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

Buckley, Seamus J. and Collins, Patrick and O'Connor, Brendan (2004) The purification and characterisation of novel dipeptidyl peptidase IV-like activity from bovine serum. International Journal of Biochemistry & Cell Biology, 36 (7). pp. 1281-1296. ISSN 1357-2725

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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.

Item Type:Article (Published)
Subjects:Biological Sciences > Biotechnology
Biological Sciences > Biochemistry
Biological Sciences > Enzymology
DCU Faculties and Centres:DCU Faculties and Schools > Faculty of Science and Health > School of Biotechnology
Research Initiatives and Centres > Irish Separation Science Cluster (ISSC)
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Copyright Information:© 2004 Elsevier
Use License:This item is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 3.0 License. View License
Funders:Enterprise Ireland, Health Research Board
ID Code:17813
Deposited On:28 Feb 2013 14:41 by Brendan O'Connor. Last Modified 27 Apr 2017 13:53

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