Subcellular localization, partial purification, and characterization of a dynorphin processing endopeptidase from bovine pituitary.
An enzyme capable of cleaving dynorphin B-29 to dynorphin B-13 is present in bovine pituitary, with 40- to 50-fold higher specific activity in the posterior and intermediate lobes than in the anterior lobe. Subcellular fractionation of bovine neurointermediate pituitary shows that this enzyme is present in the peptide-containing secretory vesicles. The enzyme has been purified 2,800-fold from whole bovine pituitaries using ion-exchange and gel filtration chromatography. Purified dynorphin-converting enzyme has a neutral pH optimum, and is subsantially inhibited by the thiol-protease inhibitor p-chloromercuriphenylsulfonic acid, but not by serine or metalloprotease inhibitors. The purified enzyme processes dynorphin B-29 at Arg14, producing both dynorphin B-14 and dynorphin B-13 in a 5:1 ratio. No other cleavages are observed, suggesting that the activity is free from other proteases and is specific for single Arg sequences. Purified enzyme also processes dynorphin A-17 at the single Arg cleavage site, generating both dynorphin A-8 and A-9 in a 7:1 ratio. The tissue distribution, subcellular localization, and substrate specificity of this enzyme are consistent with a physiological role in the processing of dynorphin B-29 and dynorphin A-17, and possibly other peptides, at single Arg residues.
Devi
L
L
Department of Pharmacology, New York University Medical Center, New York 10016.
Gupta
P
P
Fricker
L D
LD
D002731:4-Chloromercuribenzenesulfonate; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D004399:Dynorphins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010902:Pituitary Gland / Q000201:enzymology* / Q000648:ultrastructure; D012697:Serine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors; D013379:Substrate Specificity; D014018:Tissue Distribution
D015842:Serine Proteinase Inhibitors; D002731:4-Chloromercuribenzenesulfonate; D004399:Dynorphins; D012697:Serine Endopeptidases; C054870:dynorphin-converting endopeptidase