The silicatein propeptide acts as inhibitor/modulator of self-organization during spicule axial filament formation.

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Abstract

Silicateins are crucial enzymes that are involved in formation of the inorganic biosilica scaffold of the spicular skeleton of siliceous sponges. We show that silicatein acquires its structure-guiding and enzymatically active state by processing of silicatein from pro-silicatein to the mature enzyme. A recombinant propeptide (PROP) of silicatein from the siliceous demosponge Suberites domuncula was prepared, and antibodies were raised against the peptide. In sponge tissue, these antibodies reacted with both surface structures and the central region of the spicules. Using phage display expression, spicule-binding 12-mer peptides were identified that are rich in histidine residues. In the predicted tertiary structure of PROP, these histidine residues are only present in the α-helical region. The recombinant PROP was found to inhibit self-assembly of silicatein molecules. By light scattering, it was shown that, in the presence of 4 m urea, the recombinant silicatein is obtained in the mono/oligomeric form with a hydrodynamic radius of 4 nm, while lower urea concentrations promote self-aggregation and assembly of the protein. Finally, it is shown that the enzymatic activity of silicatein is abolished by PROP in silicatein samples that predominantly contain mono- or oligomeric silicatein particles, but the enzyme is not affected if present in the filamentous aggregated form. It is concluded that the functions of silicatein, acting as a structural template for its biosilica product and as an enzyme, are modulated and controlled by its propeptide.

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Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose.

Jan Kyhse-Andersen (1984)

A simple, horizontal device for rapid electrophoretic transfer of proteins from several polyacrylamide gels simultaneously is described. Up to six 'TRANS-UNITS' consisting of soaked filter paper on either side of polyacrylamide gel/nitrocellulose sheets that are separated by dialysis membranes are stacked between graphite plate electrodes. The only buffer reservoir in the apparatus is that in stacked, soaked filter paper. A special buffer system based on the isotachophoresis theory was developed for this purpose. The electrophoretic transfer was performed with equal efficiency in all TRANS-UNITS of the stack. Only traces of a few proteins remained in the polyacrylamide gel after transfer. With this apparatus, 50 protein bands from a human serum protein sample (diluted 1 : 100) were detected by immunoblotting with the retainment of the high resolution of the SDS-PAGE technique. The apparatus provided a constant current density of 0.8 mA/cm2 during the 1-h transfer time at 21 degrees C, irrespective of the number of TRANS-UNITS. The apparatus generated 1-5 W in joule heat, depending on the number of TRANS-UNITS in the stack.

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Pattern formation by local self-activation and lateral inhibition.

H Meinhardt, A Gierer (2000)

In 1972, we proposed a theory of biological pattern formation in which concentration maxima of pattern forming substances are generated through local self-enhancement in conjunction with long range inhibition. Since then, much evidence in various developmental systems has confirmed the importance of autocatalytic feedback loops combined with inhibitory interaction. Examples are found in the formation of embryonal organizing regions, in segmentation, in the polarization of individual cells, and in gene activation. By computer simulations, we have shown that the theory accounts for much of the regulatory phenomena observed, including signalling to regenerate removed parts. These self-regulatory features contribute to making development robust and error-tolerant. Furthermore, the resulting pattern is, to a large extent, independent of the details provided by initial conditions and inducing signals. Copyright 2000 John Wiley & Sons, Inc.

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Silicatein filaments and subunits from a marine sponge direct the polymerization of silica and silicones in vitro.

Jens S. Christiansen, Tomoko K. Shimizu, G Stucky … (1999)

Nanoscale control of the polymerization of silicon and oxygen determines the structures and properties of a wide range of siloxane-based materials, including glasses, ceramics, mesoporous molecular sieves and catalysts, elastomers, resins, insulators, optical coatings, and photoluminescent polymers. In contrast to anthropogenic and geological syntheses of these materials that require extremes of temperature, pressure, or pH, living systems produce a remarkable diversity of nanostructured silicates at ambient temperatures and pressures and at near-neutral pH. We show here that the protein filaments and their constituent subunits comprising the axial cores of silica spicules in a marine sponge chemically and spatially direct the polymerization of silica and silicone polymer networks from the corresponding alkoxide substrates in vitro, under conditions in which such syntheses otherwise require either an acid or base catalyst. Homology of the principal protein to the well known enzyme cathepsin L points to a possible reaction mechanism that is supported by recent site-directed mutagenesis experiments. The catalytic activity of the "silicatein" (silica protein) molecule suggests new routes to the synthesis of silicon-based materials.