Solubilization and partial characterization of a tumor‐rejection antigen from an ultraviolet light‐induced murine tumor

Tumor rejection antigen (TRA) of an ultraviolet‐light‐induced murine skin tumor was solubilized, fractionated and partially characterized. Subcellular fractions were prepared by differential centrifugation of tumor cells that had been ruptured via nitrogen cavitation. Only the 110,000‐g membrane fraction induced significant tumor protection, as determined by in vivo immunization and challenge assays. Extraction of the membrane fraction with 3 M KCI resulted in solubilization of material that could induce in vivo tumor‐rejection immunity. Both the membrane fraction and soluble extract had a limited effective dose range. The KCI extract was separated on a Sepharose, CL‐6B column in the presence of 6 M guanidine‐HCI. Only one of five fraction pools (molecular weight range of 76,000–127,000 daltons) was immunogenic. It contained at least eight protein bands by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE), but no lipid components. This immunogenic Sepharose fraction was chromatographed on a Sephadex G‐150 column. Each of the four Sephadex fraction pools was immunogenic. One protein component was common to each of those fractions. It migrated as a single 76,000‐dalton band on SDS‐PAGE and contained [¹⁴C]‐leucine and [³H]‐glucosamine that had been incorporated during cell growth. These results suggest that the TRA of this tumor is a 76,000‐dalton glycoprotein.