Proteolytic degradation of rat growth hormone-releasing factor(1-29) amide in rat pituitary and hypothalamus.
The identification of peptide bonds vulnerable to tissue peptidases is a valuable approach to design peptide agonists which exhibit a longer duration of action than the native molecules. Therefore, the kinetic of disappearance of rat growth hormone-releasing factor (rGRF(1-29)NH2) and the identification of its metabolites were studied in rat pituitary and hypothalamus. Synthetic rGRF(1-29)NH2 (10 microM) was incubated (0-120 min, 37 degrees C) in the presence of a pituitary (237 +/- 51 micrograms protein/ml) or hypothalamus homogenate (576 +/- 27 micrograms protein/ml). Using analytical high pressure liquid chromatography (HPLC), apparent half-lives of 22 +/- 3 min and 25 +/- 4 min were found in pituitary and hypothalamus, respectively. In both tissues, three degradation products, all less hydrophobic than the native peptide, were detected and isolated by preparative HPLC. The identification of the purified metabolites was ascertained by amino acid analysis, sequencing and chromatography with synthetic homologs. These results indicate that the main sites of cleavage in the pituitary and hypothalamus are Lys21-Leu22 (trypsin-like cleavage site), Leu14-Gly15 and Tyr10-Arg11 (chymotrypsin-like cleavage sites). TLCK and leupeptin did not affect the formation of fragment (1-21)OH while TPCK blocked the cleavage of Leu14-Gly15. The low affinity of fragment (1-21)NH2 for pituitary GRF binding sites suggests that hydrolysis of the Lys21-Leu22 bond inactivates rGRF(1-29)NH2 in this target tissue.
Boulanger
L
L
Department of Biochemistry, University of Montréal, Notre-Dame Hospital Research Center, Canada.
Lazure
C
C
Lefrançois
L
L
Gaudreau
P
P
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D010450:Endopeptidases / Q000378:metabolism*; D013007:Growth Hormone-Releasing Hormone / Q000378:metabolism*; D007030:Hypothalamo-Hypophyseal System / Q000378:metabolism; D007031:Hypothalamus / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism*; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D010902:Pituitary Gland / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D018013:Receptors, Neuropeptide; D017981:Receptors, Neurotransmitter / Q000378:metabolism; D018017:Receptors, Pituitary Hormone-Regulating Hormone
D000596:Amino Acids; D010446:Peptide Fragments; D010455:Peptides; D018013:Receptors, Neuropeptide; D017981:Receptors, Neurotransmitter; D018017:Receptors, Pituitary Hormone-Regulating Hormone; D013007:Growth Hormone-Releasing Hormone; C059148:somatotropin releasing hormone (1-29); D010450:Endopeptidases; C048250:somatotropin releasing hormone receptor