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

The discovery of a potentially novel proline-specific peptidase from bovine serum is presented which is capable of cleaving the dipeptidyl peptidase IV (DPIV) substrate Gly-Pro-MCA. The enzyme was isolated and purified with the use of Phenyl Sepharose Hydrophobic Interaction, Sephacryl S300 Gel Filtration, and Q-Sephacryl Anion Exchange, producing an overall purification factor of 257. SDS PAGE resulted in a monomeric molecular mass of 158 kDa while Size Exclusion Chromatography generated a native molecular mass of 328 kDa. The enzyme remained active over a broad pH range with a distinct preference for a neutral pH range of 7-8.5. Chromatofocusing and Isoelectric Focusing revealed the enzyme’s isoelectric point to be 4.74. DPIV-like activity was not inhibited by serine protease inhibitors but was by the metallo-protease inhibitors, the phenanthrolines. The enzyme was also partially inhibited by Bestatin. Substrate Specificity studies proved that the enzyme is capable of sequential cleavage of bovine β- Casomorphin and Substance P. The peptidase cleaved the standard DPIV substrate, Gly-Pro-MCA with a KM of 38.4 μM, while Lys-Pro-MCA was hydrolysed with a KM of 103 μM. The DPIV- like activity was specifically inhibited by both Diprotin A and B, non-competitively, generating a Ki of 1.4x10-4 M for both inhibitors. Ile-Thiazolidide and Ile-Pyrrolidide both inhibited competitively with an inhibition constant of 3.7x10-7 M and 7.5x10-7 M respectively. It is concluded that bovine serum DPIV-like activity share many biochemical properties with DPIV and DPIV-like enzymes but not exclusively, suggesting that the purified peptidase may play an important novel role in bioactive oligopeptide degradation

Inhibition of prolyl oligopeptidase with a synthetic unnatural dipeptide

A new inhibitor, containing a linked proline-piperidine structure, for the enzyme prolyl oligopeptidase (POP) has been synthesised and demonstrated to bind covalently with the enzyme at the active site. This provides evidence that covalent inhibitors of POP do not have to be limited to structures containing five-membered N-containing heterocyclic rings