Prolidase from bovine intestine: purification and characterization

Area of Science:

• Biochemistry
• Enzymology
• Proteomics

Background:

• Prolidase (EC 3.4.13.9), also known as iminodipeptidase, plays a role in peptide metabolism.
• Understanding prolidase function requires detailed characterization of the purified enzyme.

Purpose of the Study:

• To highly purify prolidase from bovine small intestine cytosol.
• To characterize the biochemical and physical properties of the purified bovine prolidase.

Main Methods:

• Enzyme purification using multiple chromatography techniques (DEAE-Toyopearl, Sephadex G-150, PCMB-T-Sepharose, hydroxyapatite).
• Homogeneity assessment via disc gel electrophoresis.
• Enzyme activity assays at varying pH and substrate specificity determination.
• Stability studies at different temperatures and pH.
• Inhibition studies with specific reagents (PCMB, DFP, PMSF, metal chelators).
• Molecular weight determination using gel filtration and SDS-PAGE.
• Amino acid composition analysis.

Main Results:

• Highly purified, homogeneous prolidase was obtained.
• Optimal activity was observed at pH 7.2 with Gly-Pro substrate.
• The enzyme demonstrated stability between pH 5.5-8.5 and moderate thermal stability.
• Prolidase was inactivated by p-chloromercuribenzoate (PCMB) but not by DFP, PMSF, or metal chelators.
• Molecular weight estimates indicated a dimeric structure (116,000 by gel filtration, 56,000 by SDS-PAGE).
• The enzyme specifically hydrolyzed X-Pro dipeptides.

Conclusions:

• Bovine prolidase is a dimeric enzyme with specific substrate preferences and stability characteristics.
• The enzyme's sensitivity to PCMB suggests the involvement of sulfhydryl groups in its active site.
• This purified and characterized prolidase provides a foundation for further functional and structural studies.