Fukushima Hideto, Yoon Sung Ho, Watabe Shugo
Laboratory of Aquatic Molecular Biology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo, Tokyo 113-8657, Japan.
J Agric Food Chem. 2003 Jul 2;51(14):4089-95. doi: 10.1021/jf0211985.
Fast skeletal light meromyosins (LMMs) of white croaker and walleye pollack were prepared in our expression system using Escherichia coli and determined for their polymer-forming ability and thermodynamic properties by using sodium dodecyl sulfate polyacrylamide gel electrophoresis and differential scanning calorimetry (DSC), respectively. White croaker LMM formed dimer by heating at 80 degrees C and showed only a single peak at 32.1 degrees C of temperature transition in DSC. On the other hand, walleye pollack LMM hardly formed polymer and showed four peaks at 27.7, 30.5, 35.8, and 43.9 degrees C. When Cys525 of white croaker LMM was replaced by alanine, this point-mutated LMM showed no change in its DSC profile but formed no dimer upon heating, suggesting a possible role of Cys525 in dimer formation. On the other hand, walleye pollack LMM where Cys491 was substituted by alanine changed its DSC profile, showing four peaks at 27.9, 29.1, 38.4, and 43.9 degrees C. However, this point-mutated LMM formed no dimer upon heating as in the case of native LMM. These results suggest that cysteine residue(s) participates in thermal gel formation of LMM when it locates in a suitable position of the sequence.
我们利用大肠杆菌在表达系统中制备了白姑鱼和狭鳕鱼的快速骨骼肌轻肌球蛋白（LMMs），并分别通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳和差示扫描量热法（DSC）测定了它们的聚合物形成能力和热力学性质。白姑鱼LMM在80℃加热时形成二聚体，在DSC中温度转变仅在32.1℃出现一个单一峰。另一方面，狭鳕鱼LMM几乎不形成聚合物，在27.7、30.5、35.8和43.9℃出现四个峰。当白姑鱼LMM的Cys525被丙氨酸取代时，这种点突变的LMM的DSC图谱没有变化，但加热时不形成二聚体，这表明Cys525在二聚体形成中可能起作用。另一方面，Cys491被丙氨酸取代的狭鳕鱼LMM改变了其DSC图谱，在27.9、29.1、38.4和43.9℃出现四个峰。然而，这种点突变的LMM加热时也像天然LMM一样不形成二聚体。这些结果表明，半胱氨酸残基位于序列的合适位置时参与LMM的热凝胶形成。
