Study on the aggregation mechanism of walleye pollack myosin

银鱼Theragra chalcogramma是日本食品加工业的主要来源之一。在本研究中，热诱导的狭鳕肌球蛋白的聚集机制进行了研究，通过使用分离的狭鳕肌球蛋白，HMM，和LMM。圆二色谱分析和α-糜蛋白酶消化结果表明，狭鳕LMM N端2/3和C端1/3区域的α-螺旋在50 ℃热处理5 min后分别发生可逆和不可逆的去折叠，其LMM区域的变性过程与分离的LMM相似。然后，通过ANS-荧光光谱法研究了热处理过程中肌球蛋白、HMM和LMM的疏水性变化。温度高于40℃时，肌球蛋白和HMM的ANS荧光显著增强，而LMM的ANS荧光几乎没有变化。这表明热处理引起的肌球蛋白疏水性的增加主要是由于HMM区域的变性。伴随着疏水性的增加，浊度的肌球蛋白和HMM显着增加，但是，LMM没有显示出增加。这些结果表明，肌球蛋白和HMM在热处理时形成聚集体，然而，LMM在相同处理时不能聚集，尽管LMM的α-螺旋的某些部分不可逆地展开。另一方面，发现LMM与肌球蛋白一起在40℃加热10 min时与肌球蛋白形成共聚集体，而它与HMM不形成共聚集体。因此，LMM似乎通过与肌球蛋白尾部区域的相互作用与肌球蛋白形成共聚集体。在此基础上，提出了狭鳕肌球蛋白热处理聚集的模型。

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.