Study on the aggregation mechanism of walleye pollack myosin

黑鳕鱼是日本食品加工业的主要来源之一。在本研究中，以分离的鳕鱼肌球蛋白、HMM和LMM为原料，研究了其热诱导聚集机制。经圆二色谱分析和α-胰凝乳酶消化后发现，经50℃热处理5min后，LMM的N端2/3区和C端1/3区的DNA螺旋分别可逆和不可逆地展开。完整的鳕鱼肌球蛋白的LMM区在热处理后表现出与分离的LMM相似的变性过程。然后，用ANS-荧光光谱仪研究了肌球蛋白、HMM和LMM在热处理过程中疏水性的变化。当温度高于40℃时，肌球蛋白和隐形肌球蛋白的ANS荧光显著增强，而肌球蛋白和隐形肌球蛋白的ANS荧光几乎没有变化。这表明热处理提高肌球蛋白的疏水性主要是由于HMM区的变性所致。随着疏水性的增加，肌球蛋白和HMM的混浊度显著增加，而LMM的混浊度没有增加。这些结果表明，肌球蛋白和HMM在热处理时形成聚集，但LMM不能在相同处理下聚集，尽管LMM的某些a-螺旋不可逆转地展开。另一方面，在40℃的温度下加热10min，肌球蛋白与肌球蛋白形成共聚体，而与HMM不形成共聚体。因此，LMM似乎通过与肌球蛋白尾部区域的相互作用而与肌球蛋白形成共聚集体。在这些结果的基础上，我们提出了一个热处理的鳕鱼肌球蛋白聚集的示意图模型。

Walleye pollack Theragra chalcogramma is one of the major sources for food processing industries in Japan. In the present study, the heat-induced aggregation mechanism for the walleye pollack myosin was investigated by using isolated pollack myosin, HMM, and LMM. According to the circular dichroism spectrometry and α-chymotryptic digestion of the walleye pollack LMM, the α-helices of N-terminal 2/3 and C-terminal 1/3 regions of the LMM were revealed to be reversibly and irreversibly unfolded, respectively, by the heat-treatment at 5O℃ for 5 min. The LMM region of intact pollack myosin showed similar denaturation process to the isolated LMM upon heat-treatment. Then, the changes in hydrophobicity of the myosin, HMM, and LMM during heat-treatment were investigated by means of ANS-fluorescent spectrometry. The ANS-fluorescence of myosin and HMM showed significant increase at temperatures higher than 40℃, however, that of LMM showed practically no change. This indicates that the increase in hydrophobiclty of the myosin by the heat-treatment is mainly due to the denaturation of HMM region. Concomitantly with the increase in hydrophobicity, turbidity of the myosin and HMM significantly increased, however, that of the LMM showed no increase. These results indiccate that the myosin and HMM form aggregates upon the heat-treatment, however, the LMM is incapable of aggregating upon the same treatment although some parts of the a-helices of the LMM unfolds irreversibly. On the other hand, it was found that the LMM formed coaggregates with myosin when heated at 40℃ for 10 min together with myosin, while it did not form coaggregates with the HMM. Thus, the LMM seemed to form coaggregates with the myosin through the interaction with the tail regions of myosin. Based on these results, we presented a schematic model for the pollack myosin aggregation by the heat-treatment.