The effects of modification of the COOH-terminal regions of bovine thyrotropin and its subunits.

Removal of a limited number of amino acid residues at the COOH termini of the peptide chains of the glycoprotein hormone, thyrotropin (TSH) shows that the integrity of these regions is important both in subunit-subunit interactions and in uivo biological response. Treatment of the native hormone with a mixture of carboxypeptidases A and B leads to loss of half of the biological activity with the removal of -Lys-Ser-COOH from the cx chain and -Tyr-Met-COOH from the p chain. No other alterations to the molecule were detected. Although modification of the hormone does not lead to detectable dissociation, once separated the subunits isolated from the modified hormone do not recombine. The treated p subunit will, however, recombine with native LY subunit as judged by gel electrophoresis but no biological activity is recovered. Treatment of isolated subunits leads to further and well defined hydrolysis at their COOH termini with -Tyr-TyrLys-Ser-COOH removed from the LY chain and -Lys-Ser-TyrMet-COOH removed from the p. Significant recombinations of these materials with each other or native TSH-a! and TSHp did not occur (as expected from the results with the subunits from treated hormone) but both TSH-a and luteinizing hormone (LH)-a! after enzymic hydrolysis yielded recombinants, though inactive, with native LH-0. These data indicate significant differences in the regions of the two hormone specific subunits which are in contact with the cy subunit. Immunological cross reactivity of all products of enzymatic treatment appears unchanged. Measurement of circular dichroism showed conformational changes resulting from the alterations at the COOH termini only in the case of the a! subunit where a strong positive band at 235 to 240 nm decreased markedly after treatment with the carboxypeptidases. Comparisons of the circular dichroism spectra of intact TSH and its subunits and those of luteinizing hormone (LH) were also made. The spectrum of intact TSH was not the sum of the spectra of its corresponding (Y and /3 subunits, in agreement with data reported by others for LH, and was very similar to that of bovine LH with the addition of a large maximum at 230 to 240 nm which results from the high tyrosine content of TSH-0 versus LH-/3.