Naturally Occurring Forms of Thyrotropin with Low Bioactivity and Altered Carbohydrate Content Act as Competitive Antagonists to More Bioactive Forms

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Abstract
We have examined the interaction of certain forms of mouse (m) tumor and bovine (b) pituitary TSH with standard bTSH on the activation of adenylate cyclase in human thyroid membranes. Tumor extract, serum from tumor-bearing mice, culture medium from dispersed cell incubations, and two preparations of purified bTSH (Sigma and Pierce) were fractionated on Sephadex G-100 (1.2 X 200 cm). For each fraction, TSH bioactivity was measured by stimulation of adenylate cyclase activity in human thyroid membranes, and immunoactivity was determined by RIA. On G-100, Pierce bTSH had multiple immunoactive components with partition coefficients (Kav) of 0.28-0.32 and ratios of biological over immunological activity (B/I) of 0.59-1.42. Sigma bTSH, mouse tumor, serum, and medium were even more heterogeneous (Kav = 0.23-0.32), with a lower range of B/I (0.04-1.0). When single doses (125-2000 ng) of those fractions with the highest Kav (0.30-0.32) and lowest B/I (0.04-0.51) were mixed with multiple doses (200-10,000 microU) of Armour TSH standard (B/I = 1), there was 30-56% inhibition of adenylate cyclase activity stimulation. Double reciprocal plots showed competitive inhibition for the low B/I forms from all sources, except for a medium form which showed mixed inhibition. The medium form had the highest inhibitory activity. There were no inhibitors in G-100 fractions from the Kav regions devoid of TSH immunoactivity or from the same Kav regions of normal mouse serum. To determine the chemical differences between different forms, affinity chromatography on Concanavalin A, wheat germ agglutinin, and soybean agglutinin was employed. Compared with the apparent higher molecular weight form with higher B/I, the apparent lower molecular weight form with lower B/I contained decreased amounts or availability of alpha-mannose and increased amounts or availability of beta-N-acetyl-D-galactosamine and/or beta-galactose; both forms appear to contain similar beta-N-acetyl-D-glucosamine residues, presumably in the inner core.

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