Purification and partial characterization of a murine melanoma-associated antigen.

A murine B16 melanoma-associated antigen was detected and partially purified from spent culture medium and intracellular material. The antigen was also purified and partially characterized from the detergent extract of cells cultured in the presence of [3H]glucosamine done or together with [‘4C]leucine. During purification, the melanoma-associated antigen activity was monitored by a double antibody antigen binding assay (Bystryn, J.-C., Schenkein, I., Baur, S., and Uhr, J. W. (1974) J. Nat. Cancer Inet 62, 1263). A radiochemically pure preparation of this glycoprotein antigen was obtained by gel filtration on Sepharose CG4B and Sepharose CL-GB, ion exchange chromatography on DEAESepharose, and chromatography on concanavalin ASepharose. Gel filtration on a calibrated Sepharose CL6B column in the presence of detergent gave an apparent molecular weight of 375,000. Prolonged treatment with sodium dodecyl sulfate and 2-mercaptoethanol followed by gel filtration or acrytamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated a subunit molecular weight in the range of 44,000 to 52,000. The tritium label in the antigen was distributed mainly between sialic acid and N-acetylglucosamine, with a small amount in N-acetylgalactosamine. Pronase digestion of antigen followed by fractionation on a Bio-Gel P-10 column yielded several glycopeptides. The results of the action of exoand endoglycosidases on these glycopeptides and their behavior on lectin affinity columns suggested the presence of both complex (N-acetyllactosaminyl type) and simple (oligomannosy1 type) oligosaccharides linked via N-acetylglucoermine to asparagine. The glycopeptide fractions isolated after proteolytic digestion were devoid of antigenic activity. Significant antigen activity was detected in the nuclei isolated from the labeled cells.