Proteins of calcified endoskeleton:: II Partial amino acid sequences of endoskeletal proteins and the characterization of proteinaceous organic matrix of spicules from the alcyonarian, Synularia polydactyla
Proteins of calcified endoskeleton:: II Partial amino acid sequences of endoskeletal proteins and the characterization of proteinaceous organic matrix of spicules from the alcyonarian, Synularia polydactyla
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DOI：
10.1002/pmic.200401130
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发表时间：
2005-03-01
期刊：
影响因子：
3.4
通讯作者：
Uehara, T
中科院分区：
文献类型：
--
作者：
Rahman, MA;Isa, Y;Uehara, T
骨骼中的钙化有机物质含有蛋白质-多糖复合物，在生物钙化的调节中起关键作用。然而，关于基质蛋白在钙化过程中的作用的信息仍然是未知的。出于这个原因，我们研究了有机基质的内骨骼骨针从alcyonarian珊瑚，Synularia polydactyla，分析蛋白质与它们的序列，并通过分子方法研究功能特性。从殖民地中分离的针状体用扫描电镜（SEM）进行鉴定。可溶性有机基质占骨针重量的0.04%。通过记录实验设计中pH的下降，揭示了基质对CaCO 3沉淀的抑制作用。在电泳之前，我们对从针状体的可溶性有机基质中提取的蛋白质的分析揭示了分子量丰富的蛋白质。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析的制剂显示了7条带的蛋白质的表观分子量为109，83，70，63，41，30和22 kDa。电洗脱后的蛋白在Tricine-SDS-PAGE上电泳，然后转移到聚偏氟乙烯（PVDF）膜上，并对其N-末端进行测序。两个条带的蛋白质的约70和61 kDa的成功进行N-末端氨基酸测序。对于钙结合蛋白的检测，通过Ca-45放射自显影对提取物进行Ca 2+覆盖分析。发现109和63 kDa的钙结合蛋白具有放射性。高碘酸希夫染色表明，83和63 kDa的蛋白质被糖基化。对被认为在钙化过程中起重要作用的碳酸酐酶的测定显示出低水平的活性。这些研究结果表明，内骨骼骨针的alcyonarian珊瑚有富含蛋白质的有机基质，这可能与钙化过程。
Calcified organic substances in the skeleton contain a protein-polysaccharide complex taking a key role in the regulation of bio-calcification. However, information concerning the matrix proteins in alcyonarian and their effect on calcification process is still unknown. For this reason, we have studied the organic matrix of endoskeletal spicules from the alcyonarian coral, Synularia polydactyla, to analyze the proteins with their sequences and investigate the functional properties by a molecular approach. The separated spicules from the colony were identified by scanning electron microscope (SEM). The soluble organic matrix comprised 0.04% of spicule weight. By recording decline of pH in the experimental design, the inhibitory effect of the matrix on CaCO3 precipitation was revealed. Prior to electrophoresis, our analysis of proteins extracted from the soluble organic matrix of the spicules revealed an abundance of proteins in molecular weight. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the preparations showed seven bands of proteins with an apparent molecular mass of 109, 83, 70, 63, 41, 30 and 22 kDa. The proteins were electrophoresed on Tricine-SDS-PAGE after electro-elution treatment, and then transferred to polyvinylidene difluoride (PVDF) membranes and their N-termini were sequenced. Two bands of proteins of about 70 and 61 kDa successfully underwent N-terminal amino acid sequencing. For the detection of calcium binding proteins, a Ca2+ overlay analysis was conducted on the extract by Ca-45 autoradiography. The 109 and 63 kDa calcium binding proteins were found to be radioactive. Periodic acid schiff staining indicated that 83 and 63 kDa proteins were glycosylated. An assay for carbonic anhydrase, which is thought to play an important role in the process of calcification revealed low level of the activity. These findings suggest that the endoskeletal spicules of alcyonarian corals have protein-rich organic matrices, which might be related to the calcification process.