Phosphatidylcholine biosynthesis in the anaerobic protozoon Entodinium caudatum.

In an extension of the work of Broad & Dawson (1975) on phospholipid biosynthesis in the anaerobic protozoon Entodinium caudatum we have characterized a number of aspects of phosphatidylcholine biosynthesis which, in this organism, is exclusively through the phosphorylcholine, CDP-choline pathway. Initial studies have centred on the subcellular distribution of the enzymes involved in the overall incorporation of choline into phosphatidylcholine and on the cofactors needed for the incorporation. As well, information on the site of action of an inhibitor of phosphatidylcholine biosynthesis (hemicholinium-3) has been obtained. These studies were seen as a necessary prerequisite to the examination of the role of phosphatidylcholine biosynthesis in membrane biogenesis in this organism. E. caudatum cells, suspended in ice-cold 100~-Tris-HC1 buffer (pH 8.3) were ultrasonicated for 6 0 s (KB80/1 ultrasonic bath; Kerry, Basildon, Essex, U.K.) and centrifuged brie5y (500g; 2min) to obtain a sediment from which the plasma membrane fraction was separated. The supernatant fraction was then centrifuged at various speeds (6OOOg for l0min; 144OOOg for 60min) to sediment three other membrane fractions of different particle sizes. In this way four membrane fractions and a soluble supernatant fraction were obtained. Each of the membrane fractions as well as the supernatant contained choline kinase (EC 2.7.1.32). CDP-cholinel ,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2) was confined to the membrane fractions and cholinephosphate cytidylyltransferase (EC 2.7.7.1 5 ) was located in the soluble fraction. No single cell fraction could incorporate choline into phosphatidylcholine; this could be achieved only by a reconstituted system consisting of any of the membrane fractions and the supernatant. However, the plasma membrane fraction (plus added supernatant) was by far the least active in synthesizing phosphatidylcholine. Cofactors known to be necessary for phosphatidylcholine biosynthesis de nouo in other cell systems were examined in detail in the reconstituted synthesizing system. In the absence of CTP and Mgz+ in particular, no synthesis could be observed; only very low concentrations were needed to induce a large stimulation of phosphatidylcholine biosynthesis. A requirement for ATP could also be shown. An examination of the intermediates of phosphatidylcholine biosynthesis accumulating during the reaction indicated that the conversion of phosphorylcholine into CDP-choline is probably the ratelimiting step in the overall incorporation of choline into phosphatidylcholine. Hemicholinium-3 has been shown to be a potent inhibitor of choline kinase (Spanner & Ansell, 1973; Broad & Dawson, 1974). The effect of this compound on various enzymes involved in phosphatidylcholine biosynthesis wasexamined. It could beshown that concentrations that produced almost complete inhibition of phosphorylcholine formation from choline, had no effect on the conversion of either phosphorylcholine into CDP-choline or of CDP-choline into phosphatidylcholine. These findings indicate that hemicholinium inhibits biosynthesis of phosphatidylcholine by preventing the phosphorylation of choline. Caz+ ions at low concentrations also inhibited the incorporation of choline into phosphatidylcholine in the reconstituted system in a similar way to its actions in the reconstituted liver system (Roberts & Bygrave, 1973) even in the presence of high concentrations of Mgz+.