An electron cytochemical demonstration and biochemical analysis of adenosine triphosphatase activity in cancer cell plasma membrane

1. The studies of structure and function of the plasma membranes of cancer cells is extremely important for the elucidation of specificity of phenotypes of cancer cells. In order to bring this subject to light, plasma membranes, mitochondria, microsomes and nuclei have been isolated from the AH 130 ascites carcinoma cells and rat liver cells. The electron cytochemical observations and biochemical assays of M g²+-Na+-K+-ATPase, ADPase, AMPase, and β-glycerophosphatase activities have been carried out before and after the fixation with glutaraldehyde. 2. M g²+-ATPase and Mg²+-N a +-K +-ATPase are present in the isolated plasma membranes, mitochondria and microsomes in both AH 130 cells and rat liver cells. ADPase and AMPase of the mitochondria and microsomes show far lower activities than those of the corresponding enzymes found in rat liver plasma membrane. ADPase and AMPase of AH 130 cell fraction exhibit activity much lower or zero. Generally, enzymatic activity of the AH 130 cell fraction is much lower than that of rat liver cell fraction. 3. Mg²+-Na+-K+-ATPase is completely abolished by 5% glutaraldehyde fixation while it shows less effect on Mg²+-ATPase in the plasma membrane. ADPase and AMPase activities of the mitochondria and microsomes are completely inhibited by glutaraldehyde fixation. AMPase of the plasma membrane of rat liver is completely abolished while ADPase activity is not affected in any way. 4. Only Mg²+-ATPase can be demonstrated electron cytochemically. Cytochemical reaction products of Mg²+-ATPase are located at the outer layer of the plasma membrane of the AH 130 cells and rat liver tissue. In the isolated membrane fractions it is located at the inner layer. 5. ρ-Chloromercuribenzoate has only a slight effect on Mg²+-ATPase and Mg²+-Na+-K+-ATPase activities of the rat liver membrane, while it inhibits these enzyme activities in the AH 130 cell membrane. NaF (1 mM) and NaN3 (1 mM) inactivate ADPase of the rat liver plasma memo brane. 6. In these experimental conditions, nonenzymatic hydrolysis of ATP by lead ions is not recognized. 7. It seems most reasonable to conclude that cytochemical electron microscopic demonstration of Mg²+-ATPase after fixation with glutaraldehyde may serve as the absolute marker for the plasma membrane of ascites hepatoma and liver cells.