BIOCHIMIE, 1978, 60, 743-753.

Ribonucleic acid synthesis in rat liver during fatty acid stimulated secretion of very low density lipoproteins. Alain RAISONNIER, Marie-ElisabeCh BOUMA, Colette SALVAT and Recaredo INFANTE . (25-3-1978).

Unite de Recherches d'Hipatologie de I'I.N.S.E.R.M. (U-9) and Laboratoire de Biochimie, U.E.R. de Midecine Saint-Antoine, 186, rue du Fg St-Antoine, F-75571 Paris Cedex 12, France.

Rdsumd.

Summary.

La production des lipoprot6ines de tr6s b a s s e densit6 par le foie d6pend de la disponibilit6 en acides qras pour la cellule. EUe est stimul6e par la captation d'acides qras libres du plasma ou par l'augraentation de la lipocj6n6se et, est inhib6e par l'actinomycine D suqq6rant l'exisfence d'une r6qulation de la synth6se des apoprot6ines au niveau de la synth~se des RNA. Cette hypoth6se a 6t6 v6rifi6e dens le foie isol6 et perfus6 et chez le rat in v i v o avec ou sans stimulation par un apport suppl6mentaire en acides qras. L'incorporation de [14C] orotate dans les RNA des polyribosomes du foie est auqment6e de 60 p. cent dens les rotes de rats surcharq6s en acides qras par rapport aux t6moins. Cette auqmentation est principalement le r6sultat d'une plus active incorporation dens les polyribosomes li6s et ceux comportant au moths 6 ribosomes ; elle n'est pas le r6sultat d'une inhibition de la ribonucl6ase. La diqestion du RNA polyribosomal par la RNase (4.10 -10 M enzyme, 0°C, 3 h) montre que la radioactivit6 des RNA hydrolys6s est deux lois plus forte chez les animaux surcharq6s en acides qras que dens les rotes t6moins. Lorsque l'on purifie partiellement p a r chromatoqraphie d'affinit6 les RNA riches en poly(A), la r6cup6ration en masse et en radioactivit6 est deux fois plus importante que dans les rotes t6moins, lorsque le foie a 6t6 stimul6. Une synth6se nouvelle de RNA semble donc n6cessaire au d6clenchemeat p a r les acides qras de la production des VLDL.

Production of v e r y low density lipoproteins b y the liver depends on the cellular availability of fatty acids. It is stimulated b y the uptake of free fatty acids from the plasma and b y increased lipoqenesis a n d is inhibited b y actinomycin D, suqqestinq that RNA synthesis is involved in the requlation of apolipoprotein synthesis. This hypothesis has been investiqated in rats in v i v o and in isolated perfused livers with a n d without stimulation b y fatty acid overload : [14C] orotate incorporation in liver polyriboso. real RNA is 60 per cent qreater in stimulated livers as compared to controls. This increase is primarily due to a hiqher incorporation in bound polysomes and in those containinq at least six ribosomes a n d does not result from the inhibition of ribonuclease. RNase diqestion of polysomal RNA (4.10 -l° M enzyme, 0°C, 3 h) shows that there is twice as much radioactivity in the hydrolyzed RNA of stimulated livers as compared to controls. After partial purification of poly A-rich RNA b y affinity chromatoqraphy, the mass yield and radioactivity are increased b y 100 per cent in stimulated livers as compared to controls. In conclusion, de n o v o RNA synthesis seems to be n e c e s s a r y for fatty acid stimulation of VLDL production.

0 To w h o m all correspondence should be addressed. Part of this work was reported at the F o r u m International Francophone de Recherches en Gastroentdrologie, Sherbrooke (Can.) in June, 1975, and published in abstract form in Biol. Gaslroenterol. (1975), 8, 169.

Abbreviations. RNase : Ribonuel6ase. Poly (A)-RNA : Ribonueleic acid with a polyadenylie sequence. TKM : T r i s - KC1 - MgCl~ b u f f e r . LDL : Low density lipoproteins (d: 1.063-1.006 g/l). VLDL : Very low density lipoproteins (d < 1.006 g / l ) . Enzymes. Glueose-6-phosphatase E.G. 3.1.3.9. N A D H - e y t C - o x y d o r e d u c t a s e E.G. 1.6.99.2. Ribonucleas,e A E.C. 2.7.7.16.

744

A . R a i s o n n i e r a n d coll.

Introduction. The i n c r e a s e of p l a s m a free fatty a c i d levels in vivo or in isol,ated p e r f u s e d l i v e r prep,arations is f o l l o w e d b y a s h a r p rise of pl~asma t r i a c y l g l y c e r o l c o n c e n t r a t i o n s [1-5]. In the same conditi,ons K a y a n d Ent.enman [6] d e m o n s t r a t e d an i n c r e a s e of t r i a c y l g l y c e r o l - r i c h v e r y low dens.try l i p o p r o t e i n s (VL.DL, d < 1.006 g/l), su,ggesting an e n h a n c e d s e c r e t i o n of these ] i p o p r o t e i n s b y the liver. In 1968, it was s h o w n [7-8] that w h e n the perfusate of isolated livers w,as s u p p l e m e n t e d w i t h fatty a c i d s c o m p l e x e d to albumin, the net synthesis of VLDL r e l e a s e d in the p erfusat.e w a s e n h a n ced as e s t i m a t e d b y r a d i o a c t i v e l e u c i n e a n d fatty a c i d inc,orporations. This in,creased VLDL synthesis seemed to be specific, for u.either the conc e n t r a t i o n n o r the r a d i o a c t i v i t y of h i g h d e n s i t y l i p o p r o t e i n s (d < 1.21 g/l) or ~albumin differed from controls. Conversely, in isolated r a t l i v e r a g l y c e r o l overl o a d [9] is follow.ed b y ,a d e c r e a s e d ~LDJ~ synthe_ sis p r e s u m a b l y due to a d e p l e t i o n ~f ,an i n t r a c e l l u tar f a t t y acid p o o l w h i c h ,is s u p p o s e d to c o n t r o l apo-VLDL synthesis. This i n h i b i t i o n can be r e v e r s e d 'when the p e r fusate is s u p p l e m e n t e d in fatty a c i d s [10]. E l e c t r o n microscopi, c studi.es s h o w e d t h a t enh a n c e d VLDL secretio~n w a s a c c o m p a n i e d b y the filling of smooth ,endoplasmic ret,iculum vesicl'es a n d the Golgi a p p a r a t u s of liver cells b y n u m e r o u s s p h e r i c a l o s m i o p h i l i c p,articles w h i c h w e r e chem i c a l l y a n d i m m u n o l o g i c a l l y identified as VLDL m o l e c u l e s [11]. P u r o m y c i n , a,n i n h i b i t o r of mRNA t r a n s l a t i o n , s u p p r e s s e s h e p a t i c s e c r e t i o n of VLDL, suggesting that de novo s y n t h e s i s of apoprotei.n is necess'ary for l i p o p r o t e i n p r o d u ~ i o n [12]. F u r t h e r more, a c t i n o m y c i n D [:8] a n d a~amanitiu [13], i n h i b i t o r s of RNA p o l y m e r i z a t i o n , p r e v e n t e d the fatty a c i d stimu~at~ion of h e p a t i c s e c r e t i o n of VLDL. Therefore, "~ve suggested t h a t s y n t h e s i s of n e w RNA molecules is r e q u i r e d for VL;D.L p r o d u c t i o n after fatty a c i d overload. E x p e r i m e n t s w i t h transcripti.on i n h i b i t o r s , h o w e v e r , must be int.erpreted w i t h c a u t i o n in view of r e c e n t d a t a c o n c e r n i n g o t h e r effects of a c t i n o m y c i n D on RNA t u r n o v e r . In 1968, F M o o n a el aI. [14] s h o w e d that the YLD~L + LDL c o n c e n t r a t i o n in r a t s e r u m decreaBIOCItIMIE, 1978, 60, n ° 8.

s,e,d in vivo after a d m i n i s t r a t i o n of actin~omycin D. Simultaneously, [l~C].orotic a c i d i n c o r p o r a t i o n in T C A - p r e c i p i t a b l e m a t e r i a l of the l i v e r w a s i n h i b i t e d 95 p e r cent a n d [3H]lysin.e i n c o r p o r a t i o n in VLDL -4- LDL d e c r e a s e d 50 p e r cent w i t h i n 8 hours. In p e r f u s e d livers p r e t r e a t e d ~vith actinom y c i n D, [3H]lysin.e .in,corporation in VLDL apol i p o p r o t e i n s w a s 83 p e r c e n t t h a n controls. Actin o m y c i n D adde,d to the p e r f u s a t e d i d "~ot affect the basa,l rate of V:LDL + LD'L secretion. E x p e r i m e n t s u s i n g a c f i n o m y c i n D in o r d e r to d e m o n s t r a t e the p a r t i c i p a t i o n of t r a n s c r i p t i o n in an e x p e r i m e n t a l m o d e l of p r o t e i n s y n t h e s i s are open to c r i t i c i s m . This is because of c e r t a i n sideeffects .of the drug, such as p o l y s o m a l disaggregatio.n [15] w h i c h is ind,ependent oi the n o r m a l c a t a b o l i s n l of c y t o p l a s m i c mRNA f o l l o w i n g transc r i p t i o n i n h i b i t i o n [16]; m o r e o v e r , a c t i n o m y c i n D actiwates r i b o n u c l e a s e s [17], i'n~erferes "with the t r a n s p o r t of n u c l e o t i d e p r e c u r s o r s [18], r e t a r d s m e s s e n g e r r i b o n u c l e o p r o t e i n t r a n s f e r from the nucleus to the c y t o p l a s m [19, 20] and fin~dly, p r o longs c y t o p l a s m i c a.nd p o l y s o m a l mBN'A half-lives [21, 22]. A n y of these side effects could s t i m u l a t e the a p p a r e n t t u r n o v e r rate of h e p a t i c RNA [8]. The p r e s e n t e x p e r i m e n t s involve ,a m o r e d i r e c t a p p r o a c h to this p r o b l e m b y e s t i m a t i n g RNA synthesis b y the incorporatio,n of active p r e c u r s o r s into rat liver p o l y s o m e s after stimul.ation of ¥ L D L s y n t h e s i s b y a fatty a c i d o v e r l o a d . The pcrlysomal RNA content was i n c r e a s e d u n d e r these c o n d i t i o n s a n d analysis b y RNase digestio,n and affinity c h r o m a t o g r a p h y sho~ved t h a t n e w l y s y n t h e s i z e d RNA w a s prim,arily of the m e s s e n g e r type.

Materials and Methods. Chemicals and radioactive precursors.

Sprague Dawley rats (Charles River, France) weighing 250-3!50 g and maintained on a standard diet were used in all experiments. [6-14C] orotic acid (specific radioactivity 56. mCi/ mmoIe) was. purchased from the C.E.A., Saclay, France. Deoxycholic acid, R~ibonuclease A (bovine pancreas) and polyuridylic acid were obtained from Sigma (Saint Louis, Mo. U.S.A.) I4uman serum albumin (Cohn's fraction V) was obtained from the Centre National de Transfusion Sanguine (Paris, France). Other reagents included oleic acid (Mlerck, Darmstadt, Ciermany) ribonuclease-frce sucrose (B. D. H. Poole, England) and CN.Br-activated Sepharose 4B (Pharmacia, Uppsala, S~weden).

RNA synthesis in rat liver during VLDL secretion. Stimulation of VLDL production. Two different techniqucs VLDL s y n t h e s i s .

were

used

to

stimulate

1) I s o l a t e d r a t l i v e r s w e r e p e r f u s e d [23] w i t h 33 p e r c e n t (v/v) r a t b l o o d d i l u t e d w i t h K r e b s - R i n g e r b i c a r b o n a t e b u f f e r , p H 7.35, 0.75 p e r c e n t ( w / v ) g l u cose, 4.4 p e r c e n t (w/x0 h u m a a s e r u m a l b u m i n a n d 100 btmoles of oleic a c i d c o m p l e x e d to a l b u m i n [24]. C o m p l e x e d oleic acid w a s e l i m i n a t e d perfusates. 2) U n a n a e s t h e t i z e d r a t s w e r e f e d fresh cream containing more than ceride i.e. m o r e t h a n 2 m E q f a t t y tube. Animals were exsanguinated C o n t r o l r a t s w e r e f e d 2 m l of gastric tube.

from control

w i t h 2 m l of t h i c k 30 p e r c e n t t r i g l y acids, w i t h g a s t r i c after three hours. 0.15 M NaG1 w i t h

Radioactive labeling of nucleic acids. T h e i n c o r p o r a t i o n of [6-14C] orotic acid w a s p r e f e r r e d to 32p i n c o r p o r a t i o n , s i n c e P m e t a b o l i s m is a l t e r e d d u r i n g l i p i d d i g e s t i o n [25]. 100 IxCi of 6-[14C] o r o t a t e w e r e a d m i n i s t e r e d to livers simultaneously with the fatty acid overload. A f t e r o n e h o u r of i n c o r p o r a t i o n , t h e specific r a d i o a c t i v i t i e s of a c i d - s o l u b l e n u c l e o t i d e s i n t h e cell s a p a n d n u c l e i a n d t h e d i s t r i b u t i o n of r a d i o a c t i v i t y in u r a c i l a n d c y t o s i n e w e r e c a l c u l a t e d [26, 27] b y r a d i o activity and phosphate determinations after thin layer c h r o m a t o g r a p h y [28]. I n e x p e r i m e n t s in vivo, 50 t~Ci of [6-14C] o r o t a t e w e r e i n j e c t e d i n t r a p e r i t o n e a l l y ').5 hours, a f t e r t h e f a t t y m e a l a n d 1 h o u r b e f o r e t h e r a t s w e r e sacrificed by exsanguination.

Preparation of polysomes. Rat liver polysomes were prepared with the proced u r e d e s c r i b e d b y H e r r i m a n et al. [29]. A f t e r a t h o r o u g h w a s h i n g w i t h 0.15 M NaC1 to r e m o v e blood, t h e l i v e r w a s h o m o g e n i z e d i n T K M b u f f e r (5.10'-2 M T r i s - H C l , p H 7.4, 2.5.19-2 ~ KC1 a n d 5.10-~ M MgCl.) c o n t a i n i n g 0.25 ~ s u c r o s e . T h e s u p e r n a t a n t of a 10 009' )< g, 2@ m i n c e n t r i f u g a t i o n w a s r e c o v e r e d . The postmitochondrial supernatant contained only 50 p e r c e n t of t h e m i c r o s o m e s of t h e c r u d e h o m o g e n a t e , b u t w a s f r e e of n u c l e i a n d m i t o c h o n d r i a . T h e

745

microsome yield was determined with two enzyme m a r k e r s : g l u c o s e - 6 - p h o s p h a t a s e (E.C. 3.1.3.9.) a n d NA~DH-cytochrome C - o x i d o r e d u c t a s e (E.C. 1.6.99.2.) [30, 31]. M i c r o s o m e s w e r e f u r t h e r s o l u b i l i z e d i n Na d e o x y c h o l a t e (final c o n c e n t r a t i o n 1.3 g / 1 0 0 m l ) a n d 6.6 m l of t h i s s o l u t i o n w e r e l a y e r e d o v e r a d i s c o n t i n u o u s s u c r o s e g r a d i e n t ( 3 5 m l of 2.0 M s u c r o s e in T K M a n d 1 m l of 1.(} M s u c r o s e in TKM). A f t e r 17 h o u r s of c e n t r i f u g a t i o n a t 14ti,000 × g t h e p e l l e t o b t a i n e d w a s r e s u s p e n d e d in 0.25 M s u c r o s e i n TKM. Free cytoplasmic polysomes were separated from t h o s e b o u n d to t h e e n d o p ] a s m i c m e m b r a n e s as f o l l o w s : 6.6 m l of t h e p o s t m i t o e h o n d r i a l s u p e r n a t a n t ( b e f o r e t h e a d d i t i o n of d e o x y c h o l a t e ) w e r e l a y e r e d o v e r 4.5 m l of 2.0 1~ s u c r o s e in T K M a n d c e n t r i f u g e d f o r 17 h o u r s a t 145,0@0 × g. T h e p o l y s o m e s r e t a i n e d at t h e i n t e r f a c e of t h e t w o s o l u t i o n s w e r e a l m o s t e n t i r e l y m e m b r a n e - b o u n d . T h e pellet, on t h e c o n t r a r y , c o n t a i n e d a b o u t 65 p e r c e n t f r e e p o l y s o m e s a n d 35 p e r c e n t b o u n d - p o l y s o m e s .

Analysis of polysomes. P o l y s o m e profiles w e r e o b t a i n e d b y l a y e r i n g 1 to 2 m g of t h e s u s p e n s i o n s o n l i n e a r s u c r o s e g r a d i e n t s (0.3 M to 1.0 M s u c r o s e i n TKM) a n d c e n t r i f u g i n g a t 70,000 × g f o r 3 h o u r s [32]. C o n c e n t r a t i o n of t h e h e a v i e s t f r a c t i o n s at t h e b o t t o m of t h e t u b e s w a s p r e v e n t e d b y a c u s h i o n of 2.0 M s u c r o s e . T h e r a d i o a c t i v i t y of g r a d i e n t f r a c t i o n s a n d of t o t a l p o l y s o m e s w e r e d e t e r m i n e d b y m i x i n g w i t h 10 m l of B r a y ' s s o l u t i o n [33] f o l l o w e d b y c o u n t i n g i n a l i q u i d s c i n t i l l a t i o n ~ s p e c t r o m e t e r (SL 30, I n t e r t e c h n i q u e , France).

Ribonuclease digestion of RNA. A v e r y m i l d h y d r o l y s i s o f t h e p o l y s o m a ] I~NA b y r i b o n u c l e a s e (E.C. 2.7.7.16, S i g m a ) w i t h t h e t e c h n i q u e of T o m i n a g a et al. [21] w a s u t i l i z e d . R i b o n u e l e a s e (1 n g / m g r i b o s o m a l p r o t e i n , 4.10 ~10 M) w a s a l l o w e d to r e a c t f o r 3 h o u r s a t 0°C. T h e r a d i o a c t i v i t y of t h e a c i d precipitated RNA was determined before and after digestion.

Extraction and determination of nucleic acids. Affinity chromatography, at

RNA c o n c e n t r a t i o n w a s d e t e r m i n e d b y a b s o r p t i o n 260 n m [34]. More p r e c i s e m e a s u r e m e n t s w e r e

TABLE I.

[14C]leucine incorporation in VLDL apoprotein, albumin and liver protein in per[used liver preparations. Time oI periusion rain.

Control cpm. 10 -~

Stimulated liver cpm. t0 -~

Apo-VLDL

30 60 90

8 1 . 3 ___~ 1 6 . 1 2 4 0 . 7 ___. 3 1 . 2 3 8 5 . 6 -4- 3 4 . 0

9 6 . 3 -4- 2 2 . 0 3 1 9 . 1 -4- 4 2 . 6 4 6 6 . 6 --t- 4 6 . 5

Albumin

90

5 157 --i- 6 7 9 . 2

5 4 3 1 . 0 -4- 7 2 5 . 2

Liver protein

90

52 750 ~___ 16 475

43 780 ----t- l l 935

M e a n s -4- S.E.M., f r o m 6 p e r f u s i o n s p e r g r o u p . I n c o r p o r a t i o n w a s c a l c u l a t e d as r a d i o a c t i v i t y i n V L D L or a l b u m i n i n t h e t o t a l p e r f u s a t e a n d in TCA p r e e i p i t a b l e p r o t e i n s of whole liver.

BIOCHIMIE, 19'78,

60, n ° 8.

51

A. R a i s o n n i e r a n d coil.

746

10-2 M Tris-H'C1, p H 7.4, 0.5 p e r c e n t d o d e c y l s u l f a t e ) a t 4°C. P o l y ( A ) - c o n t a i n i n g RS~,A h y b r i d i z e s to poly(U) u n d e r t h e s e c o n d i t i o n s [40]. A f t e r w a s h i n g t h e c o l u m n w i t h t h e s a m e Tris-HC1 b u f f e r b u t w i t h o u t LiC1 at 15°C, R N A w a s e l u t e d w i t h t h e s a m e b u f f e r b y i n c r e mental temperature increases until hybrid melting t e m p e r a t u r e (50°C) w a s r e a c h e d .

TABLE II.

[~C]orotic acid incorporation (100 ~Ci ; 1 hr) into acid soluble nucleotides of subcellular fractions from perfused livers and relative distribution of radioactivity between Uracil (U) and cytosine (C),

P u r i f i e d p o l y ( A ) - R N A was, t h u s r e c o v e r e d .

Cytosol Nuclei Totalliver

I U C

Controls

Stimulated livers

19 122 30 402 8 7 . 4 per c e n t 12.6 per cent

18 757 33 329 8 6 . 3 per c e n t 13.7 per cent

R N A w a s a n a l y z e d in t u b e s c o n t a i n i n g 35 m l of l i n e a r s u c r o s e g r a d i e n t s ~0.3 to 0.9 M) o v e r a 5 m l c u s h i o n of 2 M' s u c r o s e to a v o i d t h e s e d i m e n t a t i o n of t h e h e a v i e s t p a r t i c l e s . T h e t u b e s w e r e c e n t r i f u g e d at 25,000 r p m a t 4°C f o r 17 h i n a n S W 27 r o t o r in a S p i n c o L 5(~ u l t r a c e n t r i f u g e . N o n - r a d i o a c t i v e t o t a l r a t l i v e r R N A w a s a d d e d to the tubes and used as internal molecular weight markers.

R a d i o a c t i v i t y is e x p r e s s e d as c p m p e r 0.5 m g n u cleotides.

E l u t i o n w a s p e r f o r m e d w i t h a n ISCO a p p a r a t u s . Optical density (A~o)was continuously recorded and t h e e f f l u e n t w a s c o l l e c t e d i n 1.2 m l f r a c t i o n s f o r r a d i o a c t i v i t y d e t e r m i n a t i o n b y s c i n t i l l a t i o n in B r a y ' s s o l u t i o n [331.

o b t a i n e d w i t h t h e o r c i n o l m e t h o d [36] p r e v i o u s l y calib r a t e d w i t h a p h o s p h o r u s a s s a y [36]. Nucleic acids were extracted fronl polysomal fract i o n s w i t h p h e n o l - c h l o r o f o r m [37, 38]. R N A o b t a i n e d w a s m a d e u p t o 0.4 M NaC1, 5.10-3 M M gCl~ a n d 1 per cent :sodium dodecyl sulfate and was then prec i p i t a t e d a t - - 2 0 ° G w i t h 2 v o l u m e s of e t h a n o l : it c o u l d be s t o r e d a s s u c h f o r s e v e r a l m o n t h s .

Ultrastructural examinations. Liver biopsies were sampled during experiments for histological examination after staining with Masson's trichrome. O t h e r s f r a g m e n t s w e r e r a p i d l y sliced a n d d o u b l y fixed w i t h o s m i c acid a n d g l u t a r a l d e h y d e . T h e y w e r e t h e n e m b e d d e d i n ]~pon, c u t i n u l t r a t h i n s e c t i o n s , contrasted with lead citrate-uranyl acetate and observed and photographed with a Siemens Elmiskop electron microscope.

R N A c o n t a i n i n g a t e r m i n a l poly(A) s e q u e n c e w a s i s o l a t e d f r o m t o t a l p o l y r i b o s o m a l P~NA b y a f f i n i t y chromatography on Sephadex 4B-poly(U) columns. P o l y ( U ) w a s b o u n d to S e p h a r o s e w i t h t h e m e t h o d of L i n d b e r g [39]. P o l y s o m a l R N A w a s t h e n l o a d e d o n t h e c o l u m n i n h i g h i o n i c s t r e n g t h b u f f e r (0.1 M LiC1,

TABLE I I I .

Specific radioactivity of rat liver polysomes. Labelling procedure W h o l e p o l y s o m e s (8)

Controls

(in vivo)

440 ~

W h o l e p o l y s o m e s (3) ( p e r f u s e d liver)

Stimulated livers

61

680 i - 165 (p ~ 0 . 0 0 1 ) 1 060 -t- 337 (p ~ 0 . 0 5 )

750 -I- 148

P o l y s o m e s w e r e o b t a i n e d f r o m r a t l i v e r s a f t e r 3 h of i n t r a g a s t r i c a d m i n i s t r a t i o n of s a l i n e or a f a t t y l i q u i d m e a l or a f t e r 1 h of p e r f u s i o n w i t h s t a n d a r d or f a t t y a c i d s u p p l e m e n t e d p e r f u s a t e . [14C]orotic a c i d i n c o r p o r a t i o n b e g a n 1 h o u r b e f o r e p o l y s o m e p r e p a r a t i o n . R e s u l t s a r e e x p r e s s e d a s c p m p e r Awo u n i t . M e a n s ___ S.E.M. n u m b e r of a n i m a l s i n b r a c k e t s . TABLE IV.

RNase activity in liver homogenates from control and fatty acid fed rals using yeast RNA (27,000 daltons) as substrate. V max lag RNA. mn -1. m g -I

Km RNA m y . ml -I

Km RNA t0-SM

Control

186

1,18

4,4

F a t t y acid overload

180

1,17

4,3

V m a x . w a s c a l c u l a t e d f o r 1 m g of h o m o g e n a t e p r o t e i n .

BIOCHIMIE, 1978, 60, n ° 8.

R N A s y n t h e s i s in rat liver d u r i n g V L D L secretion.

F16. l. - - Section of liver tissue, three hours after experimental fatty meal. S o m e VLDL p a r t i c l e s are seen in t h e s m o o t h - s u r f a c e d e n d s of the r o u g h e n d o p l a s m i c r c t i c u l u m (RER). F i n a l m a g n i f i c a t i o n 56.060 × ; b a r : 0.5 ~t.

BIOCHIMIE, 1978, 60, n ° 8.

747

A. Raisonnier

748

and coll.

Fro. 2. - - Section of liver tissue, three hours after e x p e r i m e n t a l f a t t y meal. T y p i c a l a s p e c t s of VLDL s e c r e t i o n a r e s e e n i n s m o o t h e n d o p l a s m i c r e t i c n l u m (SER) a n d in Golgi a p p a r a t u s (G). F i n a l m a g n i f i c a t i o n I~0L0:0' × ; b a r = 0.5 ~.

BIOCHIMIE,

1978, 60, n ° 8.

R N A s y n t h e s i s in rat liver d u r i n g V L D L secretion.

749

w h e n the results were calculated as r a d i o a c t i v i t y per g of fresh tissue or per A2e,o unit. F u r t h e r m o r e , c o m p a r a b l e results ~'ere o b t a i n e d in the intact

Results.

STIMULATION OF VLDL BIOSYNTHESIS. TABLE V. Three h o u r s after the a n i m a l s h a d been fed a fatty meal, the mitky a p p e a r a n c e of lhe mesenteric l y m p h a t i c vessels i n d i c a t e d a marked absorption of lipids. E l e c t r o n m i c r o g r a p h s of liver sections from these a n i m a l s showed n u m e r o u s VLDL particles in the Golgi apparatus a n d endop l a s m i c r e t i c u l u m vesicles, occasio~n,ally i n close p r o x i m i t y to r i b o s o m e - r i c h regions (fig. 1 and 2), a d m i t t e d l y i n d i c a t i v e of active lipopro~ein production. According to p u b l i s h e d data [8], fatty acid overload i.n perfused livers increases the biosynthesis of VLDL apolipopeptides as judged by [14C] leucine in.corporation. The specific r a d i o a c t i v i t y of [I¢C]}eucine isolated from liver extracts of fatfed or fasted a n i m a l s was s i m i l a r ( m e a n : 6,106 c p m / m g of leucine). L e u c i n e i n c o r p o r a t i o n in VLDL apoproteins, however, i n c r e a s e d in fed-rat livers (tabte I), but tot.al liver p r o t e i n an,d plasma a l b u m i n r a d i o a c t i v i t y were u n c h a n g e d . OROT1C ACID INCORPORATION IN POLYSOMAL RNA. Uptake of [6-14Clorotic acid a n d p y r i m i d i n e metaboHsni by cells are a p p a r e n t l y unaffected dur i n g the s t i n m l a t i o n of VLDL p r o d u c t i o n in the perfused liver (table II). The yield of total polysomal RNA frouI the livers of fatty acid overloaded and control groups was siinidar. Radioactivity in polysonles d e t e r m i n e d one h o u r after orotic acid injection was significantly increased in the stimulated livers (table III),

Specific radioactivity (cpm per A2~o unit) of free or bound polysomes after 1 hour of [l~C]orotic acid incorporation in control rats or 3 hours of incorporation after a fatty meal. Control Whole polysomes

580

960

Membrane-bound poiysomes

486

1 204

Free polysomes (')

51~7

830

(*) Bound polysomal contamination of the free fraction was 30 per cent. TABLE VI.

Distribution of total radioactivity among short, medium and long polysomes after incorporation of [~C]orotic acid for I hour. Controls

Stimulated livers

299

358

,~,~

Whole polysomes (from perfused liver) 3 to 6 ribosomes 7 and more ribosomes

126 85

61 135 162

Whole polysomes (from in oivo experiments)

656

961

1,2 ribosomes 3 to 6 ribosomes 7 and more ribosomes

295 '290 11)1

291 491 254

1 , 2 ribosomes

Results are given in cpm per A=,~ unit.

TABLE VII.

Radioactivity of entire polysomes from conlrols and fatty acid overloaded (in viva or in perfusion) rat livers, before and after mild ribonaclease digestion (#.10 -~o M). In viva

Perlusioas

Controls

Fatty meal

Controls

Fatty acid overload

Whole polysumes

837

I 308

897

i 392

Polysomes after digestion

449

382

331

278

Digested fraetion

388

926

566

1 114

Polysome preparation and RNase digestion were according to Tominaga [21]. Results are expressed as epm per A..~ounit of entire polysomes before incubation.

BIOCHIMIE, 19'78, 60, n ° 8.

Stimulated livers

A. R a i s o n n l e r a n d coil.

750

c 3m

A26o

0,5

2,0 1.5

0,3

/j,"'"\

t

/...\

O,4

1000

1.0

cpm

A260

1500

200

-100 0,2

500

0.5

7.--?.::.,.."'-.....'"

0

0,1

29S

18S

0

4.5S

29S

18S

4,5S

0

C 3m

A26o :

t i

2£

" I,

I.:: I l::'

,.... •

1,5

::

,

:t

i

:. , I~ :.l-,' I

,"

: : :I : :"

t: "

t

:

: ,l "

0,5

A26 o

1500

ii ' I

t

~

"

1000

2 O0

0,4

• •

% ', ",

'A-/ ,.~"

0,5

',,

'

cpm

~'

.

0,1 12'

.

,

~.

.=;

50

,-

0

29S 18 S 4.5 S RNA profiles after ultracentrifugation in sucrose density gradients (see M a t e r i a l a n d M e t h o d s ) . FIG. 3 . -

I~NA w a s o b t a i n e d f r o m c o n t r o l ( u p p e r figure) a n d f a t t y acid o v e r l o a d e d (lower figure) p e r f u s e d l i v e r s a n d w a s t h e n p a r t l y d i g e s t e d b y R N a s e a c c o r d i n g to T o m i n a g a [21]. O p t i c a l d e n s i t y ( ) was continuousl y r e c o r d e d a n d r a d i o a c t i v i t y w a s m e a s u r e d i n collect e d f r a c t i o n s f r o m n a t i v e (. . . . . ) a n d d i g e s t e d ( . . . . ) RNA.

29S

18S

affinity chromatography. Non-radioactive total polysom a l R N A w a s a d d e d to t h e g r a d i e n t as m o l e c u l a r weight and optical density marker ( ).

TABLE V I I I .

Controls optical density Total RNA from polysomes

R e c o v e r y at total RNA

500

C

elution

115

Stimulated livers epm

optical density

62 737

115

48 444 87,2

from

0.336 0.29 per c e n t

3 392 1 782 2.84 per c e n t

68.7

0.890 I). 77 per c e n t

cpm 83 178 56 684 3 818 5 567 6.69 per c e n t

R e s u l t s a r e e x p r e s s e d a s c p m p e r 115, A ~ o f t o t a l R N A o f t h e e l u t e d f r a c t i o n . R N A w a s l a b e l e d in vivo f o r 1 h o u r ( a f t e r i n t r a v e i n o u s i n j e c t i o n of [ l ~ C l o r o t i c acid.

BIOCHIM1E, 1978, 60, u ° 8.

0

Fit~. 4. - - Poly(A)-RNA radioactivity (-.-.-.) profile after ultracentrifugation in sucrose density gradients upper part control liver, lower part fatty acid overloaded perfused liver. P o l y ( A ) - R N A w a s i s o l a t e d b y

A ~ o and total radioactivity of the RNA fractions eluted f r o m poly(U) Sepharose columns.

4u C f r a c t i o n 15 ° C f r a c t i o n 50 ° C f r a c t i o n R N A p o l y (A)

4,5S

RNA synthesis in rat liver during VLDL secretion.

751

r a t a n d in isolated l i v e r p r e p a r a t i o n s : [14C] o r o t i c a c i d i n c o r p o r a t i o n into h e p a t i c RNA was i n c r e a sed 56 p e r cent after fatty a c i d overload. Since the s p e c i f i c r a d i o a c t i v i t y of n u c l e o t i d e s w a s unchanged, i t m a y be c o n c l u d e d that fatty acid overload e n h a n c e s RNA synthesis.

RNA S P E C T R U M AND D I S T R I B U T I O N OF RNAs~ HYDROLYSIS.

T O T A L R I B O N U C L E A S E ACTIVITY IN LIVER

W e f o u n d t h a t the e n h a n c e m e n t of 1~C i n c o r p o r a t i o n in different zones of l h e RNA p r o f i l e was not regular. N e w l y s y n t h e s i z e d m a t e r i a l in stinmla¢ed livers was f o u n d in the 17S region a n d to tess er extents in the 2~S region (fig. 3).

CELLS.

T h e acfivi,ty of e x o g e n o u s R,Nase a d d e d to liver h o m o g e n a t e s was ,determined in o r d e r to detect an eventt~al difference of c y t o p l a s m i c RNase inhib i t o r b e t w e e n c o n t r o l a n d fatty a c i d o v e r k ) a d e d livers. As s h o w n in table IV, t h e r e w a s no significant difference. P A R T I C I P A T I O N OF B O U N D POL'YSOMES IN BIO S~rNTHESIS.

The s e p a r a t i o n of b o u n d and free p o l y s o m e s revealed l.arge differences in p o l y s o m a l associated r a d i o a c t i v i t y b e t w e e n the two groups of a n i m a l s (table V). The i n c r e a s e of o r o t i c acid i n c o r p o r a tion p r i m a r i l y aff.ected (~9 p e r cent) the p o l y r i b o somes b o u n d to the e n d o p l a s m i c re¢iculum.

RNA s p e c t r u m a n a l y s i s before h y d r o l y s i s show e d that [ l a C ] o r o t i c a c i d i n c o r p o r a t i o n w a s stim u l a t e d in the 10S to 26S region of g r a d i e n t s from o v e r l o a d e d livers. These l~NAs w e r e good substrates for RNase u n d e r our c o n d i t i o n s .

RNA FRACTIONATION ON POLY (U)-SEPHAROSE. The passage of total p o l y s o m a l RNA t h r o u g h a poly(U) S e p h a r o s e c o l u m n led to tile s e p a r a t i o n of poly(A) RNA a n d thus to the d e t e r m i n a t i o n of its concentration and radioactivity. W h e n equal amounts of total RNA from c o n t r o l or fatty a c i d o v e r l o a d e d livers w e r e f r a c t i o n a t e d on the column, the y i e l d of poly(A)-RNA w a s s i g n i f i c a n t l y h i g h e r in the l a t t e r (table VIII). DISTRIBUTION OF POLY(A)-I~NA.

RADIOACTIVITY DISTRIBUTION IN POLYSOME PROFILES. The r a d i o a c t i v i t y in p o l y s o m e s of differents size classes is s h o w n in table VI. The r a d i o a c t i v i t y i n c r e a s e in the w h o l e p o l y somes of s t i m u l a t e d livers ~,as f o u n d to be m a i n l y in the m e d i u m a n d long polysomes. The r a d i o a c t i v i t y in the free r i b o s o m e s from stim u l a t e d livers was u n c h a n g e d in the in vivo exper i m e n t s and was s u r p r i s i n g l y l o w e r in the isolated p e r f u s e d livers. S E N S I T I V I T Y OF NE%VLY S Y N T H E T I S F ~

RNA

TO RNAsE. A v e r y m i l d digestion of p o l y s o m a l RNA w i t h a minima~l a m o u n t of pa'ncreatic r i b o n u c l e a s e (4.10 -10 M enzyme, 0°C, 3 h) led to the d i s a p p e a r a n c e of most of t h e [14C] o r o t i c a c i d r a d i o a c t i v i t y from the polysomes. The r a d i o a c t i v i t y in the nonhydro,lyzed f r a c t i o n w a s s i m i t a r in t r e a t e d and c o n t r o l li~ers, but t h e r a d i o a c t i v i t y of the RNase digested f r a c t i o n was h i g h e r (70-80 p e r cent of total r a d i o a c t i v i t y ) in livers a c t i v e l y p r o d u c i n g VLDL t h a n in the controls. [l*C]orotic acid inc o r p o r a t i o n d u r i n g V,LDL s t i m u l a t i o n w.as 100140 p e r cent g r e a t e r in this f r a c t i o n in conlparison ¢o ,control livers (t~ble VH).

BIOCHIMIE, 1978,

60, n ° 8.

Sucrose g r a d i e n t analysis of the poly(A)-RNA o b t a i n e d b y the affinity column s h o w e d a heterogeneous d i s t r i b u t i o n of r a d i o a c t i v i t y w i t h maximal s p e c i f i c a c t i v i t y in the 18S region (fig. 4). O v e r l o a d e d l i v e r y i e l d saveral f r a c t i o n s w i t h h i g h e r r a d i o a c t i v i t y t h a n c o n t r o l s e s p e c i a l l y in the high m o l e c u l a r weight regions (fig. 4).

Discussion.

A c t i n o m y c i n D i n h i b i t s the fatty a c i d stimulation of apo-VLDL p r o d u c t i o n !8], suggesting that s y n t h e s i s of new RNA molecules is i n v o l v e d in the process. The i n v o l v e m e n t of a t r a n s c r i p t i o n a l mec h a n i s m in fatty a c i d stimul.ated livers can be d i r e c t l y d e m o n s t r a t e d only by the study of RNA s y n t h e s i s from l a b e l e d p r e c u r s o r s , such as orotic acid. A c c o r d i n g to T o m i n a g a from the r,adioactivity i n c o r p o r a t e d in the p o l y s o m a l RNA after 4 h of l a b e l l i n g in vivo, only 25 p e r cent w e r e f o u n d in the RNase-digested fraction (mainly c o m p o s e d of mRNA). In our in vivo e x p e r i m e n t s , after 1 h of i,n c o r p o r a t i o n 4.6 p e r cent and 71 p e r cent of the p o l y s o m a l r a d i o a c t i v i t y w e r e r e c o v e r e d respecti-

A. R a i s o n n i e r a n d coll.

752

v,ety in the RNase d i g e s t e d f r a c t i o n f r o m c o n t r o l and o v e r l o a d e d livers (table VII). In the p e r f u s e d livers these values w e r e respect i v e l y 63 p e r cent a n d 80 p e r cent of the p o l y s o real radi,oactivity. I n d e e d , as it could be e x p e c t e d a h i g h e r p r o p o r t i o n of the label was f o u n d in the rapic~ly-labeled R.NA .(identified by T o m i n a g a as mRNA) after 1 h than at 4 h of i n c o r p o r a t i o n . The d i s t r i b u t i o n of mRNA in the p o l y s o m a l p r o file shows .a h i g h e r conte'nt in lo~ng polysom~es, and Iow content in m o n o m e r s or dimers, w h i c h corr e s p o n d to free or i n a c t i v e ribosomes. R.adio,activily was signifi,ca.ntly i n c r e a s e d in med i u m a n d long e n d o p l a s m i c r e t i c u l u m - b o u n d ribosomes, w h i c h are i n v o l v e d in p l a s m a p r o t e i n synthesis. The f r a c t i o n of p o l y s o m a l RNA h y d r o l y s e d b y ribonuclease under very mild conditions had prop e r t i e s previous~ly d e s c r i b e d [21] : after digestion, the p o l y s o m e sedimentatio~n p r o f i l e s h o w e d only m o n o m e r s a n d dimers, i n d i c a t i n g that the RNA l i n k i n g the r i b o s o m a l units to form p o l y s o m e s h a d been digested. The s p e c i f i c r a d i o a c l i v i t y of the digested fraction w a s 25 times greater than that of the r e m a i n i n g f r a c t i o n ; the (A + U) : (G + C) ratio of the RNA f r a c t i o n d e c r e a s e d from 0,96 to 0.68 d u r i n g digestion, i n d i c a t i n g a relative i n c r e a s e of rRNA in the m e d i u m . Moreover, the digested f r a c t i o n c o n t a i n e d a r e l a t i v e l y h i g h p r o p o r t i o n of a,d e n y l i c acid. It sho'u,l,d be noted that i~n our e x p e r i m e n t s the r a t i o of a b s o r b a n c e of 18S/29S r e m a i n e d constant. As a result of the different sensitivities of these two f r a c t i o n s to l~Nase, it m a y be concluded that t h e r e was no h y d r o l y s i s of rRNA. F i n a l l y , the p r o f i l e s of the digested f r a c t i o n o b t a i n e d after c e n t r i f u g a t i o n on sucrose g r a d i e n t s s h o w e d a h e t e r o g e n e o u s d i s t r i b u t i o n of the digested m a t e r i a l in the zones of high m o l e c u l a r weight, w i t h a p e a k at 18S c o r r e s p o n d i n g to the m o l e c u l a r w e i g h t of sermn a l b u m i n mRNA. These a r g u m e n t s s u p p o r t t h e vie'w th,at t h e digested fraction consists of mRNA molecules. L,abeled RNA s y n t h e s i z e d d u r i n g fatty ,acid o v e r l o a d is contain e d in this f r a c t i o n a n d c o n f o r m s to the same c r i teria. If .the RNA f r a c t i o n p r o d u c e d d u r i n g VLDL synthesis is messenger, it s h o u l d contain, as all k n o w n e u k a r y o t i c messengers a long t e r m i n a l p o l y a d e n y l i c sequence. The b i n d i n g of this fraction to p o l y ( U ) - c o n t a i n i n g filters or affinity columns s h o w e d that the RNA s y n t h e s i z e d d u r i n g

BIOCHIMIE,

1978, 60, n ° 8.

fatty a c i d o v e r l o a d d o e s i n d e e d contai,n such a sequence. The h i g h specific r a d i o a c t i v i t y of this poly(A)-RNA i n d i c a t e d it c o n t a i n e d r a p i d l y synthesized RNA. ~ e c o v e r y of mRNA b y affinity c h r o m a t o g r a p h y was p r o b a b l y not c o m p l e t e a n d RNase digested f r a c t i o n c o n t a i n e d some contami, nation w i t h ribon u c l e i c ,acids others than mRNA. Owing to these t e c h n i c a l l i m i l a l i o n s w e do not e x p e c t e d to obtain q u a n t i t a t i v e results on the a m o u n t of mRNA, present i,n the RNA extracts. H o w e v e r , these results p r o v i d e s v a l u a b l e i n f o r m a t i o n on the e n h a n c e d s p e c i f i c r a d i o a c t i v i t y of RNA of m e s s e n g e r t y p e d u r i n g the stimul,ation of VLD,L p r o d u c t i o n in the Hver c o m p a r e d -with the c o n t r o l liver. This f r a c t i o n or the p o l y s o m e s w h i c h c o n t a i n it, w a s able to i n c r e a s e a m i n o acid i n c o r p o r a t i o n b y a cell-free system [41] in,to acid p r e c i p i t a b l e m a c r o l n o l e c u l e s [.423 i n c l u d i n g a p o l i p o p r o t e i n s [43]. The p r e s e n t e x p e r i m e n t s c o n t r i b u t e new data c o n c e r n i n g an i n c r e a s e d synthesis of h i g h molec u l a r w e i g h t mRNA duri,ng the fatty a c i d - i n d u c e d b i o s y n t h e s i s of V.LDL~apolipoproteins in the rat liver. W e p o s t u l a t e that, the n e w l y s y n t h e s i z e d RNA p r e s u m a b l y codes for one o r m o r e VLDL a p o p r o teins a n d thus ,enables the l i v e r cell to r a p i d l y mobitize .the excess of t r i ~ y c e r i d e s f o r m e d after a fatty a c i d overload, such as those o b i a i n e d in vivo .after i n t e s t i n a l a b s o r p t i o n of fat or p e r i p h e ral fatty acid m o b i l i s a t i o n . E x p e r i m e n t s n o w in p r o g r e s s are e x p l o r i n g the s p e c i f i c i t y of these m~NAs. If it is c o n f i r m e d that l i p o p r o t e i n s y n t h e s i s is u n d e r t r a n s c r i p t i o n a l control, c e r t a i n genetic aspects in the p a t h o l o g y of p l a s m a l i p o p r o t e i u c o u l d be better u n d e r s t o o d .

Acknowledgement.

We greatly appreciate the helpful discussion and advice from Professor J. Polonovski and the skilfull technical assistance of Misses C. Rey and J. lnfante.

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