| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2002: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2001: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2000: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Research Abstract |
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
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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
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