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Using RIAs for six regions within proopiolipomelanocortin (proOLMC), gel filtration, and electrophoresis, we studied pituitary peptides in a normal horse and one with Cushing's disease caused by a pars intermedia adenoma. Almost all immunoreactive (IR) ACTH (78%) was 4,500 mol wt (4.5K) ACTH in normal pars distalis, but it was almost 100% corticotropin-like intermediate lobe peptide (CLIP) in normal pars intermedia. alpha MSH and beta MSH were found mainly in pars intermedia: equal concentrations of the beta MSH precursors, beta-lipotropin (beta LPH) and gamma LPH, were found in pars distalis. Most IR-beta-endorphin (IR-beta END) was found as beta END in pars intermedia, but roughly equal concentrations of beta END and its precursor, beta LPH, were found in pars distalis. A 33K molecule containing IR-ACTH, IR-gamma 3MSH, and IR-beta END, presumed to be proOLMC, and a variety of 15-27K presumed biosynthetic intermediates were found in both normal pars distalis and pars intermedia. The pars intermedia adenoma causing Cushing's syndrome contained high IR-peptide concentrations. Several differences in precursors were noted, including the presence of three larger presumed precursors (38.5K, 47K, and 63K) that had both ACTH and beta END immunoreactivities and both deletions and additions of 15-27K intermediates. The Cushing's horse's plasma peptides reflected tumor concentrations; 4.5K ACTH was modestly elevated, but the concentrations of CLIP, alpha MSH, beta MSH, gamma LPH, and beta END were dramatically increased. About 20% of plasma IR-ACTH and 5% of IR-beta MSH and IR-beta END were found as high molecular weight forms. Normal processing of horse proOLMC appears to be similar to that in other species, but may be altered in pars intermedia tumors of horses with Cushing's disease, the plasma of which contains disproportionately increased concentrations of pars intermedia proOLMC peptides.

A continuous line (DMS-79) of human pulmonary small cell carcinoma cells was shown to secrete immunoreactive adrenocorticotropin (ACTH), lipotropin, and P-endorphin concomitantly into the culture medium. Gel filtration of the culture medium demonstrated at least five components: high molecular weight material(s) that had ACTH, lipotropin, and fl-endorphin immunoreactivities and materials similar to ACTH, B-lipotropin, y-lipotropin, and P-endorphin in their immunoreactivities and apparent molecular weights. The same components were observed when gel filtration was carried out in 6 M guanidine-HCI, and the high molecular weight material(s) appeared to consist of more than one component, with molecular weights in the range of 15,000-40,000. Immune affinity chromatography of the high molecular weight component(s) from gel filtration with a specific anti.l-24)ACTI serum demonstrated that the ACTH, lipotropin, and P-endorphin immunoreactivities were possessed by the same molecule(s), suggesting that ACTH, lipotropins, and P-endorphin were derived from a common, high molecular weight precursor. It has been reported that mouse pituitary tumor cell line AtT-20/D-16v secretes an adrenocorticotropin (ACTH)-like component, a f3-lipotropin (,3LPH)-like component, a (3-endorphin (3End)-like component (1-3) and a high molecular weight (Mr 31,000) glycopeptide, designated Mr 31,000 ACTH (4). This Mr 31,000 ACTH is thought to be a common precursor for the ACTH, f3LPH, and 3End produced by this cell (2,3,5).That such a common precursor also exists in man remains to be demonstrated, although there is already some evidence for the existence of a similar biosynthetic pathway (6)(7)(8)(9)(10)(11)(12)(13)(14)(15) (1 g/liter) and Trasylol (300 kallikrein inactivator units/ml) to buffer A; (b) labeled hACTH was purified by Sephadex G-50 fine (Pharmacia) gel filtration; and (c) separation of antibody-bound hormone from free hormone was achieved by precipitation with a second antibody.(ii) Human (3-Endorphin RIA. Anti-ovine fEnd serum RB100-11/15 (19) was used for the hfEnd RIA. Synthetic hflEnd (Bachem, Inc., Torrance, CA) was used for iodination and as standard; that used for iodination was stored at -70°in 5 mM HCl and that for standard, at -70°in buffer C [0.02 M Na2HPO4/KH2PO4, pH 7.4/0.1 M NaCl containing 1 g of USP gelatin, 100 mg of bovine serum albumin (Miles), and 100 mg of Merthiolate per liter]; further dilutions were made in buffer D (buffer C containing 1 ml of Triton X-100 per liter). 125I-Labeled hf(End was prepared by the method described for hACTH (18); a specific activity of 650 ,uCi/,ug was obtained. It was purified by Sephadex G-50 fine gel filtration prior to each assay. Mixtures were incubated for 3 days at 40; separation of antibody-bound hormone from free hormone was achieved by precipitation with a second antibody.(iii) Human LPH RIA. The hLPH RIA procedure, using antiserum R-3 (20) and synthetic (37-58)hLPH ["human ,Bmelanotropin (f3-MSH)," Ciba-Geigy] as tracer and standard Abbreviations: hACTH, huma...