Maruyama K, Kanemaki N
USDA Agricultural Research Service, Avian Physiology Laboratory, Beltsville, Maryland 20705.
Poult Sci. 1991 Aug;70(8):1748-57. doi: 10.3382/ps.0701748.
The appearance of myosin isoforms in skeletal muscles of turkey embryos, poults, and toms was studied, using monoclonal antibodies raised against myosin isoforms in chicken fast-twitch muscle (Pectoralis). The myosin extract was prepared by repeated salt extraction-precipitation. The reactivity of monoclonal antibodies with turkey myosin isoforms was tested by an enzyme linked immunosorbent assay using alkaline phosphatase-conjugated antibody and detection by color development with p-nitrophenyl phosphate. Detection was also effected by protein slot blotting using peroxidase-conjugated antibody and color development with 3,3'-diaminobenzidine tetrahydrochloride. The monoclonal antibody AB8 was found to be specific for the adult myosin isoform, present in Pectoralis muscle of 14-day-old and adult turkeys and adult chickens. Subsequent peptide mapping also indicated that the adult myosin isoform of turkey Pectoralis muscle was nearly identical to the adult isoform from chickens. The monoclonal antibody 2E9 reacted with the myosin extract only from poults at ages of 7 days and 14 days posthatch, indicating that 2E9 is specific for the neonatal myosin isoform. The reactivity of 2E9 was noted with the muscle of the mixed fiber type (the thigh muscle group) as well as with the fast-twitch muscle (Pectoralis). Monoclonal antibodies EB 165 and AG6 were found to react with the myosin extract from all ages tested. Based on the reactivity with monoclonal antibodies, it was concluded that myosin in turkey muscles existed as at least three discrete isoforms that were expressed sequentially in the course of muscle development.
利用针对鸡快肌(胸肌)中肌球蛋白亚型制备的单克隆抗体,研究了火鸡胚胎、雏火鸡和成年雄火鸡骨骼肌中肌球蛋白亚型的出现情况。通过反复盐提取-沉淀制备肌球蛋白提取物。使用碱性磷酸酶偶联抗体的酶联免疫吸附测定法,并用对硝基苯磷酸显色检测单克隆抗体与火鸡肌球蛋白亚型的反应性。还通过使用过氧化物酶偶联抗体的蛋白质狭缝印迹法并用3,3'-二氨基联苯胺四盐酸盐显色进行检测。发现单克隆抗体AB8对成年肌球蛋白亚型具有特异性,存在于14日龄和成年火鸡以及成年鸡的胸肌中。随后的肽图谱分析还表明,火鸡胸肌的成年肌球蛋白亚型与鸡的成年亚型几乎相同。单克隆抗体2E9仅与孵化后7天和14天的雏火鸡的肌球蛋白提取物发生反应,表明2E9对新生儿肌球蛋白亚型具有特异性。注意到2E9与混合纤维类型的肌肉(大腿肌群)以及快肌(胸肌)都有反应。发现单克隆抗体EB 165和AG6与所有测试年龄的肌球蛋白提取物都发生反应。基于与单克隆抗体的反应性,得出结论:火鸡肌肉中的肌球蛋白至少以三种不同的亚型存在,它们在肌肉发育过程中依次表达。