Solubilization, purification and characterization of lysoplasmalogen alkenylhydrolase (lysoplasmalogenase) from rat liver microsomes

Abstract Alkenylhydrolase (EC 3.3.2.2; EC 3.3.2.5) has been purified 200-fold to a specific activity of 8.0 μmol/min per mg from rat liver microsomes with 51% of the activity recovered. Purification was accomplished by solubilization of the membrane-associated enzyme with octylglucoside and chromatographic resolution on sequential DEAE cellulose and hydroxylapatite (HPLC) columns in the presence of octylglucoside. The partially purified enzyme, specific for the 2-deacylated plasmalogen, lysoplasmalogen (1-alk-1′-enyl- sn -glycero-3-phosphocholine or ethanolamine), had no hydrolytic activity with intact plasmalogens or 1-acyl- sn -glycero-3-phosphoethanolamine. Kinetic analyses of enzymic activity demonstrated apparent K m values of 5.5 and 42 μM for 1-alk-1′-enyl- sn -glycero-3-phosphocholine and 1-alk-1′-enyl- sn -glycero- 3-phosphoethanolamine, respectively. The V max values were 11.7 and 13.6 μmol/min per mg with the choline and ethanolamine substrates, respectively. The optimal pH range was between 6.6 and 7.1 with both substrates; the energy of activation for the purified enzyme was 15 200 cal. The enzyme required no cofactors and was unaffected by low millimolar concentrations of Ca 2+ , Mg 2+ or EDTA. It was inhibited by the sulfhydryl-reacting reagent, p -chloromercuribenzoate. Mono- or diradylglycerophospholipids or sphingomyelin did not affect the enzymic activity at 37° C. Activity of the purified enzyme, destroyed by freezing at −20°C, was preserved if stored at this temperature in the presence of 300–600 μM diradylglycerophosphocholine or 50% glycerol. A continuous spectrophotometric assay, adapted in our laboratory for the assay of liver alkenylhydrolase, faciitated this purification. This is the first reported purification of alkenylhydrolase.