390

Synthesis of 6-PhosphatidyI-L-Ascorbic Acid by Phospholipase D Akihiko N a g a o * , Nobuaki Ishida a n d Junji Terao National Food Research Institute, Ministryof Agriculture, Forestryand Fisheries,Tsukuba, Ibaraki, 305 Japan P h o s p h o l i p a s e D (EC 3.1.4.4) of Streptomyces s p e c i e s w a s found to catalyze t r a n s p h o s p h a t i d y l a t i o n to Lascorbic acid from p h o s p h a t i d y l c h o l i n e ( P C ) in a biphasic reaction system. The product w a s i d e n t i f i e d as 1,2-diacyl-sn-glycero-3-phospho-6'-L-ascorbic acid (PA-AsA) by m a s s s p e c t r o m e t r y and nuclear m a g n e t i c r e s o n a n c e spectroscopy. The optimal pH o f t r a n s p h o s phatidylation w a s 4.5 and the rate o f PA-AsA formation i n c r e a s e d as c o n c e n t r a t i o n s o f L-ascorbic acid increased. The c o n v e r s i o n o f PC to PA-AsA w a s greater than 80%. PA-AsA w a s found to be more r e s i s t a n t to hydrolysis by p h o s p h o l i p a s e D than w a s PC.

Lipids 26, 390-394 11991). Phospholipase D (PLD) catalyzes the hydrolysis of the terminal p h o s p h o e s t e r bond of phospholipids a n d can bring a b o u t t r a n s p h o s p h a t i d y l a t i o n (1,2). By using the t r a n s p h o s p h a t i d y l a t i o n reaction with PLD, the head group of phospholipids can be modified easily. Thus, phosphatidylglycerol can be p r o d u c e d from phosphatidylcholine and glycerol by catalysis with PLD (3,4). The t r a n s p h o s p h a t i d y l a t i o n technique also has been shown to be useful for p r e p a r i n g phosphatidylserine and plasmenylethanolamine (5-10). In addition, novel types of phospholipids with various physiological or t h e r a p e u t i c activities can be synthesized by catalysis with PLD. Thus, Shuto et al. (11) synthesized phosphatidylnucleosides with antileukemic activity, and Brachwitz et al. (12) p r e p a r e d O-alkylglycerophospho-L-serine with cytostatic activity. We have been working on the cellular defense system against oxygen toxicity, and especially against m e m b r a n e lipid peroxidation (13-15). The defense against active oxygen radicals at the interface between m e m b r a n e lipids and the aqueous p h a s e is of considerable importance because the a t t a c k of active oxygen radicals originating in the aqueous p h a s e is believed to be one of the initiation steps of m e m b r a n e lipid p e r o x i d a t i o n (16,17). To prevent active oxygen radicals f r o m attacking m e m b r a n e lipids, it seems effective to arrange an a p p r o p r i a t e antioxidant on the surface of a m e m b r a n e . One possible a p p r o a c h would be to substitute the polar head group of a m e m b r a n e phospholipid with a functional group having antioxidant activity. L-Ascorbic acid, one of the import a n t antioxidants in the cellular defense against active oxygen radicals (18), is an excellent candidate for a polar head group substitution t h a t would p r o d u c e phospholipid with antioxidant activity. Moreover, L-ascorbic acid has a p r i m a r y h y d r o x y group available for t r a n s p h o s phatidylation by PLD. In this paper, the synthesis of phosphatidyl-L-ascorbic acid by phospholipase D is described. *To whom correspondence should be addressed. Abbreviations: COSY, correlation spectroscopy;, FAB, fast atom bombardment; HPLC,high performance liquid chromatography; MS, mass spectrometry;, NMR, nuclear magnetic resonance spectroscopy; PA, phosphatidic acid; PA-AsA, 6-phosphatidyl L-ascorbic acid; PC, phosphatidylcholine; PLD, phospholipase D; TLC, thinlayer chromatography. LIPIDS,Vol. 26, No. 5 (199'1)

MATERIALS AND METHODS Phospholipase D f r o m Streptomyces lydicus (78.3 U / m g protein) was obtained from Honen Co. (Yokohama, J a p a n ) . The two PLD p r e p a r a t i o n s from Streptomyces species (1130 and 391 U / m g protein) and PLD from cabbage (0.67 U / m g protein) were p u r c h a s e d from Toyo Jozo Co. (Tokyo, J a p a n ) , Sigma Chemical Co. (St. Louis, MO) a n d Boehringer Mannheim GmbH (Mannheim, Germany), respectively. Phosphatidylcholine (PC) from egg yolk (99% purity) and dimyristoyl PC were p u r c h a s e d from Sigma. Thin-layer c h r o m a t o g r a p h y (TLC) plates (Art. 5721) were p u r c h a s e d from Merck ( D a r m s t a d t , Germany). Other chemicals were reagent grade. Enzyme reaction. The enzyme reaction was carried out in a biphasic system consisting of a diethyl ether and an aqueous p h a s e using an 8-mL vial. The s t a n d a r d reaction m i x t u r e contained 1 mL of 10 mM egg yolk PC in diethyl ether, 0.8 mL of 1 M L-ascorbic acid, 0.05 mL of the PLD solution which contained 90 U of PLD from S. lydicus in 1 mL of 10 mM sodium acetate buffer, pH 5.1, and 0.15 mL of deionized water. The pH of 1 M L-ascorbic acid was adjusted to 4.5 with KOH. The enzyme reaction was initiated by the addition of the enzyme a n d the m i x t u r e was shaken (120 r p m ) at 37~ The reaction was s t o p p e d by adding 0.2 mL of 6 N HC1. The p r o d u c t was e x t r a c t e d three times f r o m the reaction m i x t u r e with 4 mL of c h l o r o f o r m / m e t h a n o l (2:1, v/v). The combined chloroform layer was w a s h e d with 5 mL of deionized w a t e r and e v a p o r a t e d in a r o t a r y evaporator. The e x t r a c t was dissolved in 5 mL of n - h e x a n e / i s o p r o p a n o l (4:1, v / v ) and then centrifuged. The s u p e r n a t a n t was analyzed by TLC and high p e r f o r m a n c e liquid c h r o m a t o g r a p h y (HPLC). PLD units were d e t e r m i n e d by the m e t h o d of Kato et al. (19). The hydrolytic activity of the PLD with PC or 6phosphatidyl-L-ascorbic acid (PA-AsA) as s u b s t r a t e was determined by measuring the a m o u n t of phosphatidic acid (PA) formed. The reaction m i x t u r e contained 5 #mol of egg yolk PC or PA-AsA p r e p a r e d from egg yolk PC in 1 mL of diethyl ether and 4.5 U of the PLD ofS. lydicus in 1 mL of 0.1 M p o t a s s i u m citrate buffer, pH 6.0. The reaction was s t o p p e d by adding 0.2 mL of 6 N HC1, and PA was e x t r a c t e d as described above and analyzed by HPLC. TLC analysis. The lipid e x t r a c t of the reaction m i x t u r e was subjected to thin-layer c h r o m a t o g r a p h y using the developing solvent c h l o r o f o r m / m e t h a n o l / w a t e r (65:35:5, by vol). Phospholipids were visualized with Dittmer's reagent or iodine vapor. Substances with reducing activity were detected using ferric ion reagent (20). HPLC analysis. HPLC was p e r f o r m e d on a Shimadzu (Shimadzu Co., Kyoto, J a p a n ) LC-5A equipped with a Rheodyne 7125 m a n u a l loop injector (20 #L), a Shimadzu SPD-2A variable wavelength detector (AUFS 0.01) a n d a Shimadzu C-R3A d a t a processor. PA-AsA was analyzed on a silica gel column (YMC-Pack SIL-06, 4.6 • 150 mm, YMC Co., Kyoto, J a p a n ) with n-hexane/isopropanol/10% phosphoric acid (85:15:0.4, by vol) serving as eluent at a flow r a t e of 2 mL/min. The column t e m p e r a t u r e was maintained at 40~ for the analysis of PA-AsA. The retention time of PA-AsA was 3.3 min. PC was analyzed on

391

SYNTHESIS OF 6-PHOSPHATIDYL-L-ASCORBICACID t h e s a m e c o l u m n w i t h n-hexane/isopropanol/10% p h o s p h o r i c a c i d (20:30:5, b y vol) s e r v i n g as e l u e n t a t a flow r a t e of 1.2 m L / m i n . The r e t e n t i o n t i m e o f PC w a s 3.7 min. P A w a s a n a l y z e d on SHIM-PACK FLC-NH2 c o l u m n (4.6 X 50 mm, S h i m a d z u ) w i t h n-hexane/isopropanol/lO% p h o s p h o r i c a c i d (80:20:0.5, b y vol) s e r v i n g as e l u e n t a t a flow r a t e o f 2 m L / m i n . T h e r e t e n t i o n t i m e o f P A w a s 2.0 min. PA-AsA, PC a n d P A w e r e m o n i t o r e d a t 245, 206 a n d 206 nm, respectively. S a m p l e w a s i n j e c t e d using a s a m p l e l o o p o f 20 ~L. Spectral analyses. I n f r a r e d s p e c t r a w e r e m e a s u r e d on p o t a s s i u m b r o m i d e p e l l e t s u s i n g a S h i m a d z u FTIR-4200 i n f r a r e d s p e c t r o p h o t o m e t e r . Mass s p e c t r a w e r e m e a s u r e d in t h e n e g a t i v e f a s t a t o m b o m b a r d m e n t ( F A B ) mode with a JEOL (Japan Electronic Optics Laboratory Co., Tokyo, J a p a n ) JMS-HX 100. T h e FAB g u n w a s o p e r a t e d a t 6 KV; x e n o n a t o m s w e r e u s e d to ionize t h e s a m p l e in t h e d e s o r p t i o n m o d e f r o m a t r i e t h a n o l a m i n e m a t r i x . 1H a n d 13C n u c l e a r m a g n e t i c r e s o n a n c e (NMR) s p e c t r a w e r e m e a s u r e d w i t h a J E O L GSX-270 F r NMR s p e c t r o m e t e r a t 270 a n d 67.9 MHz, r e s p e c t i v e l y . S a m p l e s w e r e d i s s o l v e d in C D C l n / C D 3 0 D (2:1, v / v ) ; t e t r a m e t h y l s i l a n e w a s u s e d as i n t e r n a l s t a n d a r d .

w a s 2 M. A c o n v e r s i o n of 93.6% w a s a t t a i n e d a f t e r 24 hr. P A - A s A w a s p u r i f i e d on a silicic a c i d c o l u m n using t h e s a m e s o l v e n t m i x t u r e as for TLC; t h e i s o l a t e d yield o f P A - A s A w a s 54.2% r e l a t i v e to PC used. The i n f r a r e d spectrum of the purified PA-AsA indicated the presence o f a p h o s p h a t e e s t e r a n d of t h e d o u b l e b o n d o f L - a s c o r b i c a c i d (KBr, c m 1:1738 Uc=o, i 6 0 0 Vc=c, 1231 PP=O, 1109 vFoc). P A - A s A s h o w e d t h e e x p e c t e d UV s p e c t r u m for t h e L - a s c o r b i c a c i d moiety. The FAB m a s s s p e c t r u m of PAA s A s h o w e d a [M+Na-2H- ion a t m/z 771, a [M-H]- ion a t m/z 749, a c a r b o x y l a t e a n i o n a t m/z 227, a n d a p h o s p h a t i d a t e a n i o n a t m/z 591 (Fig. 1). 13C NMR c h e m i c a l shift a s s i g n m e n t s for P A - A s A in CDC13/CD3OD (2:1, v / v ) w e r e m a d e b a s e d on t h e c h e m i -

RESULTS

A n e w p r o d u c t d e r i v e d f r o m egg y o l k PC a n d L - a s c o r b i c a c i d w a s p r o p o s e d b y c a t a l y s i s w i t h PLD ofS. lydicus. The p r o d u c t (Rf 0.25) w a s s e p a r a t e d f r o m PC (Rf 0.35) a n d P A (Rf 0.41) b y TLC. The f u n c t i o n gave a b l u e s p o t w i t h D i t t m e r ' s r e a g e n t a n d a r e d s p o t w i t h ferric ion r e a g e n t indicating the presence of a phospholipid with reducing activity. The p h o s p h o l i p i d w a s t e n t a t i v e l y i d e n t i f i e d a s phosphatidyl-L-ascorbic acid (PA-AsA) having an ester l i n k a g e b e t w e e n e i t h e r t h e 5- o r 6 - h y d r o x y g r o u p o f La s c o r b i c a c i d a n d t h e p h o s p h a t i d y l moiety. The structure of PA-AsA was confirmed by spectral a n a l y s i s o f d i m y r i s t o y l PA-AsA. P A - A s A w a s s y n t h e s i z e d f r o m 1 , 2 - d i m y r i s t o y l - s n - g l y c e r o - 3 - p h o s p h o c h o l i n e a n d La s c o r b i c a c i d u s i n g t h e S a m e p r o c e d u r e a s d e s c r i b e d in Materials and Methods, except that the concentration of L - a s c o r b i c a c i d in t h e a q u e o u s p h a s e o f r e a c t i o n m i x t u r e

TABLE 1 13C NMR C h e m i c a l Shifts of PA-AsA, PC and L-Ascorbic Acid

PA-AsA Dimyristoyl glycerol moiety CHzO 62.31 CHO 70.04 a CH2OP 65"288 CO 174.21 173.80 CH2 34.43-22.93 CH3 14.19 Choline moiety CH2OP CH2N (CH3)3N L-Ascorbic acid moiety C-1 C-2 C-3 C-4 C-5 C-6

Dimyristoyl PC

L-Ascorbic acid

63.01 70.74 a 63.93 b 174.30 173.93 34:55-22.96 14.21 59.34 b 66.79 54.39 172.43 119.05 153.45 76.40 70.15 62.96

172.03 119.15 152.62 75.67 68.12 a 67.34 b

a3JcP = 8.25 HZ. b2Jcp : 5.50 HZ.

100 148

227

80 749 .I I/I r

C o~

60'

40.

._> 20

o

297

loo

20o

591 [

367

LJ L ~o

z.oo mlz

'"

~o

~

~

'16o

'1"

BGo

FIG. 1. N e g a t i v e ion FAB m a s s s p e c t r u m of dimyristoyl PA-AsA.

LIPIDS,Vol. 26, No. 5 (1991)

392

A. NAGAOETAL.

-5 4 E v

"o 3 o.

'-;0' .........'.........'~....

1

o .c 2 o.

o ,

.

.

.

"

. 8 o. .

",.o

'

'

";

PPM FIG. 2. 13C N M R s p e c t r u m o f d i m y r i s t o y l PA-AsA.

cal shifts of dimyristoyl PC and L-ascorbic acid in the same solvent (Table 1). The z3C chemical shift assignments for dimyristoyl PC were consistent with those reported for dioctanoyl PC (e.g., ref. 21). The assignments for L-ascorbic acid were also based on literature values (22). The 13C chemical shift assignments for PA-AsA were confirmed by DQF correlation spectroscopy (COSY) and 1H-13C COSY experiments. The 13C chemical shifts of the dimyristoyl glycerol moiety of PA-AsA essentially resembled those of dimyristoyl PC. The 13C chemical shifts of the L-ascorbic acid moiety of PA-AsA were essentially those of L-ascorbic acid, except for the C-5 and C-6 shifts. The C-6 signal of PA-AsA showed a significant downfield shift relative to that of L-ascorbic acid, while the C-5 signal was slightly shifted upfield. This 13C chemical shift pattern for carbons proximal to phosphate were previously observed for other phosphoesters such as 5'-(3sn-phosphatidyl) nucleosides (11) and L-ascorbic acid 6phosphate (23). Moreover, the 13C-31pcouplings observed (Fig, 2) for C-6(J=5.5Hz) were smaller than those for

lO(1 v

5o f

4

6

8

pH FIG. 3. E f f e c t o f pH on t h e s y n t h e s i s o f P A - A s A a n d on t h e h y d r o l y s i s to PA. T h e r e a c t i o n w a s d o n e a s d e s c r i b e d in Materials and Methods, except that the pH was varied. The pH of the a q u e o u s m e d i u m w a s a d j u s t e d t o 3.0, 4.0, 4.5, 5.0 a n d 5.5 w i t h KOH; to 6.0 w i t h 0.1 M s u c c i n i c acid-KOH buffer; a n d to 7.0 a n d 8.0 w i t h 0.1 M Tris-HC1 b u f f e r . T h e r e a c t i o n w a s t e r m i n a t e d a f t e r 30 min. R e l a t i v e a c t i v i t i e s w e r e c a l c u l a t e d b a s e d on t h e a m o u n t o f P A - A s A f o r m e d at pH 4.5 (100%). Q, PA-AsA; a n d 9 PA.

LIPIDS,Vol. 26, No, 5 (1991)

I

i

I

i

I 2 Concentration of L-ascorbic acid(M) FIG. 4. E f f e c t o f L - a s c o r b i c acid c o n c e n t r a t i o n on t h e s y n t h e s i s o f PA-AsA. T h e r e a c t i o n w a s t e r m i n a t e d a f t e r 30 rain. I , PA-AsA; a n d O, PA.

C-5(J=8.25Hz). Based on the 2Jcp and 3Jcp values reported for various phospholipids (24), our data strongly suggest that the phosphatidyl moiety in PA-AsA is linked v / a the h y d r o x y group at C-6 of L-ascorbic acid r a t h e r than v / a C-5. Thus, PLD of S. lydicus catalyzes transphosphatidylation from PC to L-ascorbic acid to form 1,2-diacyl-snglycero-3-phospho-6'-L-ascorbic acid. Figure 3 shows the effect of pH of the reaction mixture on the synthesis of PA-AsA and on the hydrolysis of PC to PA by PLD of S. lydicus. The optimum pH for PA-AsA synthesis was in the range of 4.5 to 5.0. The optimum pH for the hydrolysis of PC to PA in the presence of Lascorbic acid was 7.0. Therefore, the pH of the reaction mixture of subsequent experiments was kept at 4.5. The effect of L-ascorbic acid concentration on the synthesis of PA-AsA is shown in Figure 4. PA-AsA formation markedly increased as the concentration of Lascorbic acid increased. The a m o u n t of PA formed decreased only slightly as the concentration of L-ascorbic acid increased. Hence, higher concentrations of Lascorbic acid clearly favored conversion of PC to PA-AsA. Among the organic solvents tested for use in the transphosphatidylation, ethyl acetate gave the same level of PA-AsA as did diethyl ether. However, twice as much PA was p r o d u c e d when ethyl acetate was used. Other solvents such as n-hexane, cyclohexane, chloroform, acetonitrile and tert-butanol gave lower conversions to PA-AsA and produced a higher ratio of PA to PA-AsA. Thus, diethyl ether appeared most suitable as organic phase for the synthesis of PA-AsA. The synthesis of PA-AsA proceeded in a linear fashion during the initial 30 min and then gradually reached a plateau. After 6 hr, conversion of PC to PA-AsA reached about 80%. PA was also formed, but at a m u c h lower level. PC decreased rapidly and entirely disappeared after prolonged reaction. After 24 hr, PA-AsA started to decrease gradually while PA increased (Fig. 5). The susceptibility of PA-AsA to hydrolysis by PLD was compared with that of PC in the biphasic reaction system (Fig. 6). PC was rapidly hydrolyzed to release PA, where as PA-AsA was more resistant to hydrolysis. The initial rate of hydrolysis of PA-AsA was about 6% of that of PC in this condition.

393 SYNTHESIS OF 6 PHOSPHATIDYLL ASCORBICACID TABLE 2 Synthesis of P A - A s A by Different P h o s p h o l i p a s e s D

PLD-Aa

PLD-Bb

PLD-Cc

PLD=Dd

#mol 30 min reaction

PA AsA PA

2.86 0.50

3.25 1.36

4.33 1.36

0 0.44

7.79 1.82

6.33 2.86

---

0 0.47

8.85 2.07

6.22 3.24

7.16 3.46

0 0.48

5 hr reaction

PA-AsA PA 24 hr reaction 0

2

4

6

8

24

48

72

Time(hr) FIG. 5. Time course for the synthesis of PA-AsA. Q, PA-AsA; i~, PC; and O, PA.

o

E

19

4 _----------O

3

m g

"o

2

m e-

(3. m o r

I

0_ I

2

4

6

8

Time(hr) FIG. 6. Hydrolysis of PA-AsA and PC to PA. 0 , PA-AsA; and O, PC.

The synthetic activities of four different p h o s p h o l i p a s e D were c o m p a r e d using the s a m e r e a c t i o n conditions (see Materials a n d Methods) e x c e p t t h a t in the case of cabbage PLD the r e a c t i o n m i x t u r e c o n t a i n e d 40 mM CaC12 (Table 2). PLD-A s h o w e d t h e highest conversion of PC to PA-AsA. On t h e o t h e r hand, PA-AsA could not be synthesized a n d PC could not be significantly hydrolyzed by cabbage PLD u n d e r the conditions tested. However, PC was completely hydrolyzed by cabbage PLD within 30 min, when the 0.8 M L-ascorbic acid was r e p l a c e d by 80 mM s o d i u m a c e t a t e buffer, pH 4.5. This m a y suggest t h a t t h e activity of cabbage PLD is s u p p r e s s e d at the higher Lascorbic acid c o n c e n t r a t i o n . DISCUSSION

The p r e s e n t s t u d y d e m o n s t r a t e s t h a t 6-phosphatidyl-Lascorbic acid can be p r o d u c e d in high yield by utilizing the t r a n s p h o s p h a t i d y l a t i o n activity of p h o s p h o l i p a s e D. M a x i m u m conversion of PC to PA-AsA was 93.6%. This value is similar to the conversion r e a c h e d for p h o s p h a t i d y l e t h a n o l a m i n e to p h o s p h a t i d y l g l y c e r o l (97.3%) a n d for PC to p h o s p h a t i d y l g l y c e r o l (83%) by this enzyme (25,26). PLD of Actinomycetes has been shown to have a b r o a d s u b s t r a t e specificity for a c c e p t o r alcohols including gera-

PA-AsA PA

aPLD of Streptomyces lydicus (Honen Co.). bpLD of Streptomyces sp. (Toyo Jozo Co.). cPLD of Streptomyces sp. (Sigma Co.). dpLD of cabbage (Boehringer Mannheim GmbH). niol, glucose a n d nucleosides (27-29). L-ascorbic acid was found to also be a good a c c e p t o r alcohol for t h e p h o s p h o l i p a s e D of t h r e e different Streptomyces species. T r a n s p h o s p h a t i d y l a t i o n vs. hydrolysis v a r i e d among t h e phospholipases. The t r a n s p h o s p h a t i d y l a t i o n activity of cabbage PLD in r e s p e c t to L-ascorbic acid r e m a i n s u n k n o w n b e c a u s e activity is s u p p r e s s e d in the p r e s e n c e of ascorbic acid at high c o n c e n t r a t i o n s . However, it is likely t h a t cabbage PLD does not exhibit t r a n s p h o s p h a t i d y l a t i o n activity in r e s p e c t to L-ascorbic acid, b e c a u s e cabbage PLD w a s s h o w n to catalyze t r a n s p h o s p h a t i d y l a tion to several p r i m a r y alcohols, b u t not to sucrose, glucose, galactose (2) a n d nucleosides (29). It has been r e p o r t e d t h a t the o p t i m a l pH was 6.0 for the hydrolysis of egg yolk PC (solubilized with Triton X-100) by PLD from S. lydicus. However, in the p r e s e n t study, o p t i m a l f o r m a t i o n of PA o c c u r r e d at pH 7.0 in the p r e s e n c e of L-ascorbic acid. This difference m a y b e due to t h e different physical s t a t e s of PC which would affect t h e enzyme r e a c t i o n at the e t h e r / w a t e r interface. Shuto et al. (29) r e p o r t e d t h a t t h e o p t i m u m pH for the synthesis of 5'p h o s p h a t i d y l - n u c l e o s i d e s by PLD was equal to t h e pK of nucleoside bases. Similarly, t h e o p t i m u m pH for t h e synthesis of PA-AsA (pH 4.5) was a b o u t equal to t h e pK (4.17) of L-ascorbic acid. It a p p e a r s t h a t PLD has a higher afffmity for the non-ionized form of L-ascorbic acid. J u n e j a et al. (30) r e p o r t e d t h a t t h e initial r a t e of t r a n s p h o s p h a t i d y l a t i o n from PC to e t h a n o l a m i n e by several p h o s p h o l i p a s e s D, including S. lydicus, r e a c h e d a m a x i m u m at a b o u t 0.3 M e t h a n o l a m i n e . At higher conc e n t r a t i o n s of e t h a n o l a m i n e , the initial r e a c t i o n r a t e decreased. The r a t e of hydrolysis of PC was also low at higher c o n c e n t r a t i o n s of ethanolamine. Both t r a n s p h o s p h a t i d y l a t i o n a n d hydrolysis activities of cabbage PLD d e c r e a s e d at higher c o n c e n t r a t i o n s of a c c e p t o r alcohol, such as glycerol, choline, ethanolamine, m e t h a n o l a n d e t h a n o l (6,8). By c o n t r a s t , the r a t e of f o r m a t i o n of PAAsA was not s u p p r e s s e d at higher c o n c e n t r a t i o n s of Lascorbic acid a n d t h e Km value for ascorbic acid was ca. 1 M in the p r e s e n t study. A similar d e p e n d e n c e of t r a n s p h o s p h a t i d y l a t i o n on the c o n c e n t r a t i o n of an a c c e p t o r alcohol was observed in the case of L-serine (6,31). The activity of PLD was not d e p r e s s e d at higher c o n c e n t r a t i o n s of L-serine a n d m a x i m u m conversion to PS was a t t a i n e d at the s a t u r a t i o n level of L-serine. LIPIDS,Vol. 26, No. 5 (1991)

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A. NAGAO ETAL. S e v e r a l solvents, s u c h as d i e t h y l e t h e r , c h l o r o f o r m a n d e t h y l a c e t a t e , h a v e b e e n u s e d to f a c i l i t a t e d i s p e r s i o n s o f p h o s p h o l i p i d s in t h e t r a n s p h o s p h a t i d y l a t i o n m i x t u r e . S u r f a c t a n t s s u c h as T r i t o n X-100 a n d SDS h a v e a l t e r n a t e ly b e e n used. It also h a s b e e n r e p o r t e d t h a t c h l o r o f o r m c a n be a s u i t a b l e s o l v e n t to d i s p e r s e d i m y r i s t o y l PC for t h e s y n t h e s i s o f p h o s p h a t i d y l - L - h o m o s e r i n e (9) a n d p h o s p h a t i d y l n u c l e o s i d e s (29). In t h e s y n t h e s i s o f PA-AsA, h o w e v e r , c h l o r o f o r m r e s u l t e d in low y i e l d s a n d high PAt o - P A - A s A r a t i o s . Diethyl e t h e r w a s f o u n d to be t h e b e s t s o l v e n t for t h e t r a n s p h o s p h a t i d y l a t i o n r e a c t i o n o f La s c o r b i c acid. A l t h o u g h d i m y r i s t o y l PC d o e s n o t dissolve c o m p l e t e l y in d i e t h y l e t h e r , 93.6% o f t h e PC w a s c o n v e r t e d to PA-AsA. C o n v e r s i o n to P A - A s A r e a c h e d a m a x i m u m ( a b o u t 80%) a f t e r 24 hr. T h e r e a f t e r , P A - A s A d e c r e a s e d g r a d u a l l y w h i l e P A i n c r e a s e d slowly. It is likely t h a t t h e d e c r e a s e o f P A - A s A is d u e to its h y d r o l y s i s to P A a f t e r c o n s u m p t i o n o f PC. This is c o n s i s t e n t w i t h t h e f a c t t h a t P A - A s A c a n be h y d r o l y z e d to P A b y PLD, a l t h o u g h t h e r a t e of h y d r o l y s i s is m u c h s l o w e r t h a n t h a t of PC. T h e r e s i s t a n c e o f P A - A s A to h y d r o l y s i s b y t h e PLD f a v o r s t h e s y n t h e s i s o f PA-AsA. It also s e e m s u n n e c e s s a r y to s t o p t h e r e a c t i o n a t a specific t i m e p o i n t a s p r o l o n g e d r e a c t i o n d o e s n o t signifi c a n t l y d i m i n i s h final P A - A s A levels. The p h o s p h o l i p a s e D o f S t r e p t o m y c e s c a t a l y z e t h e t r a n s p h o s p h a t i d y l a t i o n to L - a s c o r b i c a c i d a n d t h e PAA s A f o r m e d r e t a i n s t h e r e d u c i n g a c t i v i t y of L - a s c o r b i c acid. We h a v e c o n f i r m e d t h e a n t i o x i d a n t a c t i v i t y o f PAA s A in t h e p e r o x i d a t i o n o f m e t h y l l i n o l e a t e in a p o l a r s o l v e n t a n d o f egg y o l k p h o s p h a t i d y l c h o l i n e in l i p o s o m a l s u s p e n s i o n (32).

ACKNOWLEDGMENT The authors wish to thank Mr. K. Shinbo of Honen Co. for kindly supplying PLD and Dr. H. Oe of Eisai Co. for FAB mass Spectra.

REFERENCES 1. Yang, S.F., Freer, S., and Benson, A.A. (1967) J. Biol. Chem. 242, 477-484. 2. Dawson, R.M.C. (1967) Biochem. J. 102, 205-210. 3. Lee, S.Y.,Hibi, N., Yamane, T., and Shimizu, S. (1985) J. Ferment. Tech'nol. 63, 37-44.

4. Juneja, L.R., Hibi, N., Yamane, T., and Shimizu, S. (1987) Appl. Microbiol. Biotechnol. 27, 146-151. 5. Jezyk, P.F., and Hughes, H.N. (1973) Biochim. Biophys. Acta 296, 24-33.

LIPIDS,Vol. 26, No. 5 (1991)

6. Comfurius, P., and Zwaal, R.F.A. (1977) Biochim. Biophys. Acta

488, 36-42. 7. Eibl, H., and Kovatchev, S. (1981) Methods Enzymol. 72, 632639. 8. Achterberg, V., Fricke, H., and Gercken, G. (!986) Chem. Phys. Lipids 41, 349-353. 9. Shuto, S., Imamura, S., Fukukawa, K., Sakakibara, H., and Murase, J. (1987) Chem. Pharm. Bull. 35, 447-449. 10. Wolf, W.A., and Gross, R.W. (1985) J. Lipid Res. 26, 629-633. 11. Shuto, S., Ito, H., Ueda, S., Imamura, S., Fukukawa, K., Tsujino, M., Matsuda, A., and Ueda, T. (1988) Chem. Pharm. Bull. 36, 209-217. 12. Brachwitz, H., Langen, P., Dube, G., Schildt, J., Paltauf, F., and Hermetter, A. (1990) Chem. Phys. Lipids 54, 89-98. 13. Terao, J., Shibata, S.S., and Matsushita, S. (1988) Anal. Biochem. 169, 415-423. 14. Terao, J. (1989) Biochim. Biophys. Acta 1003, 221-224. 15. Terao, J. (1989) Lipids 24, 659-661. 16. Kellogg, E.W., and Fridovich, I. (1977)J. Biol. Chem. 252, 67216728. 17. Niki, E. (1987) Chem. Phys. Lipids 44, 227-253. 18. Bendich A., Machlin, L.J., Scandurra, O., Burton, G.W., and Wayner, D.D.M. (1986)Adv. Free Radical Biol. Med. 2, 419-444. 19. Kato, S., Kokusho, Y., Machida, H., and Iwasaki, S. (1984) Agric. Biol. Chem. 48, 2181-2188. 20. Tsugo, T., Yamauchi, K., and Kanno, C. (1968) Nippon Nogeikagaku Kaishi 42, 367-377. 21. Mufeed, M.B., and LaPlanche, L.A. (1990) Chem. Phys. Lipids 54, 99-113. 22. LeRoy, F.J., and William, C.J. (1972) Carbon-13NMR Spectra:A Collection of Assigned, Coded, and Indexed Spectra, pp. 171, John Wiley & Sons, Inc., New York. 23. Liao, M.L., Wang, X.Y., Chung, C., Liang, Y.T., and Seib, P.A. (1988) Carbohydrate Res. 176, 73-77. 24. Murari, R., Abd E1-Rahman, M.M.A.,Wedmid, Y., Parthasarathy, S., and Baumann, W.J. (1982) J. Org. Chem. 47, 2158-2163. 25. Shinbo, K., Yano, H., and Miyamoto, Y. (1989) Agric. Biol. Chem. 53, 3083-3085. 26. Shinbo, K., Yano, H., and Miyamoto, Y. (1990) Agric. Biol. Chem. 54, 1189-1193. 27. Kokusho, Y., Kato, S., Machida, H., and lwasaki, S. (1987) Agr/c. Biol. Chem. 51, 2515-2524. 28. Shuto, S., Imamura, S., Fukukawa, K., and Ueda, T. (1988) Chem. Pharm. Bull. 36, 5020-5023. 29. Shuto, S., Ueda, S., Imamura, S., Fukukawa, K., Matsuda, A., and Ueda, T. (1987) Tetrahedron Lett. 28, 199-202. 30. Juneja, L.R., Kazuoka, T., Yamane, Y., and Shimizu, S. (1988) Biochim. Biophys. Acta 960, 334-341. 31. Juneja, L.R., Kazuoka, T., Goto, N., Yamane, Y., and Shimizu, S. (1989) Biochim. Biophys. Acta 1003, 277-283. 32. Nagao, A., and Terao, J. (1990) Biochim. Biophys. Res. Commun. 172, 385-389. [Received August 1, 1990; and in revised form December 17, 1990; Revision accepted February 25, 1991 ]