Kirkeby S, Moe D, Bøg-Hansen T C, van Noorden C J
Department of Oral Function, Faculty of Health Sciences, University of Copenhagen, Denmark.
Histochemistry. 1993 Dec;100(6):415-21. doi: 10.1007/BF00267821.
Biotin carboxylases in mammalian cells are regulatory enzymes in lipogenesis and gluconeogenesis. In this study, endogenous biotin in skeletal and cardiac muscle was detected using avidin conjugated with alkaline phosphatase and applied in high concentrations to muscle sections. The avidin binding was subsequently visualized by histochemical demonstration of the alkaline phosphatase activity. All cardiac muscle cells showed high affinity for avidin with only the nuclei and the intercalated discs remaining unstained. In skeletal muscle a diffuse reaction could be detected in the sarcoplasm of the muscle fibres. A granular reaction was noted in the same fibres that showed activity for succinic dehydrogenase. The specificity of the coloured reaction product in the muscle sections was investigated and is suggested to be caused by avidin binding to biotin moieties in mitochondria and the cytosol. Mitochondrial and cytosolic preparations of skeletal muscle were electrophoresed in sodium dodecyl sulphate gels. After blotting and incubation with conjugated avidin, two bands with molecular weights of 75 kDa and 130 kDa respectively were evident in the mitochondrial preparation. It is suggested that the 75-kDa band represents comigration of the biotin-containing subunits of propionyl-CoA carboxylase and methylcrotonyl-CoA carboxylase. The 130-kDa band may represent the biotin-containing pyruvate carboxylase. In the cytosolic preparation a 270-kDa band was stained in blots that had been incubated with conjugated avidin; this band is suggested to represent acetyl-CoA carboxylase. A 190-kDa cytosolic band might be a cleavage product of acetyl-CoA carboxylase. We propose that using alkaline phosphatase-conjugated avidin it is possible to detect the mitochondrial and cytosolic biotin-dependent carboxylases in striated muscle.
哺乳动物细胞中的生物素羧化酶是脂肪生成和糖异生过程中的调节酶。在本研究中，使用与碱性磷酸酶偶联的抗生物素蛋白检测骨骼肌和心肌中的内源性生物素，并将其高浓度应用于肌肉切片。随后通过碱性磷酸酶活性的组织化学显示来观察抗生物素蛋白的结合情况。所有心肌细胞均显示出对抗生物素蛋白的高亲和力，只有细胞核和闰盘未被染色。在骨骼肌中，可在肌纤维的肌浆中检测到弥漫性反应。在显示琥珀酸脱氢酶活性的相同纤维中观察到颗粒状反应。对肌肉切片中显色反应产物的特异性进行了研究，提示其是由抗生物素蛋白与线粒体和胞质溶胶中的生物素部分结合所致。将骨骼肌的线粒体和胞质溶胶制剂在十二烷基硫酸钠凝胶中进行电泳。印迹并与偶联的抗生物素蛋白孵育后，线粒体制剂中明显出现两条分别为75 kDa和130 kDa的条带。提示75 kDa条带代表丙酰辅酶A羧化酶和甲基巴豆酰辅酶A羧化酶含生物素的亚基共迁移。130 kDa条带可能代表含生物素的丙酮酸羧化酶。在胞质溶胶制剂中，与偶联的抗生物素蛋白孵育的印迹中出现一条270 kDa的条带被染色；提示该条带代表乙酰辅酶A羧化酶。一条190 kDa的胞质溶胶条带可能是乙酰辅酶A羧化酶的裂解产物。我们提出，使用碱性磷酸酶偶联的抗生物素蛋白可以检测横纹肌中线粒体和胞质溶胶中依赖生物素的羧化酶。
