Attempts toward biosynthesis of the thyrotropin-releasing hormone and studies on its breakdown in hypothalamic tissue preparations.

Attempts were made to study the reported biosynthesis of the thyrotropin-releasing hormone (TRH = pyroGlu-His-Pro-amide) by incubating extracts of freeze-dried hypothalamic tissue with radioactively labeled precursor amino acids. Chromatographic analysis indicated a fast incorporation of radioactivity into many metabolites, including one that initially co-migrated with TRH. However, on two-dimensional chromatography, such coincidence disappeared and thus a biosynthesis of TRH could not be confirmed. A very fast degradation of TRH by serum, as well as by brain tissue preparations, was observed and was studied in detail because it could be a cause of difficulties encountered in detecting an in vitro synthesis. In hypothalamic and cortical tissue preparations, on incubation with TRH labeled with [3H]proline, fast formation of radioactively labeled deamido-TRH and liberation of prolineamide and free proline were found. On incubation of serum with labeled TRH there was a similar rapid breakdown, but different products were yielded. Degradation of TRH by serum has been reported to be strongly inhibited by pyroGlu-His-OCH3, a dipeptide analogue of TRH (10). The peptidolytic cleavage of TRH by brain enzymes, yielding proline and prolineamide as split products, was also effectively reduced using comparatively high concentrations of the dipeptide ester without, however, preventing TRH deamidation. Presuming deamido-TRH to be a biosynthetic intermediary, we decided to continue studying the synthesis of TRH with hypothalamic tissue preparations in the presence of inhibitory concentrations of the dipeptide ester, aiming at the isolation of deamido-TRH. Using [14C]proline as the label, it appeared that rather large amounts of radioactively labeled deamido-TRH, which was identified as such by vigorous purification, could be isolated from such incubates. However, only proline was incorporated, but labelled histidine or glutamic acid were not, and ATP addition was, if anything, inhibitory. Therefore, this proline incorporation could not have been due to de novo synthesis. Since the inhibiting pyroGlu-His-methyl ester was rapidly split during incubation, and, therefore, presumably inhibited the tissue peptidase by competition, we have concluded that ester-derived peptidase-bound dipeptide had reacted with [3H]proline in reverse to form the radioactive deamido-TRH in a process unrelated to biosynthesis.