Katoh T, Numata T, Konishi K, Furuya K, Yazawa M
Graduate School of Science, Hokkaido University, Sapporo, 060, Japan.
Anal Biochem. 1997 Nov 1;253(1):78-84. doi: 10.1006/abio.1997.2346.
The exchange of 17-kDa essential light chain (LC17) in smooth muscle myosin was carried out by incubating myosin with a 10-fold molar excess of exogenously added LC17 over the corresponding endogenous light chain in the presence of trifluoperazine and 4.5 m ammonium chloride. Porcine aorta myosin contains two kinds of LC17 isoform, LC17nm and LC17gi, while chicken gizzard myosin contains only one kind of LC17 isoform. As LC17gi can be separated from LC17nm and gizzard LC17 by urea-gel electrophoresis, LC17nm in aorta myosin and LC17 in gizzard myosin were exchanged with LC17gi and LC17gi in aorta myosin was exchanged with LC17nm, and the degree of exchange was estimated by urea-gel electrophoresis. Under the optimal conditions (6 and 10 degrees C for aorta and gizzard myosin, respectively), nearly 90% of exchange, which is close to the theoretical value, was achieved for the former combinations, and a slightly lower exchange was obtained for the latter. The LC17-exchanged myosins contained stoichiometric amounts of the heavy and light chains and retained the original nature in the phosphorylation-dependent actin-activated ATPase activity, 6S-10S conformational transition, and filament assembly.
在三氟拉嗪和4.5 M氯化铵存在的情况下，将肌球蛋白与外源添加的LC17（相对于相应的内源性轻链，摩尔过量10倍）一起孵育，实现平滑肌肌球蛋白中17-kDa必需轻链（LC17）的交换。猪主动脉肌球蛋白含有两种LC17同工型，即LC17nm和LC17gi，而鸡砂囊肌球蛋白仅含有一种LC17同工型。由于LC17gi可通过尿素凝胶电泳与LC17nm和砂囊LC17分离，因此将主动脉肌球蛋白中的LC17nm和砂囊肌球蛋白中的LC17与LC17gi进行交换，将主动脉肌球蛋白中的LC17gi与LC17nm进行交换，并通过尿素凝胶电泳估计交换程度。在最佳条件下（主动脉和砂囊肌球蛋白分别为6℃和10℃），前一组的交换率接近90%，接近理论值，后一组的交换率略低。LC17交换后的肌球蛋白含有化学计量的重链和轻链，并在磷酸化依赖性肌动蛋白激活的ATP酶活性、6S-10S构象转变和细丝组装方面保留了原始特性。
