Demonstration and characterization of tumor-associated antigenic components from cell membranes of a UV-induced mouse sarcoma using the macrophage-electrophoretic-mobility (MEM) test

Purification of tumor-associated antigenic material from the ascites sarcoma cells was carried out by extraction with 3 M KCl and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The antigenic activity was assayed in the MEM test. Extraction of whole tumor cells with 3 M KCl results in preparations with low antigenic activity which on SDS-PAGE show a very heterogeneous composition of more than 2o protein bands. In comparing the antigenic activity of different subcellular fractions obtained by differential centrifugation of the homogenate, the antigenic activity could be attributed to the cell membrane fraction. With this cell membrane fraction, three extraction media - 3 M KCl, 2% Triton X-100, and 5% sodium cholate - were tested for their ability to extract selectively the antigenic membrane proteins. The Triton X-100 treatment sobubilized the greatest amount of membrane protein. Both Triton X-100 and sodium cholate, however, produced very heterogeneous protein extracts. In contrast, 3 M KCl extracted selectively three membrane components: a glycoprotein of high molecular weight and two low-molecular weight carbohydrate-free proteins. Removal of the KCl in the presence of Triton X-100 precipitates the carbohydrate-free proteins, while the glycoprotein remains in solution. Testing the components of the KCl extracts in the MEM test after isolation by preparative SDS-PAGE revealed antigenic activity only with the glycoprotein component. (author)