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

除了一些淡水鳐鱼外，海洋鲨鱼体内含有一种最典型的蛋白质变性剂尿素，其含量在0.2 ~ 0.6 M之间，以抵抗海水的高渗透压。然而，含尿素鲨鱼体内的蛋白质即使在尿素存在的情况下也能像硬骨鱼体内的蛋白质一样维持其生理功能。海洋鲨鱼的蛋白质被认为是通过一些分子机制来防止变性的。本研究的目的是分析淡水鳐肌球蛋白的初级结构，并与安魂鲨进行比较，以验证上述关于海洋鲨鱼肌球蛋白耐尿素分子机制的假设。安曲鲨肌纤维Mg^<2+>- atp酶活性随尿素浓度的升高而逐渐降低，在尿素浓度为0.3 M左右达到最大值，随后随尿素浓度的升高而逐渐下降。另一方面，即使在低浓度下，添加更多的尿素也会降低鲤鱼的活性，并随着尿素浓度的增加而迅速下降。淡水鳐鱼的活动接近安魂鲨和鲤鱼的相对活动的平均值。这些结果表明，从淡水射线中提取的肌球蛋白是研究尿素抵抗性的合适材料。根据淡水鳐骨骼肌肌球蛋白的cDNA核苷酸序列，推导出骨骼肌肌球蛋白的氨基酸序列，预测出1933个氨基酸残基。本研究对淡水鳐肌球蛋白中的1788个氨基酸残基与安魂鲨和其他硬骨鱼的残基进行了比较。残基785-793位于肌球蛋白重链S1区必需的轻链结合位点。与其他硬骨鱼相比，安魂鲨和淡水鳐肌球蛋白氨基酸序列表现出极强的亲水性，但797-799残基表现出疏水性。685-693残基位于S1区ATP和肌动蛋白结合位点附近。该淡水鳐肌球蛋白序列显示了鲤鱼和鲨鱼之间的亲水性。该区域靠近atp酶活性域，与尿素耐受性相关。所得结果阐明了海洋鲨鱼尿素抗性的部分分子机制。少

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