Cloning and sequencing of cDNA for fish slow skeletal muscle myosin light chains

各种各样的鱼有两种类型的骨骼肌，普通（白色）和暗色（红色）肌肉。高等脊椎动物的普通肌肉与快肌相对应，暗肌与慢肌相对应。在高等脊椎动物中，两种骨骼肌肌球蛋白都由两条约200kDa的重链和四条约20kDa的轻链组成。鱼类快速骨骼肌肌球蛋白有三种轻链，可分为两类，一类是由两个异源组分（按分子量递减顺序为A1和A2）组成的碱基轻链，另一类是由5,5′-二硫代-双-2-硝基苯甲酸（DTNB）组成的碱基轻链。另一方面，众所周知，鱼类慢肌肌球蛋白与快肌肌球蛋白在轻链组成上是不同的。前肌凝蛋白一般只有两种轻链（D1和D2），心肌肌凝蛋白也是如此。然而，关于鱼类慢骨骼肌肌球蛋白轻链的结构特征的信息是不可用的。本研究分别对斑马鱼慢骨骼肌D1轻链和D2轻链的cDNA克隆进行了克隆、测序和鉴定。与A1轻链一样，D1轻链在n端也有一部分差异肽。这一结果强烈提示慢速骨骼肌肌球蛋白D1轻链与快速骨骼肌肌球蛋白A1轻链具有相同的功能。D2轻链也发现有丝氨酸残基，表现出与快肌DTNA轻链一样的磷酸化。本研究的结果是首次报道鱼类慢肌肌球蛋白轻链的完整初级结构。

Various species of fish have two types of skeletal muscle, the ordinary (white) and dark (red) muscles. It has been suggested that ordinary muscle corresponds to fast muscle of higher vertebrates, while dark muscle corresponds to slow muscle. Both skeletal muscle myosins consist of two heavy chains of approximately 200kDa and four light chains of approximately 20kDa as in the case of higher-vertebrate counterparts. The fish fast skeletal muscle myosin has been shown to have three kinds of light chains, which are classified into two groups, one pair of alkali light chains consisting of two heterologous components (A1 and A2 in the order of decreasing molecular weight) and another pair of 5,5'-dithio-bis-2-nitrobenzoic acid (DTNB) light chains. On the other hand, it is well-known that fish slow muscle myosin differs from fast muscle myosin in light chain composition. The former myosin generally has only two kinds of light chain (D1 and D2) as does cardiac myosin. However, information on structural characterization of fish slow skeletal muscle myosin light chains is not available at all. This study described cloning, sequencing, and characterization of cDNA clones encoding D1 and D2 light chains of striped jack Caranx delicatissimus and horse mackerel Trachurus japonicus slow skeletal muscles, respectively. D1 light chain was found to have a part of the difference peptide portion in the N-terminal region, as was detected in A1 light chains. This result strongly suggests that D1 light chain in slow skeletal muscle myosin possess the same function as A1 light chain in fast skeletal muscle myosin. D2 light chain was also found to have serine residue, showing phosphorylation as was detected in DTNA light chain of fast muscle. The results in this study were the first known reports on the complete primary structure of fish slow muscle myosin light chains.