High-performance liquid chromatographic system for the separation of dynorphin A (1-17) fragments and its application in enzymolysis studies with rat nerve terminal membranes.
A high-performance liquid chromatographic method capable of separating a large number of C- and N-terminal degradation fragments of dynorphin A (1-17) (dyn 1-17) in 1 h has been developed. The system has been applied to study the metabolism profiles of various dyn 1-17-derived peptides following in vitro incubation with rat striatum and spinal cord nerve terminal membranes. In addition to the removal of the N-terminal amino acid Tyr, major sites of cleavage between the following amino acids could be established: Leu5-Arg6 in dyn 1-7 (formation of dyn 1-5); Arg6-Arg7 and Leu5-Arg6 in dyn 1-8 (formation of dyn 1-6 and dyn 1-5, respectively); Arg7-Ile8 in dyn 1-9 (formation of dyn 1-7) and Arg9-Pro10 in dyn 1-10 (formation of dyn 1-9). Studies with inhibitors of the enzymes involved show that dyn 1-5 is formed directly from dyn 1-8 via an endopeptidase insensitive to the angiotensin-converting enzyme inhibitor MK 422 acting on the scissile Leu5-Arg6 bond in dyn 1-8. The method circumvents the use of [3H]Tyr-labelled dynorphins, which have the inherent drawback that fragments lacking the N-terminal Tyr cannot be detected. Owing to the high resolution, also for the larger dynorphins dyn 1-14, dyn 1-15 and dyn 1-16, the chromatographic system should prove especially useful in the elucidation of the enzymolysis pattern of dyn 1-17. Furthermore, the method offers a way to evaluate simultaneously the selectivity of new enzyme inhibitors for several cleavage sites in the same assay.
Seyfried
C A
CA
Department of Neurochemistry, Biological Research, E. Merck, Darmstadt, F.R.G.
Tobler
P
P
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004399:Dynorphins / Q000032:analysis*; D009411:Nerve Endings / Q000201:enzymology*; D010446:Peptide Fragments / Q000032:analysis*; D051381:Rats; D011919:Rats, Inbred Strains; D013116:Spinal Cord / Q000166:cytology
D010446:Peptide Fragments; D004399:Dynorphins