Abstract
Dynorphin B (Dyn B-13, also known as rimorphin) is generated from Dyn B-29 (leumorphin) by the cleavage at a single Arg residue. An enzymatic activity capable of processing at this monobasic site has been previously reported in neurosecretory vesicles of the bovine pituitary and pituitary-derived cell lines. This enzyme termed "the dynorphin-converting enzyme" (DCE) has been purified to apparent homogeneity from the neurointermediate lobe of the bovine pituitary using hydrophobic chromatography on phenyl-Sepharose, preparative isoelectrofocusing in a granulated gel between pH 4 to 6.5, and non-denaturing electrophoresis on 5% polyacrylamide gel. DCE exhibits a pI of about 5.1 and a molecular mass of about 54 kDa under reducing conditions. DCE is a metallopeptidase and exhibits a neutral pH optimum. Specific Inhibitors of soluble metallopeptidases such as enkephalinase (EC 3.4.24.11) or enkephalin generating neutral endopeptidase (EC 3.4.24.15) do not inhibit DCE activity indicating that DCE is distinct from these two enzymes. Cleavage site determination with matrix-assisted laser desorption ionization time of flight (MALDITOF) mass spectrometry shows that DCE cleaves the Dyn B-29 N terminus to the Arg14 generating Dyn B-13 and Dyn B-(14-29). Among other peptides derived from Dyn B-29, DCE cleaves only those peptides that fit the predicted "consensus motif" for monobasic processing. These data are consistent with a broader role for the dynorphin converting enzyme in the biosynthesis of many peptide hormones and neuropeptides by processing at monobasic sites.
Highlights
The majority of the neuropeptides are synthesized as larger precursors that undergo endoproteolysis at specific sites [1]
Purification of the dynorphin converting enzyme” (DCE) from Bovine Pituitary—We used a selective assay that allows specific detection of Dyn B-13 to isolate the DCE; this assay makes use of Dyn B-29-(9 –22), a Dyn B-29-derived peptide that is not recognized by DCE and protects the Dyn B-29 and Dyn B-13 from hydrolysis by other peptidases [21]
We chose bovine neurointermediate lobe (NIL) as the source of DCE since previously we had shown that in the NIL the DCE is contained within the neuropeptide containing secretory vesicles and that in the NIL DCE is at a higher specific activity as compared with the anterior pituitary
Summary
The majority of the neuropeptides are synthesized as larger precursors that undergo endoproteolysis at specific sites [1]. Several neuropeptideprocessing enzymes have been identified in mammalian cells [2,3,4,5,6] Endoproteases such as furin, PC1, and PC2 are thought to preferentially cleave peptide precursors at multibasic residue cleavage sites [4, 7]. DCE is a peptide processing enzyme that converts Dyn B-292 into Dyn B-13 by cleavage at single basic residue Arg14 [5, 6]. In the brain and pituitary, the distribution of the DCE activity generally matches that of Dyn B-13 [10, 11] These data suggest that DCE is physiologically involved in the processing of dynorphin peptides at single arginine cleavage sites. These data support the premise that DCE is a monobasic processing endoprotease involved in the generation of Dyn B-13
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