Poly(silicate)-metabolizing silicatein in siliceous spicules and silicasomes of demosponges comprises dual enzymatic activities (silica polymerase and silica esterase) Werner E. G. Mu¨ller1, Ute Schloßmacher1, Xiaohong Wang2, Alexandra Boreiko1, David Brandt1, Stephan E. Wolf3, Wolfgang Tremel3 and Heinz C. Schro¨der1 1 Institut fu¨r Physiologische Chemie, Abteilung Angewandte Molekularbiologie, Universita¨t, Mainz, Germany 2 National Research Center for Geoanalysis, Beijing, China 3 Institut fu¨r Anorganische Chemie, Universita¨t, Mainz, Germany
Keywords poly(silicate); silica esterase; silica polymerase; silicatein; sponges Correspondence W. E. G. Mu¨ller, Institut fu¨r Physiologische Chemie, Abteilung Angewandte Molekularbiologie, Universita¨t, Duesbergweg 6, 55099 Mainz, Germany Fax: +49 6131 39 25243 Tel: +49 6131 39 25910 E-mail:
[email protected] Website: http://www.biotecmarin.de/
Siliceous sponges can synthesize poly(silicate) for their spicules enzymatically using silicatein. We found that silicatein exists in silica-filled cell organelles (silicasomes) that transport the enzyme to the spicules. We show for the first time that recombinant silicatein acts as a silica polymerase and also as a silica esterase. The enzymatic polymerization ⁄ polycondensation of silicic acid follows a distinct course. In addition, we show that silicatein cleaves the ester-like bond in bis(p-aminophenoxy)-dimethylsilane. Enzymatic parameters for silica esterase activity are given. The reaction is completely blocked by sodium hexafluorosilicate and E-64. We consider that the dual function of silicatein (silica polymerase and silica esterase) will be useful for the rational synthesis of structured new silica biomaterials.
(Received 22 October 2007, revised 13 November 2007, accepted 26 November 2007) doi:10.1111/j.1742-4658.2007.06206.x
Silicon is the second most common element in the Earth’s crust [1]; it possesses semi-metallic as well as metalloid properties. Silicon exists in nature in combination with oxygen as silicate ions or as silica; silica has no negative charge, while silicate anions carry a negative net electrical charge, which is counterbalanced by cations. Free silica ⁄ silicate is found both in the crystalline state (such as quartz) and in the amorphous state (such as opal). Silica ⁄ silicate is widely used in industry and medicine for the fabrication of poly(silicate), e.g. in amorphous glasses, ceramics, paints, adhesives, catalysts and photonic materials [2,3]. Furthermore, poly(silicate) is an important new material in nano(bio)technology [4,5]. This multidisciplinary research field is concerned with bio- and electronic engineering at nanometer, molecular and cellular levels
[4]. Currently, production of silica require high temperature conditions and extremes of pH [6]. Hydrated amorphous silica, e.g. in the form of opal, has superb properties such as low density and high porosity. In nature, amorphous silica can be produced by diatoms by passive deposition onto an organic matrix. Siliceous sponges (Demospongiae) have the exceptional ability to synthesize silica enzymatically via silicatein [7,8]. Based on its protein sequence, silicatein is related to the proteinases of the cathepsin class [9]. Silicatein has been isolated from a number of siliceous sponges, e.g. Tethya aurantium or Suberites domuncula [9,10]. If the enzyme is isolated from the skeletal elements of these animals, the spicules, it can be used in vitro to catalyze polycondensation of a wide variety of alkoxides, as well as ionic and organometallic
Abbreviations BAPD-silane, bis(p-aminophenoxy)-dimethylsilane; EDTA, ethylenediaminetetraacetic acid; E-64, L-trans-epoxysuccinyl-leucylamido(4guanidino)butane; MOPS, [3-(N-morpholino) propanesulfonic acid]; PoAb, polyclonal antibodies; SEM, scanning electron microscopy; TEM, transmission electron microscopy; TEOS, tetraethoxysilane.
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precursors, to the corresponding metal oxides; these processes occur at standard, ambient temperature and pressure and neutral pH [11]. Using site-directed mutagenesis, those amino acids critical to the condensation of tetraethoxysilane (TEOS) have been determined; the catalytic triad is histidine (His), asparagine (Asn) and serine (Ser) [4,12]. In the active center of silicatein, the hydroxyl group of Ser26 and the imidazole of His165 (catalytic diad) have been shown to play key roles in the condensation of TEOS [11]. It has been proposed that these functionalities participate in the formation of a transitory pentavalent silicon species, stabilized through a donor bond to the imidazole nitrogen [11]. Using a nitrilotriacetic acid-terminated alkanethiol, which had been successfully self-assembled onto gold surfaces, silicatein could be immobilized on matrices; it was found to retain its enzymatic function, allowing the polycondensation of monomeric silicon alkoxides to form silica structures on surfaces [13]. It was shown that silicatein is the main component of the axial filament of the spicules [9,10]. Later, this enzyme was also detected in the extraspicular space, where it contributes to the appositional growth of these skeletal elements [14,15]. Silicatein uses either organofunctional silanes [9] or orthosilicate (W. E. G. Mu¨ller, unpublished results) for the synthesis of poly(silicate). As seawater has a low content of silicate (about 5 lm), the sponges have to transport silicate actively into their cells, via a putative Na+ ⁄ HCO3) [Si(OH)4] co-transporter [16]. Intracellularly, silicate is stored in silicasomes, organelles with a high content of silicate [17]. These results were obtained using a sponge tissue culture system (termed a primmorph system) [18] that comprises a special form of 3D cell aggregates composed of proliferating and differentiating cells. Primmorphs allow the investigation of spicule formation under controlled conditions [19]. Based on electron microscopic studies presented previously [17], it appears that the silicasomes are intracellular granules that can release their content by exocytosis to the mesohyl. The existence of silicatein in silicasomes with high silica levels implies that silicatein might be involved in the storage of silica in these organelles, presumably controlling the gel–sol state of silicate. From diatoms, it is known that silicate is deposited in special organelles, the silica deposition vesicles, which, in addition to high levels of silicate, also contain organic components of unknown function, e.g. mannose [20–22]. It may be assumed that these molecules prevent polycondensation of silicate. As silicate – at neutral pH – polycondenses at concentrations above 1 mm to poly(silicate) [23], it may be postulated that (organic) molecules, e.g. silicatein, contribute to stabilization of the sol state of
Silicatein comprises dual enzymatic activities
silica. One mechanism for the gel to sol conversion could be hydrolysis of the oxygen bridge of the polymerized ⁄ polycondensed silicic acid. The linkage between silicate or tetrahedral silica units in poly(silicate) is an ester-like bond. In order to test whether silicatein – in addition to being a poly(silicate)-forming enzyme (silica polymerase) – also functions as an silica esterase, we studied its hydrolytic function on bis(p-aminophenoxy)-dimethylsilane (BAPD silane). This compound comprises two ester-like bonds between silicon and p-aminophenol and two methyl silane linkages (Fig. 1). In line with a previous study [9], we propose that hydrogen bonding between the imidazole nitrogen of the conserved His and the hydroxyl of the active-site Ser increases the nucleophilicity of the Ser oxygen, facilitating attack of the hydroxy group on the silicon atom of the substrate. This reaction can be monitored spectroscopically on the basis of the release of p-aminophenol. The experimental data show that, in addition to its silica polymerase activity, silicatein also comprises a silica esterase function, thus supporting the concept that silicatein is involved in stabilization of the sol state of biogenic silica. The esterase reaction can be completely blocked by sodium hexafluorosilicate and by the cysteine proteinase inhibitor E-64 (l-trans-epoxysuccinyl-leucylamido(4-guanidino)butane) [24]. For these
Silicatein Ester-like bond
Ser
O
Silane bond
His
H N CH3
O
Si
–
N
O +
H
CH3
H2 N
NH2
+H
+
CH3
OH
+ H2 N
HO
Si
OH
CH3
Fig. 1. Proposed silicatein-a-mediated reaction mechanism for hydrolysis of bis(p-aminophenoxy)-dimethylsilane which contains two silicic ester-like (blue) and two silane bonds (red). In the catalytic center of silicatein, the serine (Ser) oxygen makes a nucleophilic attack on the silicon, resulting in displacement of p-aminophenol and formation of a (alkoxyl)-monosilane. This reaction is facilitated by hydrogen bonding between the imidazole nitrogen of the conserved histidine (His) and the hydroxyl of the Ser.
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studies, resulting in elucidation of a new activity of silicatein as a silica esterase, we used recombinant silicatein-a from the demosponge S. domuncula [10].
Results Presence of silicatein in the spicules and cell organelles, the silicasomes Sections through primmorphs were exposed to antibodies to silicatein, PoAb-aSILIC, and analyzed by the transmission electron microscopy immunogold labeling A
B
C
D
E
F
G
H
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technique. As expected, strong signals were seen in the axial filament within the sponge spicule (Fig. 2A), the site hitherto proposed for major occurrence of the enzyme [14,25]. The images also show, however, dense accumulation of gold grains in the extraspicular space, reflecting dense packaging of silicatein molecules there also. The silicatein molecules are arranged around the spicules in concentric rings (Fig. 2B). A closer view of the axial canal in the center of the spicule reveals localization of silicatein in the axial filament as well as within the silica shell surrounding the spicule (Fig. 2C). Controls show that pre-immune serum does
Fig. 2. Localization of silicatein in spicules and in intra- and extracellular vesicles by TEM immunogold labeling. (i) Association of silicatein with spicules. (A) Strong antibody reactions are seen within the axial canal (ac) in the axial filament (af), which is surrounded by the spicule (sp); in addition, a silica vesicle (siv) within one concentric ring (ri) is present. (B) Strong antigen–antibody reactions are also seen on the concentric rings (ri) surrounding a spicule (sp). (C) In the axial canal (ac), high levels of signals are seen in and on the axial filament (af), as well as the inner rim of the silica spicule (sp). (D) Control: incubation of a section with pre-immune serum; no reaction is seen within the axial canal (ac) and around the spicule (sp). (ii) Intracellular localization of silicatein in vesicles. (E,F) The cells around the spicules, the sclerocytes (sc), are filled with vesicles, which strongly react with antibodies. These vesicles are termed silicasomes (sis). (iii) Extracellular localization of silicatein in vesicles. (G) In the extracellular space (ex-s), the silica vesicles (siv) can still be seen. (H) These silica vesicles (siv) frequently remain intact within rings ⁄ cylinders (ri).
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A 70
429.3
Me
60
Intensity (%)
not react with structures within or around the spicules (Fig. 2D). Likewise, the adsorbed PoAb-aSILIC preparation, pre-incubated with recombinant silicatein, did not react either (as shown previously [14]). Strong reactions of PoAb-aSILIC are also seen in vesicles of the sclerocytes, the cells surrounding the spicules (Fig. 2E,F). These intracellular vesicles, termed silicasomes, are rich in silica [17], and are additionally densely filled with the enzyme. Extracellularly (Fig. 2G), the silica vesicles fuse with the concentric ring structures around the spicule (Fig. 2H). These silica vesicles often remain as intact entities within the rings ⁄ cylinders, reacting positively to anti-silicatein (Fig. 2H).
Silicatein comprises dual enzymatic activities
MeO
50
503.2
30 20
577.2
10
Catalytic function of silicatein: silica esterase activity (catabolic enzyme) The temperature optimum was found to be in the range 20–25 C; the temperature coefficient (Q10) decreases by 2.5-fold above 25 C and increases by 2.9-fold below 25 C. Silica esterase activity was routinely determined at 20 C using a substrate range between 20 and 250 lm of BAPD silane. After cleavage of one of the silica ester bonds, the concentration of the released product p-aminophenol was determined at a wavelength of 300 nm, which is in the trailing edge of the main absorption bands under the conditions used. Another maximum is recorded at 230 nm (Fig. 4). The molar absorption coefficient (e at k = 300 nm) was determined [26] to be 2096.6 LÆmolÆ cm)1, in enzyme reactions with BAPD silane (20, 100 and 200 lm; non-saturating conditions). The Michaelis constant (Km) was determined using this value [27],
623.3
461.2
0 400
500
600
700
800
900
1000
m/z
Intensity (%)
Synthesis of polymerized polysiloxane derivatives of silicic acid, was performed using silicatein and dimethyldimethoxysilane as substrate. After an incubation period of 1 h, the sample was analyzed by MALDIMS. As shown in Fig. 3B, a stepwise 74–75 Da increase in mass is recorded above an m ⁄ z of 500, which is due to stepwise polymerization of -Si(Me)2-Ounits to the starter silane substrate. Under the incubation conditions used, synthesis of oligomers with 11 -Si(Me)2-O- units could be detected. If silicatein is absent from the sample, no signals above an m ⁄ z of 500 Da are seen (Fig. 3A). This result suggests that silicatein, via its silica polymerase activity, lowers the activation energy for the polymerization ⁄ condensation reaction, resulting in successive addition of monomeric silica units.
OMe
Me
40
Me
B 70
Catalytic function of silicatein: silica polymerase (anabolic enzyme)
Si
n = 11
O
60
Si
50
Me
n = 10 n=9 n=8
40
n=7
30
n =6
20
475.1
503.2 577.2 623.2
697.2
771.4
847.0
925.2
10 0 400
500
600
700
800
900
1000
m/z Fig. 3. MALDI-MS spectrum of the products formed from dimethyldimethoxysilane in the absence (A) or presence of 4.5 lgÆmL)1 silicatein (B). The mass distributions differ significantly. In the presence of silicatein (B), a distinct increase in chain length can be observed. The distance of 74–75 Da between each individual peak corresponds to the mass of a single Si(Me)2-O unit; oligomeric polymerization of 11 units can be resolved. In contrast, no polymerization products are observed in the absence of silicatein.
and was calculated to be 22.7 lm. In comparison, the Km value for human recombinant cathepsin L (EC 3.4.22.15), the enzyme closest related to silicatein, expressed in Escherichia coli, was 1.1 lm, using the substrate benzyloxycarbonyl-Phe-Arg-4-methylcoumarin-7-amide [28]. The turnover value (molecules of converted substrate per enzyme molecule per second) for silicatein in the silica esterase assay was 5.2. Although this catabolic de-polymerization reaction may be substantially different from the cleavage of peptide bonds by human cathepsin L, the human enzyme shows only a slightly higher turnover value of 20 using the same substrate [29]. The specificity of the reaction was determined in two series of experiments. First, silicatein was replaced in the assay by the same amount of BSA. Under otherwise identical conditions, no significant increase in absorbance was seen at either 300 or 230 nm over
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1.8 20 min
1.6
Absorption
1.4 1.2 1.0 0.8
8 6 4 2
0.6 0.4 0.2 0 220
0
240
260
280
300
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340
The dissolution process of spicules, the tylostyles, can also be followed in vivo in tissue of S. domuncula. Spicules were isolated from tissue [14] and analyzed by scanning electron microscopic (SEM) analysis. In this study, the pointed ends of the spicules were compared (Fig. 5). Intact spicules have a smooth surface (Fig. 5A), and the tips of the spicules are closed. During the decomposition process in vivo, their diameters decrease and the lamellar organization becomes overt (Fig. 5B). In later phases, the surface of the spicules becomes wrinkled due to exposure of the silica nanoparticles; the axial canal opens and exposes the axial filament (Fig. 5C).
Wavelength (nm)
Discussion Fig. 4. Change of absorption spectra during incubation of silicatein in the presence of 140 lM bis(p-aminophenoxy)-dimethylsilane substrate as described in Experimental procedures. At time zero, the absorbance at k = 300 nm is very low. The absorbance increases steadily during the subsequent 20 min of incubation. The molar absorption coefficient (e) at 300 nm is indicated.
2–60 min incubation periods (20 C). Second, a direct interaction between the ester-like substrate BAPD silane (50 lm) and the silicate monomer sodium hexafluorosilicate (1 mm) was studied in the reaction with silicatein. In previous studies, sodium hexafluorosilicate has been proven to induce growth of sponge cells in culture and to cause differential gene expression in vivo and in vitro [10,30]. After addition of a 20-fold molar excess of sodium hexafluorosilicate with respect to the ester-like substrate BAPD silane, complete suppression of the ester-like activity of the enzyme was determined in the photometric test used here. Alternatively, the Ser proteinase inhibitor E-64 was added to the reaction mixture; at a concentration of 10 lm, an inhibition of the esterase activity > 95% was determined. A
B
Sponges have to cope with an energetically highly expensive chain of reactions to form their siliceous spicules. The first barrier is the uptake of dissolved silicic acid from the surrounding aqueous environment; usually only low concentrations of silicic acid, of approximately 5 lm, exist in seawater [31]. The uptake of silicic acid is probably mediated by an ATP-consuming pump ⁄ transporter [16]. It is unknown whether the inorganic silicic acid monomers are converted intracellularly to organosilicate units. The subsequent process requires intracellular transport of the silicic acid, or derivatives of it, to the organelles (silicasomes) in which initial formation of the spicules proceeds. In the spicule-forming cells, the sclerocytes, the first layers of the silica shell of the spicules are formed around the silicatein-based axial filament in specific organelles [14]. The prerequisite for intracellular initiation of spicule synthesis is preferential accumulation of silicic acid in special organelles. Recently, such vesicles with a high silica content, the silicasomes, have been identified in sclerocytes [17]. It is expected that, in silicasomes C
Fig. 5. Stepwise dissolution of tylostyle spicules (sp) in tissue from Suberites domuncula (SEM analysis). The intact spicules (pointed terminus of the spicules) have a smooth surface (A). (B) Progressive decomposition of the spicules is followed by appearance of the lammelar organization. Finally, the surfaces of the spicules become wrinkled, and silica nanoparticles can be seen on the surface (C); the axial canal opens and the axial filament becomes visible.
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where the silicic acid content is high, self-polymerization or self-condensation processes are facilitated. Polymerization ⁄ condensation of monomeric silicic acid to poly(silicate) is a random process [23] that results in the formation of 3D condensed silica polymers or nuclei. Our experimental results show that silicic acid co-exists with silicatein in these silicasomes. Based on these microscopic analyses, we wished to determine whether silicatein could function as a silica esterase, allowing hydrolysis of the silicate ester bonds with simultaneous release of water. The data summarized here demonstrate that silicatein does indeed have such activity; it mediates cleavage of silicate ester bonds in the BAPD silane substrate. Furthermore, it is shown that silicatein also exhibits silica-polymerizing activity, as previously proposed [9]. We have demonstrated that the polymerizing growth of the silica chains, mediated by the silica polymerase activity of silicatein, involves stepwise addition of single silica monomeric units. This finding implies that silicatein has two different enzymatic properties, a silica esterase activity and a polymerizing ⁄ polycondensing activity (silica polymerase). At present, we are working on elucidation of the molecular switch controlling these dual enzymatic functions; initial data indicate that low-molecularweight compound(s) direct silicatein to either the catabolic or the anabolic reaction. Enzymatic parameters of the silica esterase activity were determined. The Michaelis constant (Km 22.7 lm) and the turnover value (5.2 molecules of converted substrate per enzyme molecule per second) for the silica esterase catalytic reaction of silicatein are similar to those that have been determined for the related hydrolytic enzyme cathepsin L [28,29]. Future studies are required to determine whether silica polymerase and silica esterase function in principle by the same mechanism. Both reactions are initiated by a nucleophilic attack by the hydroxyl group of the Ser residue in the catalytic center of the enzyme. In the silica polymerase reaction, this step may be followed by a condensation reaction, which could be facilitated by proton transfer to the His residue in the catalytic center. The content of the silicasomes is released into the extracellular space [17], and transported from there to the spicules. As shown here, the extracellular silica vesicles that contain silicatein fuse with the appositionally growing spicules in diametral direction, and probably also in the axial direction [2]. These data show that silicatein is transported in silicasomes into the extracellular space; there, the silica vesicles contribute to the appositionally growing spicules. Furthermore, our data allow development of a functional model that contributes to understanding of the growth of the siliceous
Silicatein comprises dual enzymatic activities
spicules which apparently lack a template or matrix. The simultaneous release of silicic acid and silicatein into the concentric rings around the growing spicules, which gives rise to lamellar formation of the spicules [30], allows a controlled polymerization ⁄ condensation process for silicic acid mediated by silicatein along galectin strings ⁄ nets [25]. The shape of the poly(silicate) product is probably additionally tailored by collagen sheathing [19]. A schematic outline of the localization and transport of silicatein in the extraspicular space is given in Fig. 6. The data summarized here provide, for the first time, enzyme kinetic data for silicatein, which will A
B
Fig. 6. Localization of silicatein in the extraspicular space. (A) The spicules (sp), formed from poly(silicate) (sia) are surrounded by sclerocytes (sc) that harbor special organelles, the silicasomes (sis), that are rich in silicatein (red circles) and silicic acid (blue circles). In the center of the spicules runs the axial filament (af), which is built up from silicatein molecules. (B) The silicasomes are released from the sclerocytes and transported into the extracellular space, from where these silica vesicles (siv) are translocated to the ring structures surrounding the growing spicules (sp). The silica vesicles, harboring silicatein and monomeric silicic acid, fuse with the concentric rings (ri) that are present around the spicules. There the silicatein molecules become associated with the ring sheet, while the poly(silicate) (sia) remains in the siliceous lamellae that are formed within the rings.
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render possible the rational application of silicatein in the fabrication of (new) biomaterials based on layered silica, of titania and of zirconia [32]. This view is based on the finding of a dual role for silicatein as an anabolic (silica polymerase) and catabolic enzyme (silica esterase), allowing the formation of controlled silica structures. In addition, patterning of poly(silicate) is modulated by self-assembly of silicatein molecules in an organized, fractal manner [33,34]; the fractal pattern probably dictates the initial shape of the spicules [34]. The finding that silicatein catalyzes two reactions, acting as silica polymerase and silica esterase, provides this enzyme with advantageous properties, e.g. for production of a flexible shell around organisms after bioencapsulation with silica. Recently, this feature has been utilized to encapsulate bacteria [35]: E. coli were transformed with the silicatein gene, and, after expression of silicatein and subsequent incubation with silicic acid, the bacteria had been encapsulated with bio-silica, a viscous cover, which did not reduce their growth properties [35].
Electron immunogold labeling Polyclonal antibodies (PoAb-aSILIC) were used that had been raised against recombinant silicatein-a from S. domuncula [14]. Primmorph samples were treated with 0.1% glutaraldehyde ⁄ 3% paraformaldehyde in 0.1 m phosphate buffer (pH 7.4). After 2 h, the material was dehydrated in ethanol and embedded in LR-White resin (Electron Microscopy Sciences, Hatfield, PA, USA). Slices were cut 60 nm thick and blocked with 5% BSA in NaCl ⁄ Pi, and then incubated with the primary antibody PoAb-aSILIC (1 : 1000) for 12 h at 4 C. After three washes with NaCl ⁄ Pi ⁄ 1% BSA, sections were incubated with a 1 : 1000 dilution of the secondary antibody (1.4 nm nanogold anti-rabbit IgG) for 2 h. Sections were processed as described previously [14]; enhancement of the immunocomplexes was performed using silver [36]. The samples were examined by transmission electron microscopy (TEM) using a Tecnai 12 microscope (FEI Electron Optics, Eindhoven, the Netherlands). As controls, pre-immune serum or PoAb-aSILIC, adsorbed to recombinant silicatein [15], were used.
Silicatein
Experimental procedures Materials Dimethyldimethoxysilane (C4H12O2Si, relative molecular mass 120.22) and BAPD silane (C14H18N2O2Si, relative molecular mass 274.39) were obtained from ABCR GmbH (Karlsruhe, Germany), bovine serum albumin (Cohn fraction V) from Roth (Karlsruhe, Germany), sodium hexafluorosilicate from Sigma-Aldrich (Taufkirchen, Germany), and p-aminophenol from Riedel de Hae¨n (Seelze, Germany).
Sponges and primmorphs Specimens of the marine sponge S. domuncula (Porifera, Demospongiae, Hadromerida) were collected in the Northern Adriatic near Rovinj (Croatia), and then kept in aquaria in Mainz (Germany) at a temperature of 17 C for more than 5 months. From these animals primmorphs, a 3D cell system [10,18,19] was prepared. Primmorphs were kept at 17 C in natural seawater (enriched with 60 lm of silicate), supplemented with 1% RPMI-1640 medium (GIBCO, Karlsruhe, Germany). The primmorphs were used for analysis approximately 20 days later [14].
Scanning electron microscopy
MALDI analysis Silicatein-a (4.5 lgÆmL)1; 210 pmolÆmL)1 using a molecular mass of 21 329 Da [10]) in MOPS buffer was covered with a layer of dimethyldimethoxysilane dissolved in diethyl ether (10 lmolÆmL)1) in the ratio 10 : 1 (v ⁄ v). Samples were taken after incubating the assays for 1 h at 20 C, with shaking. The aqueous layer, containing decomposition products, silicatein and buffer, was removed, and the organic phase, which contained only the substrate and the siloxane polymer, was dried using Na-sulfate to avoid further decomposition. Finally, the products were characterized by means of MALDI-MS [38,39] performed in a Finnigan MAT mass spectrometer 8230 (Midland; Canada). In a control assay, the reaction was performed in the absence of silicatein.
Esterase activity
The SEM analysis of spicules was performed using a Zeiss DSM 962 digital scanning microscope (Zeiss, Aalen, Germany) as described previously [14].
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Recombinant silicatein-a was prepared in E. coli as described previously [10,37]. The enzyme was stored at a concentration of 2 mgÆmL)1 in 20 mm MOPS [3-(N-morpholino) propanesulfonic acid] buffer (pH 7.5, 50 mm Na-acetate, 1 mm EDTA). This purified recombinant silicatein-a preparation was used to raise antibodies (PoAbaSILIC) [37]; it has been shown that such antibodies reacted specifically with the purified silicatein [2,15,25].
The assay is based on the concentration-dependent increase in the UV absorption at a wavelength of 300 nm
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of the degradation product p-aminophenol that results from hydrolysis of the substrate BAPD silane [40]. The contribution of the degradation product p-aminophenol to the UV ⁄ vis spectra was realized by the same phase transfer principle as mentioned above. During continuous stirring of the assays in Suprasil mixing cuvettes (Hellma QS-110, Mu¨llheim, Germany), the reaction was studied at 20 C within the absorbance range of 220–800 nm using a Varian Cary 5G UV-Vis-NIR spectrophotometer (Mulgrave, Australia). Typical reactions (3.5 mL assays) contained 0.4 lgÆmL)1 silicatein-a in 20 mm MOPS buffer; BSA (50 lg) was used in controls. As substrate, concentrations of BAPD silane of 20–200 lm (from a 2 mm stock solution in diethyl ether) were used. Where indicated, sodium hexafluorosilicate (1 mm) was added to the reaction mixture containing BAPD silane (20 or 200 lm) and silicatein. In one series of experiments, E-64 (10 lm) was added to the reaction mixture. In controls, silicatein was replaced by BSA (4.5 lgÆmL)1 assay). Kinetic determinations were commenced 30 s after addition of the components.
Acknowledgements This work was supported by grants from the European Commission, the Deutsche Forschungsgemeinschaft, the Bundesministerium fu¨r Bildung und Forschung Germany (Center of Excellence project BIOTECmarin), the National Natural Science Foundation of China (grant number 50402023) and the International Human Frontier Science Program. S. E. W. is the recipient of a Konrad Adenauer fellowship.
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