Studies of a Melanoma Tumor-Associated Antigen Detected in the Spent Culture Medium of a Human Melanoma Cell Line by Allogeneic Antibody. III. Physicochemical Properties

Abstract

A melanoma tumor-associated antigen (TAA), isolated from spent culture medium of human melanoma cell line UCLA-SO-M14, was purified to mean homogeneity to determine its physical and biochemical nature. Gel filtration and native polyacrylamide gel electrophoretic analyses of the ¹²⁵ I-labeled melanoma TAA revealed that the antigen had a molecular weight in the range of 180,000–190,000. However, ultracentrifugation of melanoma ¹²⁵ I-labeled TAA in a 5–20% sucrose density gradient revealed a sedimentation coefficient to be 4.96±0.24. Melanoma ¹²⁵ I-labeled TAA focused at a pH of 6.5 by isoelectric focusing. No carbohydrates were detectable by various colorometric methods in the highly purified melanoma TAA fraction, and melanoma TAA failed to bind with several lectins. Its antigenic activity was destroyed by the proteolytic enzymes but was not affected by the glycosidic enzymes or phospholipase A₂. The results suggested that the melanoma TAA was most likely a lipoprotein that had a molecular weight of 180,000–190,000, a sedimentation coefficient of approximately 5, and a pl value of 6.5. The protein portion apparently formed the antibody binding site(s).