A simple procedure for the purification of calmodulin bound to membranes; calmodulin bound to the particulate fraction of AH-66 hepatoma ascites cells.
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.
D000818:Animals; D001201:Ascites / Q000378:metabolism; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002147:Calmodulin / Q000302:isolation & purification / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D002850:Chromatography, Gel; D008099:Liver / Q000378:metabolism; D008114:Liver Neoplasms, Experimental / Q000378:metabolism*; D008297:Male; D011485:Protein Binding; D051381:Rats
D002135:Calcium-Binding Proteins; D002147:Calmodulin