The Degradation of Corticotropin-releasing Factor by Enzymes of the Rat Brain Studied by Liquid Chromatography–Mass Spectrometry

Action of Three Ectopeptidases on Corticotropin-Releasing Factor: Metabolism and Functional Aspects

“…In the bioactive region of the molecule (5-19), we found cleavages by purified ACE and recombinant neprilysin at positions 7-8, 17-18, and 18-19. In the carboxyl region of CRF, we also found cleavage sites at 20-21, 26-27, 27-28, 36-37, and 39-40. Although our recovered fragments do not match those of Kertscher et al (1998) exactly (owing most likely to different specific peptidases involved in CRF catabolism by whole brain), our results do support the idea of three major areas of cleavage susceptibility within the CRF molecule. Additionally, using purified ectopeptidases we have identified four cleavage sites in the highly conserved, bioactive region of the molecule where a single cleavage would inactivate CRF.…”

“…We did not find cleavage between Ser 33 and Asn 34 by the enzymes analyzed in this study. The current work complements and extends the work of Kertscher et al (1998). Using LC/MS and MS/MS techniques, this group characterized CRF degrading activity in both membrane and cytosol preparations from whole rat brain.…”

“…In an early study , Smith et al (1987) reported that CRF exists in at least five different molecular forms , CRF [36][37][38][39][40][41] , CRF , CRF , and an undetermined moiety) in the ovine tuberoinfundibular system and pituitary. Kertscher et al (1998) used highperformance liquid chromatographyFmass spectrometry (LC/MS) and MS/MS techniques to characterize the degradation of CRF by whole rat brain preparations. These investigators identified 13 CRF fragments in membrane preparations and concluded that the main enzymatic attack is on the central portion of the CRF molecule (positions 11-17).…”

Astressin Analogues (Corticotropin-Releasing Factor Antagonists) with Extended Duration of Action in the Rat

“…We also noted that further deletions (residues 8-14) produced antagonists such as astressin {cyclo (30)(31)(32)(33) were ca. twice and 1/100 as potent as astressin, respectively, suggesting a putative turn that encompasses residues 30-33 (previous paper: Koerber et al J. Med.…”

“…While oCRF and hCRF have been used in the clinic, CRF antagonists | Abbreviations: IUPAC rules are used for nomenclature of peptides including one-letter codes for amino acids. Also: Ac, acetyl; ACTH, adrenocorticotropin hormone; adx, adrenalectomized; astressin, cyclo- (30)(31)(32)(33)[DPhe 12 ,Nle 21,38 ,Glu 30 ,Lys 33 ]hCRF(12-41); Boc, tert-butyloxycarbonyl; BOP, benzotriazolyloxy-tris(dimethylamino)phosphonium hexafluorophosphate; BSA, bovine serum albumin; CD, circular dichroism; CRF, corticotropin-releasing factor (o, ovine; h, human); CSF, cerebrospinal fluid; CZE, capillary zone electrophoresis; DCM, dichloromethane; DIC, diisopropylcarbodiimide; DMF, dimethylformamide; FBS, fetal bovine serum; Fmoc, 9-fluorenylmethoxycarbonyl; HBTU, O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate; HF, hydrogen fluoride; HOBt, 1-hydroxybenzotriazole; IA, intrinsic activity; ic, intracisternal; LSIMS, liquid secondary ion mass spectrometry; MBHA, 4-methylbenzhydrylamine; NMP, N-methylpyrrolidinone; NMR, nuclear magnetic resonance; OFm, O-fluorenylmethyl; SAR, structure-activity relationships; TBTU, O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate; TEAP 2.25, 4.5, and 6.5, triethylammonium phosphate, pH 2.25, 4.5, and 6.5; TFA, trifluoroacetic acid; TFE, trifluoroethanol.…”