Characterization of the Immunochemical Forms of Calcitonin Released by a Medullary Thyroid Carcinoma in Tissue Culture

Immunoreactive calcitonin released by a medullary thyroid carcinoma in tissue culture has been found to exhibit heterogeneity when analyzed by gel chromatography and radioimmunoassay, in a pattern analogous to that seen in the circulation of the patient from whom the neoplasm was removed. To examine the cause of the heterogeneity, the immunoreactive material released by the tumor into tissue culture medium was further analyzed by gel electrophoresis in the presence of the protein denaturant 8 M urea, by gel chromatography after reduction and alkylation, by affinity chromatography on concanavalin A-agarose, and by bioassay in a renal adenylyl cyclase system of enhanced sensitivity. The results suggest that the larger immunochemical forms of calcitonin described in the circulation of patients with medullary thyroid carcinoma may be released directly from the neoplasm and need not derive from peripheral metabolism of the monomer. It could be demonstrated that a major proportion of the immunochemical enlargement is dependent upon intermolecular disulfide bridge formation whereas aggregation or non-convalent protein binding account for a smaller component of the heterogeneity. In view of the absence of binding of the immunoreactive material to the lectin agarose, carbohydrate side chains, at least of the alpha-d glucosyl variety, do not seem to contribute significantly to calcitonin enlargement. Additionally, the studies indicate that, at least by in vitro assay, the larger immunochemical forms of calcitonin, representing the majority of the immunoreactivity released by a medullary thyroid carcinoma, are biologically inactive.