Regulation of Activities of Cytidine 5′-Diphospho-Choline: 1-O-Alkyl-2-Acetyl-sn-Glycerol Cholinephosphotransferase, an Enzyme Responsible for de novo Synthesis of Platelet-Activating Factor, by Membrane Phospholipids

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Cytidine 5′-diphospho (CDP)-choline: 1-O-alkyl-2-acetyl-sn-glycerol cholinephosphotransferase (AAG-CPT), an enzyme responsible for de novo synthesis of platelet-activating factor (PAF), was solubilized from porcine spleen microsomes using digitonin. Although the activity of the solubilized enzyme was relatively stable, further purification by sequential chromatography on Toyopearl HW-65 gel filtration and diethylaminoethyl (DEAE)-Toyopearl 650 caused a remarkable decrease in enzyme activity, which was partially recovered by the exogenously addition of phospholipids such as egg phosphatidylcholine, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine, dioleoylphosphatidylglycerol, and dioleoylphosphatidylserine. In contrast, dioleoylphosphatidic acid (DOPA) showed an inhibitory effect on enzyme activity. In addition, lysophospholipids such as monooleoylphosphatidylcholine, monooleoylphosphatidylethanolamine, monooleoylphosphatidylglycerol, and monooleoylphosphatidic acid showed an inhibitory effect on the enzyme activity. When DOPA was concomitantly added with DOPC, the enzyme activity reactivated by DOPC decreased with the ratio of DOPA to DOPC. Furthermore, we examined whether phosphatidic acid (PA) or lysophospholipids had an inhibitory effect on AAG-CPT activity under more physiological conditions. Treatment of microsomes with exogenously added phospholipase D or phospholipase A₂ resulted in a decrease in AAG-CPT. In addition, when endogenous phospholipase D was activated by fatty acids such as oleic acid and arachidonic acid to generate PA in the porcine microsomes, the enzyme activity was significantly inhibited. The molecular weight of the enzyme solubilized from porcine spleen microsomes was estimated to be 440 kDa based on gel-filtration column chromatography on Toyopearl HW-65, suggesting that this enzyme formed a complex with other protein molecules and membrane phospholipids, and that these phospholipids were necessary to maintain the enzyme activity. Our results indicate that environmental membrane phospholipids containing PA and/or lysophospholipids are important factors in the regulation of the enzyme for de novo synthesis of PAF.