Nakajo S, Hayashi K, Nakaya K, Nakamura Y
J Biochem. 1983 Jan;93(1):149-57. doi: 10.1093/oxfordjournals.jbchem.a134148.
We have demonstrated the identity of calmodulin tightly bound to the particulate fractions of AH-66 hepatoma cells and normal liver with authentic soluble calmodulin and have compared the particulate calmodulin content of AH-66 cells with that of normal liver. Calmodulin bound to the particulate fractions of the hepatoma and normal liver cells was purified to electrophoretic homogeneity by a simple procedure which involves solubilization of the particulate fractions with LIS, extraction of the solubilized solution with 37.5% phenol, gel filtration, and affinity chromatography on Fluphenazine-Sepharose. There were no detectable differences between the particulate calmodulin thus purified and authentic soluble calmodulin of rat brain. The particulate calmodulin in the hepatoma and normal liver cells was assayed based on its ability to activate calmodulin-deficient phosphodiesterase after partial purification of calmodulin from the particulate fractions by solubilization with LIS and extraction with phenol as described above. In addition, the particulate calmodulin content in the hepatoma and normal liver cells was also measured after solubilization of the particulate fractions with Lubrol PX. The results obtained by these two different procedures indicate that calmodulin content in the particulate fraction as well as in the soluble fraction of the hepatoma is significantly higher than that in the corresponding fractions of normal liver.
我们已证实，与AH-66肝癌细胞和正常肝脏的微粒部分紧密结合的钙调蛋白，与纯可溶性钙调蛋白具有一致性，并比较了AH-66细胞和正常肝脏中微粒钙调蛋白的含量。通过一种简单的程序，将与肝癌细胞和正常肝脏细胞微粒部分结合的钙调蛋白纯化至电泳均一性，该程序包括用LIS溶解微粒部分、用37.5%苯酚萃取溶解后的溶液、凝胶过滤以及在氟奋乃静-琼脂糖上进行亲和层析。如此纯化得到的微粒钙调蛋白与大鼠脑的纯可溶性钙调蛋白之间未检测到差异。按照上述方法，先用LIS溶解微粒部分，再用苯酚萃取，对钙调蛋白进行部分纯化后，根据其激活钙调蛋白缺陷型磷酸二酯酶的能力，测定肝癌细胞和正常肝脏细胞中的微粒钙调蛋白。此外，在用Lubrol PX溶解微粒部分后，也测定了肝癌细胞和正常肝脏细胞中的微粒钙调蛋白含量。通过这两种不同程序获得的结果表明，肝癌细胞微粒部分和可溶性部分中的钙调蛋白含量均显著高于正常肝脏相应部分中的钙调蛋白含量。
