Solubilization, purification and characterization of lysoplasmalogen alkenylhydrolase (lysoplasmalogenase) from rat liver microsomes.
Alkenylhydrolase (EC 3.3.2.2; EC 3.3.2.5) has been purified 200-fold to a specific activity of 8.0 mumol/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 Km values of 5.5 and 42 microM for 1-alk-1'-enyl-sn-glycero-3-phosphocholine and 1-alk-1'-enyl-sn-glycero-3-phosphoethanolamine, respectively. The Vmax values were 11.7 and 13.6 mumol/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 Ca2+, Mg2+, Mn2+ 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 degrees C. Activity of the purified enzyme, destroyed by freezing at -20 degrees C, was preserved if stored at this temperature in the presence of 300-600 microM diradylglycerophosphocholine or 50% glycerol. A continuous spectrophotometric assay, adapted in our laboratory for the assay of liver alkenylhydrolase, facilitated this purification. This is the first reported purification of alkenylhydrolase.
Jurkowitz-Alexander
M
M
Department of Physiological Chemistry, Ohio State University, Columbus 43210.
Ebata
H
H
Mills
J S
JS
Murphy
E J
EJ
Horrocks
L A
LA
D000818:Animals; D002729:Chloromercuribenzoates / Q000494:pharmacology; D002845:Chromatography; D003840:Deoxycholic Acid / Q000494:pharmacology; D005960:Glucosides / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008246:Lysophospholipids / Q000378:metabolism; D008862:Microsomes, Liver / Q000201:enzymology*; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology; D051381:Rats; D012995:Solubility; D013053:Spectrophotometry; D013379:Substrate Specificity; D013816:Thermodynamics; D020245:p-Chloromercuribenzoic Acid
D002729:Chloromercuribenzoates; D005960:Glucosides; D008246:Lysophospholipids; C021215:lysoplasmalogens; D003840:Deoxycholic Acid; C018619:octyl-beta-D-glucoside; D011092:Polyethylene Glycols; D020245:p-Chloromercuribenzoic Acid; D017830:Octoxynol; D006867:Hydrolases; C118173:lysoplasmalogenase