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控机制，免疫电镜实验被认为是有效和有意义的。本研究首先从贻贝（Mytilus edulis）的前背牵肌（abbm）中分离纯化MCBP-450，获得MCBP-450抗原，然后制备MCBP-450抗体。基于首次报道发现该蛋白的方法（Yamanobe and Sugi, Biochim）。Biophys。Acta 1149:166-174, 1993)， SDS-PAGE分离得到分子量为450 kDa的蛋白。将钙指示剂quin2应用于样品(Tatsumi et al., Ana；生物化学。254:126-131,1997)。然后，通过检测荧光的发射，证明了蛋白质具有结合钙离子的能力。可以合理地推断样品中含有MCBP-450。然而，所得的蛋白数量较少，难以直接用于抗体的生产。因此，作者试图检测该蛋白的氨基酸序列，以便在体外合成一些肽作为免疫抗原。纯化样品透析后的SDS-PAGE未见450 kDa对应的明显条带，但有~ 100 kDa对应的显著条带。用Procise 494HT蛋白测序系统检测了~ 100 kDa蛋白中酶切片段的3个肽段的氨基酸序列，分别确定了12个氨基酸的序列。利用NCBInr文库对这些序列与多种已知蛋白序列进行同源性分析，结果表明，这些序列与多种细胞和组织的α-肌动蛋白具有较高的同源性（~ 90%）。提示MCBP-450分子的一部分与α-丝氨酸的某些部分非常相似。作为初步实验，我们还在免疫电镜下检测了一种钙结合蛋白calsequestrin在ABRM纤维中的细胞内定位，并证实了calsequestrin在肌浆网管腔中的定位。进一步的实验揭示MCBP-450的细胞内定位目前正在进行中。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