Amphipathic lipid-bound protein antigens in mouse bladder carcinomas detected by a monoclonal antibody.

Abstract

We have found organic solvent soluble proteins that contain tightly bound fatty acids. They were demonstrated in mouse bladder carcinomas by a monoclonal rat antibody, 33E7C. The antigens recognized by antibody 33E7C copurified with lipids when extracted from bladder carcinomas with a mixture of chloroform-methanol. The antigens remained in the chloroform phase after solvent partition with water. Chromatographic analysis using an alpha-hydroxy-propylated Sephadex G-50 (LH60) column led to the isolation of a group of proteins binding to antibody 33E7C. Four proteins (10K, 18K, 25K, and 40K daltons) were found on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after radioimmunoprecipitation with antibody 33E7C. After preparative SDS-PAGE, the purified antigens were soluble in both water and a mixture of chloroform-methanol (2:1) but were freed from phospholipids and glycolipids. The antigens have been characterized by the presence of tightly bound fatty acids with the following findings: (1) They were metabolically colabeled by incubating bladder carcinoma cells with [9,10-3H]palmitic acid and [14C]leucine, followed by radioimmunoprecipitation assay and by SDS-PAGE. (2) After hydrolysis of proteins in NaOH-methanol, [3H]palmitic acid methyl ester was separated from the 40K and 18K proteins by thin-layer chromatography.