Biologic and Immunologic Characterization and Physical Separation of ACTH and ACTH Fragments in the Ectopic ACTH Syndrome

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Abstract

Extracts of tumors from 32 patients with the ectopic ACTH syndrome were subjected to simultaneous bioassay and radioimmunoassays for ACTH. Radioimmunoassays were performed using three antisera, one of which reacts with the extreme N-terminal 1-13 amino acid sequence of ACTH, the second with the N-terminal 1-23 sequence of the ACTH molecule, and the third with the C-terminal 25-39 amino acid sequence of ACTH. There was, in general, good correlation between bioactivity and N-terminal ACTH immunoreactivity. However, there were large excesses of both extreme N-terminal and C-terminal immunoreactive materials in most tumor extracts, which were not found in extracts of three human pituitaries. Three tumor extracts were subjected to molecular sieve chromatography on Sephadex G-50 fine resin. The bioactive ACTH eluted in the same fractions as pituitary ACTH (mol wt≃4,500 daltons) and reacted equally in all three ACTH radioimmunoassay systems. The bioactive tumor ACTH was neutralized by incubation with the C-terminal antiserum, indicating it has an intact C-terminal sequence of amino acids. The next several fractions from the Sephadex column contained a material, mol wt≃3,100, which was biologically inactive and had C-terminal immunoreactivity but no N-terminal or extreme N-terminal immunoreactivity. Incubation with the N-terminal 1-23 ACTH antiserum did not adsorb these C-terminal fragments, indicating they lacked an intact sequence of amino acids in this region. A smaller ACTH fragment (mol wt≃1,800 daltons) eluted in still later fractions and reacted with the extreme N-terminal antiserum but not with the N-terminal or C-terminal antisera. It had no steroidogenic activity, but appeared to have significant melanocyte-stimulating activity. It is concluded that, in addition to an ACTH similar, if not identical, to pituitary ACTH, tumors of patients with the ectopic ACTH syndrome contain both N-terminal and C-terminal ACTH fragments.

Authors

David N. Orth, Wendell E. Nicholson, William M. Mitchell, Donald P. Island, Grant W. Liddle