Preferential synthesis of diacyl and alkenylacyl ethanolamine and choline glycerophospholipids in rabbit platelet membranes.

In rabbit platelet membranes, the contents of alkenylacyl phospholipids (plasmalogen) were 56% of phosphatidylethanolamine and 3% of phosphatidylcholine. This uneven distribution of plasmalogens in each phospholipid class could be attributed to the different substrate specificity of ethanolaminephosphotransferase (EC 2.7.8.1) and cholinephosphotransferase (EC 2.7.8.2). The properties of the enzymes were studied, using endogenous diglycerides and CDP-[3H]ethanolamine or CDP-[14C]choline as substrates. The newly formed phospholipids were mainly diacyl and alkenylacyl and only rarely alkylacyl type. The ratios of the labeled alkenylacyl to diacyl type of phospholipids clearly varied with the concentrations of CDP-ethanolamine or CDP-choline. When 1, 10, and 30 microM CDP-[3H]ethanolamine were used, the labeled phospholipids contained 53, 37, and 27% of the alkenylacyl type, respectively. The apparent Km for CDP-ethanolamine to synthesize alkenylacyl and diacyl types were 2.2 and 8.1 microM. On the other hand, when 1, 10, and 30 microM CDP-[14C]choline were used, the labeled lipids contained 10, 17, and 24% alkenylacyl type, respectively. The apparent Km for CDP-choline to synthesize alkenylacyl and diacyl types were 24 and 4.3 microM. Further, the syntheses of diacyl type of phosphatidylethanolamine and the alkenylacyl type of phosphatidylcholine were markedly inhibited by unlabeled CDP-choline and CDP-ethanolamine, respectively. The two enzymes had opposite substrate specificities, and ethanolaminephosphotransferase showed a high preference to plasmalogen synthesis, especially in the presence of CDP-choline.

intestine (lo), fat cells (ll), lung (12), and platelets (13). Although in most mammalian tissues the linkage to the 2position of the glycerol in glycerophospholipids is only the acyl type, three linkage types are known to be acyl, alkyl, and alkenyl bonds, in the 1-position. These three types of glycerophospholipids have different distributions of phosphatidylethanolamine and phosphatidylcholine in various mammalian tissues including platelets (14), neutrophils (14,15), brain, kidney, lung, and testis (16).
In platelets, the alkenylacyl type of phospholipids (plasmalogen) is found dominantly in phosphatidylethanolamine and scarcely in phosphatidylcholine (14,17). Alkenylacylglycerophosphoethanolamine (alkenylacyl-GPE2) contains a large amount of arachidonic acid in the 2-position of glycerol and may play an important role as a major source for arachidonic acid releasing during platelet activation (18).
In an attempt to explain the uneven distribution of alkenylacyl phospholipids in phosphatidylethanolamine and phosphatidylcholine, the activities of both transferases in the syntheses of alkenylacyl and diacyl type of phospholipids in rabbit platelet membranes were examined, using endogenous diglycerides and labeled CDP-ethanolamine or CDP-choline as substrates. We found that the transferases have different substrate specificities for diglycerides and different K,,, values for CDP-bases to synthesize the alkenylacyl and diacyl type of phosphatidylethanolamine or phosphatidylcholine.

Plasmalogen Phospholipids in Rabbit Platelets
column was washed with 1.2 ml of distilled water, and then phosphoryl-[l-3H]ethanolamine was eluted with 4 ml of distilled water. The fractions containing labeled phosphorylethanolamine were lyophilized. The labeled phosphorylethanolamine was utilized for the synthesis of labeled CDP-ethanolamine, using partially purified phosphorylethanolamine cytidyltransferase. The reaction mixture (2 ml) contained 50 mM Tris acetate (pH 7.5), 3 mM CTP, 5 mM MgCl,, 0.45 mCi of phosphoryl[l-3H]ethanolamine, and phosphorylethanolamine cytidyltransferase (6.5 mg). After a 1-h incubation at 37 "C, the mixture was directly applied on the column (0.7 X 11 cm, AG1-X8, formate form). After the unreacted phosphorylethanolamine had been washed out with 5 mM formic acid, CDP-ethanolamine was eluted with 8 ml of 40 mM formic acid and lyophilized. To completely remove the contaminant CMP and unreacted phosphorylethanolamine, labeled CDP-ethanolamine was further purified by polyethyleneimine cellulose (solvent system, 50 mM formic acid) followed by cellulose thin layer chromatography (solvent system, n-butyl alcohobacetic acidHzO, 5:2:3, v/v) (13). The specific activity of the thusobtained CDP-[l-3H]ethanolamine was 2900 cpm/pmol, as determined from the amount of CDP-ethanolamine (the absorption of CDP-ethanolamine at 280 nm) and the radioactivity (determined by liquid scintillation spectrometer).
Preparation of Rabbit Platelet Membranes-Washed rabbit platelets and platelet membranes were prepared as described (21). The 105,000 X g pellet was washed in 0.25 M sucrose containing 1 mM EGTA and 10 mM 2-mercaptoethanol, using a Teflon-glass homogenizer, and was centrifuged at 105,000 X g for 60 min. Finally, the sediments were suspended in the same sucrose solution and stored at -80 "C until use. The activities of the enzymes were well preserved for at least 2 months. The membrane protein was determined according to Lowry et al. (22), using bovine serum albumin as a standard.
Assay of Ethanoluminephosphotransferase and Cholinephosphotransferase-The standard reaction mixture (final volume, 0.2 ml) contained 50 mM HEPES buffer (pH 7.5), 5 mM MgC12, 0.25 mM EGTA, various concentrations (0.5-50 pM) of radioactive CDP-ethanolamine or CDP-choline, and 150 pg of platelet membrane proteins. The incubations were carried out for 5 min at 37 "C. Reactions were terminated by the addition of 3 ml of ice-cold ch1oroform:methanol (1:2) and 0.6 ml of H20. Phospholipids were extracted according to Bligh and Dyer (23). The chloroform layer was partitioned by adding 1 ml each of chloroform and 1% KC1. After washing with 1 ml of 50% methanol containing 0.5% KC1 and further with 1 ml of 60% methanol, the chloroform layer was carefully removed. An aliquot was dried at 80 "C, and radioactivity was measured in 10 ml of scintillation mixture using a liquid scintillation spectrometer. The radiolabeled phospholipids were separated by two-dimensional thin-layer chromatography on silica gel plates and identified by their co-migration with authentic standards. The solvent for the first dimension was ch1oroform:methanol:acetic acid (65:25:10) and the second was chloroform:methanob88% formic acid (65:25:10) (24). The radioactivity (96-98%) in the chloroform extracts migrated as phosphatidylethanolamine or phosphatidylcholine.
To determine subclasses of glycerophospholipids (diacyl-, alkenylacyl-, and alkylacyl-GPE or -GPC), two systems were used. The first system was stepwise alkaline and acid methanolysis described by Pries et al. (25). After the chloroform layer was evaporated under a stream of N2, samples were treated with 1 ml of 0.1 M methanolic KOH for 30 min at 40 "C. Excess alkali was then neutralized with ethylformate for 5 min at 40 "C. After the partition with a Folch mixture, the upper layer was withdrawn and the lower layer washed with fresh upper phase. The radioactivity in the combined upper aqueous layer was calculated as diacyl-GPE or -GPC. The lower organic layer was dried under reduced pressure and treated with 1 M HCl in 50% aqueous methanol for 30 min at 40 "C. After the partition with a Folch mixture, the upper layer was withdrawn and the lower layer was washed with fresh upper phase. The radioactivity in the combined upper layer was calculated as alkenylacyl-GPE or -GPC. The radioactivity in the lower layer was as alkylacyl-GPE or -GPC. The water-soluble deacylated radiolabeled products of both alkaline and acid hydrolysis migrated to the same position as sn-glycero-3phosphorylethanolamine (Rf, 0.37) and sn-glycero-3-phosphorylcholine (Rf, 0.31) on silica gel thin-layer chromatography using as a solvent system n-butyl a1cohol:pyridine:HzO (1:l:l). The second system was analysis on two-dimensional thin layer chromatography with HCl fume treatment (26). The thin layer plate was developed with chloroform methanol, 15 N NH,OH (65254). The plate was dried and exposed to the fumes from 12 N HCl for 10 min. It was dried again and developed in the second direction with chloroform, meth-anol, 15 N NH,OH (100:50:12). Each spot was visualized in I, vapor and scraped for counting of radioactivity or for phosphorus analysis (27).
The determinations of alkenylacyl phospholipid by the two methods were within 5% limits. The radioactivities in alkylacyl phospholipids, which were kept in the organic phase after the stepwise methanolysis, were less than 3 % of the total labeled phosphatidylethanolamine and less than 2% of the total labeled phosphatidylcholine under our assay conditions (0.5-50 p~ CDP-ethanolamine or CDPcholine, and incubation for 5-20 min). Therefore, the thin layer chromatography system was routinely used for separation of alkenylacyl phospholipids, and diacyl plus alkylacyl fractions were regarded as diacyl phospholipids.

Contents of Plasmalogen in Phosphatidylethanolamine and
Phosphatidylcholine of Rabbit Platelet Membranes-The phospholipids extracted from the platelet membrane preparation were separated by two-dimensional thin layer chromatography with HCl fume treatment. As indicated in Table   I, the distribution of alkenylacyl glycerophospholipids in platelet membranes was totally different and the contents of the alkenylacyl type in each phospholipid class were 56.4 and 3.3%, respectively, by phosphorus analysis. These values are not identical to but are essentially similar to reported data (14, 17).
pH Dependence of Ethanolaminephosphotransferase and Cholinephosphotransferase Activities-The activities of the transferases were examined at various pH, using HEPES and glycine buffer systems. As shown in Fig. 1, the pH dependence of the two enzymes was slightly different. The optimal pH of ethanolaminephosphotransferase was around 7.5 and that of cholinephosphotransferase was around 7.0. However, the optimal pH for the alkenylacyl and diacyl types of phospholipid synthesis was much the same in each enzyme, and the ratios of alkenylacyl and diacyl phospholipid syntheses were fairly constant at various pH values. in the alkenylacyl-GPC. At higher concentrations (10 and 30 p~) of CDP-choline, the percentage of alkenylacyl-GPC formation was increased to 15 and 24%, respectively. Mg2+ and Mn2+ are effective metal cofactors for both ethanolaminephosphotransferase and cholinephosphotransferase (1). Therefore, kinetic analyses of both transferases were carried out using Mg2' and Mn2+ as metal cofactors. As shown in Fig. 3, A and C, the apparent K,,, values for CDP-ethanolamine to synthesize alkenylacyl-GPE were clearly lower than the values to synthesize diacyl-GPE, with both Mg2+ and Mn2+. The apparent K,,, values for CDP-ethanolamine to synthesize alkenylacyl-GPE and diacyl-GPE were 2.2 and 8.1 pM with M$+ and 1.0 and 4.5 pM with Mn2+, respectively.

Effects of the Concentrations of CDP-Ethanolamine or CDP-Choline on Alkenylacyl and Diacyl Phospholipids Syntheses
Whereas, as shown in Fig. 2, B and D, the apparent K,,

Effects of CDP-Choline and CDP-Ethanolamine on Other
Transferases-The effects of CDP-bases on other transferase activities to produce alkenylacyl and diacyl types of phospholipids were examined. As shown in Fig. 4, the formations of alkenylacyl-GPE and diacyl-GPC with Mn2+ were slightly stimulated by 10 p~ CDP-choline and CDP-ethanolamine, respectively, and were inhibited by higher concentrations of the CDP-bases. In case of M$+, these stimulatory effects were remarkable. In contrast, the formations of diacyl-GPE and alkenylacyl-GPC were inhibited, dose dependently, by CDP-choline and CDP-ethanolamine, respectively, with both Mn2+ and M e . Kinetic analyses of the inhibitory effects on the formation of diacyl-GPE and alkenylacyl-GPC are shown in Fig. 5. The profiles of inhibitory effects were noncompetitive.

DISCUSSION
To investigate enzymic properties of ethanolaminephosphotransferase and cholinephosphotransferase diglycerides were added exogenously to the assay mixtures as lipid acceptors in most of the previous reports. Three analogous diglycerides, diacyl, alkenylacyl, and alkylacyl glycerol, were demonstrated to be the substrates for ethanolaminephosphotransferase and cholinephosphotransferase (3, 7, 8, 13). Although the enzyme activities were fairly low, some workers found in various tissues, including platelets, significant activities of the transferases to form diacyl, alkenylacyl, or alkylacyl phospholipids, detected without addition of exogenous diglycerides  (12,13,28,29). Addition of diglycerides with some detergents may alter the enzyme conformation and lipid environments, and the choice of detergent and its concentration is most critical for enzyme activities (5, 10,29). Therefore, in order to clarify which endogenous diglyceride, diacyl, alkenylacyl, or alkylacyl glycerol, is utilized as the substrate for ethanolaminephosphotransferase and cholinephosphotransferase in rabbit platelet membranes, diglycerides or detergents were not added to the assay mixture in the present study.

Plasmalogen Phospholipids in Rabbit Platelets
Ansell and Metcalfe (7) reported that with brain microsomes, K , for CDP-ethanolamine in the presence of diacyl glycerol (260 p~) was almost the same as that in the presence of alkenylacyl glycerol (220 p M ) . Strosznajder et al. (8) also reported that in brain synaptosomes, K , values for CDPethanolamine and CDP-choline to synthesize diacyl-GPE, alkenylacyl-GPE, diacyl-GPC, and alkenylacyl-GPC were 290, 270, 350, and 200 pM, respectively, in the presence of corresponding diglycerides. In human platelet homogenates, K , for CDP-ethanolamine was 160 p~, with the addition of diacyl glycerol (13). All these values are much higher than those determined in the present study. However, since the concentrations of these endogenous diglycerides were not determined and those in isolated membranes might be insufficient for both transferase activities, the K , values for CDPethanolamine and CDP-choline determined in this study were apparent K,,, values.
The addition of CDP-choline or CDP-ethanolamine to other phosphotransferase assay mixtures demonstrated the preferential synthesis of alkenylacyl and diacyl type of phospholipids (Fig. 4). It has been reported that CDP-choline and CDP-ethanolamine inhibit competitively other transferases in rat liver (5) and Plasmodium krwwlesi-infected erythrocytes (30). In fat cells, the inhibitory effect of CDP-choline on ethanolaminephosphotransferase was noncompetitive (11). In those reports, the enzyme activities were examined with exogenously added diglycerides. In the present study, diacyl-GPE and alkenylacyl-GPC formations were noncompetitively inhibited by CDP-choline and CDP-ethanolamine, respectively. Surprisingly, alkenylacyl-GPE and diacyl-GPC formations were stimulated by the other CDP-bases, and the stimulation was remarkable when Mg2+ was used as a cofactor. Although the mechanisms involved are not clear, at least with the co-existence of CDP-ethanolamine and CDP-choline, endogenous alkenylacyl and diacyl glycerol were more preferentially utilized for the syntheses of phosphatidylethanolamine and phosphatidylcholine, respectively. It is possible that the ratio of the two glycerol derivatives in the microenvironment of the transferases as well as the different affinity of two glycerol derivatives for the enzymes determines the ratio of the products. In rat liver, Kanoh and Ohno (5) demonstrated not only that ethanolaminephosphotransferase and cholinephosphotransferase were different enzymes but also that cholinephosphotransferase was partially separated into M$+-requiring and Mn2+-requiring components. Therefore, the different ratio of the two products (diacyl to alkenylacyl phospholipids), found using different metal cofactors with or without the co-existence of CDP-ethanolamine and CDPcholine, may be attributed to two or more different transferases in rabbit platelet membranes.
In many mammalian tissues (16), including platelets (17), alkenylacyl phospholipids are found mainly in phosphatidylethanolamine. Alkenylacyl-GPE is postulated to be synthesized by desaturation of alkylacyl-GPE, the immediate precursor (31). Both transferases can catalyze the synthesis of alkenylacyl phospholipids from alkenylacyl glycerol, with CDP-ethanolamine or CDP-choline (3, 7). In rat liver, the two transferases were demonstrated to have different specificities for diglycerides depending on the fatty acid side chains not only at the 2-, but also at the 1-position of the glycerol (4, 6). However, it has not been demonstrated that the enzymes have different specificities for alkenylacyl and diacyl glycerols in mammalian tissue. Recently, Smith (28) demonstrated the different selectivity of the phosphotransferases of tetrahymena for diacyl glycerol and alkylacyl glycerol and suggested that this selectivity accounted for the high content of alkylacyl-GPC in tetrahymena. Although the content of alkylacyl-GPC has been reported to constitute more or less the same percent as that of alkenylacyl-GPE in platelet membranes (13,14), the incorporation of ['4C]choline into alkylacyl-GPC was found only slightly under our assay conditions. This discrepancy is not clear now, but the presence of endogenous alkylacyl glycerol in rabbit platelet membranes may be very low as compared to alkenylacyl glycerol. More experiments are needed to clarify whether or not the uneven distribution of alkylacyl phospholipid in phosphatidylethanolamine and phosphatidylcholine is attributed to the different enzymic properties of ethanolaminephosphotransferase and cholinephosphotransferase.
It is not known whether alkenylacyl glycerol is formed by de nouo pathways in platelets. Possible sources of diglycerides are de m u o synthesis, degradation of pre-existing phospholipids by phospholipase C, phosphatidic acid phosphatase, or reverse reactions of ethanolaminephosphotransferase and cholinephosphotransferase. If diglycerides are supplied from degradation of phospholipids, these phosphotransferases do not catalyze the net synthesis of phospholipids. However, as described above, ethanolaminephosphotransferase preferentially synthesized the alkenylacyl type and cholinephosphotransferase preferentially synthesized the diacyl type, in particular with the co-existence of CDP-choline and CDP-ethanolamine. In the rabbit platelet membrane preparations used, the content of alkenylacyl type was 56% of the phosphatidylethanolamine and was only 3% of phosphatidylcholine. The preference of the enzymes no doubt plays some important role in regulating the uneven alkenylacyl phospholipid distribution in rabbit platelets.