Abstract
Previously reported experiments (Winand, R.J., and Kohn, L.D. (1970) J. Biol. Chem. 245, 967-975; Kohn, L.D., and Winand, R.J. (1971) J. Biol. Chem 246, 6570-6575) have demonstrated that partial pepsin digestion of bovine thyrotropin preparation yields a fragment of the thyrotropin molecule which is exophthalmogenic but has negligible or no thyroid-stimulating activity. In the present report this exophthalmogenic derivative of the thyrotropin molecule is shown to contain two major polypeptide components with approximate molecular weights of 14,000 and 6,000. Amino acid analyses, carbohydrate analyses, and tryptic digestion experiments indicate that this exophthalmogenic factor is composed of an intact or nearly intact beta subunit of thyrotropin and an NH2-terminal fragment of the alpha subunit of thyrotropin. Neither polypeptide component of the exophthalmogenic factor has the in vivo exophthalmogenic activity of the intact structure. In vitro the intact exophthalmogenic derivative of the thyrotropin molecule can bind to the thyrotropin receptor on thyroid membranes less efficiently than thyrotropin but significantly better than either its own polypeptide components or the alpha or beta subunits of thyrotropin. The exophthalmogenic factor and its parent thyrotropin molecule can stimulate adenylate cyclase activity in retro-orbital tissue membranes from guinea pigs, a mammalian model of exophthalmos; its polypeptide components have little or no such activity.
Highlights
Based on the primary sequence of the CYsubunit of TSH previously reported by Liao and Pierce [3], we suggest that the COOH terminus of the fragment of the cu-TSH subunit remaining in the exophthalmogenic factor structure is between arginine 46 and valine 53
Partial Pepsin Digests of Purified TSH Preparations--When chromatographed on Sephadex G-100 (Fig. 1, top), the exophthalmogenic derivative of the TSH molecule eluted after TSH
The present report describes the subunit structure and a possible primary sequence of an exophthalmogenic derivative of the TSH molecule which can be formed by partial pepsin digestion of TSH preparations
Summary
In previous reports [1, 2] we demonstrated that homogeneous bovine thyrotropin preparations were exophthalmogenic and that partial pepsin digestion of these preparations could yield an exophthalmogenic factor which lacks significant thyroidstimulating ability This exophthalmogenic derivative of the TSH’ molecule was isolated by electrofocusing and was shown to have a molecular weight of 20,000 to 22,000 when evaluated by electrophoresis on gels containing sodium dodecyl sulfate; TSH analogously evaluated had a molecular weight of 27,000 to 30,000. Whereas the exophthalmogenic factor appeared to be composed of two polypeptide chains, one with a molecular weight of approximately 14,000 and the other with a molecular weight of approximately 6,000, TSH is composed of two 13,000 to 14,000 molecular weight polypeptide chains, the cy and p subunits, which have different amino acid sequences [3] These data suggested that the exophthalmogenic derivative was composed of a major portion of one TSH subunit but only of a fragment of the second. Based on the primary sequence of the CYsubunit of TSH previously reported by Liao and Pierce [3], we suggest that the COOH terminus of the fragment of the cu-TSH subunit remaining in the exophthalmogenic factor structure is between arginine 46 and valine 53
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