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Elucidation of the molecular mechanism of urea-resistibility, comparing the structural difference between freshwater ray and marine shark myosin

阐明尿素抵抗力的分子机制，比较淡水鳐鱼和海洋鲨鱼肌球蛋白的结构差异

基本信息

批准号：
14560171
负责人：
KANOH Satoshi
金额：
$ 2.24万
依托单位：
Mie University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

项目摘要

Marine sharks except for some freshwater rays contain urea, one of the most typical protein denaturants, in the range of 0.2 to 0.6 M to exert resistancy against high osmolarity of seawater. However, proteins in urea-containing sharks can maintain their physiological functions even in the presence of urea as proteins of teleost in the absence of urea. The proteins from marine sharks are supposed to be protected from denaturation by some molecular mechanisms. The objective of this study was to analyze the primary structure of freshwater ray myosin and to compare those of requiem shark to test the above assumption on the molecular mechanism underlying urea-resistibility of marine shark myosin.The myofibrillar Mg^<2+>-ATPase activity of requiem shark was gradually decreased with increasing urea concentrations, showing maximal value at around 0.3 M urea and then declined gradually with increasing urea concentrations. On the other hand, the activity of carp was decreased by the addition of … More urea even at a low concentration and declined rapidly with increasing urea concentrations. The activity of freshwater ray was nearly the average of the relative activities of requiem shark and carp. These results indicated that the myosin from freshwater ray is suitable material for the research of the urea-resistibility.The amino acid sequence of skeletal muscle myosin from freshwater ray was deduced from cDNA nucleotide sequence, predicting 1933 amino acid residues. In this study,1788 amino acid residues from freshwater ray myosin were compared with those of requiem shark and other teleost. The residues 785-793 located in essential light chain binding site in the myosin heavy chain S1 region. This amino acid sequence of requiem shark and freshwater ray myosin showed extreme hydrohilicity comparing with those of other teleost, but the residues 797-799 showed hydrophobicity. The residues 685-693 located around the ATP and actin-binding site in S1 region. This sequence of freshwater ray myosin showed the hydropathy between carp and shark. This region is close to the ATPase activity domain, and the relevance to the urea-resistibility was indicated. The obtained results elucidate a part of molecular mechanism of urea-resistibility of the marine shark. Less

除一些淡水鱼外，海洋鲨鱼含有0.2至0.6M的尿素，这是最典型的蛋白质变性剂之一，可抵抗海水的高渗透压。然而，在没有尿素的情况下，即使在尿素存在的情况下，含有尿素的鲨鱼中的蛋白质也可以作为硬骨鱼的蛋白质保持其生理功能。海洋鲨鱼的蛋白质被认为是通过某种分子机制来保护其免受变性的。本研究的目的是分析淡水射线肌球蛋白的一级结构，并与安息鲨的一级结构进行比较，以验证上述关于海洋鲨鱼肌球蛋白耐尿素的分子机制的假设。随着尿素浓度的增加，安息鲨的肌原纤维镁离子-ATP酶活性逐渐降低，在0.3M尿素附近出现最大值，然后随着尿素浓度的增加而逐渐下降。另一方面，…的添加降低了鲤鱼的活性尿素浓度较低时仍较高，且随尿素浓度的增加而迅速下降。淡水鱼的活性接近安息鲨和鲤鱼相对活性的平均值。这些结果表明，淡水鱼肌球蛋白是研究其耐尿素能力的理想材料。根据该基因的核苷酸序列，推导出淡水鱼骨骼肌肌球蛋白的氨基酸序列，预测了1933个氨基酸残基。本研究比较了淡水鱼肌球蛋白与安息鲨等硬骨鱼的1788个氨基酸残基。残基785-793位于肌球蛋白重链S1区的基本轻链结合部位。与其他硬骨鱼相比，安息鲨和淡水鱼肌球蛋白的氨基酸序列表现出极强的疏水性，但797-799残基表现出疏水性。残基685-693位于三磷酸腺苷和肌动蛋白结合部位附近，位于S1区。这一淡水射线肌球蛋白序列显示了鲤鱼和鲨鱼之间的水生现象。该区域位于ATPase活性区域附近，表明该区域与抗尿素能力有关。这些结果解释了海洋鲨鱼耐尿素的部分分子机制。较少