The cationic composition and pH in the moulting fluid of Porcellio scaber (Crustacea, Isopoda) during calcium carbonate deposit formation and resorption

Before moulting, terrestrial isopods resorb calcium carbonate (CaCO₃) from the posterior cuticle and store it in sternal deposits. These consist mainly of amorphous calcium carbonate (ACC) spherules that develop within the ecdysial space between the anterior sternal epithelium and the old cuticle. Ions that occur in the moulting fluid, including those required for mineral deposition, are transported from the hemolymph into the ecdysial space by the anterior sternal epithelial cells. The cationic composition of the moulting fluid probably affects mineral deposition and may provide information on the ion-transport activity of the sternal epithelial cells. This study presents the concentrations of inorganic cations within the moulting fluid of the anterior sternites during the late premoult and intramoult stages. The most abundant cation is Na⁺ followed by Mg²⁺, Ca²⁺ and K⁺. The concentrations of these ions do not change significantly between the stages whereas the mean pH changed from 8.2 to 6.9 units between mineral deposition in late premoult, and resorption in intramoult, respectively. Measurements of the transepithelial potential show that there is little driving force for passive movements of calcium across the anterior sternal epithelium. The results suggest a possible role of magnesium ions in ACC formation, and a contribution of pH changes to CaCO₃ precipitation and dissolution.     

Effects of Organic Matter on Solubilities and Crystal Form of Carbonates

The results of a study of the role of organic compounds in theformation of carlxmate crystals in marine biological systemsare reported. In an increasing concentration of certain organiccompounds which complex calcium ions, the proportion of aragonitedecreases and that of calcite increases. In increasing concentrationsof magnesium ions the proportion of aragonite increases andthat of calcite and vaterite decreases. When the influence oforganic compounds is greater or smaller than that of magnesiumions, only calcite or only aragonite is formed, respectively.Organic compounds forming a strong complex with calcium ionscause the formation of magnesium-rich calcite, and with an increasein temperature and the concentration of magnesium ions, themagnesium carbonate content of precipitated magnesian calciteincreases. When the influence of organic compounds is almostequivalent to that of magnesium ions, in increasing or decreasingtemperatures, the proportion of calcite decreases or increases,respectively, and the proportion of aragonite increases or decreases,respectively. The concentration of magnesium ions in the bodyfluids of marine calcareous organisms seems to differ littlefrom that of other organisms, and seems to be similar to thatof sea water. Only the presence of certain organic compoundsbrings about the formation of the carbonate crystals observedin marine biological systems. The very important role of organicmatter in the formation of crystals found in skeletal carbonatesis emphasized.     

Carbonate Precipitation of Bacterial Strains Isolated from Sediments and Seawater: Formation Mechanisms

This article presents a research study on carbonate formation in solid and liquid media by Thalassospira sp., Halomonas sp., Bacillus pumilus, and Pseudomonas grimontii, four bacterial strains isolated from sediments and deep seawater. As part of this study, we analyzed carbonic anhydrase activity, pH, adsorption of calcium and magnesium ions, and total organic and inorganic carbon. The geochemical program PHREEQC was also used to calculate the mineral saturation indexes in all the cultures. The minerals formed were studied with X-ray diffraction, X-ray dispersive energy microanalysis, and scanning electron microscopy. In addition, all four bacterial strains were found to induce carbonate precipitation and to have carbonic anhydrase activity. Sterile control experiments did not precipitate carbonate. In solid M1 and B4 media, all of the strains precipitated magnesium calcite, whereas in the liquid media, they precipitated different percentages of magnesium calcite, aragonite, and monohydrocalcite. In both cases, small amounts of amorphous precipitates were also produced. This article discusses carbonate formation and the possible role played by metabolic activity, bacterial surfaces and carbonic anhydrase in this process. Finally, the results obtained lead to a hypothesis regarding the importance of carbonate precipitation for the survival of bacteria populations in certain habitats.     

How corals made rocks through the ages

Hard, or stony, corals make rocks that can, on geological time scales, lead to the formation of massive reefs in shallow tropical and subtropical seas. In both historical and contemporary oceans, reef‐building corals retain information about the marine environment in their skeletons, which is an organic–inorganic composite material. The elemental and isotopic composition of their skeletons is frequently used to reconstruct the environmental history of Earth's oceans over time, including temperature, pH, and salinity. Interpretation of this information requires knowledge of how the organisms formed their skeletons. The basic mechanism of formation of calcium carbonate skeleton in stony corals has been studied for decades. While some researchers consider coral skeletons as mainly passive recorders of ocean conditions, it has become increasingly clear that biological processes play key roles in the biomineralization mechanism. Understanding the role of the animal in living stony coral biomineralization and how it evolved has profound implications for interpreting environmental signatures in fossil corals to understand past ocean conditions. Here we review historical hypotheses and discuss the present understanding of how corals evolved and how their skeletons changed over geological time. We specifically explain how biological processes, particularly those occurring at the subcellular level, critically control the formation of calcium carbonate structures. We examine the different models that address the current debate including the tissue–skeleton interface, skeletal organic matrix, and biomineralization pathways. Finally, we consider how understanding the biological control of coral biomineralization is critical to informing future models of coral vulnerability to inevitable global change, particularly increasing ocean acidification.     

Biomineralization by photosynthetic organisms: Evidence of coevolution of the organisms and their environment?

Biomineralization is widespread among photosynthetic organisms in the ocean, in inland waters and on land. The most quantitatively important biogeochemical role of land plants today in biomineralization is silica deposition in vascular plants, especially grasses. Terrestrial plants also increase the rate of weathering, providing the soluble substrates for biomineralization on land and in water bodies, a role that has had global biogeochemical impacts since the Devonian. The dominant photosynthetic biomineralizers in today's ocean are diatoms and radiolarians depositing silica and coccolithophores and foraminifera depositing calcium carbonate. Abiotic precipitation of silica from supersaturated seawater in the Precambrian preceded intracellular silicification dominated by sponges, then radiolarians and finally diatoms, with successive declines in the silicic acid concentration in the surface ocean, resulting in some decreases in the extent of silicification and, probably, increases in the silicic acid affinity of the active influx mechanisms. Calcium and bicarbonate concentrations in the surface ocean have generally been supersaturating with respect to the three common calcium carbonate biominerals through geological time, allowing external calcification as well as calcification in compartments within cells or organisms. The forms of calcium carbonate in biominerals, and presumably the evolution of the organisms that produce them, have been influenced by abiotic variations in calcium and magnesium concentrations in seawater, and calcium carbonate deposition has probably also been influenced by carbon dioxide concentration whose variations are in part biologically determined. Overall, there has been less biological feedback on the availability of substrates for calcification than is the case for silicification.     

Calcification in insects: The dwelling-tube and midgut of machaerotid larvae (Homoptera)

The dwelling-tubes of machaerotid larvae consist of a mineralized organic scaffolding of mucofibrils. The mineral component accounts for 85 per cent of the dry weight and is composed of calcium, ferrous iron, manganese, magnesium, potassium, sodium, phosphate, carbonate, and chloride and of these the major ions are calcium and carbonate. Ferric iron in the form of ferritin is probably also present.Calcium, manganese, magnesium, and phosphate are derived from spherites secreted by a specialized region of the midgut. Calcium and phosphate are present in the spherites, probably as amorphous tricalcium phosphate. Subsequent to secretion the spherites are slowly dissolved and the calcium is incorporated into the dwelling-tube as calcium carbonate. Thus it appears that within the dwelling-tube calcium phosphate is converted to calcium carbonate.Ferritin and ferrous iron are secreted by another specialized region of the midgut and are also incorporated into the dwelling-tube.     

Biomineralization Studies on Cellulose Membrane Exposed to Biological Fluids of Anodonta cygnea

The present work proposes to analyse the results obtained under in vitro conditions where cellulose artificial membranes were incubated with biological fluids from the freshwater bivalve Anodonta cygnea. The membranes were mounted between two half ‘Ussing chambers’ with different composition solutions in order to simulate epithelial surfaces separating organic fluid compartments. The membrane surfaces were submitted to two synthetic calcium and phosphate solutions on opposite sides, at pH 6.0, 7.0 or 9.0 during a period of 6 hours. Additional assays were accomplished mixing these solutions with haemolymph or extrapallial fluid from A. cygnea, only on the calcium side. A selective ion movement, mainly dependent on the membrane pore size and/or cationic affinity, occurred with higher permeability for calcium ions to the opposite phosphate chamber supported by calcium diffusion forces across the cellulose membrane. In general, this promoted a more intense mineral precipitation on the phosphate membrane surface. A strong deposition of calcium phosphate mineral was observed at pH 9.0 as a primary layer with a homogeneous microstructure, being totally absent at pH 6.0. The membrane showed an additional crystal phase at pH 7.0 exhibiting a very particular hexagonal or cuttlebone shape, mainly on the phosphate surface. When organic fluids of A. cygnea were included, these crystal forms presented a high tendency to aggregate under rosaceous shapes, also predominantly in the phosphate side. The cellulose membrane was permeable to small organic molecules that diffused from the calcium towards the phosphate side. In the calcium side, very few similar crystals were observed. The presence of organic matrix from A. cygnea fluids induced a preliminary apatite–brushite crystal polymorphism. So, the present results suggest that cellulose membranes can be used as surrogates of biological epithelia with preferential ionic diffusion from the calcium to the phosphate side where the main mineral precipitation events occurred. Additionally, the organic fluids from freshwater bivalves should be also thoroughly researched in the applied biomedical field, as mineral nucleators and crystal modulators on biosynthetic systems.     

Polyanion-mediated mineralization — a kinetic analysis of the calcium-carrier hypothesis in the phytoflagellatePleurochrysis carterae

Summary Polyanions are postulated intermediates in biomineralization because they sequester large numbers of calcium ions and occur in high concentrations at mineralizing foci in distantly related organisms. In this study mineral ion and polyanion metabolism was examined inPleurochrysis carterae to determine whether polyanions function as intermediate calcium-carriers during coccolith (mineralized scale) formation. In this organism mineralization occurs intracellularly in coccolith-forming saccules, and mature coccoliths are extruded through the plasma membrane into the coccosphere. The polyanions (acidic polysaccharides known as PS-1 and PS-2) are synthesized in medial Golgi cisternae and transported to the coccolith-forming saccule prior to the onset of mineral deposition; they also cover the mineral surface of mature coccoliths. Pulse-chase experiments with⁴⁵Ca²⁺ and¹⁴CO₃ ^– show the calcium uptake into the coccolith-forming saccule is much slower than carbonate uptake. The extended intracellular half-life of calcium ions destined for the coccosphere suggests that calcium is initially sequestered in more distal Golgi elements (perhaps in association with the polyanions) and enters the coccolith-forming saccule only after passage through the endomembrane system. This is consistent with previous cytochemical studies showing that the polyanions are complexed with calcium prior to mineral deposition. It has been suggested that polyanions may be degraded at the mineralization front in order to free calcium ions for precipitation with available carbonate or phosphate ions. However, this study demonstrates that the polyanions are not degraded; essentially all PS-1 and PS-2 are eventually secreted with the mineral phase into the coccosphere. The kinetics of mineral ion and polyanion secretion are consistent with a polyanion-mediated calcium transport; however, the manner in which calcium might be sequestered by and freed from the polyanions is still obscure.Abbreviations PS-1/2/3 polysaccharide 1/2/3 - EDTA ethylenediaminetetraacetic acid - TCA trichloroacetic acid     

The role of calcium in buffering soils

Abstract. Calcium is the soluble cation that occurs in largest amount in most soils. It does not take part directly in the proton transfer reactions involved in pH-buffering, but it provides the cation charge balance for these reactions. It is also the complementary cation in formulations of chemical potential for many other ions in soils. The presence of free calcium carbonate in calcareous soils. The presence of free calcium carbonate in calcareous soils ensures a very high soil buffer capacity; d AB/ d pH ≃ 1000 Eq. m⁻³.
In acid mineral soils, dissolution and precipitation of aluminium ions contribute to the buffering processes, but most of the buffering in non-calcareous soils is caused by specific ion adsorption at variable-charge sites, in particular those associated with the dissociation of humus acids. Typical buffer capacity values of non-calcareous soils vary from 10 Eq. m⁻³ for sandy soils to 100 Eq. m⁻³ for peats. The pH changes associated with buffering are produced by leaching of calcium from soil, or by adding calcium to soil in liming materials.     

Coral calcification: autoradiography of a scleractinian coral Galaxeafascicularis after incubation in 45Ca and 14C

 The uptake of ⁴⁵Ca and/or ¹⁴C by the skeleton of coral colonies has been commonly used to investigate the processes of calcification. This study reports the differential uptake of these tracers within different regions of the skeleton and tissues of individual corallites and polyps of the hermatypic coral Galaxea fascicularis. Incubation in ⁴⁵Ca in the light resulted in 80 percent of the ⁴⁵Ca taken up being deposited in the skeleton. Autoradiography of transverse and longitudinal slices of freeze-substituted polyps and corallites showed that in the light ⁴⁵Ca was incorporated into the exsert septa, the outside of the thecal walls of the corallite and the inner edges of the septa. Incorporation did not occur in the costae. The radioactivity in the skeleton was considerably greater than in the tissues. In the dark, or in the presence of the photosynthetic inhibitor Diuron, ⁴⁵Ca was taken up by the exsert septa and was patchily distributed in the corallite walls which suggests that it was not a result of isotopic exchange. The differential incorporation of ⁴⁵Ca onto the exsert septa was confirmed by scintillation counting. Negligible radioactivity remained in the extrathecal coelenteron after a brief 5 min rinse in non-radioactive seawater. Only 0.1% of ¹⁴C taken up in the light was incorporated into the skeleton and this was confirmed by autoradiography. In the presence of Diuron or in the dark, very little ¹⁴C was incorporated into tissues or skeleton and in autoradiographs was either not evident in the skeleton or the distribution was similar to that seen in autoradiographs of ⁴⁵Ca uptake. These results show that the deposition of ⁴⁵Ca, and therefore calcium carbonate, occurs at specific loci on the skeleton of a corallite. In the dark, deposition occurs specifically at the growing points of the corallite. Differential deposition of calcium carbonate within individual corallites has not been previously reported. Accepted: 27 May 1997     

Carbonic anhydrases in anthozoan corals—A review

Coral reefs are among the most biologically diverse and economically important ecosystems on the planet. The deposition of massive calcium carbonate skeletons (biomineralization or calcification) by scleractinian corals forms the coral reef framework/architecture that serves as habitat for a large diversity of organisms. This process would not be possible without the intimate symbiosis between corals and photosynthetic dinoflagellates, commonly called zooxanthellae. Carbonic anhydrases play major roles in those two essential processes of coral’s physiology: they are involved in the carbon supply for calcium carbonate precipitation as well as in carbon-concentrating mechanisms for symbiont photosynthesis. Here, we review the current understanding of diversity and function of carbonic anhydrases in corals and discuss the perspective of theses enzymes as a key to understanding impacts of environmental changes on coral reefs.     

Reproducing Authigenic Carbonate Precipitation in the Hypersaline Lake Acıgöl (Turkey) with Microbial Cultures

In the present study, laboratory precipitation experiments using similar water chemistry and two different bacterial cultures from Lake Ac?göl sediments, a hypersaline lake in Turkey, were performed to reproduce mineral assemblages similar to those found in the lake. Two different bacterial cultures induce various calcium/magnesium carbonates precipitation under all the experimental conditions (solid vs. liquid): Hydromagnesite, dypingite, huntite, monohydrocalcite, and aragonite. The geochemical program PHREEQC was used to calculate the mineral saturation indexes in the cultures and in lake water. Carbonate mineral assemblages identified in the experiments seem to be independent of the type of microorganisms but rather controlled by the chemical composition and physical conditions of the media. The relative amounts of monohydrocalcite, hydromagnesite, and dypingite are controlled by varying sulfate concentration from 0 to 56 mM. This demonstrates a kinetic effect that could similarly affect the mineral assemblage in the lake. Also the spherical morphology of hydromagnesite points to growth of these minerals under partial inhibition in the brine under high concentrations of ions and organic polymers produced by the microbial communities. As reproduced by the culture experiments, the authigenic carbonate mineral assemblage of Lake Ac?göl most likely results from interplay of ionic composition of the brine and microbial effects.     

Carbonate and Phosphate Precipitation by Chromohalobacter marismortui

The ability of Chromohalobacter marismortui to precipitate carbonate and phosphate minerals has been demonstrated for the first time. Mineral precipitation in both solid and liquid media at different salts concentrations and different magnesium/calcium ratios occurred whereas crystal formation was not observed in the control. The precipitated minerals were studied by X-ray diffraction, scanning electron microscopy and EDX, and were different in liquid and solid media. In liquid media aragonite, struvite, vaterite and monohydrocalcite were precipitated forming crystals and bioliths. Bioliths accreted preferentially close to organic pellicles, whereas struvite preferentially grows in microenvironments free of such pellicles. Magnesian calcite, calcian-magnesian kutnahorite, “proto-dolomite” and huntite were formed in solid media. The Mg content of the magnesian calcite and of Ca-Mg kutnahorite also varied depending on the salt concentration of the culture media. This is the first report on bacterial precipitation of Ca-Mg kutnahorite and huntite in laboratory cultures. The results of this research show the active role played by C. marismortui in mineral precipitation, and allow us to compare them with those obtained previously using other taxonomic groups of moderately halophilic bacteria.     

Occurrence and diversity of lipids in modern coral skeletons

Coral skeletons are composite acellular structures, in which organic macromolecules are intimately associated with mineral phases. Previous studies focussed on proteins and sugars of the soluble organic matrices extracted from the skeletons. Here we report the occurrence of diverse lipids which were extracted from the aragonitic skeletons of seven modern coral species. Using thin layer chromatography, we show that these lipids differ in quantity and composition between the species. Higher proportions of sterols and sterol esters in skeleton extracts as compared to a much higher abundance of waxes and triglycerides in previously studied extracts from scleractinian soft tissues suggest a specific, although not yet determined, role in biomineralisation. The occurrence of intraskeletal lipids along with other organic components should also be taken into account when using coral skeletons as bone allografts, as well as in fossilisation processes.     

Absorbability of calcium from calcium-bound phosphoryl oligosaccharides in comparison with that from various calcium compounds in the rat ligated jejunum loop

Calcium-bound phosphoryl oligosaccharides (POs-Ca) were prepared from potato starch. Their solubility and in situ absorbability as a calcium source were investigated by comparing with the soluble calcium compounds, calcium chloride and calcium lactate, or insoluble calcium compounds, calcium carbonate and dibasic calcium phosphate. The solubility of POs-Ca was as high as that of calcium chloride and about 3-fold higher than that of calcium lactate. An in situ experiment showed that the intestinal calcium absorption rate of POs-Ca was almost comparable with that of the soluble calcium compounds, and was significantly higher (p<0.05) than that of the insoluble calcium groups. Moreover, the total absorption rate of a 1:1 mixture of the calcium from POs-Ca and a whey mineral complex (WMC) was significantly higher (p<0.05) than that of WMC alone. These results suggest that POs-Ca would be a useful soluble calcium source with relatively high absorption in the intestinal tract.     

Soluble proteins control growth of skeleton crystals in three coralline demosponges

Summary Calcified demosponges (coralline sponges, sclero-sponges), the first metazoa producing a carbonate skeleton, used to be important reef building organisms in the past. The relatives of this group investigated here,Spirastrella (Acanthochaetetes) wellsi, Astrosclera willeyana andVaceletia cf.crypta, are restricted to cryptic niches of modern Pacific coral reefs and may be considered as “living fossils’. They are characterized by a basic biologically controlled metazoan biomineralization process. Each of the investigated taxa forms its calcareous basal skeleton in a highly specialized way. Moreover, each taxon secretes distinct Ca²⁺-binding macromolecules which were entrapped within the calcium carbonate crystals during skeleton formation. Therefore these Ca²⁺-binding macromolecules were also described as intracrystalline macromolecules. When isolated and separated by SDS polyacrylamide gel electrophoresis, the organic skeleton matrix of the three species revealed to be composed of a respective distinct array of EDTA-soluble proteins. A single protein of 41 kDa was detected inS. wellsi, two proteins of 38 and 120 kDa inA. willeyana, and four proteins of 18 kDa, 30 kDa, 33 kDa, and 37 kDa inVaceletia sp. When run on IEF gel, the Ca²⁺-binding proteins gave staining bands at pH values between 5.25 and 5.65. As proved by anin vitro mineralization assay, the extracted proteins effectively inhibit CaCO₃ and SrCO₃ precipitation, respectively, in a saturated solution. Biochemical properties and behavior of the extracted proteins strongly suggest that they are involved in crystal nucleation and skeleton carbonate formation within the calcified sponges studied here.     

An evolutionary fast‐track to biocalcification

The ability to construct mineralized shells, spicules, spines and skeletons is thought to be a key factor that fuelled the expansion of multicellular animal life during the early Cambrian. The genes and molecular mechanisms that control the process of biomineralization in disparate phyla are gradually being revealed, and it is broadly recognized that an insoluble matrix of proteins, carbohydrates and other organic molecules are required for the initiation, regulation and inhibition of crystal growth. Here, we show that Astrosclera willeyana, a living representative of the now largely extinct stromatoporid sponges (a polyphyletic grade of poriferan bauplan), has apparently bypassed the requirement to evolve many of these mineral‐regulating matrix proteins by using the degraded remains of bacteria to seed CaCO₃ crystal growth. Because stromatoporid sponges formed extensive reefs during the Paelozoic and Mesozoic eras (fulfilling the role that stony corals play in modern coral reefs), and fossil evidence suggests that the same process of bacterial skeleton formation occurred in these stromatoporid ancestors, we infer that some ancient reef ecosystems might have been founded on this microbial–metazoan relationship.     

On the Composition of “Viscol” and the Employment of Gum Arabic for Mounting Media

“Viscol”, a water soluble permanent mounting medium hardening through evaporation of water under the cover glass, has been analyzed. It proves to consist of a mixture of gum arabic, glycerol, phenol and water and is especially suitable for simple botanical preparations. The use of gum arabic for hardening permanent mounting media is reviewed. Instead of a glycerol-phenol-water mixture a lactophenol, potassium acetate or zinc chloride solution mixed with gum arabic may be used for a permanent mounting medium. However, gum arabic contains calcium, magnesium and potassium ions giving crystals with the solutions mentioned. In the case of lactophenol and potassium acetate, the calcium and magnesium ions must be removed beforehand, which is done by precipitation with sodium or potassium carbonate. In the case of zinc chloride the potassium ions must be removed, which is done by dialysis with zinc chloride. It is pointed out that the same principles may be used for a great variety of different mounting media.     

不溶性基质中天冬氨酸丰富的蛋白在珊瑚的钙质骨片形成中的重要作用

一般认为, 酸性蛋白在控制矿物的形成和发展中发挥重要作用。因此, 在不溶性有机基质中鉴定酸性蛋白对于理解珊瑚中个体蛋白的功能是非常重要的一步。在短指多型软珊瑚(Sinularia polydactyla)的可溶性和不溶性基质层中分析蛋白组分表明, 在不溶性基质和可溶性基质层中天冬氨酸的含量分别是61%和29%。利用体外分析法发现, 基质蛋白诱导碳酸钙形成非晶态析出相先于其形成钙质的结晶态。利用X-射线衍射来鉴定骨片上结晶态的碳酸钙, 结果表明钙质的多晶态呈现强反射。傅利叶变换红外光谱分析表明珊瑚基质中富含天冬氨酸的蛋白和多醣的结构。在不溶性基质组分中用钙离子结合分析显示一个分子量为109 kD的蛋白质可以与形成骨片的钙离子结合, 这一过程对骨片形成非常重要。在对生物钙化过程中起重要作用的碳酸酐酶的分析中显示了此酶的新颖的活性。以上结果显示珊瑚中不溶性基质内的富含天冬氨酸的蛋白在生物矿化调控过程中起重要作用。     

Nautilin-63, a novel acidic glycoprotein from the shell nacre of Nautilus macromphalus

In molluscs, and more generally in metazoan organisms, the production of a calcified skeleton is a complex molecular process that is regulated by the secretion of an extracellular organic matrix. This matrix constitutes a cohesive and functional macromolecular assemblage, containing mainly proteins, glycoproteins and polysaccharides that, together, control the biomineral formation. These macromolecules interact with the extruded precursor mineral ions, mainly calcium and bicarbonate, to form complex organo-mineral composites of well-defined microstructures. For several reasons related to its remarkable mechanical properties and to its high value in jewelry, nacre is by far the most studied molluscan shell microstructure and constitutes a key model in biomineralization research. To understand the molecular mechanism that controls the formation of the shell nacreous layer, we have investigated the biochemistry of Nautilin-63, one of the main nacre matrix proteins of the cephalopod Nautilus macromphalus. After purification of Nautilin-63 by preparative electrophoresis, we demonstrate that this soluble protein is glycine-aspartate-rich, that it is highly glycosylated, that its sugar moieties are acidic, and that it is able to bind chitin in vitro. Interestingly, Nautilin-63 strongly interacts with the morphology of CaCO(3) crystals precipitated in vitro but, unexpectedly, it exhibits an extremely weak ability to inhibit in vitro the precipitation of CaCO(3) . The partial resolution of its amino acid sequence by de novo sequencing of its tryptic peptides indicates that Nautilin-63 exhibits short collagenous-like domains. Owing to specific polyclonal antibodies raised against the purified protein, Nautilin-63 was immunolocalized mainly in the intertabular nacre matrix. In conclusion, Nautilin-63 exhibits 'hybrid' biochemical properties that are found both in the soluble and insoluble proteins, rendering it difficult to classify according to the standard view on nacre proteins. DATABASE: The protein sequences of N63 appear on the UniProt Knowledgebase under accession number P86702.