Neurochemical Research, Vol. 13, No. i, 1988, pp. I-8
The Incorporation of Monomethylethanolamine and Dimethylethanolamine in Fetal Brain Aggregating Cell Culture Francine Dainous I and Julian N. Kanfer I (Accepted March 19, 1987)
Fetal rat brain aggregating cell cultures were exposed to varying concentrations of [3H]monomethylethanolamine (MME) and [3H] dimethylethanolamine (DME). The rate of labeling of water-soluble comPOunds was more rapid and the amount of radioactivity present was greater than in the lipids. After a 72 hour incubation in the presence of millimolar concentrations of these nitrogenous bases, the major water-soluble products were the phosphorylated form of the bases. Little label was associated with the free bases or their cytidyl derivate. In the phospholipids, 97% of the radioactivity was recovered in phosphatidylmonomethylethanolamine (PMME) and 3% in phosphatidyldimethylethanolamine (PDME) or 95% in PDME and 5% in phosphatidylcholine (PC) after growth in presence of [3H]MME and [3H]DME respectively. The rate of formation of the radioactive products increased as function of the concentration of the nitrogenous base added up to 4 mM, the highest concentration employed. There was no significant difference in the pattern of labeling with cells grown in media devoid of methionine or choline. The turnover of the water-soluble metabolites was more rapid than in the phospholipids where an apparent half-life of 24 hours was calculated. KEY WORDS: Aggregating nerve cell culture; monomethylethanolamine; dimethylethanolamine; phospholipid-N-methyltransferase.
INTRODUCTION The m e m b r a n e phospholipid composition of cultured mammalian cells can be modified by the inclusion in the growth medium of methylated amino alcohols which are choline analogs (1-3).
1 Department of Biochemistry, Faculty of Medicine, University of Manitoba 770 Bannatyne Avenue, Winnipeg, Canada R3E 0W3. Abbreviations: PMT, phospholipid-N-methyltransferase; AdoMet, S-adenosyl-L-methionine; EA, ethanolamine; MME, N-monomethylethanolamine; DME, N,N-dimethylethanolamine; CH, choline; PE, phosphatidylethanolamine; PMME, phosphatidylmonomethylethanolamine; PDME, phosphatidyldimethylethanolamine; PC, phosphatidylcholine; PS, phosphatidylserine; CAPS, cyclohexylaminopropane sulfonic acid.
There are alterations of the phospholipid head groups when these base analogues are present in nerve cell cultures (4, 5). It was proposed that the incorporation of the base analogues, M M E or DME, occurs by the usual de novo pathway for the synthesis of phospholipids (6, 7). H o w e v e r , these compounds can be incorporated into their corresponding phospholipids by an exchange reaction (8). Dimethylethanolamine (deanol) has been used as a pharmaceutical agent for treating tardive dyskinesia (9) and minimal brain disfunction syndrome (10) because it was suggested that deanol enhances choline and acetylcholine formation in the brain (11). Although this suggestion was disputed (12) it was shown that D M E is an efficient substitute of choline for phospholipid synthesis (13). We recently reported (14) that the inclusion of 0354-3190/88/0100-01506.00/0 9 1987 Plenum Publishing Corporation
MME and DME in the culture medium of aggregating fetal brain cells leads to the formation o f the corresponding phospholipids. These phospholipids are absent from cells grown in the unsupplemented media. Moreover these N-methylated phospholipids were effective substrates for the phospholipidN-methyltransferase activities of treated cells. In the present study we investigated the in vivo synthesis of phospholipid and water soluble phosphate ester from [3H]monomethylethanolamineand [3H]dimethylethanolamine added to brain cell cultures.
EXPERIMENTAL PROCEDURE Sprague-Dawley time-pregnant rats were obtained from Charles River Laboratories. [3H-methyl]dimethylethanolamine (28 x 106 cpm/Dxmol) and [3H-methyl]monomethylethanolamine (24.9 x 106 cprrdtxmol) were synthesized in this laboratory (8) and were radiochemically pure. MME and DME were obtained from Aldrich Chemical Co. (Milwaukee, Wisc.). Phosphatidylethanolamine, phosphatidylN-monomethylethanolamine, phosphatidyl-N,N-dimethylethanolamine, phosphatidylcholine, sphingomyelin, lysophosphatidylcholine, Dulbecco's Eagle modified medium (DMEM) cat. No. 320-1965, choline-free DMEM, methionine-free DMEM and fetal calf serum Cat. No. 240-6290 were obtained from Gibco Inc. (Burlington, Ontario, Canada). Alkaline phosphatase type III-N from E. Coli and phosphodiesterase I type II from Crotalus ademanteus were purchased from Sigma Chemical Co. (St. Louis, Missouri). Silica gel thin layer plates were from E. Merck (Darmstadt, W. Germany). Most of the routine biochemicals were obtained from Sigma Chemical Co. (St. Louis, MO.) All other reagents were of analytical grade. Culture of Fetal Rat Brain Aggregates. Cerebral hemispheres from 16 day Sprague-Dawley rat embryos were dissociated mechanically into single cells and allowed to reaggregate under constant rotation as previously described (14). Each third day 2 ml of the culture medium were removed and replaced with 2 ml of fresh growth medium. The aggregates were harvested after 18-20 days in culture. The culture medium employed was Dulbecco's modified Eagle medium (DMEM) with high glucose, no pyruvate, L-methionine at 30 mg/1, choline chloride at 4 rag/ I and containing 15% fetal calf serum. The same medium lacking choline chloride or lacking L-methionine was employed in some experiments as described. The methionine-free or choline-free medium contained 15% of fetal calf serum that had been dialized against 200 volumes of 137 mM NaC1 for 24 hours prior to use. Incorporation of [3H]MME and [3H]DME. The incorporation of these radioactive precursors was performed with cells that had been maintained for 2 weeks in culture. A 2.5 ml portion of the growth medium was removed from each culture and replaced with 1 ml of fresh medium containing [3H~MME (960 cprrd nmol) Or [3H]DME (1080 cpm/nmol) at a final concentration of 2 mM, unless otherwise indicated. The incubations were terminated by sedimenting the cells at 1,000 rpm for 5 min, and washing them three times with 9% NaC1. In some cases, the incubation medium containing the radioactive base was removed and re-
Dainous and Kanfer placed by 3 ml of fresh medium containing the corresponding non-radioactive base and the cells were maintained in culture for 1-4 days. Lipid Analysis. The cells were harvested by centrifugation, washed with 9% NaC1 and disrupted by sonication. The lipids were extracted according to the Folch partitioning procedures as previously described (14). Aliquots of the final chloroform layer were taken for the determination of the total radioactivity and for the analysis of total lipid phosphorus content by the Bartlett procedure (15). The remaining samples and appropriate phospholipid standards were applied to a precoated silica gel TLC plate. The solvent system was chloroform:methanol:28% ammonium hydroxyde (70:30:4, by vol) (16) for incubations with [3H]DME and propanol: propionic acid: chloroform:water (3:2: 2: 1, by vol) (17) for incubations with [3H]MME. Analysis of Water Soluble Compounds. The upper layer from the Folch partitioning procedure obtained after incubations with [3H]MME was evaporated to dryness under nitrogen, 1 ml of methanol was added to the residue and the tubes were sonicated for 20 s. The solvent was evaporated and the residue was extracted three times in 1 ml of ethanol. The combined ethanolic supernatants obtained after centrifugation were evaporated to dryness, the residue was suspended with 10OM of ethanol and a 50 ~1 aliquot was applied to a silica gel TLC plate using methanol:0.6% NaCl:28% ammonium hydroxyde (50:50:5, by vol) as solvent (16). MME and phosphoryl-MME chemically synthesized (6) were used as standards and were visualized by exposure to iodine vapour. The presumed CDP-MME was visualized under short wave ultraviolet light. The silica gel was removed from the plate and transferred to a scintillation vial for the determination of the radioactivity. Phosphatase and Phosphodiesterase Treatment. Aliquots from the pooled upper phases from the Folch partitioning procedures obtained after incubations with [3H]DME were taken for total radioactivity determination. The remaining samples were divided into four equal aliquots and each separately evaporated to dryness under nitrogen. Two ml of 10 mM sodium phosphate buffer pH 7.4 were added to the first aliquot which was extracted with sodium tetraphenylboron for an estimation of free base content (17). The remaining samples received two hundred p~l of 0.6M CAPS buffer pH 8.5. The second sample was incubated with 0.1 unit of phosphatase alkaline type III-N from E. Coli dissolved in 50 txl of CAPS buffer at 37~ for 2 hours in order to hydrolyze any phosphorylated base and allow for an estimate of its quantity. The third sample was incubated with 0.01 unit of phosphodiesterase I type II from Crotalus adamanteus venom dissolved in 50 ixl of CAPS buffer which should hydrolyze CDP derivatives. The fourth sample was incubated with both alkaline phosphatase and phosphodiesterase to estimate the amount of the phosphoryl and nucleotide derivatives. The reactions were stopped by cooling on ice. The volumes were adjusted to 2 ml with 20 mM sodium phosphate buffer pH %4. The extraction of nitrogenous bases from the 4 samples was based on the method of Fonnum (17). The reaction mixtures were transferred to a scintillation vial and the free [3H]DME released by the enzyme treatments was extracted with 2 ml of acetonitrile containing 5 mg/ml of sodium tetraphenylboron. Ten mt of toluene scintillation mixture containing 0.5% (w/v) of 2,5-diphenyloxazole and 0.02% (w/v) of 1,4-bis(2-(5-Phenyloxazolyl) benzene were added, The vials were shaken gently f o r a minute and the two layers allowed to separate for 10 rain before counting.
MME and DME Incorporation
The results represent the average of 3-5 independent determinations _+ SD. ~=-
Time-course of [3H]MME and [3H]DME incorporation into water-soluble products and phospholipids. Fetal rat brain aggregating cultures were grown in medium containing labeled MME for different time intervals and the radioactivity incorporated into the lipids plateaued at about 48 hours (Figure 1A). The principal labeled phospholipid was phosphatidylmonomethylethanolamine (PMME) which contained more than 80% of the total lipid bound
5 - "
l ~ \\\ \ "\
,~176 I 9 =-
t i m e (hours)
/.~/./'J 2 4
Fig. 2. Time course of [3H]DME incorporation into water-soluble and phospholipid of intact cells. Cells grown for 14 days in culture were incubated in medium containing 2 mM of [SH]DME (Specific activity 1180 cpm/nmol). The isolation and analysis of water-soluble products and phospholipids are described in Experimental Procedure. Each value represents the mean from 35 different culture vials _+ SD. (A) incorporation into lipids. (@) total lipids; ([2) PDME; (A) PC. (B) incorporation into water-soluble products. (9 total watersoluble products; (A) DME; (V) phosphoryl-DME; (11) CDPDME.
E time [hours)
Fig. 1. Time course of [3H]MME incorporation into water soluble and phospholipids of intact cells. Cells grown for 14 days in culture were incubated in medium containing 2 mM of [3HJMME (Specific activity 960 cpm/nmol). The isolation and analysis of water-soluble products and phospholipids are described in Experimental Procedure, Each value represents the mean from 3 to 5 different culture vials -z_ SD. (A) incorporation into lipids (O) total lipids; (V) PMME; (E3) PDME; (A) PC (B) incorporation into water-soluble products (o) total water-soluble products; (&) MME; (V) phosphoryl-MME; (11) CDP-MME.
radioactivity. Little label was associated with either phosphatidyldimethylethanolamine (PDME) or phosphatidylcholine (PC). The magnitude of radioactivity present in the total water-solubles (Fig. 1B) was considerably greater than that present in lipids and a plateau was reached by 24 hours. The majority of the radioactivity was present in a material chromatogramming like phosphorylmonomethylethanolamine (PhMME). There was less radioactivity associated with MME and the presumed CDP-monomethylethanolamine (CDP-MME). The appearance of radioactivity in lipids and PDME following incubation with [3H]DME was lin-
Dainous and Kanfer
ear up to 24 hours and a plateau was maintained for the next 48 hours. There was minimal labeling of PC (Figure 2A). M a x i m u m labeling of the water solubles occurred by 24 hours and there was about a 50% decrease o f the radioactivity by 72 hours. The same pattern was observed with presumed phosphoryldimethylethanolamine (Ph-DME) (Figure 2B). The a m o u n t of radioactivity present in both D M E and CDP-dimethylethanolamine was low and nearly c o n s t a n t throughout the experiment.
Effect of Varying the Concentrations of [3H]MME and [3H]DME on the Labeling of WaterSolubles and Phospholipids. The amount of radioactivity present in products in the presence of varying concentrations of [3H]MME and [3H]DME was estimated after an 8 hour incubation.
/0.125 0.5 0.050 0,25
[3H] OME (raM)
[="I OUE (ram
0.005 ] 0,25 0.5 0125
!il i 50 I
~/84 0.25 0.5 0.125
2 [3H] MME (raM)
Fig. 3, Incorporation of different concentrations of [3H]MME into water soluble and phospholipids of intact cells. Cells grown 14 days in culture were incubated for 8 hours with DMEM medium containing different concentrations of [3H]MME (Specific activity 1050cpm/nmol). Each value represents the mean -4- SD from the 3 to 4 different culture vials. (A) incorporation into lipids (I) total lipids; (V) PMME; ([~) PDME; (&) PC. (B) incorporation into water-soluble products (9 total watersotubles; (/',) MME; (T) phosphoryl-MME; (I) CDP-MME.
Fig. 4. Incorporation of [3H]DME at different concentrations into water-soluble and phospholipid fractions of intact cells. Cells grown 14 days in culture were incubated for 8 hours with DMEH medium containing different concentrations of [3H]DME (Specific activity of 1080cpm/nmol), Each value represents the mean _+ SD from 3 to 4 different culture vials. (A) incorporation into phospholipids, (O) total lipids; () PDME; (A) PC. (B) incorporation into water soluble products. (9 total watersoluble products; (A) DME; (T) phosphoryl-DME; (I)CDPDME.
The amount of radioactivity present in P M M E (Figure 3A) and P h - M M E (Figure 3B) continued to increase up to 4 mM [3H]MME, the highest concentration employed. The quantity of radioactivity in P D M E and PC (Figure 3A) was slight at 4 mM M M E suggesting that N-methyltransferase activity is relatively inactive under these conditions. There was a slight increase of the labeling of M M E and C D P - M M E with increasing base content in the medium. The appearance of [3H]DME in DME, P h - D M E and C D P - D M E (Figure 4B) was linear with respect to the concentration of [3H]DME employed from 0.050 to 1 mM and an apparent saturation for P D M E
MME and DME Incorporation
Table I. Distribution of Radioactivity F r o m [3H]MME Into Phospholipids and Water-Soluble C o m p o u n d s by Cell Aggregates Grown in Different G r o w t h M e d i u m
Total water soluble
1577 - 133 1114 - 188 1652 -+ 163
823 - 8 607 - 34 820 -4- 67
CDPPh-MME MME Total lipids PMME (nmol of [3H]MME incorporated/~zmol phospholipid)
1 Day Control Met Ch
429 • 40 314 • 14 428 +_ 6
293 - 62 268 --- 11 256 • 12
395 - 40 390 • 47 573 • 23
303 • 32 313 • 18 455 • 18
16.0 • 0.5 15.3 • 3.1 16.4 - 1.1
10.7 - 1.2 9,1 --- 1.1 11.2 - 0.7
94.0 • 12 67 -+ 15 74 - 8
101 • 12 126 - 4 144 • 21
18I • 4 204 • 9 298 • 21
125 • 18 147 -+ 10 222 - 22
6,4 • 0.1 9.0 --- 0.8 12.6 • 0.3
7 , 6 - 1.4 7.1 • 0.3 16.7 _+ 5.7
73 59 111
3.8 4.3 7.6
4.4 2.9 7,4
3.0 • 0.7 2.9 • 0.5 2.8 --- 0.4
3.2 • 0.4 2.1 - 0.7 4.1 -4- 0.1
2 Days Control Met Ch
262-+ 3 201 -+ 20 274 • 42
109 • 26 47 -+- 6 33 -+ 0.9
3 Days Control Met Ch
93 57 149
35 21 57
27.5 26.0 27.0
22.0 32,5 45.0
12.0 7.7 16.0
16.0 10.8 25.0
128 34 199
5 Days Control Met Ch
46.1 43.6 44.4
8.6 8.7 9.6
55.8 • 4.8 55.5 • 5.6 55.8 • 3.5
17.6 • 2.1 20.7 --- 3.9 24.8 • 1.9
14 d a y old cell aggregates were incubated for 24 h o u r s in their respective growth m e d i u m containing 2 m M of [3H]MME. After 1 day t h e incubation m e d i u m w a s r e m o v e d and replaced with the s a m e m e d i u m containing 2 m M of nonradioactive M M E for 4 days. Analysis o f w a t e r soluble c o m p o u n d s a n d phospholipids were as described in the text. Control = cells grown and incubated in D M E M M e t = cells g r o w n and incubated in D M E M in L - m e t h i o n i n e free m e d i u m Ch = cells g r o w n and incubated in D M E M in choline free m e d i u m E a c h value is the m e a n _+ SD o f 3 - 4 separate determinations
(Figure 4A) was reached at ! mM. The rate and amount of labeling of PC was slight with both labeled precursors. There was relatively low labeling of the presumed CDP-DME and CDP-MME. Effect of Various Growth Media on MME and DME Incorporation. After labeling with [3H]MME for 24 hours, 25% of the isotope was recovered in lipids (Table I) and most was present in PMME. In cells grown in medium devoid of choline, the labeling of lipids and PMME was 145% and 150% respectively as compared to control values. There was no significant differences in labeling in cells grown in medium devoid methionine as compared to controls. The conversion of PMME to PDME or PDME to PC was very low in the three growth medium. The radioactivity associated with MME and PhMME was reduced in cells grown in the absence of methionine, after 1 day the radioactive medium was removed and replaced by DMEM medium containing non-radioactive MME and this resulted in a rapid decline in the radioactivity present in MME and Ph-MME by 2 days. The decrease was less pronounced with the phospholipids and at 2 days, PMME was still the major product. In cells labeled with [3H]DME for 24 hours
there was no significant difference between the 3 media in the labeling of DME, Ph-DME and presumed CDP-DME of the water-solubles (Table II). In contrast, the labeling of lipids and PDME was greater in cells grown in the absence of methionine or choline as compared to control. There was very little labeling of PC suggesting that the methylation activity was relatively low. When these cells were transferred into non radioactive medium, there was a rapid decrease in the quantity of radioactivity in DME, Ph-DME and CDP-DME during the first 24 hours. The PDME radioactivity was decreased by one half in 24 hours.
DISCUSSION Deletions or supplementations of the nitrogenous bases MME and DME in tissue culture media have been employed to investigate regulatory aspects of the phospholipid-N-methylation activities (18, 19). Different laboratories (1, 7, 20, 21) showed that the addition of these nitrogenous bases to the growth medium of cell culture affects the compo-
Dainous and Kanfer
Table II. Distribution of Radioactivity From [3H]DME Into Phospholipids and Water-Soluble Compounds by Cell Aggregates Grown in Different Growth Medium
Total water soluble
396 • 31 299 -+ 25 352 _+ 4
104 _+ 15 108 _+ 12 85 • 3.1
229 • 18 216 • 14 203 -+ 8
7.3 -+ 1.1 19.9 _+ 1.6 13.8 _+ 0.7
62.3 _+ 8.9 45.1 +- 5.7 44.8 -- 4.9
1.3 +_ 0,1 4,5 -+ 1.5 3,4 _+ 0,2
5.6 + 0.7 11.4 +- 2.4 9.5 • 0.1
12.3 _+ 1.0 25.3 _+ 1.8 28.9 -+ 5.6
1.7 • 0.3 6.3 -4- 0.5 6.4 -+- 0.4
7.5 _+ 2.4 16.9 • 3.5 24.4 • 4.8
2.7 • 1.7 6.1 _+ 0.7 6.0 _+ 1.9
Ph-DME CDP-DME Total lipids (nmol of [3H]DME incorporated/~mol phospholipid)
196 _+ 30 279 _+ 15 402 _+ 13
166 _+ 24 228 ~- 3 348 _ 26
6.8 _+ 0.7 6.8 - 0.2 11,7 +_ 0.6
2,1 +_ 0.1 2.6 _+ 0.2 1.2 _+ 0.1
115 _+ 7 116 _+ 2 144 _+ 9
86 _+ 8 101 _+ 16 121 _+ 9
5.2 _+ 1,1 9.5 _+ 2,2 4.7 _+ 0.5
6.6 • 1.4 11.6 -+ 1.2 14.4 -+ 1.1
0.4 • 0.1 2.8 _+ 0.1 0.6 _+ 0.1
64 • 5.7 95 _+ 7.2 110 -4- 14
47.6 _+ 3.1 60.2 _+ 12 62.6 _+ 9
7.6 • 1.6 9.6 • 1.4 7.4 -:_ 1.6
4.4 + 0.8 11.1 -+ 3.4 10.2 • 1.05
22.5 _+ 7.7 37.5 _ 12.3 54.6 _+ 1.5
17.1 + 6.4 26.6 • 1.6 35.8 _+ 0.7
5.0 • 1.8 7.0 • 1.6 8.0 _+ 0.1
1 Day Control Met Ch
2 Days Control Met Ch
3 Days Control Met Ch
5 Days Control Met Ch
14 day old cell aggregates were incubated for 24 hours in their respective growth medium supplemented with 2 mM of [3H]DME. After 1 day the incubation medium was removed and replaced by the same medium containing 2 mM of non radioactive D M E for 4 days, Analysis of water-soluble products and phospholipid were as described in the text, Control = cells grown and incubated in DMEM Met = cells grown and incubated in L-methionine free medium Ch = cells grown and incubated in choline free medium E a c h value is the mean _+ SD of 3-4 separate determinations
sition of cellular phospholipids. MME and DME are precursors of PMME and PDME which might serve as intermediates in the endogenous synthesis of choline. We previously examined (14) the effects on cellular phospholipids by the growth of aggregating brain cell cultures in the presence of 4 mM of MME or DME for two weeks. The transport of radioactive DME was studied in nerve cell cultures prepared from rat embryos and a saturable component with an apparent Km of 28 IxM was observed (22). MME and DME were shown to inhibit competitively the high affinity choline uptake (22). The phosphorylation of DME in brain tissue was initially suggested by Ansell and Spanner (4). There is no measurable cellular pool of MME or DME since they are not usually found in nervous tissues. Therefore, the specific activity of the phospholipids and water-soluble products would be essentially identical to the initial specific activity of the labeled bases. In the present study we demonstrated the rapid conversion of both [3H]MME and [3H]DME into their corresponding water-soluble phosphate esters but the amount of unmodified bases remained rather
low (Figure 1B and 2B). This indicates that these compounds are rapidly phosphorylated after they enter the cells. Phosphoryl-MME and phosphorylDME formation were maximum after 24 hours. The decrease of labeled phosphoryl-bases between 24 and 72 hours associated with the increase in the labeling of the corresponding phospholipids suggests that the phosphoryl-bases are precursors of their corresponding phospholipids. It is possible that these phosphorylated bases have a storage function. The rate of incorporation of radioactivity from either [3H]MME or [3H]DME into phospholipids was slower than the incorporation into water-soluble products (Figures 1 and 2) and PMME and PDME respectively were the major labeled phospholipid representing 80-85% of the lipid-bound radioactivity. The phospholipids possessing one or two additional methyl group(s) had relatively little label, indicating the very low conversion by the methyltransferases. The growth of cells in a methionine free medium although affecting the size and number of aggregates did not result in a significant difference of the labeling of products compared to cell growth in methionine-supplemented medium. Methionine is
MME and DME Incorporation an essential amino acid present at a concentration of 0.2 mM in the culture medium. The reduction of extracellular methionine might reduce the levels of S-adenosylmethionine (SAM), the donor of methyl groups for transmethylation reactions. A stimulation of phospholipid methylation with increased methionine concentrations in the growth medium was reported in nerve cell culture (7) and rat diaphragm preparations (23). The growth of cells in a choline-free medium resulted in a significant increase of labeling of phospholipids after incubation with [3H]MME but the percentage of methylated phospholipids remained the same as compared to controls. In vivo choline deficiency has been used to investigate the regulation of phospholipid-N-methylation activities. The results have been controversial, since one group reported reductions (24) and another reported increased levels of activities (25). We observed (14) that PDME and PMME were present after growth of the aggregating brain cultures in the presence of the corresponding bases. The availability of the radioactive bases [3H]MME and [3H]DME (8), which had not been previously available allowed us to attempt an examination of the pathway utilized for the incorporation of these bases. The laboratory of Ansell (26) suggested that the labeled DME of unknown purity injected into rat brains was converted into PDME via the de novo pathway. In vitro support for this possibility was obtained in rat brain dispersion (6). Previous results obtained with rat brain microsomes (27) indicated that the incorporation of these bases into their corresponding phospholipids could occur by a base exchange reaction and a possible linkage between the products of the base-exchange reaction becoming substrates for the phospholipid-N-methyltransferase activities. The present data cannot unequivocally discriminate between an incorporation of the radioactive bases MME and DME into their corresponding phospholipids by a base exchange pathway or the de novo biosynthetic pathway. However, it is apparent that the amount of conversion of PMME and PDME by transmethylation mechanism is very low.
ACKNOWLEDGMENT This work was supported by a grant from the Medical Research Council of Canada.
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