Allemand D, TambuttE E, Girard JP, Jaubert J
Observatoire Oceanologique Europeen, Centre Scientifique de Monaco, Avenue Saint Martin, MC-98000 Monaco, Principality of Monaco, Commissariat a l'Energie Atomique - LDG, BP 12, F-91680 Bruyeres-Le-Chatel Cedex, France and Laborat.
J Exp Biol. 1998 Jul;201 (Pt 13):2001-9. doi: 10.1242/jeb.201.13.2001.
The kinetics of organic matrix biosynthesis and incorporation into scleractinian coral skeleton was studied using microcolonies of Stylophora pistillata. [14C]Aspartic acid was used to label the organic matrix since this acidic amino acid can represent up to 50 mol % of organic matrix proteins. External aspartate was rapidly incorporated into tissue protein without any detectable lag phase, suggesting either a small intracellular pool of aspartic acid or a pool with a fast turn-over rate. The incorporation of 14C-labelled macromolecules into the skeleton was linear over time, after an initial delay of 20 min. Rates of calcification, measured by the incorporation of 45Ca into the skeleton, and of organic matrix biosynthesis and incorporation into the skeleton were constant. Inhibition of calcification by the Ca2+ channel inhibitor verapamil reduced the incorporation of organic matrix proteins into the skeleton. Similarly, organic matrix incorporation into the skeleton, but not protein synthesis for incorporation into the tissue compartment, was dependent on the state of polymerization of both actin and tubulin, as shown by the sensitivity of this process to cytochalasin B and colchicin. These drugs may inhibit exocytosis of organic matrix proteins into the subcalicoblastic space. Finally, inhibition of protein synthesis by emetin or cycloheximide and inhibition of N-glycosylation by tunicamycin reduced both the incorporation of macromolecules into the skeleton and the rate of calcification. This suggests that organic matrix biosynthesis and its migration towards the site of calcification may be a prerequisite step in the calcification process. On the basis of these results, we investigated the effects of tributyltin (TBT), a component of antifouling painting known to interfere with biomineralization processes. Our results have shown that this xenobiotic significantly inhibits protein synthesis and the subsequent incorporation of protein into coral skeleton. This effect was correlated with a reduction in the rate of calcification. Protein synthesis was shown to be the parameter most sensitive to TBT (IC50=0.2 micromol l-1), followed by aspartic acid uptake by coral tissue (IC50=0.6 micromol l-1), skeletogenesis (IC50=3 micromol l-1) and Ca2+ uptake by coral tissue (IC50=20 micromol l-1). These results suggest that the mode of action of TBT on calcification may be the inhibition of organic matrix biosynthesis.
利用鹿角杯形珊瑚的微型群体研究了有机基质生物合成及整合到石珊瑚骨骼中的动力学过程。使用[14C]天冬氨酸标记有机基质，因为这种酸性氨基酸在有机基质蛋白中所占比例可达50摩尔%。外部天冬氨酸迅速整合到组织蛋白中，未检测到任何延迟期，这表明要么细胞内天冬氨酸池较小，要么该池的周转速度较快。经过20分钟的初始延迟后，14C标记的大分子整合到骨骼中的过程随时间呈线性。通过将45Ca整合到骨骼中测量的钙化速率以及有机基质生物合成和整合到骨骼中的速率是恒定的。Ca2+通道抑制剂维拉帕米对钙化的抑制作用降低了有机基质蛋白整合到骨骼中的量。同样，有机基质整合到骨骼中，但不是整合到组织区室中的蛋白质合成，取决于肌动蛋白和微管蛋白的聚合状态，这一过程对细胞松弛素B和秋水仙碱的敏感性表明了这一点。这些药物可能会抑制有机基质蛋白向亚钙化细胞间隙的胞吐作用。最后，依米丁或环己酰亚胺对蛋白质合成的抑制以及衣霉素对N-糖基化的抑制降低了大分子整合到骨骼中的量和钙化速率。这表明有机基质生物合成及其向钙化部位的迁移可能是钙化过程中的一个先决步骤。基于这些结果，我们研究了三丁基锡（TBT）的影响，TBT是一种已知会干扰生物矿化过程的防污涂料成分。我们的结果表明，这种外源性物质显著抑制蛋白质合成以及随后蛋白质整合到珊瑚骨骼中。这种作用与钙化速率的降低相关。蛋白质合成被证明是对TBT最敏感的参数（IC50 = 0.2微摩尔/升），其次是珊瑚组织对天冬氨酸的摄取（IC50 = 0.6微摩尔/升）、骨骼生成（IC50 = 3微摩尔/升）和珊瑚组织对Ca2+的摄取（IC50 = 20微摩尔/升）。这些结果表明，TBT对钙化的作用方式可能是抑制有机基质生物合成。
