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目的:探讨骨骼肌碳酸酐酶Ⅲ(CAⅢ)是否本身具有磷酸酶活性。方法:用双向电泳结合Western blot方法将分离的大鼠骨骼肌蛋白转移至硝酸纤维膜,利用镜像原理,识别与CAⅢ抗体发生免疫反应的抗原在双向电泳图谱中的确切位置。在此基础上,以同样的方法与原理,对在固相膜上的磷酸酶活性进行原位染色,观察CAⅢ是否磷酸酶活性染色呈阳性。此外,通过磷酸酶抑制剂实验及酶催化反应动力学实验,论证CAⅢ的磷酸酶活性染色特异性及CAⅢ具有磷酸酶的功能。结果:CAⅢ抗体反应显示,利用镜像原理能从复杂的双向电泳图谱中容易识别CAⅢ蛋白;磷酸酶活性染色表明,鉴定的CAⅢ具有磷酸酶活性。磷酸酶特异抑制剂实验表明,CAⅢ的磷酸酶活性染色具有特异性;酶催化反应动力学研究证实,CAⅢ蛋白具有确凿的磷酸酶功能。结论:骨骼肌的CAⅢ本身具有磷酸酶活性,说明CAⅢ参与了细胞的磷酸化——去磷酸化过程,提示重症肌无力(MG)患者骨骼肌收缩无力和易疲劳可能涉及了MG的非免疫机制。另外,本研究创建的双向电泳结合Western blot方法可被借鉴用于“功能蛋白质组学”的研究。
Objective: To investigate if the skeletal muscle carbonic anhydrase Ⅲ (CA Ⅲ) itself has phosphatase activity. METHODS: The isolated rat skeletal muscle protein was transferred to nitrocellulose membrane by two-dimensional electrophoresis and Western blot. The mirror image principle was used to identify the exact position of the antigens reacted with CA Ⅲ antibody in two-dimensional gel electrophoresis. On this basis, using the same method and principle, in situ staining of the phosphatase activity on the solid-phase membrane to observe whether CAⅢ is positive for phosphatase activity. In addition, through the experiments of phosphatase inhibitor and enzyme-catalyzed reaction kinetics, the staining specificity of phosphatase activity of CA Ⅲ and the phosphatase activity of CA Ⅲ were demonstrated. Results: The CA Ⅲ antibody reaction showed that the CA Ⅲ protein could be easily identified from the complex two-dimensional electrophoresis map by the mirror image principle. The phosphatase activity showed that the identified CA Ⅲ had phosphatase activity. Phosphatase-specific inhibitor experiments show that, CA Ⅲ phosphatase activity staining is specific; enzyme catalytic kinetics studies have confirmed that CA Ⅲ protein has a clear phosphatase function. CONCLUSION: CAⅢ of skeletal muscle itself has phosphatase activity, indicating that CA Ⅲ is involved in the phosphorylation-dephosphorylation of cells, suggesting that the weakness and fatigue of skeletal muscle in patients with myasthenia gravis (MG) may involve the non-immune mechanism of MG . In addition, the two-dimensional electrophoresis combined with Western blot method created in this study can be used for the study of “functional proteomics”.