Post-proline cleaving enzyme (prolyl endopeptidase) from bovine brain

Area of Science:

• Biochemistry
• Enzymology
• Neuroscience

Background:

• Post-proline cleaving enzyme (prolyl endopeptidase, EC 3.4.21.26) plays a role in processing peptide bonds involving proline.
• Understanding the properties of this enzyme is crucial for various biological processes, including neuropeptide metabolism.

Purpose of the Study:

• To purify and characterize the prolyl endopeptidase from bovine brain.
• To determine its enzymatic properties, substrate specificity, and physicochemical characteristics.
• To compare the bovine brain enzyme with those from other sources.

Main Methods:

• Purification using DEAE-Sephadex, hydroxyapatite, PCMB-T-Sepharose chromatography, and Sephadex G-200 gel filtration.
• Enzyme activity assays using synthetic substrates (Z-Gly-Pro-2-NNap, TRH) and peptide hormones (oxytocin, vasopressin, etc.).
• Homogeneity assessment via disc gel and SDS-PAGE, molecular weight determination (ultracentrifugation, gel filtration, SDS-PAGE), isoelectric focusing, and chemical inactivation studies.

Main Results:

• The enzyme was purified ~3,700-fold and appeared homogeneous.
• It exhibited optimal activity at pH 7.5 (Z-Gly-Pro-2-NNap) and 7.2 (TRH), hydrolyzing Pro-X bonds in various peptides but not collagen or casein.
• The enzyme was inactivated by DFP, Z-Gly-Pro-chloromethyl ketone, and PCMB, indicating cysteine involvement, with a molecular weight of ~76,000 Da.

Conclusions:

• The bovine brain prolyl endopeptidase shares significant similarities in enzymatic and physicochemical properties with enzymes from lamb brain and kidney.
• Contrary to previous reports, this study confirms the enzyme's activity on oxytocin, vasopressin, and bradykinin.
• The enzyme's characterization provides valuable insights into neuropeptide degradation pathways.