Hormonal effects on the immunocytochemical location of 3',5'-cyclic adenosine monophosphate-dependent protein kinase in rat tissues.
Hormonal effects on the immunocytochemical location of 3',5'-cyclic adenosine monophosphate-dependent protein kinase in rat tissues.
复制标题
激素对大鼠组织中 3,5-环单磷酸腺苷依赖性蛋白激酶免疫细胞化学定位的影响。
DOI：
10.1210/endo-109-6-2226
复制
发表时间：
1981
期刊：
影响因子：
4.8
通讯作者：
A. Steiner
中科院分区：
文献类型：
--
作者：
Y. Koide;J. Beavo;C. L. Kapoor;W. A. Spruill;H. L. Huang;S. Levine;S. Ong;P. Bechtel;W. J. Yount;A. Steiner
使用cAMP依赖性蛋白激酶(cAMP激酶)的I型和II型调节亚基(分别为RI和RII)的均质制剂作为抗原以获得同工酶特异性抗血清。注射来自 I 型同工酶的纯催化亚基 (C) 会产生与从任一同工酶获得的 C 亚基反应的抗血清。通过免疫扩散分析和通过测量免疫沉淀后亚基的cAMP结合和磷酸转移酶活性来评估针对激酶的分离亚基产生的抗血清的交叉反应性。通过使用间接免疫荧光和免疫过氧化物酶技术，这些抗血清用于定位大鼠骨骼肌、肝脏和肾上腺中的 I 型和 II 型 cAMP 激酶亚基。免疫荧光的特异性通过纯同源抗原的抗血清的吸收来显示。在骨骼肌中，I 型和 II 型 cAMP 激酶的 R 和 C 亚基均位于肌浆网区域和周期性交叉带中。在肌节的各向同性和各向异性带区均观察到这些成分的特异性荧光。免疫过氧化物酶染色的光密度测定显示，各向同性带中的 RI、RII 和 C 亚基数量比各向异性带区域中的数量多。在肝脏中，C、RI 和 RII 亚基分布在肝细胞的细胞质和核区域以及沿着质膜；然而，在这些不同的亚细胞位点之间观察到了质的差异。对于每种抗血清，通过用同源抗原预先吸收来阻断荧光。用胰高血糖素治疗大鼠后，细胞核中 C 和 RII 的相对分布模式发生了巨大变化。在肾上腺中，RI、RII 和 C 亚基定位于细胞质和核区域，并且在用 ACTH 治疗（地塞米松抑制的）大鼠后，这些亚基发生明显的重新分布。这种免疫细胞化学方法的应用提供了一种检查和监测生物系统中 cAMP 激酶这些成分的亚细胞分布的工具。
Homogeneous preparations of type I and type II regulatory subunits (RI and RII, respectively) of cAMP-dependent protein kinase (cAMP kinase) were utilized as antigens to obtain isozyme specific antisera. Injections of pure catalytic subunit (C) from the type I isozyme resulted in antisera that reacted with C subunit obtained from either isozyme type. Cross-reactivity of the antisera raised against isolated subunits of the kinase was assessed by immunodiffusion analysis and by measuring the cAMP binding and phosphotransferase activities of the subunits after immunoprecipitation. These antisera were used to localize subunits of type I and type II cAMP kinases in rat skeletal muscle, liver, and adrenal by using indirect immunofluorescence and immunoperoxidase techniques. Specificity of the immunofluorescence was shown by absorption of the antisera with pure homologous antigens. In skeletal muscle, both R and C subunits of the type I and type II cAMP kinases were localized in the area of the sarcoplasmic reticulum and in periodic crossbands. Specific fluorescence for these components was observed in both isotropic and anisotropic band regions of the sarcomere. Densitometric determinations of immunoperoxidase staining revealed a larger amount of RI, RII, and C subunits in the isotropic band than in the anisotropic band regions. In liver, C, RI, and RII subunits were distributed both in cytoplasmic and nuclear areas and along plasma membranes of hepatocytes; however, there were qualitative differences observed among these various subcellular sites. With each antiserum, fluorescence was blocked by prior absorption with homologous antigen. After treatment of rats with glucagon, dramatic changes in the relative distribution patterns of C and RII were noted in the nucleus. In the adrenal gland, RI, RII, and C subunits were localized in both cytoplasmic and nuclear areas, and an apparent redistribution of these subunits occurred after treatment of (dexamethasone-suppressed) rats with ACTH. The application of this immunocytochemical approach provides a tool for examining and monitoring the subcellular distribution of these components of cAMP kinase in biological systems.