[46] Lysoplasmalogenase: solubilization and partial purification from liver microsomes

This chapter focuses on the process of solubilization and purification of lysoplasmalogenase from liver microsomes. In this coupled enzyme assay, the aldehyde released during the hydrolysis of the vinyl ether bond of lysoplasmalogen is reduced to the alcohol by exogenously added alcohol dehydrogenase. The reaction is followed spectrophotometrically by observing the absorbance change at 340 nm when reduced nicotinamide adenine dinucleotide (NADH) is oxidized. All procedures are carried out at 0° and five male Sprague–Dawley rats (300–380 g) are sacrificed by decapitation. The livers are removed, chilled immediately in 0.25 M sucrose, and the blood is rinsed off. Livers are minced and homogenized by using a power-drived Teflon pestle in a glass homogenizing vessel, with approximately nine strokes at 600 rpm. Lysoplasmalogenase is solubilized with octylglucoside, a detergent that is relatively nondenaturing to proteins. The enzyme appears to be lipophilic and requires the presence of octylglucoside throughout the purification procedure. The enzyme is purified 300-fold by sequential diethylaminoethyl (DEAE)-cellulose and hydroxylapatite chromatographies. The chapter describes the conditions, which stabilize the enzyme. This purification may lead to an understanding of the role of the enzyme in plasmalogen metabolism in normal and pathological conditions.