The degradation of corticotropin-releasing factor by enzymes of the rat brain studied by liquid chromatography-mass spectrometry.
The corticotropin-releasing factor (CRF; 41 amino acid residues) is a major regulatory peptide in the response to stress and is distributed over many regions of the brain. We have studied the enzymatic degradation of CRF and related peptides by the CRF-degrading enzyme(s) of the rat brain (CRF-DA) by liquid-chromatographic-mass spectrometric technique and by online tandem mass spectrometric experiments. Peptide fragments of the human/rat CRF (1-41) generated by the CRF-DA of the particulate cell fraction were separated and structurally assigned. Major sites of enzymatic attack were identified at the P1 positions Ser1, Thr11 , His13, Leu15, Arg23, Arg35, and Lys36 with Leu15 as the site of primary cleavage. The CRF-DA was shown to be dominated by a metalloendopeptidase activity inhibited by O-phenanthroline and EDTA. The cytosolic fraction generated a similar degradation pattern with a pronounced cleavage at the Arg35 position.
Kertscher
U
U
Institute of Molecular Pharmacology, Berlin, Germany.
Beyermann
M
M
Krause
E
E
Furkert
J
J
Berger
H
H
Bienert
M
M
Mehlis
B
B
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002853:Chromatography, Liquid; D003346:Corticotropin-Releasing Hormone / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology; D006801:Humans; D013058:Mass Spectrometry; D008566:Membranes / Q000201:enzymology; D010446:Peptide Fragments / Q000032:analysis; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D013347:Subcellular Fractions / Q000201:enzymology
D010446:Peptide Fragments; D011480:Protease Inhibitors; D003346:Corticotropin-Releasing Hormone