Purification of a galanin degrading 70 kDa metallo-peptidase from bovine spinal cord.
Galanin is a 29/30 amino acids long neuroendocrine peptide, acting as an inhibitory modulator in the spinal cord. Several studies show that galanin is involved in control of pain threshold and acts synergistically with morphine, hence, inhibition of galanin degradation may be a pharmaceutical target for treatment of pain. In this study we have designed an 11 amino acids long substrate based on the first 10 N-terminal amino acids of galanin (this part contains the major pharmacophores of galanin), with a N-terminal fluorescent marker, anthranilic acid, and a C-terminal internal fluorescence quencher, 3-nitrotyrosine, coupled to the epsilon-amino group of the linker Lys11. Using this substrate to detect galanin degradation, we have purified a membrane bound galanin inactivating metallo-peptidase from bovine spinal cord. This enzyme, cleaving galanin between Trp2 and Thr3, is a novel 70 kDa, Zn2+ dependent metallo-peptidase.
Juréus
A
A
Department of Neurochemistry and Neurotoxicology, Stockholm University, Sweden.
Lindgren
M
M
Langel
U
U
Bartfai
T
T
D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D002614:Chelating Agents / Q000494:pharmacology; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes; D019004:Galanin / Q000378:metabolism*; D007700:Kinetics; D008566:Membranes / Q000201:enzymology; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D012995:Solubility; D013116:Spinal Cord / Q000201:enzymology*; D013379:Substrate Specificity; D015032:Zinc / Q000378:metabolism
D002614:Chelating Agents; D005456:Fluorescent Dyes; D010446:Peptide Fragments; D011480:Protease Inhibitors; D019004:Galanin; D008666:Metalloendopeptidases; D015032:Zinc