Approach to studying proteinase specificity by continuous-flow fast atom bombardment mass spectrometry and high-performance liquid chromatography combined with photodiode-array ultraviolet detection.
Fast atom bombardment mass spectrometry (FAB-MS) and high-performance liquid chromatography using a photodiode-array ultraviolet detector were applied to study a dynorphin-converting endopeptidase from the human pituitary gland. The specificity of the enzyme was tested towards various opioid peptides derived from the prodynorphin precursor, i.e. dynorphin A, dynorphin B and alpha-neoendorphin. Peptide fragments were analysed directly by continuous-flow FAB-MS and those containing aromatic amino acids were detected independently by the photodiode-array ultraviolet detector. The results obtained suggest a similar processing of these structure-related substrates and it appears that the enzyme recognizes the dibasic stretch in their sequence. It is also clear from this study that the combination of the above techniques provides a powerful tool for studies of enzymatic conversion among the prodynorphin-derived peptides and it should be applicable to studies of similar mechanisms in other peptide systems.
Department of Pharmacology, University of Uppsala, Sweden.
J
J
Silberring
P
P
Brostedt
M
M
Thörnwall
F
F
Nyberg
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004399:Dynorphins / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D004723:Endorphins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D010902:Pituitary Gland / Q000201:enzymology*; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism*; D016339:Spectrometry, Mass, Fast Atom Bombardment / Q000379:methods*; D013379:Substrate Specificity
D004723:Endorphins; D010446:Peptide Fragments; D011498:Protein Precursors; C025681:alpha-neoendorphin; D004399:Dynorphins; C036200:rimorphin; D012697:Serine Endopeptidases; C054870:dynorphin-converting endopeptidase