Exophthalmogenic Activity of the /3 Subunit of Thyrotropin LEONARD D. KOHN1 AND ROGER J. W1NAND2 l The Section on Biochemistry of Cell Regulation, Laboratory of Biochemical Pharmacology, National Institute Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20014; of 2 The Departement de Clinique et de Semiologie Medicales, Institut de Medecine, Universite de Liege, B4000 Liege, Belgium

The a subunit of thyrotropin, luteinizing hormone, and both subunits of luteinizing hormone have no exophthalmogenic activity. (Endocrinology 96: 1592, 1975)

ABSTRACT. The exophthalmogenic activity of the /3 subunit of bovine thyrotropin is only 10% to 20% that of the thyrotropin molecule or of an exophthalmogenic factor produced by partial pepsin digestion of purified thyrotropin preparations.

N studies of experimental exophthtilmos (1-10) we have shown that purified preparations of bovine thyrotropin (TSH)1 are exophthalmogenic but that partial pepsin digestion of these purified preparations can yield an "exophthalmos-producing factor" (EPF) with no significant thyroid stimulating activity. When amino acid analyses, carbohydrate analyses, and chromatographic analyses of the peptide components of the purified EPF were compared with similar data for bovine TSH and with the primary sequence of bovine TSH as determined by Liao and Pierce (11-13), it appeared likely that the EPF was composed of an intact or nearly intact TSH B subunit but only the amino-terminal half of the TSH a subunit. The obvious possibility existed that one of the subunits of this structure, i.e., the intact or nearly intact TSH 3 subunit or the aminoterminal fragment of the TSH a subunit, could account for all of the exophthalmogenic activity in either TSH or its EPF derivative. When tested in the fish bioassay, relative activities for the intact EPF, for its 3 subunit component, and for its amino-terminal a subunit component were 100:10:1, respectively (5). The 10% level of activity of the 3 subunit component was worrisome in that this TSH 3 subunit had been exposed to pepsin and other manipulative procedures which might have resulted in sequence deletions at either end of the normal TSH 3 subunit. Since these deletions might have caused a loss in biological activity, the possibility existed that the intact 3 subunit of TSH might be significantly exophthalmogenic and

that this subunit was the nonthyroid stimulating TSH derivative in the sera of patients, which we had theorized might exist in our two-factor theory of exophthalmos (3,10). In the present report we answer one aspect of this question negatively. The intact 3 subunit of TSH has exophthalmogenic activity but only 10-15% of the activity of the thyrotropin molecule or of the EPF produced by partial pepsin digestion of purified TSH preparations. It cannot in itself account for the exophthalmogenic activity of either TSH or the EPF derived from TSH.

Received December 16, 1974. 1 Abbreviations: TSH, thyrotropin; EPF, the exophthalmogenic derivative of the thyrotropin molecule which is produced by partial pepsin digestion of TSH; and LH, luteinizing hormone.

2 The bovine TSH purified by the countercurrent procedure (11-14) was a gift of Dr. John G. Pierce, Department of Biological Chemistry, University of California Center for Health Sciences, Los Angeles, California 90024.

I

Materials and Methods Hormone and subunit preparations Bovine TSH was purified using two separate procedures previously described (1-6, 11-14). One preparation used in sequence the following steps: CM-cellulose chromatography, DEAE-cellulose chromatography, and Sephadex G100 chromatography. The second procedure used in sequence: CM-cellulose chromatography, countercurrent distribution, and Sephadex G100 chromatography.2 The amino-acid composition and carbohydrate analyses of the two preparations were effectively the same. [3H]TSH was prepared as previously described (1,2,5) using the purified TSH obtained by the first procedure. Subunits of TSH were prepared by the procedure described by Liao and Pierce (11). To ensure minimal contamination by TSH they were individually rechromatographed on Sephadex G100. Bovine luteinizing hormone (LH) and the subunits of LH were also prepared as described (14-16); the LH subunits were

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NOTES AND COMMENTS also individually rechromatographed on Sephadex G100 to minimize native hormone contamination. The exophthalmogenic derivative of the TSH molecule (EPF) was prepared by partial pepsin digestion (2) of TSH preparations purified by the first procedure; it was isolated by an electrofocusing technique (2). The polypeptide components of this structure, the intact or nearly intact TSH /3 subunit structure and the amino-terminal fragment of the TSH a subunit, were separated and isolated by chromatography on Sephadex G100 after exposure to propionic acid had dissociated the components (5). The exposure to propionic acid and the chromatographic conditions were analogous to those used to separate the subunits of TSH (11). Assays Thyroid stimulating activity was assayed by the method of McKenzie (17). Exophthalmogenic activity was detected by the method of Brouhon-Massilon (18) after a screening test to eliminate the "nonresponder" fish as described by Dedman, Fawcett, and Morris (19). Specific thyroid stimulating activity is defined in terms of international units per mg of protein; specific exophthalmogenic activity is defined as equivalent thyroid stimulating units per mg, i.e., as the

exophthalmogenic activity in a crude preparation of thyrotropin (Ambinon, Oss, Holland) having that many units of thyroid stimulating activity. Protein was determined colorimetrically with the use of recrystallized bovine serum albumin as the standard (20).

Results Table 1 compares the exophthalmogenic and thyroid stimulating activities of all the hormone preparations. As can be seen, all of the purified TSH preparations are similarly exophthalmogenic in relationship to their thyroid stimulating activities. The TSH a subunit has less than 2% of the exophthalmogenic activity of TSH whereas the TSH fi subunit has 10-15% of the exophthalmogenic activity of TSH. Since the exophthalmogenic activity of the /3 subunit component of EPF is effectively the same as that of the normal TSH /3 subunit, it is clear that its exposure to pepsin and its additional manipulation have not affected its activity significantly. Dose response curves comparing the exophthalmogenic activity of the /3 subunit and

TABLE 1. Thyroid stimulating activity and exophthalmogenic activity of TSH, EPF, LH, and their component subunits

Hormone

Preparation

Method of thyrotropin purification*

Activity Thyroid stimulating!

Exophthalmogenict

IU/mg

U/mg

TSH

3

H-labeled Nonlabeled Nonlabeled

Column chromatography Countercurrent

18 ± 3 24 ± 4 32 ± 8

21 ± 4 23 ± 4 32 ± 4

a subunit of TSH

3

H-labeled Nonlabeled Nonlabeled

Column chromatography Countercurrent

0.1 ± 0.1 0.1 ± 0.1 0.2 ± 0.2

0.3 ± 0.3 0.3 ± 0.3 0.4 ± 0.4

/3 subunit of TSH

3

Column chromatography Countercurrent

Exophthalmogenic activity of the beta subunit of thyrotropin.

The exophthalmogenic activity of the beta subunit of bovine thyrotropin is only 10% to 20% that of the thyrotropin molecule or of an exophthalmogenic ...
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