Timson D J, Trayer H R, Trayer I P
School of Biochemistry, The University of Birmingham, United Kingdom.
Eur J Biochem. 1998 Aug 1;255(3):654-62. doi: 10.1046/j.1432-1327.1998.2550654.x.
There are two isoforms (A1 and A2) of the myosin essential light chain (ELC) and consequently two isoenzymes of myosin subfragment 1 (S1), S1(A1) and S1(A2). The two isoenzymes differ in their kinetic properties with S1(A1) having a lower apparent Km for actin and a slower turnover of MgATP (k(cat)) than S1(A2). The two forms of the ELC differ only at their N-termini where A1 has an additional 40-odd amino acids that are not present in A2. The human atrial ELC (an A1-type ELC) was overexpressed in Escherichia coli and purified by ammonium sulphate fractionation and ion-exchange chromatography. The recombinant ELC had actin-activated MgATPase kinetics similar to those for rabbit skeletal S1(A1) under the same conditions. Deletion of the first 45 amino acid residues resulted in an ELC similar to the rabbit skeletal A2 isoform and, when hybridised into S1, in S1(A2)-like kinetic properties. Results obtained with an ELC mutant that lacks the first 11 residues were intermediate between these two extremes but tending towards the S1(A2)-like phenotype. The wild-type ELC (both hybridised into S1 or free in solution) could be cross-linked to F-actin, whereas the deletion mutant lacking the first 45 amino acids could not. The deletion mutant lacking the first 11 amino acids cross-linked only poorly under the same conditions, consistent with the MgATPase data. We therefore conclude that these N-terminal eleven amino acids predominantly encode an actin-binding site which modulates the kinetics of the myosin motor. Furthermore, while free A1-type ELC cross-linked to both polymeric F-actin and the monomeric G-actin:DNase-I complex, the same ELC in S1(A1) could only cross-link to F-actin. This suggests that the light chain binds to a different actin monomer than the heavy chain.
肌球蛋白必需轻链（ELC）有两种同工型（A1和A2），因此肌球蛋白亚片段1（S1）有两种同工酶，即S1(A1)和S1(A2)。这两种同工酶的动力学特性不同，S1(A1)对肌动蛋白的表观Km较低，且MgATP的周转速度（k(cat)）比S1(A2)慢。ELC的两种形式仅在其N端有所不同，其中A1比A2多了40多个不存在于A2中的氨基酸。人房性ELC（一种A1型ELC）在大肠杆菌中过表达，并通过硫酸铵分级分离和离子交换色谱法纯化。在相同条件下，重组ELC具有与兔骨骼肌S1(A1)相似的肌动蛋白激活的MgATP酶动力学特性。删除前45个氨基酸残基会产生一种类似于兔骨骼肌A2同工型的ELC，当与S1杂交时，会产生类似于S1(A2)的动力学特性。用缺失前11个残基的ELC突变体得到的结果介于这两个极端之间，但倾向于S1(A2)样表型。野生型ELC（无论是与S1杂交还是游离于溶液中）都可以与F-肌动蛋白交联，而缺失前45个氨基酸的缺失突变体则不能。在相同条件下，缺失前1个氨基酸的缺失突变体交联效果很差，这与MgATP酶数据一致。因此，我们得出结论，这些N端的11个氨基酸主要编码一个肌动蛋白结合位点，该位点调节肌球蛋白马达的动力学。此外，虽然游离的A1型ELC与聚合的F-肌动蛋白和单体G-肌动蛋白：DNase-I复合物都能交联，但S1(A1)中的相同ELC只能与F-肌动蛋白交联。这表明轻链与重链结合的肌动蛋白单体不同。
