Biomedical subjects

D F Cunningham

Publications and source records attributed to D F Cunningham.

3 recordsLinked to original sources

PubMed1998

A study of prolyl endopeptidase in bovine serum and its relevance to the tissue enzyme.

Prolyl endopeptidase (PE) belongs to a group of enzymes that specifically recognise the imino acid proline. The characterisation of bovine serum PE was undertaken so that its relationship to its tissue counterparts could be considered. Using various chromatographic methods, PE was partially purified from bovine serum. This preparation was deemed to be enzymatically pure, based on its failure to hydrolyse a wide range of fluorimetric substrates. A native molecular mass of 69.7 kDa was estimated for the enzyme. PE was optimally active at pH 8.0-8.5, demonstrated a preference for phosphate buffer and remained stable over a pH range of 5.0-9.0. A narrowly focused optimal assay temperature of 37 degrees C was evident. Functional reagent studies indicated that this enzyme was a serine protease with a cysteine residue located near or at the active site. The enzyme was also sensitive to heavy metal inhibition. Substrate specificity investigations revealed that the bioactive peptides angiotensin II, bradykinin, luliberin and substance P were hydrolysed by the enzyme preparation, but lower specificities were evident towards these peptides in comparison with the enzyme's tissue counterparts. Specific inhibitor studies, using a range of compounds previously untested against a single PE source, indicated that alpha-ketobenzothiazole was the most effective PE inhibitor, with an IC50 value of 41 pM. In conclusion, the results presented in this paper indicate that bovine serum PE shares many of the characteristics associated with its tissue counterparts, with the exception of its specificity towards certain bioactive peptides.

Angiotensin II↗

PubMed1997

Proline specific peptidases.

Proline is unique among the 20 amino acids due to its cyclic structure. This specific conformation imposes many restrictions on the structural aspects of peptides and proteins and confers particular biological properties upon a wide range of physiologically important biomolecules. In order to adequately deal with such peptides, nature has developed a group of enzymes that recognise this residue specifically. These peptidases cover practically all situations where a proline residue might occur in a potential substrate. In this paper we endeavour to discuss these enzymes, particularly those responsible for peptide or protein hydrolysis at proline sites. We have detailed their discovery, biochemical attributes and substrate specificities and have provided information as to the methodology used to detect and manipulate their activities. We have also described the roles, or potential roles that these enzymes may play physiologically and the consequences of their dysfunction in varied disease states.

Amino Acid Sequence↗

PubMed1997

Identification and initial characterisation 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.

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 fluorimetric 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).

Animals↗