Identification and Initial Characterization of a N‐Benzyloxycarbonyl‐Prolyl‐Prolinal (Z‐Pro‐Prolinal)‐Insensitive 7‐(N‐Benzyloxycarbonyl‐Glycyl‐Prolyl‐Amido)‐4‐Methylcoumarin (Z‐Gly‐Pro‐NH‐Mec)‐Hydrolysing Peptidase in Bovine Serum

Abstract

A group of enzymes exists that specifically recognises proline within proteins and peptides. Prolyl endopeptidase is one such enzyme, which cleaves on the carboxyl side of proline within peptide substrates. Its broad specificity towards bioactive peptides has led to its implication in various disease states including neurodegenerative and psychiatric disorders. This association has been based primarily on the abnormal levels of activity observed following the enzymes detection with the reportedly specific fluori‐metric substrate, 7‐(N‐benzyloxycarbonyl‐glycyl‐prolyl‐amido)‐4‐methylcoumarin (Z‐Gly‐Pro‐NH‐Mec). In this study, we report the discovery and preliminary characterisation of a Z‐Gly‐Pro‐NH‐Mec‐hydrolysing activity that is distinct from prolyl oligopeptidase (prolyl endopeptidase). Following the production of serum from bovine whole blood, Z‐Gly‐Pro‐NH‐Mec hydrolysis in serum was determined to be 7.2 U/mg protein. In the presence of 350 nM Z‐Pro‐prolinal, a specific inhibitor of prolyl endopeptidase, residual Z‐Gly‐Pro‐NH‐Mec hydrolysis of 2.6 U/mg protein was observed. This residual activity was resistant to inhibition by Z‐Pro‐prolinal at concentrations in excess of 200 times its reported Ki value for prolyl endopeptidase and could not be inhibited under conditions of prolonged incubation with the inhibitor. Following cation‐exchange chromatography, Z‐Gly‐Pro‐NH‐Mec‐hydrolysing activity was resolved into two distinct entities. The first of these activities was inhibited by Z‐Pro‐prolinal, demonstrated activity towards the thyroliberin analogue, 7‐(pyroglutamyl‐histidyl‐prolyl)‐4‐methylcoumarin (Glp‐His‐Pro‐NH‐Mec), and was catalytically enhanced under reduced assay conditions. This activity was subsequently designated prolyl endopeptidase. The second activity was totally resistant to Z‐Pro‐prolinal inhibition, demonstrated no activity towards Glp‐His‐Pro‐NH‐Mec, and was unaffected when assayed under reduced conditions. It was subsequently designated Z‐Pro‐prolinal‐insensitive Z‐Gly‐Pro‐NH‐Mec‐hydrolysing peptidase (ZIP).

Purification, identification and characterisation of seprase from bovine serum

“…In this report we have described the purification, sequencing and properties of a novel prolyl peptidase from bovine serum. Cunningham and O'Connor (1997b) reported the first observation of a second Z-Gly-Pro-AMC hydrolysing activity in bovine serum. The uniqueness of this peptidase was its ability to cleave the specific prolyl oligopeptidase fluorogenic substrate Z-Gly-Pro-AMC, but being completely distinct to this serine protease (Birney & O'Connor, 2001).…”

The purification and characterisation of novel dipeptidyl peptidase IV-like activity from bovine serum

“…However, as before, the enzymatic activity did not bind to the column indicating that at least two DPIV-like activities are present in bovine serum. The Hydrophobic Interaction Chromatography step resulted in separation of the main Gly-Pro-MCA cleaving activity from Z-Gly-Pro-MCA cleaving activity attributed to Prolyl Oligopeptidase and ZIP which is also present [20]. This was essential as both activities have similar substrate specificities.…”

Characterisation of the active site of a newly-discovered and potentially significant post-proline cleaving endopeptidase called ZIP using LC-UV-MS

“…It has been implicated in many disease states, especially depression 3 , and the enzyme also has a role in the regulation of blood pressure 4 . In recent years however, a new and distinct post-proline cleaving endopeptidase, has been identified and reported 5,6 . This new enzyme has been designated ZIP (Z-pro-prolinal-InsensitivePeptidase) 5 and preliminary investigation shows that the ZIP has the ability to cleave prolinecontaining peptides such as Substance P, LHRH, Angiotensin, Neurotensin and Bradykinin, and that it can be separated from PE by cation-exchange chromatography.…”

“…In recent years however, a new and distinct post-proline cleaving endopeptidase, has been identified and reported 5,6 . This new enzyme has been designated ZIP (Z-pro-prolinal-InsensitivePeptidase) 5 and preliminary investigation shows that the ZIP has the ability to cleave prolinecontaining peptides such as Substance P, LHRH, Angiotensin, Neurotensin and Bradykinin, and that it can be separated from PE by cation-exchange chromatography. Initial investigations on human serum have found it to contain very high levels of this ZIP activity so it is highly likely that it is actually this new ZIP enzyme (and not the reported PE) that shows significant activity changes in conditions such as depression, mania, schizophrenia and Alzheimers disease as reported by Maes and co-workers 3,7,8 .…”