Purification and characterization of 45 kDa PAF acetylhydrolase from bovine colostrum

Area of Science:

• Biochemistry
• Enzymology

Background:

• Platelet activating factor (PAF) acetylhydrolase (PAF-AH) activity is present in bovine colostrum, with higher levels detected in early colostrum (within 24 hours postpartum).
• Understanding the characteristics of this enzyme is crucial for its potential applications and for elucidating its role in biological systems.

Purpose of the Study:

• To purify and characterize the PAF-AH enzyme found in early bovine colostrum.
• To determine the enzyme's molecular weight, kinetic properties, and substrate specificity.
• To identify the type of PAF-AH present in bovine colostrum.

Main Methods:

• Purification of PAF-AH using a multi-step chromatographic approach including ammonium sulfate precipitation, butyl-Toyopearl, DEAE-Sepharose, heparin-Sepharose, hydroxyapatite, chelating-Sepharose, and Mono Q HPLC.
• Determination of molecular weight via SDS-PAGE.
• Enzyme kinetics measured by V(max) and K(m) determination.
• Inhibition assays using specific chemical reagents.
• Substrate specificity analysis by testing degradation of various phospholipids.
• Western blot analysis using an antibody against human plasma PAF-AH.

Main Results:

• A 45 kDa monomeric polypeptide with PAF-AH activity was successfully purified.
• The enzyme exhibited V(max) of 87.6 µM and K(m) of 7.96 nmol/min/mg.
• Enzyme activity was inhibited by phenylmethylsulfonyl fluoride, iodoacetamide, and p-bromophenacylbromide, indicating the involvement of serine and histidine residues.
• The purified enzyme did not degrade phospholipids with long-chain fatty acyl groups at the sn-2 position, distinguishing it from phospholipase A(2).
• The enzyme selectively hydrolyzed oxidatively modified phosphatidylcholine.
• Western blot analysis confirmed the enzyme to be plasma-type PAF-AH.

Conclusions:

• Early bovine colostrum contains a 45 kDa plasma-type PAF-AH.
• This enzyme is distinct from phospholipase A(2) and possesses a specific activity towards oxidatively modified phosphatidylcholine.
• The findings contribute to the understanding of PAF-AH in colostrum and its potential biological significance.