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.
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)
Wroblewski
V J
VJ
Department of Drug Metabolism and Disposition, Lilly Research Laboratories, A Division of Eli Lilly and Company, Indianapolis, Indiana 46285.
Kaiser
R E
RE
Becker
G W
GW
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*
D007457:Iodine Radioisotopes; D011994:Recombinant Proteins; D013006:Growth Hormone; D002918:Chymotrypsin