11.6 Regulation of Amorphous Calcium Carbonate
Calcium carbonate has three crystal polymorphs; calcite, aragonite, and vaterite in
the order of thermodynamic stability. Calcifying organisms do not necessarily
adopt the most stable calcite but often form unstable aragonite or vaterite.
Crustaceans usually deposit calcium carbonate as an amorphous form (Lowenstam
and Weiner 1989; Addadi et al. 2003). Amorphous calcium carbonate (ACC) is the
least stable form and is apt to be converted to more stable crystal. There are two
types of ACC; one is stable ACC which is maintained for a long time and the
other is a transition state to the crystalline form (Aizenberg et al. 2003). Crustacean
ACC belongs to the former type. Crustaceans may have an unknown mechanism to
induce and maintain ACC. ACC is more soluble than any other crystalline forms of
calcium carbonate. It seems reasonable that crustaceans adopt ACC as a
precipitating form, because calcium carbonate in the cuticle and gastroliths should
be dissolved easily before and after molting, respectively, as mentioned previously.
Previous works reported that crustacean calcified exoskeleton contains phosphorus, which may be related to the stability of ACC (Simkiss and Wilbur 1989).
Indeed, an in vitro experiment indicated that phosphate has an ability to inhibit
crystallization and stabilize ACC at high pH (Hikida et al. 2003). We have recently
tried to identify phosphorus-containing compounds from exoskeleton and
gastroliths. They were decalcified with a dilute acetic acid solution, and the
resulting solution was separated by ultrafiltration into two fractions, a highmolecular-weight fraction (>10 kDa) and a low-molecular-weight fraction
(<10 kDa). Both fractions were assessed for ACC inducing ability in vitro. The
result showed that the low-molecular-weight fraction induced ACC at a lower
concentration. The low-molecular-weight fraction was then passed through a cation-exchange column, from which a flow-through fraction containing phosphorus
compounds was obtained.
31 P NMR spectra of these fractions showed that they both
contained phosphate, phosphoenolpyruvate (PEP) and 3-phosphoglycerate (3PG)
with different proportions. The latter two compounds were chemically identified by
1 H and
13 C NMR, and two-dimensional NMR spectral analyses combined with
mass spectral analyses (unpublished results). These two compounds inhibited
crystallization of calcium carbonate and stabilized ACC at the concentration of
1 mM in vitro.
Interestingly, both PEP and 3-PG are intermediates of glycolysis and therefore
occur in every cell. Considering the large amount of PEP and 3-PG in the gastroliths
and cuticle, metabolism is changed in the gastrolith disk probably by the function
of the molting hormone and in the epithelial cells after molting, respectively. A part
of these compounds in the cell seem to be actively secreted by the gastrolith
disk cells and epithelial cells of the exoskeleton by an unknown mechanism.
These compounds were more potent than phosphate in induction and maintenance
of ACC, perhaps because they have an additional carboxyl group, possibly serving
as a chelator in conjunction with a phosphate group.
326
H. Nagasawa
Calcium carbonate has three crystal polymorphs; calcite, aragonite, and vaterite in
the order of thermodynamic stability. Calcifying organisms do not necessarily
adopt the most stable calcite but often form unstable aragonite or vaterite.
Crustaceans usually deposit calcium carbonate as an amorphous form (Lowenstam
and Weiner 1989; Addadi et al. 2003). Amorphous calcium carbonate (ACC) is the
least stable form and is apt to be converted to more stable crystal. There are two
types of ACC; one is stable ACC which is maintained for a long time and the
other is a transition state to the crystalline form (Aizenberg et al. 2003). Crustacean
ACC belongs to the former type. Crustaceans may have an unknown mechanism to
induce and maintain ACC. ACC is more soluble than any other crystalline forms of
calcium carbonate. It seems reasonable that crustaceans adopt ACC as a
precipitating form, because calcium carbonate in the cuticle and gastroliths should
be dissolved easily before and after molting, respectively, as mentioned previously.
Previous works reported that crustacean calcified exoskeleton contains phosphorus, which may be related to the stability of ACC (Simkiss and Wilbur 1989).
Indeed, an in vitro experiment indicated that phosphate has an ability to inhibit
crystallization and stabilize ACC at high pH (Hikida et al. 2003). We have recently
tried to identify phosphorus-containing compounds from exoskeleton and
gastroliths. They were decalcified with a dilute acetic acid solution, and the
resulting solution was separated by ultrafiltration into two fractions, a highmolecular-weight fraction (>10 kDa) and a low-molecular-weight fraction
(<10 kDa). Both fractions were assessed for ACC inducing ability in vitro. The
result showed that the low-molecular-weight fraction induced ACC at a lower
concentration. The low-molecular-weight fraction was then passed through a cation-exchange column, from which a flow-through fraction containing phosphorus
compounds was obtained.
31 P NMR spectra of these fractions showed that they both
contained phosphate, phosphoenolpyruvate (PEP) and 3-phosphoglycerate (3PG)
with different proportions. The latter two compounds were chemically identified by
1 H and
13 C NMR, and two-dimensional NMR spectral analyses combined with
mass spectral analyses (unpublished results). These two compounds inhibited
crystallization of calcium carbonate and stabilized ACC at the concentration of
1 mM in vitro.
Interestingly, both PEP and 3-PG are intermediates of glycolysis and therefore
occur in every cell. Considering the large amount of PEP and 3-PG in the gastroliths
and cuticle, metabolism is changed in the gastrolith disk probably by the function
of the molting hormone and in the epithelial cells after molting, respectively. A part
of these compounds in the cell seem to be actively secreted by the gastrolith
disk cells and epithelial cells of the exoskeleton by an unknown mechanism.
These compounds were more potent than phosphate in induction and maintenance
of ACC, perhaps because they have an additional carboxyl group, possibly serving
as a chelator in conjunction with a phosphate group.
326
H. Nagasawa