736

Interferon o /,8 Induces Changes in the Metabolism of Polyenoic Phospholipids and Diacylglycerols in the Livers of Suckling Mice Georges ZwingelsteinO, Gerard Brichona, Roger Meistera, C h a n t a l Mauryba n d Ion Gresser*,b ~ de PhysiologieGenerale et comparee, CNRS UA 6t 3, Universite Claude Bernard, 69622 Villeurbanne Cedex and bLaboratoire d'Oncologie Virale, CNRS ER 274, Institutde recherches scientifiquessur le cancer, BP8, 94802 Villejuif Cedex, France S u c k l i n g m i c e w e r e i n j e c t e d d a i l y f r o m b i r t h f o r 10 days with potent preparations of mouse interferon a/B. I n t e r f e r o n t r e a t m e n t r e s u l t e d i n a m a r k e d l y lower concentration of polyunsaturated fatty acids ( 2 0 : 4 w 6 a n d 22:6(~3) i n t h e t w o p r i n c i p a l l i v e r phospholipids, phosphatidylcholine and phosphatid y l e t h a n o l a m i n e , t h a n in l i v e r s o f c o n t r o l - t r e a t e d mice. This effect appeared to correlate with a low level of synthesis of polyunsaturated phospholipids in t h e l i v e r s o f i n t e r f e r o n - t r e a t e d m i c e . T h u s , in c o n t r o l m i c e , s y n t h e s i s o f s p e c i e s o f p o l y u n s a t u r a t e d p h o s p h o l i p i d s i n c r e a s e d m a r k e d l y in t h e f i r s t 10 d a y s o f life, w h e r e a s in l O - d a y - o l d i n t e r f e r o n treated mice, the level of synthesis of species of polyunsaturated phospholipids was comparable to t h a t in n e w b o r n m i c e . I n p a r a l l e l , a m a r k e d inc r e a s e in t h e d i a c y l g l y c e r o l c o n t e n t w i t h o u t c h a n g e o f its r e n e w a l w a s o b s e r v e d in t h e l i v e r s o f i n t e r f e r o n - t r e a t e d m i c e . We s u g g e s t t h a t i n t e r f e r o n t r e a t m e n t r e s u l t s in a n i n h i b i t i o n o f o n e o f t h e p r o c e s s e s t h a t l e a d s to a c t i v a t i o n o f t h e e n z y m a t i c systems responsible for the synthesis of species of polyunsaturated phosphatidylcholine and phosp h a t i d y l e t h a n o l a m i n e in t h e l i v e r o f s u c k l i n g m i c e . It s e e m s l i k e l y t h a t t h e s e r e s u l t s a r e r e l a t e d t o t h e i n h i b i t i o n o f l i v e r cell m a t u r a t i o n a n d t h e m a r k e d c e l l n e c r o s i s t h a t a r e o b s e r v e d in i n t e r f e r o n treated suckling mice.

Lipids 22, 736-743 (1987). Administration of potent preparations of interferon a/fl to suckling mice in the first week of life results in inhibition of growth, diffuse liver cell necrosis and death (1). Recently we showed t h a t this interferon treatment resulted in a marked increase in liver triglycerides and a decrease in the level of some hepatic phospholipids (2). Whereas the percentage of liver phospholipids (PL) (relative to liver protein) increased in the first week of life for normal suckling mice, it decreased in the livers of interferon-treated mice (2). These biochemical modifications, not observed in adult mice (2), were accompanied by the appearance of abnormal tubular aggregates arising from the endoplasmic reticulum of hepatocytes (3,4), suggesting important alterations in the lipid-protein interactions at the level of these membranes (5). There have been several reports indicating that, in vitro, interferon induced changes in the fatty acid composition of PL in cells. Thus, Chandrabose et al. (6) observed t h a t mouse interferon induced a decrease in the unsaturated fatty acid content of all the major *To whom correspondence should be addressed. Abbreviations: PL, phospholipid;NDV, Newcastle disease virus; TLC, thin layer chromatography; PC, phosphatidylcholine;PE, phosphatidylethanolamine; PS, phosphatidylserine; PI, phosphatidylinositol; DG, diacylglycerol;TG, triacylglycerol. LIPID& Vol. 22, No, 10 (]987)

PL in mouse sarcoma cells, and Apostolov and Barker (7,8) showed that h u m a n interferon reduced the C18 unsaturated fatty acid content of h u m a n fibroblasts and bovine kidney cells. These changes m a y contribute to the observed increased rigidity of the plasma membrane lipid bilayer of some interferon-treated cells (9). The results presented herein indicate t h a t in vivo, interferon induces similar biochemical modifications in suckling mice as a result of alterations in the metabolism of the polyunsaturated molecular species of liver PL. MATERIAL AND METHODS

Mice. Litters of Swiss mice were obtained from the pathogen-free colony at the Institut Recherches Scientifiques sur te Cancer, Villejuif, France. Interferon preparation. Mouse interferon a/fl was prepared from suspension cultures of mouse sarcoma C243 cells inoculated with Newcastle disease virus (NDV). The methods of production and purification have previously been described (10). Interferon was assayed by inhibition of cytopathic effect of vesicular stomatitis virus on L cells in monolayer culture (0.2 ml/well) in Falcon microplates. Units in the text are expressed as international mouse reference units. The specific activity of this purified interferon was ca. 2 • 107 u n i t s / m g protein. Control preparations consisted of supernatant from cultures of C243 cells prepared in exactly the same way as interferon except t h a t the interferon inducer, NDV, was omitted. Experimental plan. Half of the mice in each litter were marked and injected subcutaneously in the intercapsular region daily from birth to the sixth or tenth day with 0.05 ml of purified mouse interferon h a v i n g a titer of 3.2 • 106/ml or with the control preparation. Mice were killed on the sixth or the tenth day. After mice were killed, the livers were weighed; a piece was removed for histologic examination and the remaining tissue was weighed, ground with a blender in 10 ml of chloroform/methanol (1:1,v/v) and stored at -80 C. The lipid extraction was performed according to Folch et al. (11).

Separation of neutral lipids and total PL for analysis of fatty acids. Lipids were chromatographed on a silica gel (Mallinckrodt, Paris KY) column. After the column was washed with hexane/isopropyl ether (90:10, v/v), triacylglycerols were eluted by h e x a n e / isopropyl ether (70:30, v/v), diacylglycerois and all nonphosphorus-containing lipids by isopropyl ether/ methanol (95:5, v/v) and PL by methanol (93-95% recovery). Aliquots of triacylglycerol and 1,2-diacylglycerol fractions were purified by thin layer chromatography (TLC) on silica gel G 60 precoated plates (Merck, Darmstadt, FRG) in heptane/isopropyl ether acetic acid (60:40:4 v / v / v ) solvent system (12). The 1,2-diacylglycerol and triacylglycerol spots, detected under UV light after spraying with primuline reagent

737

INTERFERON AFFECTS LIVER POLYENOIC PHOSPHOLIPIDS (13), w e r e s c r a p e d i n t o s c r e w - c a p p e d t u b e s . A k n o w n a m o u n t o f 17:0 f a t t y a c i d w a s a d d e d a s a t r a c e r to each fraction, and transesterification by methanolicHC1 w a s p e r f o r m e d a s p r e v i o u s l y d e s c r i b e d (14). F a t t y m e t h y l e s t e r s w e r e a n a l y z e d u s i n g a g a s liqu i d c h r o m a t o g r a p h ( P a c k a r d 428, D o w n e r s G r o v e , I L ) w i t h a 10% S P 2330 o n 1 0 0 / 1 2 0 c h r o m o s o r b W A W c o l u m n o f 9' 1 / 1 6 " S S o p e r a t e d i s o t h e r m a l l y a t 190 C. Results are expressed as percent of total fatty acids by means of a coupled integrator (Packard instrument). The amount of fatty acid in each of the samples was calculated by comparing the surface of each of the p e a k s to t h e s u r f a c e o f t h e p e a k o f 17:0 o n t h e s a m e chromatogram.

Analysis of fatty acids in phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI). P L f r o m t h e f r a c tion previously purified by column were separated by TLC using chloroform/methanol/acetic acid/water (60:30:7:3, v / v / v / v ) a s d e v e l o p i n g s o l v e n t (15). P C , PE, PS and PI spots visualized under UV light after spraying with primuline were scraped into screwcapped tubes, and methyl esters of fatty acids were prepared as described above.

In vivo measurement of PL biosynthesis in the liver of suckling mice. S u c k l i n g m i c e w e r e k i l l e d 5,20,45 o r 60 m i n a f t e r i n j e c t i o n o f 150 ~ C i o f [2-3H] g l y c e r o l p e r 5 g o f m o u s e . L i v e r s w e r e h o m o g e n i z e d i n 20 v o l o f chlor o f o r m / m e t h a n o l (1:1, v / v ) a n d t h e p r o t e i n p r e c i p i tate, separated from the organic phase by centrifugation, was reextracted by two other extractions with this solvent mixture. The pooled organic phases were

a d j u s t e d to 2:1 c h l o r o f o r m / m e t h a n o l a n d s u b s e q u e n t l y w a s h e d a s p r e v i o u s l y d e s c r i b e d (14,16). T o t a l l i p i d phosphorus and lipid radioactivity was determined on a l i q u o t s o f l i p i d s e x t r a c t e d f r o m liver. Neutral lipids were resolved by TLC on silica gel G 60 p r e c o a t e d p l a t e s i n m o n o d i m e n s i o n with hept a n e / i s o p r o p y l e t h e r / a c e t i c a c i d (60:40:4, v / v / v ) . Lipids were detected under UV light with primuline reagent, or standards were chromatographed in parallel a n d s t a i n e d w i t h v a n i l l i n / s u l f u r i c acid s p r a y r e a g e n t . P L w e r e s e p a r a t e d b y t w o - d i m e n s i o n a l T L C o n 10 • 10 c m s i l i c a g e l p r e c o a t e d p l a t e s u s i n g s o l v e n t s prev i o u s l y d e s c r i b e d (17). T h e s p o t s o n t h e c h r o m a t o gram were visualized with the phosphorus-specific s p r a y r e a g e n t o f D i t t m e r a n d L e s t e r (18). Visualized spots were scraped into a counting vial, a n d 3 m l o f e t h a n o l / w a t e r (1:1, v / v ) a n d 8 m l o f P i c o f i u o r - 3 0 ( P a c k a r d ) w e r e s u c c e s s i v e l y a d d e d (19). A f t e r m i x i n g , e a c h v i a l w a s c o u n t e d i n a T r i c a r b spect r o m e t e r ( P a c k a r d ) . T h e c o u n t i n g e f f i c i e n c y w a s determined by the external-standard channel ratio, and the results are expressed in dpm. P r o t e i n s i n t h e d e l i p i d i z e d p r e c i p i t a t e w e r e determ i n e d b y t h e m e t h o d o f L o w r y et al. (20) a f t e r d i s s o l v ing in 1 N NaOH. Triacylglycerols were determined by enzymatic m e t h o d (21) o n a l i q u o t s o f t o t a l l i p i d a n d t r i a c y l g l y cerol chromatographic fraction dissolved in a mini m u m v o l u m e o f p r o p a n o l . T h e p r o p o r t i o n s o f 1,2diacylglycerols in neutral lipids were determined in the corresponding fraction of column chromatography by the same enzymatic method after separation of

T A B L E 1. E f f e c t o f I n t e r f e r o n on the C o m p o s i t i o n o f F a t t y A c i d s in L i v e r P h o s p h o l i p i d s o f S u c k l i n g Mice a

Age of mice 6 Days F a t t y acids 16:0 16:1 16:2 18:0 18:1 18:2~6 18:3w6 18:3oJ3 20:3 20:4~o6 20:5w3 22:3 22:5w3 22:6~3 Ew6 )2r U.I. c

10 Days

At birth (8)b

Control (4)

IFN-treated (4)

Control (5)

IFN-treated (6)

25.0 _+0.3 1.3 _+0.1 1.0 _+0.1 16.6 • 0.3 11.5 _+0.1 10.4 _+0.1 0.7 • 0.1 1.1 + 0.1 1.3 + 0.1 12.2 _+0.4 2.5 + 0.1 0.3 i 0.1 0.6 • 0.1 14.8 • 0.5 23.3 18.9 198

21.2 _+0.3 0.9 • 0.1 0.8 _+0.1 17.3 _+0.1 8.9 + 0.4 10.3 _+0.2 0.5 • 0.1 1.2 • 0.5 1.9 _+0.1 15.7 _+0.2 2.4 +_0.4 0.9 • 0.4 0.9 + 0.1 15.3 + 0.8 26.5 19.8 215

21.7 +_0.1 1.1 • 0.1 1.1 _+0.1 17.6 • 0.7 12.1 _+0.4 10.2 + 0.1 0.8 • 0.1 1.4 _+0.1 2.5 + 0.1 14.2 _+0.4 2.3 • 0.1 1.0 • 0.1 1.7 • 0.1 12.0 + 0.8 25.2 18.5 193

26.1 • 0.5 0.7 + 0.1 0.7 + 0.1 16.3 • 0.2 7.5 • 0.1 10.6 _+0.2 0,4 • 0.1 0,5 -+ 0.1 1.7 • 0.2 14.1 +_0.2 1.7 _+0.1 0.5 + 0.1 1.0 • 0.1 17.1 + 0.5 25.1 20.3 212

26.8 • 0.3 0.9 • 0.1 1.0 • 0.1 16.6 _+0.1 10.7 • 0.3 11.4 _+0.2 0.5 _+0.1 1.0 • 0.2 1.2 • 0.2 10.4 • 0.2 1.6 • 0.1 0.4 _+0.1 1.7 • 0.1 14.6 • 0.4 22.2 18.9 191

aThe results expressed as the percentage of total fatty acids are means -- SM. bNumber in parentheses indicates the number of mice. Newborn and 10-day-old mice came from the same litters, whereas 6-day-old mice came from a second experiment at a different time of year. cu.I., the unsaturation index, is the sum of the percentage multiplied by the number of double bonds for each fatty acid in the mixture. LIPIDS, Vol. 22, No, 10 (1987)

738

G. ZWINGELSTEIN ET AL. the 1,2-diacylglycerols in parallel with a known a m o u n t o f p u r i f i e d d i a c y l g l y c e r o l s b y T L C a s desc r i b e d a b o v e . T h e a m o u n t of d i a c y l g l y c e r o l s o n t h e plate was calculated from the corrected OD obtained with the standard spot of diacylglycerols on the same plate.

Separation of molecular species of radioactive PC and PE. P C a n d P E w e r e p u r i f i e d f r o m t h e P L f r a c t i o n b y p r e p a r a t i v e T L C o n 20 x 20 c m p l a t e s c o a t e d w i t h s i l i c a g e l N, H R ( M a c h e r a y - N a g e l , D t i r e n , F R G ) cont a i n i n g 1% s o d i u m c a r b o n a t e in c h l o r o f o r m / m e t h a n o l / a c e t i c a c i d / w a t e r (60:30:7:3, v / v / v / v ) (15). P C a n d P E , localized by fluorescence after plates were sprayed with primuline, were eluted from gel by extraction with a mixture of chloroform/methanol/acetic acid/ w a t e r (50:39:1:10, v / v / v / v ) (22). T h e d i f f e r e n t m o l e c u lar species of PC and PE were separated by chromatog r a p h y o n 5 • 10 c m s i l i c a g e l G 60 p r e c o a t e d p l a t e s i m p r e g n a t e d w i t h s i l v e r n i t r a t e (23,24). I n b r i e f , t h e p l a t e s w e r e i m m e r s e d f o r 30 m i n i n a s o l u t i o n o f 50 g o f s i l v e r n i t r a t e d i s s o l v e d i n 50 m l o f w a t e r , t h e n d r i e d for 10 m i n a t 110 C a n d a c t i v a t e d for 8 r a i n a t 170 C b e f o r e use. E a c h p u r i f i e d P L ( a b o u t 2 m g ) w a s s p o t t e d on each plate, and chromatography was performed in a chloroform/methanol/ w a t e r s o l v e n t s y s t e m (60: 40:7.5. v / v / v ) for P C a n d (70:40:10, v / v / v ) f o r P E . After drying, the four major separated fractions were identified by the reagent of Dittmer and Lester, scraped into counting vials and counted as described above.

RESULTS

Effect of interferon on the fatty acid composition of phospholipids in the livers of suckling mice (Table 1). P a l m i t i c (16:0), s t e a r i c (18:0), oleic (18:1), l i n o l e i c (18:2~6), e i c o s a t e t r a e n o i c (20:4oJ6) a n d d o c o s a h e x a e n o i c (22:6oJ3) a c i d s a r e t h e m a j o r f a t t y a c i d s i n t h e l i v e r P L o f s u c k l i n g m i c e ( T a b l e 1). I n t h e f i r s t 10 d a y s o f life, t h e p r o p o r t i o n o f p o l y u n s a t u r a t e d f a t t y a c i d s i n l i v e r P L i n c r e a s e d (43.7% a t b i r t h t o 47.5% for 10-dayo l d mice). T h i s o v e r a l l i n c r e a s e i n p o l y u n s a t u r a t e d acids in PL was not observed in interferon-treated s u c k l i n g m i c e . T h u s , t h e p e r c e n t a g e o f 20:4~6 a n d 22:6o~3 i n P L i n c r e a s e d b y 15% for c o n t r o l m i c e (comp a r e d to n e w b o r n m i c e ) a n d e i t h e r d e c r e a s e d f o r int e r f e r o n - t r e a t e d m i c e a t 10 d a y s or s h o w e d t h e s a m e v a l u e a s for n e w b o r n m i c e . T h e p e r c e n t a g e o f oleic a c i d d e c l i n e d w i t h a g e f o r c o n t r o l m i c e , b u t for i n t e r f e r o n - t r e a t e d m i c e i t s t a y e d a t t h e s a m e l e v e l a s for n e w b o r n m i c e . I t is i n t e r e s t i n g to n o t e t h a t t h e p o l y u n s a t u r a t e d f a t t y a c i d i n d e x o f i n t e r f e r o n - t r e a t e d 10d a y - o l d m i c e w a s c l o s e r to t h a t o f n e w b o r n m i c e t h a n it w a s to c o n t r o l 1 0 - d a y - o l d s u c k l i n g mice.

Effect of interferon on the fatty acid composition of the major liver PL of suckling mice (Table 2). I n P C , there was a clear-cut decrease in the percentage of eicosatetraenoic (20:4o~6) a n d d o c o s a h e x a e n o i c (22:6~3) a c i d s a n d a n i n c r e a s e i n t h e p e r c e n t a g e o f p a l m i t o l e i c (16:1), o l e i c (18:1), l i n o l e i c (18:2w6) a n d e i c o s a p e n t a e n o i c (20:5~o3) a c i d s i n i n t e r f e r o n - t r e a t e d

TABLE 2 E f f e c t o f I n t e r f e r o n on the Fatty Acid C o m p o s i t i o n o f P h o s p h a t i d y l c h o l i n e (PC), P h o s p h a t i d y l e t h a n o l a m i n e (PE) and P h o s p h a t i d y l s e r i n e + P h o s p h a t i d y l i n o s i t o l (PS + PI) in Liver o f 1 0 - D a y - O l d Mice a

PC Fatty acids 16:0 16:1 18:0 18:1 18:2~6 18:3~6 18:3a3 20:3w6 20:4oJ6 20:5oJ3 22.5~3 22:6~3 ~o6 ~3 ~6/~3 U.l.b

PE

PS+PI

Control

IFN-treated

Control

IFN-treated

Control

IFN-treated

34.5 _+0.9 0.9 _+0.1 13.0 _+0.2 8.7 _+0.1 12.7 _+0.2 0.4 _+0.1 0.8+0.1 2.3 + 0.1 10.8 + 0.1 1.0 _+0.1 0.6 _+0.1 13.3 _+0.2 26.2 15.8 1.7 176

35.2 + 0.8 1.2 • 0.1 13.8 • 0.2 12.8 + 0.1 13.5 _+0.1 0.5 _+0.1 1.4+0.1 1.9 • 0.3 8.1 _+0.4 1.5 • 0.2 0.9 _+0.1 8.1 • 0.1 24.0 11.8 2.0 144

22.8 + 0.3 ND 22.1 + 0.2 4.3 + 0.1 2.8 + 0.1 0.7 + 0.1 0.7+0.1 1.1 • 0.1 17.1 + 0.3 2.1 + 0.2 1.4 _+0.1 24.3 + 0.1 21.7 28.5 0.8 248

19.9 _+0.4 0.6 _+0.1 21.6 • 0.2 8.7 + 0.1 8.5 + 0.1 ND 1.3_+0.1 1.2 + 0.1 13.8 _+0.3 2.7 • 0.2 1.6 _+0.1 19.5 _+0.2 23.4 25.1 1.0 227

14.9 _+0.1 ND 39.6 _+0.7 5.5 _+0.2 2.1 _+0.1 ND 0.3_+0.1 0.7 +_0.1 21.7 _+0.1 1.0 _+0.1 0.8 + 0.1 12.0 + 0.1 24.5 14.1 1.7 181

15.2 + 0.2 ND 37.0 _+0.6 7.3 _+0.2 3.4 _+0.1 ND 1.0+ 0.6 1.6 +_0.6 18.7 _+0.3 1.3 -+ 0.1 1.6 + 0.1 11.6 _+0.5 23.6 15.6 1.5 181

aThe results are given in percentage of total fatty acids recovered and are expressed as means + SE. Four pools of two livers were analyzed: each determination was done in duplicate. Minor fatty acids as 12:0, 14:0, 14:1, 15:0, 17:0, 20:0, 20:1, 22:1 and 24:0 are not included in the table, but for the determination of total fatty acids all identified acids were included. bu.I., the unsaturation index, is the sum of the percentage multiplied by the number of double bonds for each fatty acid in the mixture. ND, Not detected. LIPIDS, Vol. 22, No. 10 (1987)

739

INTERFERON AFFECTS LIVER POLYENOtC PHOSPHOLIPIDS mice. The u n s a t u r a t i o n index decreased from 176 for control mice to 144 for interferon-treated mice. P E c o n t a i n s the h i g h e s t p e r c e n t a g e of u n s a t u r a t e d f a t t y acids of liver PL. I n t e r f e r o n t r e a t m e n t resulted in a decrease in palmitoleic, eiocosatetraenoic a n d d o c o s a h e x a e n o i c acids, a c c o m p a n i e d b y a n increase in the percent of oleic, tinoleic a n d linolenic in ~3 (18:3w3) f a t t y acids. The overall u n s a t u r a t i o n index decreased from 248 for control mice to 227 for interferon-treated mice. No c h a n g e in the u n s a t u r a t i o n index w a s observed in the fractions PS a n d PI.

Effect of interferon on the amount of fatty acids in the livers of suckling mice. The results presented above give only an imperfect picture of the extent of the biochemical c h a n g e s due to interferon t r e a t m e n t , as in the first 10 d a y s of life the liver w e i g h t increases from 83 m g at birth to 208 m g (10 days) for control mice a n d 260 m g for interferon-treated mice, a n d the a m o u n t of liver lipids increases from 3.1 m g / l i v e r at birth to 9.8 m g for control mice a n d 25 m g for interferon-treated mice (2). For this reason, we h a v e quantitated f a t t y acids in the liver P L u s i n g a n i n t e r n a l s t a n d a r d of 17:0. Thus, per liver, the total a m o u n t of palm• acid (16:0) doubled, a n d oleic (18:1) a n d linoleic (18:2o~6) acids tripled or q u a d r u p l e d in interferon-treated mice (Table 3). However, in the two g r o u p s of mice, the levels of p o l y u n s a t u r a t e d f a t t y acids in C20 a n d C22 were virtually equal; only the s u m of u n s a t u r a t e d f a t t y acids in ~6 w a s increased in interferon-treated mice as a result of the m a r k e d increase in linoleic acid (18:2~o6).

I n contrast, there w a s a m a r k e d decrease in the a m o u n t per liver of f a t t y acid present in the liver P L (i.e., 8.8~mol per liver for control mice to 5.2 pmol for interferon-treated mice). Palmitoleic (16:1), oleic (18:1), linolenic (18:3oJ6 a n d 18:3w3) a n d docosatrienoic (22:3) acids present in P L decreased ca. 20%; palm• (16:0), stearic (18:0), eicosatrienoic (20:3) a n d eicosapentaenoic (20:5) acids decreased 40%; eicosatetraenoic (20:4w6) a n d d o c o s a h e x a e n o i c (22:6~3) decreased more t h a n 50%. Only d o c o s a p e n t a e n o i c acid (22:5oJ3) increased by 8%. It c a n be seen, therefore, t h a t the p o l y u n s a t u r a t e d f a t t y acids in P L were the m o s t affected b y interferon t r e a t m e n t . (For control mice, p o l y u n s a t u r a t e d f a t t y acids in p h o s p h o l i p i d s represent 4.3 pmol per liver, w h e r e a s t h e y only constitute 2.3 ~mol for interferontreated mice).

Effect of interferon on biosynthesis of lipids in the livers of suckling mice: kinetics of incorporation of l(3)-3Hglycerol. Five m i n after injection of radioactive glycerol, 50% of the lipid r a d i o a c t i v i t y w a s present in triacylglycerides. The level of r a d i o a c t i v i t y in triacylglycerides t h e n decreased for 8-day-old control mice, w h e r e a s it increased for interferon-treated mice (Fig. 1). The r a d i o a c t i v i t y in diacylglycerides was greater in interferon-treated mice t h a n in control mice a n d decreased in parallel with time in b o t h g r o u p s (Fig. 1). The r a d i o a c t i v i t y in P L i n c r e a s e d in parallel for b o t h groups, a l t h o u g h the a m o u n t in interferon-treated mice was a b o u t h a l f t h a t of control mice (Fig. 1). A l t h o u g h there was a m a r k e d decrease in the P L content of the liver in interferon-treated suckling mice (2.1

TABLE 3 Effect of I n t e r f e r o n on the Amount of Fatty Acids in Total Lipids and Phospholipids of the Livers of 10-Day-Old Suckling Mice a Total lipids Fatty Acids 16:0 16:1 18:0 18:1 18:2w6 18:3~6 18:3~o3 20:3 20:4~6 20:5oJ3 22:3 22:5~3 22:6~3 ~6 EoJ3

Control (6) 3.1 _+0.2 0.2 + 0.1 1.7 _+0.2 2.5 _+0.4 1.5 • 0.2 0.1 + 0.1 ND 0.2 -+ 0.1 1.4 • 0.1 0.2 _+0.1 ND 0.1 • 0.1 1.5 _+0.3 3.2 • 0.3 2.0 _+0.2

IFN-treated (6) 6.1 _+0.5** 0.8 _+0.2 1.8 _+0.3 8.6 + 1.2"* 3.7 _+0.4** 0.2 -+ 0.1 ND 0.5 _+0.1 1.1 -+ 0.2 0.5 _+0.2 ND 0.4 • 0.1 1.2 _+0.2 5.3 • 0.5** 2.7 _+0.4

Phospholipids Control (6)

IFN-treated (6)

2.14 _+0.12 0.07 _+0.01 1.51 • 0.02 0.73 _+0.01 0.94 _+0.03 0.04 • 0.01 0.08 _+0.01 0.16 _+0.0t 1.33 -+ 0.01 0.18 _+0.01 0.06 • 0.01 0.08 _+0.01 1.46 _+0.06 2.31 _+0.02 1.80 • 0.04

1.32 _+0.14"* 0.05 _+0.01"* 0.91 + 0.06** 0.60 _+0.03* 0.58 _+0.05** 0.03 _+0.01 0.06 + 0.01 0.09 • 0.01"* 0.63 -+ 0.02** 0.10 _+0.01"* 0.05 -+ 0.01 0.09 • 0.01 0.71 _+0.08** 1.24 _+0.03** 0.96 _+0.05**

aThe amount ingmol/liver of each fatty acid is mean_+ SM and is determined by comparison with an internal standard of 17:0fatty acid introduced in the lipids at the time of transesterification (see Materials and Methods). Calculations take into account the surface of the peaks of fatty acids with that of standard on the same chromatogram. Number in parentheses indicates the number of mice. Values statistically different between control and IFN-treated:*, p< 0.05; **, p< 0.01. ND, not detected. LIPIDS, Vol. 22, No. 10 (1987)

740

G. ZWINGELSTEIN ET AL.

.5-

**s

TG

DG

m g liver protein doubles or triples in control mice between birth a n d 10 days, w h e r e a s the levels in interferon-treated mice are c o m p a r a b l e to those f o u n d in n e w b o r n mice.

PL

.4-

Distribution of radiolabel among the different molecular species of PC and PE (Table 5). F o r 10-day-old

E

.1

5

20

60

2O

control

20

60

60

m i n ute~

IFN - trea~ed

FIG. 1. E f f e c t o f i n t e r f e r o n t r e a t m e n t o n t h e k i n e t i c s o f 1(3)'3H-glycerol i n c o r p o r a t i o n i n t o the d i a c y l g l y c e r o l s (DG), t r i a c y l g l y c e r o l s (TG) and p h o s p h o l i p i d s (PL) o f t h e l i v e r s o f s u c k l i n g mice. E a c h v a l u e w a s t h e m e a n +_SM o f d p m / 1 0 0 m g o f w e t tissue. V a l u e s s t a t i s t i c a l l y d i f f e r e n t b e t w e e n c o n t r o l and IFN-treated; *, p < 0.05; **, p < 0.01.

control mice, the radiolabel in PC was f o u n d a l m o s t equally distributed in the low u n s a t u r a t e d (i.e., monoenoic a n d dienoic) a n d p o l y u n s a t u r a t e d (i.e., tetraenoic a n d hexaenoic) species. I n contrast, in 10-dayold interferon-treated mice, only 37% of the radioactivity was f o u n d in the p o l y u n s a t u r a t e d species, as was also found in n e w b o r n mice. For PE, in 10-day-old control mice, the m a j o r p a r t (82%) of the r a d i o a c t i v i t y was f o u n d in the p o l y u n s a t u r a t e d species. I n t e r f e r o n t r e a t m e n t resulted in a decrease in this p e r c e n t a g e to 66%, w h i c h w a s similar to the percent of r a d i o a c t i v i t y in n e w b o r n mice. I n s u m m a r y , interferon t r e a t m e n t affected preferentially the s y n t h e s i s of molecular species of PC a n d P E c o n t a i n i n g m o s t l y p o l y u n s a t u r a t e d f a t t y acids.

Effect of interferon on the composition and metabolism of 1,2odiacylglycerols and triacylglycerols. As + 0.2% of wet tissue c o m p a r e d with 3.6 + 0.3% in control mice), the specific r a d i o a c t i v i t y of P L w a s comp a r a b l e between the 2 groups a n d increased regularly between 5 a n d 60 m i n (for control mice, 450 _+ 60 d p m / # g of P L [5 min] to 560 _+40 d p m / ~ g [20 min] a n d 710 +_50 [60 min], w h e r e a s for interferon-treated mice, the values were 290 _+ 80 [5 min], 570 _+ 50 [20 min] a n d 740 _+40 [60 min]). There w a s no s i g n i f i c a n t difference in the distribution of r a d i o a c t i v i t y in the different species of PL.

Comparison of PL synthesis in the livers of newborn and lO-day-old control and interferon-treated mice. I n c o r p o r a t i o n of 3H-glycerol in liver P L w a s d e t e r m i n e d 45 m i n after injection of the tracer. As c a n be seen in Table 4, the a m o u n t of r a d i o a c t i v i t y per 100

interferon t r e a t m e n t of suckling mice resulted in a n a b n o r m a l a c c u m u l a t i o n of l(3)-3H-glycerol in 1,2-diacylglycerols a n d as these c o m p o u n d s are c o m m o n intermediates for triacylglycerol a n d phospholipid biosynthesis, we d e t e r m i n e d the c o n c e n t r a t i o n a n d the specific r a d i o a c t i v i t y of 1,2-diacylglycerols a n d their f a t t y acid composition. I n t e r f e r o n t r e a t m e n t increased the c o n c e n t r a t i o n of 1,2-diacylglycerols a b o u t four- to fivefold in the liver (from 0.4 + 0.3 p m o l / g of wet tissue in control mice to 2.1 _+ 1.0 t~mol/g in interferon-treated mice), a l t h o u g h their a v e r a g e specific r a d i o a c t i v i t y r e m a i n e d a l m o s t e q u i v a l e n t (1787 + 291 d p m / # g of diacylglycerols for control mice a n d 1591 _+ 230 d p m / u g for interferon-treated mice). Moreover, there was only a s i g n i f i c a n t decrease of 18:1

TABLE 4 R a d i o a c t i v i t y I n c o r p o r a t e d in the Liver P h o s p h o l i p i d s o f N e w b o r n Mice and 1 0 - D a y - O l d C o n t r o l and I n t e r f e r o n - T r e a t e d Mice*

10-Day-old mice Newborn (8) LPC SPH PC PS PI PE DPG PA TG

1,013 _+ 150a 454 _+ 55 b 110,160 _+ 6,082c 1,201 _+ 130d 6,738 _+ 403f 62,803 _+ 3,827g 2,768 _+ 210h 613 _+ 51i 160,066 + 21,735k

Control (6)

IFN-treated (6)

2,112 + 276 616 _+ 114b 263,976 _+22,220 2,219 + 258e 15,532 + 1,830 169,406 _+14,356 2,147 _+ 215h 1,198 _+ 134J 231,116 _+20,358k

563 _+ 172a 318 _+ 19b 105,268_+20,912c 1,211 _+ 244e,d 5,769_+ 1,096f 56,746 +_11,879g 1,611 _+ 309h 976 _+ 207ij 294,982_+59,655k

*Values are means _+SE of dpm/100 mg of liver proteins. The radioactivity in each phospholipid was measured 45 min after injection of 150 t~Ciof 1(3)-3H-glycerol/5g of mouse. Each chromatogram of labeled lipids was done in duplicate and the radioactivity was determined in each of the chromatograms. Number of parentheses indicates the number of mice per group. Values with a common superscript are not significantly different. LPC, lysophosphatidylcholine; PA, phosphatidic acid; SPH, sphingomyelin; DPG, diphosphatidylglycerol; TG, triacylglycerols. LIPIDS, Vol. 22, No, 10 (1987)

741

INTERFERON AFFECTS LIVER POLYENOIC PHOSPHOLIPIDS (from 32.5 to 26.3%) a n d a n o n s i g n i f i c a n t i n c r e a s e o f 20:4 a n d 22:6 i n t h e d i a c y l g l y c e r o l s (DG) of i n t e r f e r o n t r e a t e d m i c e ( T a b l e 6). T h e s e v a r i a t i o n s c a n n o t e x p l a i n , t h e r e f o r e , t h e int e r f e r o n - i n d u c e d d e c r e a s e i n t h e p r o p o r t i o n of p o l y u n s a t u r a t e d f a t t y a c i d s i n l i v e r PL. T h e s a m e a r g u m e n t s h o l d for t h e t r i a c y l g l y c e r o l s , w h o s e c o n c e n t r a t i o n w a s i n c r e a s e d five- to s i x f o l d b y i n t e r f e r o n t r e a t m e n t (2) a n d w h e r e o n l y a s i g n i f i c a n t i n c r e a s e of 18:1 w a s observed in the fatty acid composition between the two g r o u p s ( T a b l e 6).

DISCUSSION The experimental results presented herein emphasize t h e v e r y m a r k e d effects of i n t e r f e r o n o n l i p i d m e t a b o l i s m i n t h e l i v e r s of s u c k l i n g m i c e . I t s e e m s , t h e r e f o r e , t h a t t h e i n t e r f e r o n - i n d u c e d a l t e r a t i o n s a t t h e level of n e u t r a l l i p i d s a n d P L do n o t h a v e t h e s a m e o r i g i n . T h u s , we h a v e p r e v i o u s l y o b s e r v e d , o n o n e h a n d , a r e t e n t i o n of n e w l y f o r m e d t r i a c y l g l y c e r o l s i n t h e l i v e r w i t h o u t a n y m o d i f i c a t i o n of t h e i r f a t t y a c i d c o m p o s i tion and, on the other hand, a decrease in the total

TABLE 5 D i s t r i b u t i o n o f the l(3)-aH-Glycerol A m o n g Molecular S p e c i e s o f Liver P h o s p h a t i d y l c h o l i n e (PC) and P h o s p h a t i d y l e t h a n o l a m i n e (PE) in N e w b o r n Mice and 1 0 - D a y - O l d C o n t r o l and I n t e r f e r o n - T r e a t e d Mice a PC

PE

10-Day-old Molecular Species b

Newborn

Monoenoic Dienoic Tetraenoic Hexaenoic

30 32 21 17

10-Day-old

Control(6)c IFN-treated (6) Newborn 18 • 1 31 • 4 23 _+3 27 • 4

28 • 3* 35 • 3 19 + 2 18 _+2

17 16 31 38

Control (6) 8• 10 • 35 • 47 •

IFN-treated (6)

1 1 4 5

16 • 2* 19 • 2* 29 + 3 37 • 4

aResults are expressed as dpm percentage of the total dpm recovered from the four fractions separated on the chromatogram. bEach molecular species is defined by its most unsaturated fatty acid. CNumber in parentheses indicates number of determinations. The distribution of l(3)-3H-glycerol among PC and PE of different degrees of saturation at each time-interval was analyzed by argentation TLC. In lO-day-old suckling mice, the distribution of radiolabel did not change with time during the experimental period, and the results were the means • SM of analyses 5, 20 and 60 rain after injection of labeled glycerol (six pools of lipids from two animals). At birth, PC and PE were analyzed 45 rain after label injection (two pools of lipids from three animals each). *Value statistically different between control and IFN-treated, p ~ 0.01.

TABLE 6 Effect o f I n t e r f e r o n o n the Fatty Acid C o m p o s i t i o n o f 1 , 2 - D i a c y l g l y c e r o l and T r i a c y l g l y c e r o l in the L i v e r s o f 1 0 - D a y - O l d S u c k l i n g Mice a 1,2-Diacylglycerol Fatty acids

Triacylglycerol

Control (3)b

IFN-treated (3)

Control (6)

IFN-treated (6)

16:0 16:1

26.4 • 0.2 2.0 _+ 0.I

27.2 • 0.5 3.1 • 0.I

23.3 • 0.6 2.6 + 0.1

24.7 • 0.5 2.9 _+ 0.I

18:0 18:1 18:3~6

5.0 • 0.1 32.5 i 0.7 15.4 • 0.3 ND

4.8 • 0.1 26.3 _+0.7 14.2 • 0.5 ND

5.3 _+0.1 27.6 • 1.1 15.4 • 0.5 1.1 + 0.1

4.3 • 0.1 33.6 • 0.3 15.3 + 0.2 0.6 + 0.1

18:3~3 18:4 20:3

3.7 • 0.i ND i.I • 0.6

2.8 • 0.2 ND 1.2 • 0.I

4.6 • 0.2 I.i _+ 0.I 0.6 • 0.3

4.7 • 0.2 0.6 + 0.I I.i _+ 0.I

20:4~6 20:5oJ3 22:5~3 22:6~3

7.8 • 0.9 ND 0.4 -+ 0.1 3.2 • 0.3

10.7 • 0.8 ND 0.5 • 0.1 4.8 • 0.6

1.8 • 0.3 1.1 _+0.3 0.7 • 0.1 2.0 i 0.2

1.4 • 0.9 • 1.3 • 2.5 •

18:2e,6

0.1 0.1 0.1 0.1

aThe results expressed as the percentage of total fatty acids are means • SM. bNumber in parentheses indicates number of mice. ND, not detected. LIPIDS, Vol, 22, No, 10 (t987)

742

G. ZWINGELSTEINET AL. amount of PL (2). This latter effect was most pronounced for those PL containing polyunsaturated fatty acids in C20 and C22, which correlated with a decrease in the incorporation of 2-all-glycerol into polyunsaturated species of PL (this publication). At the same time, the concentration of precursors (1,2diacylglycerols) increases markedly without significant modifications in their renewal or in their composition in polyunsaturated fatty acids. Our present results m a y best be discussed relative to the pronounced metabolic changes that occur in the liver of normal animals during the postnatal period. Pollak and Harsas (25) observed an increase in the polyunsaturated fatty acids arachidonic (20:4oJ6) and docosahexaenoic (22:6o~3) and a decrease in the proportion of oleic acid (18:1) in the PL of liver mitochondria in 3-day-old rats as compared to newborn. Ogino et al. (26) showed that in suckling rats the polyunsaturated species of liver choline glycerophospholipids increased rapidly during the first days of life, in contrast to the decrease in the monoenoic species. We noted similar changes in the liver PL of control suckling mice in our experiments: an increase in arachidonic (20:4o~6) and docosahexaenoic (22:6oJ3) acids and a decrease in oleic acid (Tables 1 and 2). These physiological changes in the degree of unsaturation of liver PL fatty acids in the course of development in the first few days of life was markedly affected by daily administration of interferon. Thus, by the sixth to eighth day of life, interferon treatment resulted in a pronounced decrease in the percentage of polyunsaturated fatty acids in liver PL compared to control mice. In fact, the composition of PL fatty acids of these 6- or 10-day-old interferon-treated suckling mice remains similar to t h a t of newborn mice. The decrease in the polyunsaturated fatty acids in PL in the livers of interferon-treated mice appeared to be a specific effect and did not stem from a general decrease in the availability of polyunsaturated fatty acids. Thus, when one compares the ratio of fatty acid content of liver PL to the total amount of liver fatty acids, it was apparent that, for each fatty acid examined, this ratio was much lower for interferon-treated suckling mice t h a n for control mice (Table 3). Furthermore, the decrease in 18:1 and the accumulation of the 20:4 and 22:6 species in DG appears to be the result of the decreased incorporation of polyunsaturated fatty acids in the PL of interferon-treated mice. The second major effect of interferon on liver lipid metabolism concerned the amount of liver PL (2). In the neonatal period of several different animal species, selective changes occur in the activity and concentration of microsomal enzymes for the synthesis of liver PL and triacylglycerols (27-31). As shown in Table 4, interferon treatment inhibited the physiological increase in the s:}nthesis of liver PL. When the activity was expressed per 100 mg of protein, the value in 10-day-old control mice was double t h a t of newborn or interferon-treated mice. This effect of interferon on PL synthesis was especially marked for PC and PE. Induction of the synthesis of diacylglycerol-cholinephosphotransferase and diacylglycerol-ethanolaminephosphotransferase occurs in rats during the first days of life (27,31,32). This increase in the activity of the PC and PE synthesis is probably accompanied by a change in the specificity of the enzymes toward the LIPIDS,Vol. 22, No, 10 (1987)

different molecular species of diacylglycerol (33). In this regard, it has recently been reported t h a t there is a significant increase in the production of liver microsomal PC, with an increasingly higher proportion of unsaturated fatty acids in the perinatal period (26, 34,35). In parallel, there is also an increase in the activity of the PC synthesis by N-methylation of PE (27), a metabolic p a t h w a y t h a t produces molecular species with a high content of polyunsaturated fatty acids (22,36,37). It is also possible that interferon treatment resulted in an inhibition of the reacylation of lysophospholipids formed by hepatic phospholipases (38-40). In fact, the microsomal lysophospholipid acyltransferases, which can remodel PL by transferring fatty acids on C2 of the glycerol of lysophospholipids, show considerable activity toward polyunsaturated acyl-CoA (38-42). The results in Table 5 suggest t h a t interferon treatment of suckling mice inhibits this change in the molecular specificity of the biosynthetic enzymes. For example, the inhibitory effect of interferon is especially evident in the molecular species of PC and PE, having the greatest percentage of polyunsaturated fatty acids in C20 and C22. The liver content of these molecular species is about h a l f as much in 10-day-old interferon-treated mice (1.66 ~mol per liver) as in 10day-old control mice (3.28 ~mol) (Table 4). The inhibition of the synthesis of polyunsaturated PL in interferon-treated mice can be related to the ultrastructural observations of Moss et al. (3,4), who showed t h a t interferon treatment of suckling mice resulted in the rapid appearance of characteristic tubular aggregates associated with the granular endoplasmic reticulum. In the postnatal period, there is also a rapid development and increase in the endoplasmic reticulum, which is the principal site of PL synthesis in hepatocytes (25,43,44). One m a y suggest that interferon alters the normal development of the endoplasmic reticulum, which would then limit the growth of enzymatic systems necessary for the synthesis of PL, or that interferon inhibits the synthesis of those enzymes necessary for PL synthesis, which, in turn, would limit the construction of new membranes essential for the developing endoplasmic reticulum (32). Lastly, interferon t r e a t m e n t results in a very marked increase in liver diacylglycerol level. The diacylglycerol accumulation in response to hormonal stimulation has been shown using a variety of cells and hormones (45-47). Diacylglycerol serves as a substrate for lipases or as a precursor in PL and triacylglycerol synthesis. But it can also act as a second messenger and can regulate protein kinase C activity (47-48). The increase in the hepatic diacyglycerol concentration in interferon-treated suckling mice m a y alter the metabolic response of the liver to different hormonal stimuli in the postnatal period (49). Indeed, Wolfman and Macara (50) have recently observed t h a t the ras-transformed fibroblasts possess elevated diacylglycerol levels and have decreased sensitivity to phorbol ester activation of protein kinase C. In Daudi cells, which h a d been prelabeled with [23H]-glycerol, Yap et al. (51,52) have observed a very early and transient two- to threefold increase in the concentration of radiolabel in DG of interferon-treated cells. Interferon in concentration exceeding 2 I U / m l

743

INTERFERON AFFECTS LIVER POLYENOIC PHOSPHOLIPIDS i n h i b i t s t h e division of cells, a n d the degree o f i n h i b i tion c o r r e s p o n d s closely to the size of the i n c r e a s e in d i a c y l g l y c e r o l label (52). I n n e w b o r n mice, interferon t r e a t m e n t does n o t result in a n i n h i b i t i o n of liver g r o w t h (2), a n d the specific r a d i o a c t i v i t y of liver DG after injection of (1,3-3H)-glycerol a p p e a r e d identical between the two g r o u p s of mice. I n fact, in vivo, the i n c r e a s e in the c o n c e n t r a t i o n of DG reflects a more complex a c t i o n of i n t e r f e r o n on the m e t a b o l i s m of lipids in the liver. I n t e r f e r o n p r o b a b l y alters in the newborn m o u s e the h e p a t i c p a t h w a y s r e s p o n s i b l e for f o r m a t i o n a n d utilization of DG. This ensemble of results, especially the decrease in liver p o l y u n s a t u r a t e d P L a n d i n c r e a s e in DG w o u l d a p p e a r to h a v e c o n s e q u e n c e s for the m a t u r a t i o n of h e p a t o c y t e s in s u c k l i n g mice. I n m a n y respects, the h e p a t o c y t e s of 10-day-old interferon-treated suckling mice r e s e m b l e d n e w b o r n mice b i o c h e m i c a l l y (rather t h a n control 10-day-old littermates). On the o t h e r h a n d , the fact t h a t i n t e r f e r o n interferes w i t h the synthesis of c e r t a i n p o l y u n s a t u r a t e d species of P L in the liver (especially c e r t a i n species of p o l y e n o i c PC a n d PE) of s u c k l i n g mice m a y lead to a decrease in the g e n e r a l a v a i l a b i l i t y of p o l y u n s a t u r a t e d species of t h e s e P L i n d i s p e n s a b l e for the d e v e l o p m e n t of other tissues (35,53). This decrease in p o l y u n s a t u r a t e d f a t t y acids a v a i l a b i l i t y m a y be related to other interferoni n d u c e d lesions, w h i c h o n l y become m a n i f e s t later in life (54-56). ACKNOWLEDGMENTS This work was supported in part by grants from the Richard Lounsbery Foundation, the Foundation pour la Recherche M~dicale, the Fondation Simone et Cino del Duca, INSERM (contract 832 034), the DRET (contract 85.055), the CNRS and the Association pour le ddveloppement de la Recherche sur le Cancer ~ Villejuif. Thanks to the Chambre de Commerce et de l'Industrie de Toulon for the gift of gas liquid chromatography apparatus to the Laboratoire Maritime de Physiologie of Claude Bernard University. REFERENCES 1. Gresser, I., Tovey, M.G., Maury, C., and Chouroulinkov, I. (1975) Nature 258, 76-78. 2. Zwingelstein, G., Meister, R., Abdul Malak, N., Maury, C., and Gresser, I. (1985) J. Interferon Res. 5, 315-325. 3. Moss, J., Woodrow, D.F., Sloper, J.C., Riviere, Y., Guillon, J.C., and Gresser, I. (1982) Br. J. Exp. Pathol. 63, 43-49. 4. Moss, J., Woodrow, D.F., and Gresser, I., (1984) Histochemical J. 17, 33-41. 5. Coleman, R. (1973) Biochim. Biophys. Acta 300, 10-30. 6. Chandrabose, K., Cuatrecasas, P., and Pottathil, R. (1981) Biochem. Biophys. Res. Com~un. 98, 661-668. 7. Apostolov, K., and Barker, W. (1981) FEBS Lett. 126, 261-264. 8. Apostolov, K., and Barker, W. (1984) Ann. Virol. 134E, 245-256. 9. Pfeffer, L.M., Landsberger, F.R., and Tamm, I. (1981) Interferon Res. i, 613-620. 10. Tovey, M.G., Begon-Lours, J., and Gresser, I. (1974) Proc. Soc. Exp. Biol. Med. 146, 809-815. 11. Folch, J.F., Less, M., and Sloane-Stanley, G.H. (1957) J. Biol. Chem. 226, 497-509. 12. Coleman, A. and Haynes, E.B. (1984) J. Biol. Chem. 259, 8934-8938. 13. Skipski, V.P. (1975) Method Enzymol, 35B, 396-425. 14. Anderton, P., Wild, T.F., and Zwingelstein, G. (1981) Biochem. Biophys. Res. Commun. 103, 285-291.

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