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-

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

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)

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

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

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

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-

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

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+
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

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]

)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

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

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,

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,

chemical markers for osteoporosis. Calcif. Tissue Int. 59 (Suppl. 1),
S2–S9.
2. Risteli, L. & Risteli, J. (1993) Biochemical markers of bone
metabolism. Ann. Med. 25, 385–393.
3. Nawawi, H., Samson, D., Apperley, J. & Girgis, S. (1996)
Biochemical markers in patients with multiple myeloma. Clinica
Chimica Acta 253, 61–77.
4. Magnusson, P., Larsson, L., Magnusson, M., Davie, M.W.J. &
Sharp, C.A. (1999) Isoforms of bone alkaline phosphatase:
Characterization and origin in human trabecular and cortical
bone. J. Bone Mineral Res. 14, 1926–1933.
5. Whyte, M.P. (1994) Hypophosphatasia and the role of alkaline
phosphatase in skeletal mineralization. Endocr. Rev. 15, 439–461.
6. Narisawa, S., Fro
¨
hlander, N. & Millan, J.L. (1997) Inactivation of
two mouse alkaline phosphatase genes and establishment of a
model of infantile hypophosphatasia. Dev. Dynamics 208,
432–446.
7. Robison, R. (1923) The possible significance of hexosephosphoric
esters in ossification. Biochem. J. 17, 286–293.
8. Noda, M., Yoon, K., Rodan, G.A. & Koppel, D.E. (1987) High
lateral mobility of endogenous and transfected alkaline phospha-
tase: a phosphatidylinositol-anchored membrane protein. J. Cell
Biol. 105, 1671–1677.
9. Pizauro, J.M., Ciancaglini, P. & Leone, F.A. (1994) Osseous plate
alkaline phosphatase is anchored by GPI. Brazilian. J. Med. Res.
27, 453–456.
10. Anderson, H.C. (1995) Molecular biology of matrix vesicles. Clin.
Orthop. Res. 314, 266–280.

22. Pellegrino, E.D. & Blitz, R.M. (1972) Mineralization in chick
embryo. I. Monohydrogen phosphate and carbonate relationship
during maturation of the bone crystal complex. Calcif. Tissue Res.
10, 128–135.
23. Grypnas, M.D., Bauer, L.C. & Glimcher, M.J. (1984) Failure
to detect amorphous calcium phosphate solid phase in bone
mineral: a radial distribution function study. Calcif. Tissue Int. 36,
291–309.
24. Ashton, B.A. & Bromley, L. (1985) The influence of b-glycero-
phosphate on osteoblastic-like cell lines. Bone 7, 305–306.
25. Kuhn,L.T.,Wu,Y.,Rey,C.,Gerstenfeld,L.C.,Grypnas,M.D.,
Ackerman, J.L., Kim, H.M. & Glimcher, M.J. (2000) Structure,
Composition, and maturation of newly deposited calcium-phos-
phate crystals in chicken osteoblast cell cultures. J. Bone Mineral
Res. 15, 1301–1309.
26. Guicheux, J., Palmer, G., Shukunami, C., Hiraki, Y., Bonjour,
J.P. & Caverzasio, J. (2000) A novel in vitro culture system for
analysis of functional role of phosphate transport in endochondral
ossification. Bone 27, 69–74.
27. Wang, D., Canaff, L., Davidson, D., Corluka, A., Liu, H., Hendy,
G.N. & Henderson, J.E. (2001) Alterations in the sensing and
transport of phosphate and calcium by differentiating chon-
drocytes. J. Biol. Chem. 276, 33995–34005.
28. Kirsch, T., Nah, H D., Shapiro, I.M. & Pacifi, M. (1997) Regu-
lated production of mineralization-competent matrix vesicles in
hypertrophic chondrocytes. J. Cell. Biol. 137, 1149–1160.
29. Kirsch, T., Harrison, G., Worch, K.P. & Golub, E.E. (2000)
Regulatory roles of zinc in matrix vesicle-mediated minerali-
zation of growth plate cartilage. J. Bone Mineral Res. 15,
261–270.

(1989) Stimulatory effects of phosphates on bone mineralization
in vitro. Cell. Tissue Res. 257, 555–563.
38. Tennenbaum, H.C., Lineback, H., McCulloch, C.A.G., Mamuju,
H., Sukku, B. & Tarantali, M. (1987) Osteogenic-phase speci-
fic co-regulation of collagen synthesis and mineralization by
b-glycerophosphate in chick periosteal culture. Bone 12, 129–138.
39. Iwamoto, M., Shapiro, I., Yagami, K., Boskey, A.L., Leboy, P.,
Adams, S. & Pacifici, M. (1993) Retinoic acid induces rapid
mineralization and expression of mineralization-related genes in
chondrocytes. Exp. Cell. Res. 207, 413–420.
40. Chang, Y.L., Stanford, C.M. & Keller, J.C. (2000) Calcium and
phosphate supplementation promotes bone cell mineralization:
implications for hydroxyapatite (HA) enhanced bone formation.
J. Biomed. Mater. Res. 52, 270–278.
41. Hsu, H.H. (1992) Further studies on ATP-mediated CA deposi-
tion by isolated matrix vesicles. Bone Mineral 17, 279–283.
42. Radisson, J., Angrand, M., Chavassieux, P., Roux, B. & Azzar, G.
(1996) Differential solubilization of osteoblastic alkaline phos-
phatase from human primary bone cell cultures. Int. J. Biochem.
Cell. Biol. 28, 421–430.
43. Lowry, O.H., Rosebrough, N.I., Farr, A.L. & Randall, R.J. (1951)
Protein measurement with the folin phenol reactant. J. Biol. Chem.
193, 265–273.
44. Frot Coutaz, J. & Got, R. (1971) Proprie
´
te
´
s de troisglucosyltrans-
fe
´

phate to hydroxyapatite. New correlations between X-ray dif-
fraction and infrared data. Calcif. Tissue Int. 58, 9–16.
54. Paschalis, E.P., DiCarlo, E., Betts, F., Shermann, P., Mendelsohn,
R. & Boskey, A.L. (1996) FTIR microscopic analysis of human
osteonal bone. Calcif. Tissue Int. 59, 480–487.
55. Rey, C., Shimizu, M., Collins, B. & Glimcher, M.J. (1991)
Resolution enhanced Fourier transform infrared spectroscopy
study of the environment of phosphate ions in the early deposits of
a solid phase calcium phosphate in bone and enamel, and their
evolution with age: 2. Investigations in the m3PO
4
domain. Calcif.
Tissue Int. 49, 383–388.
56. Rey, C., Shimizu, M., Collins, B. & Glimcher, M.J. (1990)
Resolution enhanced Fourier transform infrared spectroscopy
study of the environment of phosphate ions in the early deposits of
a solid phase of calcium-phosphate in bone and in enamel, and
their evolution with age. I: Investigations in the m4PO
4
domain.
Calcif. Tissue Int. 46, 384–394.
57. Ou-Tang, H., Pachalis, E.P., Boskey, A.L. & Mendelsohn, R.
(2000) Two-dimensional vibrational correlation spectroscopy of
in vitro hydroxyapatite maturation. Biopolymers (Biospectro-
scopy) 57, 129–139.
58. Boskey, A.L., Guidon, P., Doty, S.B., Leboy, P. & Binderman, I.
(1996) The mechanism of b-glycerophosphate action in mineral-
izing chick limb-bud mesenchymal cell cultures. J. Bone Mineral
Res. 11, 1694–1702.
59. Feki, H.E., Rey, C. & Vignoles, M. (1991) Carbonate ions in

66. Chung, C.H., Golub, E.E., Forbes, E., Tokuoka, T. & Shapiro,
I.M. (1992) Mechanism of action of b-glycerophosphate on bone
cell mineralization. Calcif. Tissue Int. 51, 305–311.
Ó FEBS 2003 Bone GPI-bALP, roles in mineralization (Eur. J. Biochem. 270) 2089
67. Mehta, S., Reed, B. & Antich, P. (1995) Effects of high levels of
fluoride on bone formation: an in vitro model system. Biomaterials
16, 97–102.
68. Anagnostou, F., Plas, C., Nefussi, J.R. & Forest, N. (1996)
Role of b-GP-derived Pi in mineralization via ecto-alkaline
phosphatase in cultured fetal calvaria cells. J. Cell. Biochem. 62,
262–274.
69. Rattner, A., Sabido, O., Le, J., Vico, L., Massoubre, C., Frey, J. &
Chamson, A. (2000) Mineralization and alkaline phosphatase in
collagen lattices populated by human osteoblasts. Calcif. Tissue
Int. 66, 35–42.
70. Tenenbaum, H.C., McCulloch, C.A.G., Fair, C. & Birck, C.
(1989) The regulatory effect of phosphates on bone metabolism
in vitro. Cell. Tissue Res. 257, 555–563.
71. Skrtic, D. & Eanes, E.D. (1992) Effect of different phospholipid-
cholesterol membrane compositions on liposome-mediated for-
mation of calcium phosphates. Calcif. Tissue Int. 50, 253–260.
72. Hatori, M., Teixeira, C.C., Debolt, K., Pacifici, M. & Shapiro,
I.M. (1995) Adenine nucleotide metabolism by chondrocytes
in vitro: role of ATP in chondrocyte maturation and matrix
mineralization. J. Cell. Phys. 165, 468–474.
73. Fernley, H.N. (1971) Mammalian alkaline phosphatase. In: The
Enzymes, Vol. IV (ed. Boyer, P.D.). Academic Press, New York,
pp. 417–447.
74. Skrtic, D. & Eanes, E.D. (1994) Effect of 1-hydroxyethylidienne-
1,1-biphosphonate on membrane-mediated calcium phosphate