Isolation and Characterization of the Golgi Complex from Rat Ascites Hepatoma AH-130 Cells1

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Abstract
A Golgi-rich fraction has been isolated from rat ascites hepatoma AH-130 cells. Unlike the usual procedure for isolating Golgi complexes from liver and other tissues, a hypotonic solution including 2 mM CaCl 2 was used as the homogenization medium for the ascites hepatoma cells, followed by a combination of differential and discontinuous sucrose gradient centrifugations. Electron microscopic observation revealed that the isolated fraction consisted of cisternae, vesicles and tubular elements which were similar to those structures described previously for the Golgi fraction isolated from rat liver. Galactosyl- and sialyl-transferases were concentrated about 55- and 75-fold, respectively, in this fraction compared with the homogenate, indicating that these enzymes are useful markers for the Golgi complex of rat ascites hepatoma AH-130 cells, as they are for those of other normal tissues. The preparation was virtually free of cytochrome oxidase, but contained minor amounts of acid phosphatase, alkaline phosphatase and phosphodiesterase I activities. Electrophoretic analysis on sodium dodecylsulfate-polyacrylamide gels showed that the hepatoma Golgi membranes were resolved into at least 23 protein bands, which were apparently different from the electrophoretic profile of the plasma membrane isolated from the same hepatoma cells.