Intracellular Localization of Ca-binding protein MCBP-450 and the Regulatory Mechanism for Contraction in a Molluscan Smooth Muscle

为了明确钙结合蛋白MCBP-450的细胞内定位及其收缩调节机制，人们认为免疫电镜实验是有效和有意义的。第一步，在本研究中，从紫贻贝的前深海牵开肌（ABRM）中分离和纯化MCBP-450，以获得MCBP-450抗体之前的抗原。基于发现这种蛋白质的第一份报告所应用的方法（Yamanobe和Sugi，Biochim. Biophys. Acta 1149：166-174，1993），通过SDS-PAGE分离含有对应于450 kDa的分子量的蛋白质的样品。将Ca-指示剂quin 2应用于样品（Tatsumi等人，安娜254：126-131，1997）。通过荧光检测证明该蛋白具有与Ca ~（2+）结合的能力。可以合理推断样品中含有MCBP-450。然而，蛋白质产量是非常低的， ...更多信息 产量小，难以直接供应抗体生产。因此，作者试图对该蛋白的氨基酸序列进行分析，以期在体外合成多肽作为免疫抗原。纯化样品透析后的SDS-PAGE显示没有对应于450 kDa的明显条带，但对应于约100 kDa的显著条带。用Procise 494 HT蛋白质测序系统对100 kDa蛋白酶切片段的3个肽段进行了氨基酸序列测定，分别确定了12个氨基酸的序列。利用NCBInr文库对这些序列进行同源性搜索，结果表明它们与多种细胞和组织中的α-辅肌动蛋白具有很高的同源性（约90%）。结果表明，MCBP-450分子中有一部分与α-scrinin分子中的某些部分相似。作为一个初步的实验，细胞内定位的一个高贵的钙结合蛋白，calsequestrin，在ABRM纤维的免疫电镜检查，并证明了calsequestrin的肌浆网腔的本地化。目前正在进行进一步的实验以揭示MCBP-450的细胞内定位。少

To make clear the intracellular localization of a Ca-binding protein, MCBP-450, and its regulatory mechanism for contraction, it has been thought that an experiment by immuno-electron microscopy is valid and significant. For the first step, in the present study, the isolation and purification of MCBP-450 from the anterior byssal retractor muscle (ABRM) of Mytilus edulis were examined to obtain the antigen prior to producing antibody for MCBP-450. Based on the method applied by the first report finding this protein (Yamanobe and Sugi, Biochim. Biophys. Acta 1149:166-174, 1993), a sample containing protein of molecular weight corresponding to 450 kDa was separated by SDS-PAGE. Ca-indicator quin2 was applied to the sample (Tatsumi et al., Ana. Biochem. 254:126-131, 1997). Then it was proved that the protein had an ability to bind Ca ions, by detecting the emission of fluorescence. It is reasonable to conclude that the sample is containing the MCBP-450. However, the protein yielded was ver … More y small in quantity, and difficult to supply directly for antibody producing. Therefore, the author tried to examine the amino acid sequence of this protein, in order to synthesize some peptides in vitro as an antigen for immunity. SDS-PAGE after the dialysis of purified samples showed no distinct band corresponding to 450 kDa, but a remarkable band corresponding to 〜100 kDa. The amino acid sequence of three peptides fragmented enzymatically from the 〜100 kDa protein was examined with Procise 494HT Protein Sequencing System, and determind the sequences of 12 amino acids, respectively. The homology-search of these sequences to the various known protein sequences using the NCBInr Library indicated a high homology (〜90%) with α-actinin of various kinds of cells and tissues. It is suggested that a part of MCBP-450 molecule resembles closely to some parts of α-scrinin. As a preliminary experiment, intracellular localization of a noble Ca-binding protein, calsequestrin, in ABRM fibers was also examined immuno-electron microscopically, and proved the localization of calsequestrin in the lumen of sarcoplasmic reticumum. Further experiments to reveal the intracellular localization of MCBP-450 is now in progress. Less