title: Proteolysis of human growth hormone by rat thyroid gland in vitro: application of electrospray mass spectrometry and N-terminal sequencing to elucidate a metabolic pathway.
abstract: The present studies were designed to provide structural characterization of peptide metabolites of biosynthetic human growth hormone (hGH) formed by rat thyroid gland proteases in vitro. Electrospray ionization mass/spectrometry (ESI-MS) and N-terminal sequencing were used to characterize the peptide metabolites. The predominant enzyme in the thyroid gland preparations was a chymotrypsin-like serine protease which was biochemically similar to rat mast cell protease-I. Metabolic intermediates were formed by cleavage of hGH exclusively at Tyr/Phe/Leu-Xaa bonds. After a 5- or 45-min incubation of hGH with thyroid gland S9 pellet fraction, the majority of metabolites formed were two-chain variants of hGH having masses ranging from 16,002 to 22,143 Da. These metabolites were formed as a result of proteolysis in the large disulfide loop region of hGH in combination with processing at Tyr42-Ser43. Based upon the time-related appearance and structural characterization of these intermediates, a sequence of metabolic events is proposed. The initial event appears to be cleavage by the chymotrypsin-like protease between Tyr143-Ser144 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 deletion of the peptides Lys140-Tyr143 and Ser144-Phe146 from the large loop region. The identification of similar metabolites truncated by a single amino acid at the carboxyl terminus indicated the action of a carboxypeptidase on these metabolic products. After a 4.5-hr incubation, the protease isolated from the S9 pellet fraction degraded hGH to > 20 small peptides, having masses < or = 2300 Da.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Drug Metabolism and Disposition, Lilly Research Laboratories, A Division of Eli Lilly and Company, Indianapolis, Indiana 46285.
authors.forename: V J
authors.initials: VJ
authors.lastname: Wroblewski
authors.forename: R E
authors.initials: RE
authors.lastname: Kaiser
authors.forename: G W
authors.initials: GW
authors.lastname: Becker
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin / Q000378:metabolism; D013006:Growth Hormone / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007457:Iodine Radioisotopes; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D013961:Thyroid Gland / Q000201:enzymology / Q000378:metabolism*
chemical_list: D007457:Iodine Radioisotopes; D011994:Recombinant Proteins; D013006:Growth Hormone; D002918:Chymotrypsin