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

Dipeptidyl Peptidase IV as a Potential Target for Selective Prodrug Activation and Chemotherapeutic Action in Cancers

[Image: see text] The efficacy of chemotherapeutic drugs is often offset by severe side effects attributable to poor selectivity and toxicity to normal cells. Recently, the enzyme dipeptidyl peptidase IV (DPPIV) was considered as a potential target for the delivery of chemotherapeutic drugs. The purpose of this study was to investigate the feasibility of targeting chemotherapeutic drugs to DPPIV as a strategy to enhance their specificity. The expression profile of DPPIV was obtained for seven cancer cell lines using DNA microarray data from the DTP database, and was validated by RT-PCR. A prodrug was then synthesized by linking the cytotoxic drug melphalan to a proline-glycine dipeptide moiety, followed by hydrolysis studies in the seven cell lines with a standard substrate, as well as the glycyl-prolyl-melphalan (GP-Mel). Lastly, cell proliferation studies were carried out to demonstrate enzyme-dependent activation of the candidate prodrug. The relative RT-PCR expression levels of DPPIV in the cancer cell lines exhibited linear correlation with U95Av2 Affymetrix data (r(2) = 0.94), and with specific activity of a standard substrate, glycine-proline-p-nitroanilide (r(2) = 0.96). The significantly higher antiproliferative activity of GP-Mel in Caco-2 cells (GI(50) = 261 μM) compared to that in SK-MEL-5 cells (GI(50) = 807 μM) was consistent with the 9-fold higher specific activity of the prodrug in Caco-2 cells (5.14 pmol/min/μg protein) compared to SK-MEL-5 cells (0.68 pmol/min/μg protein) and with DPPIV expression levels in these cells. Our results demonstrate the great potential to exploit DPPIV as a prodrug activating enzyme for efficient chemotherapeutic drug targeting