Purification and Characterization of a Dynorphin-processing Endopeptidase

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 Arg 14 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.

Specificity of the Dynorphin‐Processing Endoprotease: Comparison with Prohormone Convertases

“…1B and Table 1). The affinity of DCE for these peptides is substantially lower than the affinity for Dyn B-29 (0.13 pw, suggesting that longer peptide substrates are preferred by the enzyme (Berman et al, 1995).…”

“…In contrast, DCE does not cleave a strictly dibasic sequence (type I1 site) as evidenced by the lack of processing at the internal Arg-Arg sites within the Dyn B-29 sequence (Berman et al, 1995). These results suggest that DCE exhibits cleavage site selectivity; this enzyme could be involved in the processing of certain bioactive peptides at sites that require cleavage at type I in addition to cleavage at the type I11 sites.…”

“…The sequence of peptide 1, RRQFKVVTRSQ, is derived from the sequence of Dyn B-29 surrounding the monobasic cleavage site. This substrate (Dyn B-29) was cleaved N-terminal to the monobasic Arg as determined by MALDI-TOF mass spectrometry (Berman et al, 1995). As shown in Fig.…”

“…A synthetic peptide, Dyn B-29-(9-22), which contains the cleavage site but does not contain a basic residue at the P4, P6, or P8 position, is not recognized by DCE Berman et al, 1995). In contrast, peptides 1 and 2, which contain Arg in the P8 position, and peptides 3-9, which contain Arg in the P4 position, are recognized by DCE ( Fig.…”

Specificity of the Dynorphin‐Processing Endoprotease: Comparison with Prohormone Convertases

“…1B and Table 1). The affinity of DCE for these peptides is substantially lower than the affinity for Dyn B-29 (0.13 pw, suggesting that longer peptide substrates are preferred by the enzyme (Berman et al, 1995).…”

“…In contrast, DCE does not cleave a strictly dibasic sequence (type I1 site) as evidenced by the lack of processing at the internal Arg-Arg sites within the Dyn B-29 sequence (Berman et al, 1995). These results suggest that DCE exhibits cleavage site selectivity; this enzyme could be involved in the processing of certain bioactive peptides at sites that require cleavage at type I in addition to cleavage at the type I11 sites.…”

“…The sequence of peptide 1, RRQFKVVTRSQ, is derived from the sequence of Dyn B-29 surrounding the monobasic cleavage site. This substrate (Dyn B-29) was cleaved N-terminal to the monobasic Arg as determined by MALDI-TOF mass spectrometry (Berman et al, 1995). As shown in Fig.…”

“…A synthetic peptide, Dyn B-29-(9-22), which contains the cleavage site but does not contain a basic residue at the P4, P6, or P8 position, is not recognized by DCE Berman et al, 1995). In contrast, peptides 1 and 2, which contain Arg in the P8 position, and peptides 3-9, which contain Arg in the P4 position, are recognized by DCE ( Fig.…”

A Quantitative Peptidomic Analysis of Peptides Related to the Endogenous Opioid and Tachykinin Systems in Nucleus Accumbens of Rats Following Naloxone-Precipitated Morphine Withdrawal

“…In addition, the C-terminal sequence (residues 16-29) of dynorphin B-29 and Leu-enkephalin was also detected in the nucleus accumbens neuropeptidome. Dynorphin B-29 was earlier found to be enzymatically released from prodynorphin 42 and this peptide was also shown to induce a dose-dependent short-lasting change on the nociceptive threshold in rats. 43 Both alpha-neoendorphin and dynorphin B were found to display an increased expression during withdrawal in the neuropeptidome of Sprague-Dawley rats but not in Wistar rats.…”

Specificity of the Dynorphin‐Processing Endoprotease: Comparison with Prohormone Convertases

“…1B and Table 1). The affinity of DCE for these peptides is substantially lower than the affinity for Dyn B-29 (0.13 pw, suggesting that longer peptide substrates are preferred by the enzyme (Berman et al, 1995).…”

“…In contrast, DCE does not cleave a strictly dibasic sequence (type I1 site) as evidenced by the lack of processing at the internal Arg-Arg sites within the Dyn B-29 sequence (Berman et al, 1995). These results suggest that DCE exhibits cleavage site selectivity; this enzyme could be involved in the processing of certain bioactive peptides at sites that require cleavage at type I in addition to cleavage at the type I11 sites.…”

“…The sequence of peptide 1, RRQFKVVTRSQ, is derived from the sequence of Dyn B-29 surrounding the monobasic cleavage site. This substrate (Dyn B-29) was cleaved N-terminal to the monobasic Arg as determined by MALDI-TOF mass spectrometry (Berman et al, 1995). As shown in Fig.…”

“…A synthetic peptide, Dyn B-29-(9-22), which contains the cleavage site but does not contain a basic residue at the P4, P6, or P8 position, is not recognized by DCE Berman et al, 1995). In contrast, peptides 1 and 2, which contain Arg in the P8 position, and peptides 3-9, which contain Arg in the P4 position, are recognized by DCE ( Fig.…”

A Quantitative Peptidomic Analysis of Peptides Related to the Endogenous Opioid and Tachykinin Systems in Nucleus Accumbens of Rats Following Naloxone-Precipitated Morphine Withdrawal

“…In addition, the C-terminal sequence (residues 16-29) of dynorphin B-29 and Leu-enkephalin was also detected in the nucleus accumbens neuropeptidome. Dynorphin B-29 was earlier found to be enzymatically released from prodynorphin 42 and this peptide was also shown to induce a dose-dependent short-lasting change on the nociceptive threshold in rats. 43 Both alpha-neoendorphin and dynorphin B were found to display an increased expression during withdrawal in the neuropeptidome of Sprague-Dawley rats but not in Wistar rats.…”

Specificity of the Dynorphin‐Processing Endoprotease: Comparison with Prohormone Convertases

“…1B and Table 1). The affinity of DCE for these peptides is substantially lower than the affinity for Dyn B-29 (0.13 pw, suggesting that longer peptide substrates are preferred by the enzyme (Berman et al, 1995).…”

“…In contrast, DCE does not cleave a strictly dibasic sequence (type I1 site) as evidenced by the lack of processing at the internal Arg-Arg sites within the Dyn B-29 sequence (Berman et al, 1995). These results suggest that DCE exhibits cleavage site selectivity; this enzyme could be involved in the processing of certain bioactive peptides at sites that require cleavage at type I in addition to cleavage at the type I11 sites.…”

“…The sequence of peptide 1, RRQFKVVTRSQ, is derived from the sequence of Dyn B-29 surrounding the monobasic cleavage site. This substrate (Dyn B-29) was cleaved N-terminal to the monobasic Arg as determined by MALDI-TOF mass spectrometry (Berman et al, 1995). As shown in Fig.…”

“…A synthetic peptide, Dyn B-29-(9-22), which contains the cleavage site but does not contain a basic residue at the P4, P6, or P8 position, is not recognized by DCE Berman et al, 1995). In contrast, peptides 1 and 2, which contain Arg in the P8 position, and peptides 3-9, which contain Arg in the P4 position, are recognized by DCE ( Fig.…”

A Quantitative Peptidomic Analysis of Peptides Related to the Endogenous Opioid and Tachykinin Systems in Nucleus Accumbens of Rats Following Naloxone-Precipitated Morphine Withdrawal

“…In addition, the C-terminal sequence (residues 16-29) of dynorphin B-29 and Leu-enkephalin was also detected in the nucleus accumbens neuropeptidome. Dynorphin B-29 was earlier found to be enzymatically released from prodynorphin 42 and this peptide was also shown to induce a dose-dependent short-lasting change on the nociceptive threshold in rats. 43 Both alpha-neoendorphin and dynorphin B were found to display an increased expression during withdrawal in the neuropeptidome of Sprague-Dawley rats but not in Wistar rats.…”