PROTEOLYSIS OF HUMAN GROWTH-HORMONE BY RAT-THYROID GLAND IN-VITRO - APPLICATION OF ELECTROSPRAY MASS-SPECTROMETRY AND N-TERMINAL SEQUENCINGTO ELUCIDATE A METABOLIC PATHWAY

The present studies were designed to provide structural characterizati on of peptide metabolites of biosynthetic human growth hormone (hGH) f ormed by rat thyroid gland proteases in vitro. Electrospray ionization mass/spectrometry (ESI-MS) and N-terminal sequencing were used to cha racterize the peptide metabolites. The predominant enzyme in the thyro id gland preparations was a chymotrypsin-like serine protease which wa s biochemically similar to rat mast cell protease-I. Metabolic interme diates were formed by cleavage of hGH exclusively at Tyr/Phe/Leu-Xaa b onds. After a 5- or 45-min incubation of hGH with thyroid gland S9 pel let fraction, the majority of metabolites formed were two-chain varian ts of hGH having masses ranging from 16,002 to 22,143 Da. These metabo lites were formed as a result of proteolysis in the large disulfide lo op region of hGH in combination with processing at Tyr42-Ser43 . Based upon the time-related appearance and structural characterization of t hese intermediates, a sequence of metabolic events is proposed. The in itial event appears to be cleavage by the chymotrypsin-like protease b etween Tyr143-Ser43 to form a two-chain hGH. This intermediate is then cleaved between Tyr42-Ser43, liberating the N-terminal peptide, Phe1- Tyr42. Two other metabolites were generated as a result of the deletio n of the peptides Lys140-Tyr143 and Ser144-Phe146 from the large loop region. The identification of similar metabolites truncated by a singl e amino acid at the carboxyl terminus indicated the action of a carbox ypeptidase on these metabolic products. After a 4.5-hr incubation, the protease isolated from the S9 pellet fraction degraded hGH to >20 sma ll peptides, having masses less-than-or-equal-to 2300 Da. The data ill ustrate the utility of combining ESI-MS and N-terminal sequencing in t he study of protein metabolism and the enzymatic pathways involved.