applied on
octyl Sepharose (CL4B) column containing 25 m
M
glycine
pH 9 and 1.5
M
Na
2
S0
4
. Elution was followed by a gradient
of decreasing sodium sulfate concentration (1.5–0
M
)and
increasing 1-propanol concentration (0–40%, respectively).
The GPI anchored enzyme fraction was dialysed against
100 m
M
Tris/HCl pH 7.4 and 1 m
M
MgCl
2
overnight.
Protein determination
Protein concentration was determined by Lowry’s method
[43] after precipitation of proteins by 10% trichloracetic
acid. It was followed by washing the precipitated material
with methanol and methylal/methanol mixture (4 : 1, v/v)

and dissolution in 1
M
NaOH [44] Bovine serum albumin
was used as a standard. A second method was used for small
quantities of proteins based on Bradford’s method [45]
modified by Read and Northcote [46].
Alkaline phosphatase activity
GPI-bALP activity was measured according to the method
of Cyboron and Wuthier [47], at 37 °C by means of a
Uvikon 810 spectrophotometer. The reaction mixture
contained 10 m
M
pNPP and 25 m
M
glycine buffer,
pH 10.4. One unit of GPI-bALP activity (U) is defined as
the amount of enzyme that is required to hydrolyze 1 lmol
of pNPP per min at 37 °C.
Mineralization process
Purified GPI-bALP (0.8 U) was added in a mineralization
solution containing Ca
2+
(2.5, 6, 7.5 and 24 m
M
, respect-
ively), 62.5 l
M
ZnCl
2
, 62.5 l

M
MgCl
2
, 37.5 m
M
NaCl,
0.05% NaN
3
and 25 m
M
Tris, pH 7.3. It was incubated at
37 °C for up to 6 days according to Golub et al. [31] and
Harrrison et al. [48]. However, the mineralization medium
did not contain inorganic phosphate to initiate the
mineralization process. Instead of inorganic phosphate,
12.5 m
M
of substrate for GPI-bALP (either b-GP, glucose
6-phosphate, glucose 1-phosphate, glucose 1,6-bisphos-
phate, AMP, ADP, ATP, or phosphocreatine) was added
in the mineralization medium. Controls without GPI-
bALP were performed under the same conditions. After
incubation at the indicated time, samples were centrifuged
(2000 g, 5 min). The mineral pellet was washed twice with
deionized water, and centrifuged under the same condi-
tions. Another washing was performed with a chloroform/
methanol (2 : 1, v/v) mixture. After centrifugation, pellets
(mineral samples) were dried under N
2
flow. Their calcium/

phosphate ratio, IR spectra and X-ray diffraction were
measured.
Ó FEBS 2003 Bone GPI-bALP, roles in mineralization (Eur. J. Biochem. 270) 2083
Calcium and phosphate determination
Mineral samples were treated with 1% HCl. Calcium was
determined by atomic absorption spectroscopy (Perkins-
Elmer 3110 spectrometer, atomization temperature: 1700–
3151 °C, wavelength: 422.7 nm, nitrous oxide-acetylene
flame). To avoid refractory aggregates, EDTA was added
to the solution to chelate calcium, thereby preventing a
reaction with phosphate. Phosphorus assay was performed
using the method of Chen et al. [49].
Fourier-transform infrared spectroscopic measurements
KBr pellets (100 mg) containing 1–2 mg of mineral samples
obtained during the mineralization process were analysed
using IR spectroscopy. IR spectra were recorded, using a
Nicolet 510 M FTIR spectrometer equipped with a DTGS
detector. Two hundred and fiftysix interferograms were
measured, Fourier transformed to yield infrared spectra at
4cm
)1
resolution. Each IR spectrum is representative of at
least three independent measurements. During data acqui-
sition, the spectrometer was continuously purged with dried
filtered air (Balston regenerating desiccant dryer, model
75-45 12 VDC).
X-ray diffractometry
Mineral samples were analysed with a Siemens D500
diffractometer using cooper Ka radiation and a highly
crystalline mineral hydroxyapatite as a standard. Diffrac-

tometer was equipped with a scintillation counter and a
geometrical goniometer (Bragg–Breteno). Diffraction angle
2h is comprised between 10° and 70°. The voltage and
current intensity were 40 kV and 30 mA, respectively. X-ray
analysis was performed in the Henri Longchambon diffrac-
tometer centre in the University of Lyon I, Villeurbanne,
France.
Results
Evidence of different steps in the GPI-bALP mediated
mineralization: effects of Ca
2+
concentration
To assess the role of Ca
2+
in initiating the mineralization
process, we modelled the biological process by using 0.8 U
of purified GPI-bALP in a mineralization medium without
exogenous phosphate but containing 12.5 m
M
b-GP as
phosphate substrate. GPI-bALP was incubated at 37 °C
in this medium at different concentrations of Ca
2+
for 1.5–
144 h. At various time intervals, the Ca/P
i
ratios were
measured. A theoretical Ca/P
i
molar ratio of 1.67 (which is

the highest among the various calcium-phosphate com-
plexes) corresponds to HA. When 2.5 m
M
Ca
2+
was added
in the incubation medium, no calcium-phosphate minerals
were obtained during 72 h of incubation. The Ca/P
i
ratio
was 0.8 at 144 h, characteristic of a poorly crystalline
structure. When the Ca
2+
concentration increased from
2.5 m
M
to 6–7.5 m
M
, the mineralization began at 48 h. At
144 h the Ca/P
i
ratio was % 1.2–1.3. At 24 m
M
Ca
2+
, HA
crystals were obtained at about 12 h with a Ca/P
i
ratio of
1.5 in the presence of anchored GPI-bALP (Fig. 1). The

presence of HA in the mineral samples was confirmed by
measuring their IR spectra and X-ray diffraction patterns
(see below). In all cases, medium lacking GPI-bALP did
not produce any mineral material.
Influence of other phosphate substrates
in the mineralization process
To examine the effects of substrates catalysed by GPI-bALP
in the production of inorganic phosphate and in the
mineralization process, different phosphate compounds
were used: b-GP, phosphocreatine, phosphate sugars and
nucleotides. In all cases, the mineralization medium without
exogenous phosphate contained 24 m
M
Ca
2+
, 12.5 m
M
phosphate substrate and 0.8 U of GPI-bALP. In the
presence of phosphocreatine, the incubation time necessary
to obtain a Ca/P
i
characteristic of HA was 24 h. Its Ca/P
i
ratio was the highest (Fig. 2A) of those obtained with
different phosphate substrates. When glucose 1-phosphate
was used as a substrate, HA mineral was formed within 12 h
of incubation (Ca/P
i
ratio, 1.32). With glucose 6-phosphate,
glucose 1,6-bisphosphate, the incubation time to produce

HA was longer, i.e. 24 and 72 h, respectively; Ca/P
i
ratios
were, respectively, 1.35 and 1.12 (Fig. 2B). Results obtained
in the presence of nucleotides were different. AMP gave
crystal identified as HA. However, the incubation time
necessary for the mineralization process was at least 72 h
and the Ca/P
i
ratio was 1.38. ATP and ADP did not
produce any organized mineral structure and their Ca/P
i
ratio did not correspond to hydroxyapatite crystals
(Fig. 2Ca,b). Although GPI-bALP from chick embryo
femurs hydrolyzed ATP and ADP, the products of hydro-
lysis did not lead to the formation of HA. No mineral
material was formed in media lacking GPI-bALP.
Mineral calcium phosphate identification by infrared
spectra
To identify the quality and the type of mineral formation
obtained in the mineralization medium during the GPI-
bALP-induced hydrolysis of different phosphate substrates
Fig. 1. Ca/P
i
ratio (m
M
/m
M
) during in vitro mineralization for different
Ca

2+
concentrations in the presence of b-GP as substrate (12.5 m
M
).
Incubation containing equal activities of purified GPI-bALP (0.8 U)
without PO
4
3–
. Mineralization solution is described in Materials and
methods. At each time point (0–144 h), an aliquot was taken and
centrifuged. Ca
2+
and P
i
were determined in the pellets corresponding
to the mineral structure. Symbols corresponding to various Ca
2+
concentrations are: n, Ca
2+
2.5 m
M
; e, Ca
2+
6m
M
; h, Ca
2+
7.5 m
M
; s, Ca

2+
24 m
M
.
2084 E. Hamade et al. (Eur. J. Biochem. 270) Ó FEBS 2003
(b-GP, phosphocreatine, phosphate sugars and nucleo-
tides), mineral deposits were analysed by IR spectroscopy as
a function of incubation time ranging from 1.5 h to 144 h.
The phosphate bands (m1, m3andm4) were used to monitor
the formation of mineral deposits. The absorption in the
550–650 cm
)1
region arises primarily from the antisymmet-
ric P-O bending mode (m4), while the absorption around the
900–1200 cm
)1
region is characteristic of symmetric (m1)
and antisymmetric (m3) P-O stretching mode [12]. Correla-
tions between IR spectral features and phosphate phases are
presented in Table 1. These correlations facilitated the
interpretation of IR spectra of mineral deposits. Distinct
mineralization steps were observed during the GPI-bALP-
induced hydrolysis of b-GP. At incubation times in the
range 1.5–3 h, two bands located at 1053 cm
)1
and
1040 cm
)1
were observed (Fig. 3, top trace). The
1053 cm

)1
band (Fig. 3, top trace) found at early incubation
time suggested the presence of a disordered phosphate
environment, characterized by a 1060 cm
)1
band [50].
Alternatively, this band may correspond to ACP, usually
identified by a m3 band at 1052–1060 cm
)1
[51,52] (Table 1).
The component band, located at 1040 cm
)1
(Fig. 3, top
trace) was tentatively assigned to a m3 vibrational mode that
Fig. 2. Molar Ca/P
i
ratio during in v it ro mineralization in the presence
of 12.5 m
M
phosphate substrates as indicated below and 24 m
M
Ca
2+
.
Experimental conditions are identical to those presented in Fig. 1. (A)
r, 12.5 m
M
creatine phosphate. (B) 12.5 m
M
Phosphate sugars. h,

glucose 1-phosphate; r, glucose 6-phosphate; n, glucose 1,6-bis-
phosphate. (C) (a) n, 12.5 m
M
AMP; r, 12.5 m
M
ADP; (b) r,
12.5 m
M
ATP.
Table 1. Assignment of some spectral features in m1, m3 and m4
phosphate regions, used to interpret infrared spectra of mineral samples.
Mineral complexes
Wavenumber
(cm
)1
) Origin Ref.
HPO
2À
4
1125–1145 HPO
2À
4
stretch [57]
HPO
2À
4
in OCP 918–924 HPO
2À
4
stretch [51]

ACP 1052–1060 m3 [51,52]
Amorphous OCP 587–600 m4 [56]
1038 m3 [53]
Brushite 1127 PO
3
stretch
in HPO
4
2–
[53]
H-bonded OH group 741 OH libration [59,60]
HA 561–563 m4 [57]
575 m4 [57]
601–603 m4 [57]
960 m1 [57]
1032 m3 [57]
1092–1094 m3 [57]
Poorly crystalline HA 1112–1116 m3 [57]
Fig. 3. Time evolution of the m1, m3andm4 bands of the IR spectrum
during mineralization process from the ACP (amorphous calcium phos-
phate) to HA (hydroxyapatite), in the presence of b-glycerophosphate.
For experimental conditions, see Materials and methods.
Ó FEBS 2003 Bone GPI-bALP, roles in mineralization (Eur. J. Biochem. 270) 2085
was not part of the apatite structure as in the case of OCP,
where a component band absorbs at 1038 cm
)1
[53].
However, our X-ray data did not indicate the presence of
crystalline OCP. After 12 h incubation time, HA was
identified. The m3 bands shifted to 1031–1032 cm

)1
and to
1092 cm
)1
(Fig. 3, middle and bottom traces), typical for
HA [54,55] (Table 1). In addition, m4 bands were resolved
into two distinct peaks located at 563 cm
)1
and 602 cm
)1
,
with a shoulder around 575 cm
)1
(Fig. 3), confirming the
presence of HA [50,56,57]. The appearance of a resolved m1
band located at 960 cm
)1
(Fig. 3) is indicative of crystallized
HA [50] but its intensity is not proportional to crystal size
[50,58]. The 741 cm
)1
band (top traces in Fig. 3) that
disappeared once crystalline apatites were formed is prob-
ably associated with hydrogen-bonded OH libration [59,60]
(Table 1). The mineral formations induced by the GPI-
bALP-induced hydrolysis of phosphocreatine, glucose
1-phosphate, glucose 6-phosphate, glucose 1,6-bisphosphate
as a function of incubation time were similar to that
produced by the GPI-bALP-induced hydrolysis of b-GP. In
all cases, the appearance of characteristic 1108–1092 cm

)1
,
1032–1031 cm
)1
, 960 cm
)1
, 602–601 cm
)1
and 563–
561 cm
)1
bands (Fig. 4A–D), confirmed the presence of
HA [50,56,57]. Nucleotides as phosphorous substrates can
also be hydrolyzed by alkaline phosphatase. There was no
crystalline HA formation during the GPI-bALP-induced
hydrolysis of ATP (Fig. 5A) or ADP (Fig. 5B) even after
144 h incubation and complete hydrolysis of nucleotide by
GPI-bALP. However, formation of HA was observed at
around 144 h during hydrolysis of AMP by GPI-bALP
(Fig. 5C). The formation of HA induced by hydrolysis of
AMP by GPI-bALP was relatively slow. ADP hydrolysis by
GPI-bALP in the mineralization medium, produced inor-
ganic phosphate which led to a mineral deposit. The IR
spectrum of the mineral deposit was different from that of
HA. Two bands located at 558 cm
)1
and at 918 cm
)1
(Fig. 5B) suggested the presence of HPO
4

2–
group in OCP
[61] (Table 1). The other bands may reveal different types of
calcium-phosphate deposits. ATP hydrolysis by GPI-bALP
in the mineralization medium produced a mineral deposit
containing also HPO
4
2–
. The IR spectrum of this mineral
deposit exhibited two bands located at 1122 cm
)1
and
918 cm
)1
bands (Fig. 5A). As mentioned above, the
918 cm
)1
band may correspond to HPO
4
2–
group in OCP.
The 1122 cm
)1
band (Fig. 5A) may be associated with an
Fig. 4. IR spectra of mineral samples obtained after 144 h incubation in
the mineralization medium containing: (A) 12.5 m
M
creatine phosphate;
(B) 12.5 m
M

glucose 6-phosphate; (C) 12.5 m
M
glucose 1-phosphate; (D)
12.5 m
M
glucose 1,6-biphosphate. For experimental conditions, see
Materials and methods.
Fig. 5. IR spectra of mineral samples obtained after 144 h incubation in
the mineralization medium containing: (A) 12.5 m
M
ATP; (B) 12.5 m
M
ADP; (C) 12.5 m
M
AMP. For experimental conditions, see Materials
and methods.
2086 E. Hamade et al. (Eur. J. Biochem. 270) Ó FEBS 2003
acid-phosphate environment or brushite (Table 1). The
556 cm
)1
band and the faint shoulder at 600 cm
)1
(Fig. 5A)
suggested the presence of octacalcium phosphate [56] in
addition to other types of complexes. Based on the inter-
pretation of its IR spectrum, the mineral deposit obtained
after hydrolysis of ATP contains a mixture of distinct types
of mineral formation such as brushite, OCP and other
phosphate species. Indeed, its IR spectrum is different
from the IR spectrum of a single chemical species such

as brushite, OCP or amorphous phosphate [50,51,56,62,63].
X-ray diffraction analysis
Mineral crystalline structures were also identified by X-ray
diffraction, after 144 h incubation of one of the eight
phosphate substrates (12.5 m
M
), in mineralization medium
containing calcium (24 m
M
) in the presence of purified chick
embryo GPI-bALP. X-ray spectra corresponding to 144 h
incubation time are presented in Fig. 6. The data shown are
in agreement with those obtained by IR spectra. Indeed,
crystalline structures observed after 144 h incubation of one
of these substrates, b-GP (Fig. 6A), phosphocreatine
(Fig. 6A), glucose 1-phosphate (Fig. 6B), glucose 6-phos-
phate (Fig. 6B), glucose 1,6-bisphosphate (Fig. 6B) were
unambiguously identified as HA in comparison with HA
standard. The 144 h incubation of the mineralization
medium containing either ATP or ADP, as a putative
phosphate donor, did not induce any organized crystalline
structure, as evidenced by the lack of X-ray characteristic
bands of HA (Fig. 6C). In contrast to this result, when
AMP was present in the mineralization medium and after
144 h incubation, the X-ray spectrum revealed the presence
of crystalline HA (Fig. 6C).
Discussion
Organic phosphate compounds that induce
mineralization
b-GP has been routinely supplemented in bone cells,

chondrocytes and cultured cell types to initiate mineraliza-
tion and to better monitor different aspects of mineral
formation [25,31–40,58,64–68]. The mechanism of minerali-
zation caused by the addition of b-GP is related to several
factorssuchashydrolysisofb-GPbyGPI-bALPtoincrease
the concentration of inorganic phosphate [31,40,58,66,68],
blocking the activity of a phosphorylated phosphatase [58]
or affecting other biological events [68]. Addition of
exogenous phosphate was often supplemented [26,28–
31,40,58,59,69–71] to facilitate the mineralization. Hexose
phosphate esters [7], phosphoethanolamine [72] and ATP
[41] could induce mineral formation; however, they were
usually not routinely supplemented in the mineralization
medium. The hydrolysis of glucose 1-phosphate, glucose
6-phosphate, glucose 1,6-bisphosphate and phosphocrea-
tine, by GPI-bALP in the mineralization medium produced
HA, in a similar manner as during the hydrolysis of b-GP by
GPI-bALP. These results suggest that supplementation of
phosphate compounds other than b-GP, may be a valuable
tool to better monitor the activity of different enzymes
participating in the mineralization process. Whether these
organic phosphates are substrates for GPI-bALP in vivo
remains to be evaluated.
Organic phosphate compounds that inhibit
mineralization
Nucleotides can be released in extracellular medium from
chondrocytes and can be rapidly degraded [72]. Under our
in vitro conditions, formation of HA was observed during
hydrolysis of AMP by GPI-bALP, at a slower rate as
compared with the other organic phosphate substrates. The

relative rate of hydrolysis of AMP by the enzyme is identical
to that of glucose 1-phosphate (value from mammalian
bALP [73]), suggesting that the rate of hydrolysis is not the
limiting factor for mineral formation. There was no
formation of any HA after 144 h incubation time in the
presence of either ADP or ATP in hydrolysing medium
containing GPI-bALP and excess of calcium (to compen-
sate the complexation of calcium by the phosphate within
nucleotides). The relative rate of hydrolysis of ADP was
Fig. 6. X-ray spectra of mineral samples obtained after 144 h incubation
in the mineralization medium. Themediumcontained(A)toptrace:
12.5 m
M
b-GP; bottom trace: 12.5 m
M
creatine phosphate; (B)
12.5 m
M
phosphate sugars: top trace: glucose 6-phosphate (G6P);
middle trace: glucose 1-phosphate (G1P); bottom trace: glucose 1,6-
bisphosphate (G1–6P); (C) 12.5 m
M
nucleotides: top trace: AMP,
middle trace: ADP, bottom trace: ATP. For experimental conditions,
see Materials and methods.
Ó FEBS 2003 Bone GPI-bALP, roles in mineralization (Eur. J. Biochem. 270) 2087
comparable to that of b-GP (1.13 vs. 1.31 for the
mammalian enzyme) while that of ATP was about 0.37
[73]. Although ADP and ATP were completely hydrolyzed
by GPI-bALP and were not inhibitors of GPI-bALP,

different types of mineral deposits such as OCP and HPO
4
2–
complexes of calcium were obtained. The lack of HA
formation is probably associated with the presence of the
pyrophosphate group within the two nucleotides, as pyro-
phosphate [36] as well as organic biphosphonates [74–76]
are known inhibitors of HA formation. Our findings are
consistent with the observation [77] that a specific ATPase,
rather than alkaline phosphatase is responsible for the ATP
dependent mineral deposits within matrix vesicles [77].
Possibly, the undefined Ca- and Pi- deposits [41,77] may
contain complexes other than HA, which could result from
ATP hydrolysis by GPI-bALP.
Conclusions
Our results indicate that the formation of HA does not
depend uniquely on the ability of GPI-bALP to hydrolyze
the organic phosphate substrate, suggesting that a nucle-
ating mechanism or interactions with the products of
hydrolysis are important factors that may affect the
mineralization process.
Acknowledgements
The authors thank Professor Slawomir Pikula for helpful scientific
discussions and for revision of the text. The authors gratefully
acknowledge the careful X-ray diffraction measurements by Dr Ruben
Vera. We also thank Dr John Carew for reviewing the English
manuscript. This work is part of PhD Thesis of Mrs Eva Hamade who
is supported by CNRS UMR 5013.
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