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First published online February 4, 2005
Journal of Experimental Biology 208, 637-646 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01428

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Arginine kinase in the demosponge Suberites domuncula: regulation of its expression and catalytic activity by silicic acid

Sanja Perovi-Ottstadt¹, Matthias Wiens¹, Heinz-C. Schröder¹, Renato Batel², Marco Giovine³, Anatoli Krasko¹, Isabel M. Müller¹ and Werner E. G. Müller^1,*

¹ Institut für Physiologische Chemie, Abteilung Angewandte Molekularbiologie, Universität, Duesbergweg 6, D-55099 Mainz, Germany,
² Center for Marine Research, `Ruder Boskovic' Institute, HR-52210 Rovinj, Croatia
³ CNR-Direzione Progetto Finalizzato Biotecnologie, Via Leon Battista Alberti 4, I-16132 Genova, Italy

View larger version (89K): [in a new window]   Fig. 1. S. domuncula arginine kinase. (A) The sponge deduced protein (AK_SUBDO) is aligned with the arginine kinase from the Cnidarian Anthopleura japonica (KARG_ANTJA, O15992; Suzuki et al., 1997), and Drosophila melanogaster (AK_DROME, AAA68172, glycocyamine kinase from Nereis virens (GLYCAM_NEREIS, AAL26699 and human creatine kinase (CK_HUMAN, AAC31758 Mariman et al., 1987). Residues conserved (similar or related with respect to their physico-chemical properties) in all sequences are shown in white on black and those in at least three sequences in black on gray. The two characteristic domains for the ATP:guanido phosphotransferases, the N-terminal domain (ATP_GUA_N) and the C-terminal catalytic domain (ATP_GUA_C), are marked. The A. japonica arginine kinase is a two-domain enzyme, so the two phosphotransferase domains are also marked for this protein. The residues involved in coordinating Mg2+ to ATP are marked [ATP]. (B) Radial, unrooted phylogenetic tree, which includes the above mentioned sequences; the two domain enzyme from A. japonica has been split into the N-terminal and C-terminal segments (KARGn_ANTJA, aa1 to aa367; KARGc_ANTJA, aa368 to aa715). Shown in addition are the deuterostomian related sequence from (1) the Holothuria Stichopus japonicus (AK_STICJA, BAA76385, (2) the insect Schistocerca americana (AK_SCHIAM, AAC478301), (3) the mollusks Solen strictus (Bivalvia) (AK_SOLSTRI, BAB91358 N terminus aa1 to aa372 and C terminus aa373 to aa724; Suzuki et al., 2002), Corbicula japonica (Bivalvia) (AK_CORBJA, BAB913571; N terminus aa1 to aa372 and C terminus aa373 to aa723; Suzuki et al., 2002) and Ensis directus (Bivalvia) (AK_ENSIS, AAM906981; N terminus aa1 to aa372 and C terminus aa373 to aa723; Compaan and Ellington, 2003), (4) the oyster Crassostrea gigas (Bivalvia) (AK_CRASGI, BAD119501), (5) Crustaceans Homarus gammarus (KARG_HOMGA, 538542), Eriocheir sinensis (AK_ERISI, AAF43437 and Artemia franciscana (KARG_ARTSF, Q95V58), and (6) the nematode Caenorhabditis elegans (AK_CAEEL, NP_492714.1). Gene duplications within the mollusks and the Cnidarian taxa are indicated (D). The scale bar indicates an evolutionary distance of 0.1aa substitutions per position in the sequence.

View larger version (29K): [in a new window]   Fig. 2. Expression of the arginine kinase gene in primmorphs is dependent on addition of silicic acid. Primmorphs were incubated in the presence (plus) or absence of silicic acid (minus) for 0-5 days. Then RNA was extracted, and identical amounts of total RNA were size-separated. After blot transfer, hybridization was performed using the probes for arginine kinase or the housekeeping gene ß-tubulin.

View larger version (120K): [in a new window]   Fig. 3. Expression of the arginine kinase gene is dependent on the presence of silicic acid. (A-D) Primmorphs were cultivated for 5 days in the absence (-; A,B) or presence of 60 µmol l-1 silicic acid (SA+; C,D). (E,F) Primmorphs were treated with silicic acid and 100 µmol l-1 DIDS and in situ hybridization performed with SDAK as the probe. Hybridized cells are stained by brown/black deposits. Magnification: x10 (A,C,E); x20 (B,D,F).

View larger version (22K): [in a new window]   Fig. 4. Arginine kinase activity in primmorphs from S. domuncula in response to silicic acid. The primmorphs were incubated for 0-5 days in the absence (white bars) of presence of 60 µmol l-1 silicic acid (SA; black bars). In a parallel series of experiments the primmorphs were coincubated with 100 µmol l-1 of DIDS in assays without (hatched) and with SA (cross-hatched). The enzyme activity is given in nmoles ATP generated min-1 10 µg-1 protein. Values are means ± S.E.M., N=5.

View larger version (156K): [in a new window]   Fig. 5. Growing spicules in sclerocytes obtained from primmorphs cultured in the presence of 60 µmol l-1 silicic acid; sequence of TEM images. (A) Two sclerocytes (scl) in the primmorph start to synthesize spicules (sp). The blunt end of one newly growing spicule (B) is associated with collagen-related fibrils (col), but not the second spicule (shown enlarged in C). The sharp end of the spicule shows inhomogeneous structure (D), suggesting less dense packing of silica in the spicule. Collagen fibers (col) surround the two spicules.