Takeuchi K, Senba S, Furukawa K, Eto M, Morita F
Osaka Prefectural College of Health Science, Habikino, Osaka, 583-0872, Japan.
J Biochem. 1999 Feb;125(2):334-42. doi: 10.1093/oxfordjournals.jbchem.a022291.
Smooth muscle myosin II contains two 17-kDa essential light chain isoforms (LC17gi and LC17nm) of which the relative contents differ among myosins. To understand the roles of LC17 isoforms in the functions of myosin, we performed an immunofluorescence microscopic examination of their localization in primary cultured cells isolated from rat aortic smooth muscle. To identify the isoforms, rabbit polyclonal antibodies were prepared against C-terminal nonapeptides corresponding to either LC17gi or LC17nm from porcine aortic smooth muscle myosin. These isoforms differ in only 5 amino acid residues within the C-terminal 9 residues. These antibodies specifically recognize each LC17 isoform on urea-PAGE of total rat aortic cell lysates. Immediately after plating, the smooth muscle cells stained heterogeneously with each antibody, indicating differing contents of LC17 isoforms among cells. On double staining 1-2 d cultures with both antibodies, LC17nm was detected diffusely throughout the cytoplasm, whereas LC17gi was concentrated in specific regions such as the cell periphery and the base of cytoplasmic processes. These results support the suggestion that myosin containing LC17gi is essential for force-generation by aortic smooth muscle and that myosin containing LC17nm may play an important role in maintaining smooth muscle tension.
平滑肌肌球蛋白II包含两种17 kDa的必需轻链同工型（LC17gi和LC17nm），其相对含量在不同的肌球蛋白之间存在差异。为了了解LC17同工型在肌球蛋白功能中的作用，我们对从大鼠主动脉平滑肌分离的原代培养细胞中它们的定位进行了免疫荧光显微镜检查。为了鉴定这些同工型，制备了针对猪主动脉平滑肌肌球蛋白中与LC17gi或LC17nm相对应的C端九肽的兔多克隆抗体。这些同工型在C端9个残基中仅5个氨基酸残基不同。这些抗体在大鼠主动脉细胞总裂解物的尿素聚丙烯酰胺凝胶电泳上能特异性识别每种LC17同工型。接种后立即观察发现，平滑肌细胞用每种抗体染色时呈现异质性，表明不同细胞中LC17同工型的含量不同。在用两种抗体对培养1-2天的细胞进行双重染色时，LC17nm在整个细胞质中呈弥散性分布，而LC17gi则集中在特定区域，如细胞周边和细胞质突起的基部。这些结果支持了这样的观点，即含有LC17gi的肌球蛋白对于主动脉平滑肌产生力至关重要，而含有LC17nm的肌球蛋白可能在维持平滑肌张力方面发挥重要作用。
