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Iron stimulation of ferritin biosynthesis
BIOCHIMIE, 1974, 56, 741-749.
Iron stimulation of ferritin biosynthesis. I. Localization of the iron effect. R. SADDI (*), R. FAGARD (*), J. FEINGOLD (~ *) and M. M. SABATIER (*).
(*) I n s t i t u t de Pathologie Mol~cnlaire ( * ~ ) , 24, rue da faubourg Saint-Jacques, 75014 Paris. (**) Groupe 1NSERM U 12, H6pital des Enfants-Malades, lZ~9, rue de S~vres, 75015 Paris. (11..1-1974). Summary. - - Studies stemming from several laboratories have shown that the increase in synthesis of ferritin brought about by iron is aetimyein D independent, thus establishing its post-transcriptional nature. Ferritin synthesis as well as its increase when tested with fractions prepared from iron injected animaIs have been realized in vitro. Ferritin synthesis thus provides a convenient model for the study of translational control of protein synthesis in mammals. This work presents the first results of a comprehensive study aiming at the analysis of the site and mechanism of iron action : (1) iron acts through a modification of the microsomal apparatus which is not mediated by a change in the soluble fraction of the cell ; (2) this modification can he obtained appreciably only in oivo ; (3) once induced, this mierosomal activation may be studied in cell-free preparations ; it results in an increase of the amount of completed ferritin molecules released into the incubation medium, whereas labelled ferritin polypeptides detached from microsomes by sonication are little if at all increased, at least in the few hours after iron injection.
INTRODUCTION. F e r r i t i n is a w a t e r soluble i r o n storage molecule w i d e l y d i s t r i b u t e d a m o n g m a m m a l s , plants and fungi. It is r e a d i l y purified o w i n g to its h i g h m o l e c u l a r w e i g h t (MW) and density, its r e l a t i v e r e s i s t a n c e to heat d e n a t u r a t i o n and r e a d y crystallization w i t h C a d m i u m ions [1~3]. Its t w o constitutive parts are easily identified in the electron m i c r o s c o p e , d i r e c t l y for the i r o n core ( d i a m e t e r 70 X), after s h a d o w i n g for the p r o t e i n shell (diam e t e r 120 A). A n u m b e r of o l i g o m e r s n a t u r a l l y coexist in solution along w i t h m o n o m e r i c ferritin [4-15]. Several i s o f e r r i t i n s h a v e been c h a r a c t e r i z e d : in a given species in different tissues, in c u l t u r e d cells in the e m b r y o n i c and n e o p l a s t i c state, i n d i c a t i n g the existence of several s t r u c t u r a l genes differently e x p r e s s e d in each cell type [8, 9, 16-19]. Other d i f f e r e n c e s h a v e been described, p r o b a b l y i n v o l v e d in different b i o c h e m i c a l roles [21}, 211. T h e p r o t e i n moiety, a p o f e r r i t i n , h a v i n g in the m o n o m e r i c state a MW of 460,0,00 daltons, is m a d e of 20 to 2,4 p r o b a b l y i d e n t i c a l subunits (**~) Groupe U 15 de l'Institut National de la Sant6 et de la Recherche M6dicale, Laboratoire Associ6 au Centre National de la Recherche Scientifique.
[22-24J. S t r u c t u r a l studies on a p o f e r r i t i n are in p r o g r e s s in s e v e r a l l a b o r a t o r i e s [25-34]. T h e a m o u n t of f e r r i t i n p r o t e i n has long been k n o w n to d e p e n d on the tissues c o n t e n t of i r o n [35, 36]. Studies s t e m m i n g f r o m s e v e r a l laboratories have s h o w n : (1) that the r e l a t i o n l i n k i n g the a m o u n t of ferr i t i n p r o t e i n to the tissue c o n t e n t of i r o n m a i n l y results f r o m an i r o n s t i m u l a t i o n of f e r r i t i n synthesis [37], as e v i d e n c e d by its c o m p l e t e i n h i b i tion by c y c l o h e x i m i d e [38]. T h e c o n t r i b u t i o n of the stabilization of the p r o t e i n m o i e t y by i r o n is still a m a t t e r of c o n t r o v e r s y ; c o n f l i c t i n g results h a v i n g been p u b l i s h e d r e c e n t l y , d e m o n s t r a t i n g [39, 40] or d e n y i n g [38] s u c h an effect of the metal ; (2) that the i r o n i n d u c e d s t i m u l a t i o n of s y n t h e sis is a c t i n o m y c i n i n d e p e n d e n t , i n d i c a t i n g a postt r a n s c r i p t i o n a l m e c h a n i s m , l o c a t e d in the cytoplasm. This p o i n t results f r o m the w o r k of s e v e r a l groups ([41, 42, 39i, p r o v i d e d doses of a c t i n o m y e i n D (8,0 > 150 ~g p e r 100 g of rat) w h i c h specifically depress DNA d e p e n d e n t RNA synthesis [43] w i t h o u t affecting p r o t e i n synthesis w e r e used,
R. Saddi, R. Fagard, J. Feingold and M. M. Sabatier.
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as i n d i c a t e d in each e x p e r i m e n t [41] b y simultaneous l a b e l l i n g of a l b u m i n as i n t e r n a l control. One must keep in m i n d h o w e v e r that this c r i t i c a l p o i n t is not c o m p l e t e l y settled yet, two groups h a v i n g f o u n d i n h i b i t i o n of the i r o n a c t i o n b y actin o m y c i n D [43 bis-45]. T h e results of one of these have been c r i t i c i z e d as t h e y w e r e i m p r o p e r l y u s i n g the a n t i b i o t i c , in too large doses k n o w n to result in aspecific i n h i b i t i o n of p r o t e i n s y n t h e s i s ; a n d i n d e e d in s u b s e q u e n t e x p e r i m e n t s , this group d i d not confirm t h e i r p r e v i o u s results ; (3) that f e r r i t i n a n d its increase, w h e n tested w i t h cell f r a c t i o n s c o m i n g f r o m i r o n i n j e c t e d animals, m a y be a c h i e v e d in vitro in cell-free systems ; the a m o u n t of l a b e l l e d m a t e r i a l r e c o v e r e d is ulinute but it p r e s e r v e s the c h a r a c t e r i s t i c s of f e r r i t i n : heat stability, e l e c t r o p h o r e t i c a n d salt i n g out p r o p e r t i e s , high m o l e c u l a r weight, antigenicity, specific s t i m u l a t i o n b y i r o n [41]. On the basis of the above d e l i n e a t e d 3 points, it a p p e a r e d that f e r r i t i n s y n t h e s i s p r o v i d e s a conv e n i e n t m o d e l for the s t u d y of t r a n s l a t i o n a l cont r o l of p r o t e i n s y n t h e s i s in m a m m a l s . The e x p e r i m e n t s p r e s e n t e d h e r e are p a r t of a c o m p r e h e n s i v e s t u d y d e v i s e d to analyze the p l a c e and mechanism by which iron enhances ferritin synthesis. T h e y s h o w that i r o n a c t i o n is p r i m a r i l y to i n t r o d u c e a m o d i f i c a t i o n of the m i c r o s o m a l app a r a t u s , a m o d i f i c a t i o n w h i c h c a n n o t be i n d u c e d to a n y a p p r e c i a b l e extent in vitro a n d results in an i n c r e a s e of the amount of c o m p l e t e d f e r r i t i n m o l e c u l e s r e l e a s e d in the i n c u b a t i o n m e d i u m . MATERIALS AND METHODS. CHEMICALS" ATP, GTP, CP a n d CPK (*) w e r e o b t a i n e d from the Sigma Chemical Co., SaintLouis (Miss.) USA, I r o n d e x t r a n (inferon) a n d I r o n s o r b i t o l c i t r a t e (jectofer r e s p e c t i v e l y f r o m L athevet (Paris) a n d from Astra (Sweden), Bovine s e r u m a l b u m i n (BSA) a n d o v a l b u m i n f r o m NBC ('Cleveland, Ohio). I4C l e u c i n e u n i f o r m l y labelled, 270 m C i / m m o l e , a n d 1¢C c h l o r e l l a h y d r o l y s a t e (algal) 30 mCi/14C nl atom, w e r e o b t a i n e d r e s p e c t i v e l y from A m e r s h a m (G.B.) a n d C.E.A. fSaclay, France).
ANIMALS: Albinos male rats of the W i s t a r s t r a i n r e c e i v i n g n o r m a l diet w e r e used t h r o u g hout. RAT
FERRITIN
-"
w e r e p e r f u s e d t h r o u g h the p o r t a l vein, r e m o v e d , h o m o g e n i z e d w i t h 3 volumes of w a t e r , e x t r a c t e d 1/2 h w i t h shaking, c e n t r i f u g e d for 30 mAn at i'0,0,00 g in a Sorvall c e n t r i f u g e ; the s u p e r n a t a n t was h e a t e d for 5 mAn at 76 ± 2°C, f r e e d of coagulated p r o t e i n s b y eentrifugation, p r e c i p i t a t e d t w i c e w i t h a m m o n i u m sulfate b e t w e e n 15 a n d 35 p. 100 c o n c e n t r a t i o n ( w / v ) , finally d i a l y z e d against a p p r o p r i a t e buffer, t i t r a t e d at 3,25,0 nm, a n d s t o r e d frozen in aliquots w h i c h w e r e t h a w e d only once ( f e r r i t i n i n d e e d is p a r t i a l l y d e n a t u r e d on r e p e a t e d freezing a n d thawing)• (2) Ferritin twice crystallized : • C r y s t a l l i z a t i o n w a s a c h i e v e d using the m e t h o d of F i n e b e r g [46] : to c a r r i e r f e r r i t i n d i a l y z e d against NaC1 6.0 raM, 2.6 l u t i d i n e 2.0 mM p H 7.2 buffer (N.L) was a d d e d an equal volume of Cadm i u m sulfate 4 p. cent ( w / v ) d i s s o l v e d in N L a n d a d j u s t e d to p H 7.2 (NIL Cd.). C r y s t a l l i z a t i o n took p l a c e in a few h o u r s a n d w a s c o m p l e t e after a few d a y s at 4°C. It w a s m o n i t o r e d b y m i c r o c o s p i c examination.
• Separation of crystals from amorphous precipitate (AP): s e d i m e n t a b l e m a t e r i a l (crystals + &P) was w a s h e d t w i c e b y c e n t r i f u g a t i o n w i t h NL Cd, r e s u s p e n d e d in the m i n i n u m volume of NL Cd a n d h o m o g e n i z e d w i t h 23 p a r t s of 2.4 M sucrose in NL Cd, w h i c h r e s u l t e d in d i s s o l u t i o n of a m o r p h o u s material, l a y e r e d over 6 ml sucrose 2.4 M in N~L Cd a n d c e n t r i f u g e d for 1 h r at 20,000 r p m in a SW 25 rotor. Crystals w e r e recov e r e d free of AP at the b o t t o m of the tube, w a s h e d in 57L Cd, dissolved in NaC1 c o m p l e x o n buffer p H 7 . 2 (NaC1 0.9 p. cent, c o i n p l e x o n 5 p. cent) a n d d i a l y z e d against the same buffer once, then 3 times against NL buffer and r e c r y s t a l l i z e d a n d r e p u r i f i e d u s i n g the same p r o c e d u r e . T w i c e c r y s t a l l i z e d Cd free r a t f e r r i t i n was finally d i a l y zed against NaCI 0.1 M, p h o s p h a t e 0.025 M p H 7.2, c o n t r o l l e d for p u r i t y b y i m m u n o e l e c t r o p h o r e s i s , using r a b b i t a n t i s e r a d i r e c t e d against r a t l i v e r soluble p r o t e i n s a n d p l a s m a p r o t e i n s (usually not a single p r e c i p i t a t i o n line o t h e r t h a n f e r r i t i n was p r e s e n t ) ; f e r r i t i n w a s finally t i t r a t e d at 3,250 m n a n d s t o r e d frozen in fractions. PREPARATION OF ANTISE'RA : antisera d i r e c t e d against t w i c e c r y s t a l l i z e d rat f e r r i t i n , BSA, a n d o v a l b u m i n w e r e p r e p a r e d in rabbits, u s i n g these p r o t e i n s m i x e d w i t h F r e u n d ' s a d j u v a n t [47].
(1) Carrier ferritin: 250 g rats w e r e i n j e c t e d i n t r a m u s c u l a r l y w i t h i r o n d e x t r a n (130, mg iron) for 15 days. After a lag p e r i o d of 15 days, livers
Preparation of the microsomal and soluble tractions :
(*) Adenosine triphosphate, guanosine triphosphate, Creatine phosphate and creatine phosphokinase.
( 1 ) I R O N S T I M U L A T I O N in v i v o : male rats weighing b e t w e e n 120 a n d 150 g, s t a r v e d overnight,
BIOCHIMIE, 1974, 56, n ° 5.
CE'LL-F~EE
INCUBATIONS
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Iron stimulation
w e r e i n j e c t e d w i t h 10 m g p e r 100 g b o d y w e i g h t with iron sorbitol citrate intraperitoneally, usually 3 and o c c a s i o n a l l y 14 hrs before b e i n g killed. Control rats r e c e i v e d saline i n s t e a d of iron. Rats w e r e killed by d e c a p i t a t i o n , bled, t h e i r livers w e r e r e m o v e d , m i n c e d and h o m o g e n i z e d in a loose fitting teflon glass h o m o g e n i z e r in 2.5 times t h e i r w e i g h t of m e d i u m (0.26 M sucrose 0.05 M Tris buffer (pH 7.8 at 20°C), 0.05 M KC1, 0,00,5 M Mg+~C12, 0.005 M glutathione adjusted to p H 7 w i t h KOH). The h o m o g e n a t e s w e r e c e n t r i fuged t w i c e at 10,000 g for 10 m i n ; after r e m o v a l of the fat layer, the p o s t m i t o c h o n d r i a l supernatants w e r e c e n t r i f u g e d in a S p i n c o {model L) TABLE I.
F e r r i t i n stability near the isoeleetrie p o i n t (pH I4.8) at various ionic strengths. pH 7.8 Ionic strengths
0.150 0.100 0.050 0.035 0.025 0.015 0.010
pH 4.8
Ferritin [remaining E 3.250nmlin solution E 3.250 nm {% of inii (tial value . ~ 06 0 0.270 0.250 0.250 0.258 o. 245 0.260
1
100 104 96 100 100 94 100
0.260 0.275 0.215 0.220 0.215 0.145 0.025
Fer.rit.in remaining in solution {% ol initial value 100 105 83 85 83 56 9.6
Rat ferritin samples were dissolved in Tris NaCI buffer at decreasing ionic strengths. Each sample was then divided into 2 halves and placed in an ice bath : one half was acidified to pH 4.8 with diluted acetic acid. After 1/2 hr standing in ice, the samples were centrifuged and the E 3,250 nm absorbancy of the supernatants recorded.
p r e p a r a t i v e u l t r a - c e n t r i f u g e at 32,500, 45,000 rpln for 45 rain in the 40 or 50 rotor. T h e m i d d l e p a r t of the s u p e r n a t a n t w a s r e c o v e r e d a n d used as such, or alternatively, ~n some e x p e r i m e n t s , rec e n t r i f u g e d at 50,000 r p m .for 1 h r 30 in the 50 t i t a n i u m r o t o r to give p a r t i c l e free supernatant. The m i c r o s o m a l (Ms) pellets of the first and, if necessary, of the s e c o n d c e n t r i f u g a t i o n w e r e m i x e d , r e s u s p e n d e d by gentle h o m o g e n i z a t i o n , f r e e d of aggregated m a t e r i a l by a s h o r t l o w speed c e n t r i f u g a t i o n ; the a b s o r b a n c y at 2,6,00, 2,800, 3,250 n m was r e a d to give a m e a s u r e of the conc e n t r a t i o n s of the Ms suspension and the supernatant f r a c t i o n s ; these w e r e adjusted to s t a n d a r d c o n c e n t r a t i o n s ; a l l o w a n c e w a s m a d e f o r the a b s o r b a n c y at 2,60.0 n m due to f e r r i t i n by subs-
BIOCHIMIE, 1974, 56, n ° 5.
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of ferritin biosynthesis• t r a c t i n g the a b s o r b a n c y at 3,250 nm from the a b s o r b a n c y at 2,60,0 nm.
×
1.6 [48]
(2) IRON ADDED in vitro : f r a c t i o n s w e r e prep a r e d using the same p r o c e d u r e f r o m n o r m a l rats. T h e y w e r e then s e p a r a t e d in t w o h a l v e s : to one was a d d e d i r o n f e r r i c a m m o n i u m c i t r a t e : 2, 10 and 22 .ag p e r ml of i n c u b a t i o n m e d i u m and to the o t h e r was a d d e d the e q u i v a l e n t q u a n t i t y of medium. • The incubation m e d i u m contained in a volume of 6.5 m l : total m i c r o s o m e s f r o m 4 to 5 g of liver, s u s p e n d e d in 2.5 ml of m e d i u m , 2.75 ml of the c o r r e s p o n d i n g s u p e r n a t a n t ( c o n t a i n i n g 100 m g of p r o t e i n ) , 6.5 uM ATP, 1.3 ~M GTP, 6.5 ~M phosp h o c r e a t i n e , 125 eg GPK, and usually 20 ~Ci 1'C leucine. T h e final ion c o n c e n t r a t i o n s w e r e : Mg ÷+ : 0.005 M; K + : 0.043 M. The m i x t u r e was i n c u b a t e d for 3/4 h r at 37°C in air, in a s h a k i n g i n c u b a t o r .
P u r i f i c a t i o n of labelled f e r r i t i n : at the end of the i n c u b a t i o n time, aliquots w e r e set a p a r t to d e t e r m i n e the s p e c i f i c a c t i v i t y (SA) of total protein used as a m e a s u r e of the b i o s y n t h e t i c activity of the i n d i v i d u a l i n c u b a t i o n s . E a c h i n c u b a tion m i x t u r e was t h e n t r e a t e d in one of the following ways : - - in some e x p e r i m e n t s , w h e r e it w a s d e c i d e d to d i s t i n g u i s h the p a r t of the s y n t h e s i z e d ferritin n a t u r a l l y r e l e a s e d in the s u p e r n a t a n t f r o m the one r e m a i n i n g attached to the ribosomes, the incubation m i x t u r e w a s c e n t r i f u g e d for 30 rain at 40,000 r p m (Spinco, Model L c e n t r i f u g e 40 rotor) and m i c r o s o i n e s and s u p e r n a t a n t w e r e t r e a t e d separately ; --in others, the w i t h o u t separation.
whole
sample
was
treated
T h e n each f r a c t i o n , w h e t h e r s e p a r a t e d or not, was h a n d l e d in the same w a y : a d d i t i o n of c a r r i e r f e r r i t i n , e x p o s u r e to US v i b r a t i o n (1.5 m n MSE s o n i c a t o r m o d e l PG 153, p r o b e n ° 25 926 setting : m e d i u m 3), h e a t i n g to 76 ± 2°C d u r i n g 5 rain. The r e s u l t i n g s u p e r n a t a n t was t h e n p r e c i p i t a t e d w i t h a m m o n i u m sulfate b e t w e e n 15 and 35 p. cent conc e n t r a t i o n ( w / v ) ; the p r e c i p i t a t e d f e r r i t i n w a s d i s s o l v e d in 2.5 ml of NaC1 0.2 M, p h o s p h a t e 0.05 M buffer p H 7.3 (*) and t i t r a t e d to p H 4.85 in ice w i t h acetic acid N/3. T h e p r e c i p i t a t e was disc a r d e d and the s u p e r n a t a n t n e u t r a l i z e d a n d cent r i f u g e d free of debris (some f e r r i t i n is lost in this a c i d i f i c a t i o n step but it w a s a s c e r t a i n e d that its SA w a s not different f r o m the one d i s p l a y e d by the acidosoluble ferritin). To each s a m p l e was (*) This high ionic strength ensures stability of the ferritin during the acidification step (table I).
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R. Saddi, R. Fagard, J. Feingold and M. M. Sabatier.
then a d d e d 0.5 ml of ~2C l e u c i n e s a t u r a t e d saline c o n t a i n i n g 400 gg o v a l b u m i n , and a first p r e c i pitate o v a l b u m i n - a n t i o v a l b u m i n w a s o b t a i n e d a n d s e p a r a t e d by c e n t r i f u g a t i o n ; 2 such p r e c i p i t a t e s w e r e o b t a i n e d b e f o r e the s p e c i f i c f e r r i t i n - a n t i f e r r i t i n p r e c i p i t a t e was p r o d u c e d by a d d i t i o n of r a b b i t anti rat f e r r i t i n in excess (it is w e l l - k n o w n that w i t h r a b b i t a n t i s e r a t h e r e is no solubilization of the antigene a n t i b o d y c o m p l e x e s in the antib o d y excess zone).
on the c l a r i f i e d a n t i f e r r i t i n p r e c i p i t a t e o b t a i n e d p e r ml thereof. In table II an e x a m p l e of the results thus obtained in one e x p e r i m e n t by the 3 t e c h n i q u e s of ferritin e s t i m a t i o n is shown. TABLE II.
Protein content of the ferritin-anti[erritin precipitates as measured by different methods. Lowry
P R O C E S S I N G AND COUNTING OF RADIOACTIVE A N T I GENE ANTIBODY P R E C I P I T A T E S ,
FERRITIN
AND P R O -
TEIN TITRATION :
--The antigene a n t i b o d y p r e c i p i t a t e s w e r e w a s h e d 3 times by c e n t r i f u g a t i o n in p h o s p h a t e buffered saline, s u s p e n d e d in 6 ml distilled water. One d r o p of p e r c h l o r i c acid (PCA) N / 3 w a s t h e n a d d e d r e s u l t i n g in the d i s s o c i a t i o n and c l a r i f i c a tion of the c o m p l e x w h i c h allows d i r e c t absorb a n c y r e a d i n g at 3,2.5.0 n m for f e r r i t i n a n d 2,800 nm for o v a l b u m i n p r e c i p i t a t e s ; --they w e r e t h e r e a f t e r p r e c i p i t a t e d by PCA N / 3 and the p r o t e i n s p r o c e s s e d by a t e c h n i q u e d e r i v e d f r o m that of S c h n e i d e r [49]; the d r y p o w d e r was t h e n dissolved in c o n c e n t r a t e d form i c acid, aliquots w e r e set apart for p r o t e i n assay by the m e t h o d of L o w r y [50], and the r e m a i n d e r w a s c o u n t e d e i t h e r in a l o w b a c k g r o u n d N u c l e a r Chicago c o u n t e r (after e v a p o r a t i n g the f o r m i c acid on steel p l a n c h e t s and w e i g h i n g ) or d i r e c t l y in an I n t e r t e c h n i q u e l i q u i d s c i n t i l l a t i o n counter, each vial c o n t a i n i n g 0.2 ml of the p r o t e i n s dissolved in f o r m i c acid, 0,8 ml of distilled w a t e r a n d 10 ml of Instagel (Packard). - - F e r r i t i n t i t r a t i o n : p r e l i m i n a r y studies h a v e s h o w n that t h e r e exists a good c o r r e l a t i o n (in a n y case sufficient for this k i n d of c o m p a r a t i v e studies) b e t w e e n p r o t e i n e s t i m a t i o n by w e i g h i n g on planchets, L o w r y estimation, and use of extinction coefficient at 3,25.0 nm on a n t i b o d y p r e c i p i tates clari~fied by the above d e s c r i b e d t e c h n i q u e : a c a l i b r a t i o n c u r v e w a s w o r k e d out u s i n g both the r e a d i n g s at 3,250 nm and the p r o t e i n estimation by the m e t h o d of L o w r y : an e x t i n c t i o n coefficient of E 3o._~0L~m nm 1 m g / m l of 9.4 Do units has been established for c r y s t a l l i z e d f e r r i t i n of m e a n i r o n c o n t e n t 20 p. cent ( w / w ) . U s i n g c a r r i e r f e r r i t i n the same coefficient m a y be used p r o v i d e d the a m o u n t of f e r r i t i n p r o t e i n that it c o n t a i n s p e r ml is established for each i n d i v i d u a l pool of c a r r i e r f e r r i t i n , by r e a d i n g the 3,250 nm absorb a n c y d i r e c t l y on the solution as such, and then
BIOCHIMIE, 1974, 56, n ° 5.
Exp 1 Normal.. i -~- F e . . . Exp 2 Normal.. +
Fe...
Weight on E 3,250nm E 3.250 n m planchels absorbancy ×2
2.47 2.85
2.15 2.90
1.21 1.325
2.42 2.65
0.98
1.10 1.25
0.49 0.54
O. 98 1.08
1.145
In this table, the results of the ferritin estimation by E 3,250 nm absorbancy readings are matched with those of the protein assay by the method of Lowry and direct weighing on steel planehets. One must take into account that Lowry titration and weighing measure the total protein (ferritin -b antiferritin) of the precipitate, when extinction at 3,250 nm only estimates ferritin moiety ; bearing this in mind, the results above presented stronly suggest that in the ferritin-antiferritin precipitate there is about the same amount of ferritin and antiferritin protein, which means that for each molecule of ferritin (MW 460,000) there exists a mean of 3 antibody molecules 7 S (MW : 160,000 X 3 -----480,000).
RBSeULTS P R E S E N T A T I O N : Results h a v e been e x p r e s s e d in 2 w a y s listed f r o m left to r i g h t in the tables :
(1) the c p m i n c o r p o r a t e d into f e r r i t i n p e r incubation i.e. by the m i c r o s o m e s of 4 g l i v e r (after d e d u c t i o n of b a c k g r o u n d counts, and t h e ones r e c o v e r e d into f e r r i t i n for the test s a m p l e prep a r e d w i t h o u t e n e r g y and p l a c e d in ice at the b e g i n n i n g of the i n c u b a t i o n p e r i o d i.e. about 15 c p m each). This figure has also been c o r r e c t e d to the i n i t i a l l y a d d e d a m o u n t of c a r r i e r f e r r i t i n , u s i n g the a b s o r b a n c y at 3,250 n m of the c l a r i f i e d p r e c i p i t a t e at the end of the p u r i f i c a t i o n p r o c e d u r e : this c o r r e c t i o n accounts for the losses of material during purification ; (2) the last figure takes into a c c o u n t for the sake of c o m p a r i s o n the b i o s y n t h e t i c a c t i v i t y of the i n d i v i d u a l i n c u b a t i o n (each p r e p a r e d f r o m a d i f f e r e n t rat), m e a s u r e d by the s p e c i f i c activity (SA) of total p r o t e i n s at t h e end of the i n c u b a tion ; this c o r r e c t i o n brings the f e r r i t i n c p m p e r i n c u b a t i o n to the one w h i c h w o u l d h a v e been f o u n d for a h y p o t h e t i c a l SA for total p r o t e i n of 2,000 c p m / m g .
Iron s t i m u l a t i o n of ferritin biosynthesis. (3) the data h a v e been statistically t r e a t e d by analysis of the v a r i a n c e and by the t. test of Student. RESULTS. F o r c l a r i t y each result w i l l be p r e s e n t e d a n d discussed s e p a r a t e l y in a series of tables a n d figures. (1) I r o n s t i m u l a t i o n c a n n o t be a p p r e c i a b l y o b t a i n e d w h e n the metal is d i r e c t l y a d d e d in vitro to a cell-free system p r e p a r e d f r o m a normal rat liver.
745
F r o m results s h o w n in table III, it is clear that no s t i m u l a t i o n w a s o b t a i n e d by the d i r e c t a d d i tion of i r o n in the i n c u b a t i o n m e d i u m . (In some e x p e r i m e n t s , instead, i n h i b i t i o n w a s observed). The d i s c r e p a n c y b e t w e e n these results and those of p r e v i o u s p r e l i m i n a r y e x p e r i m e n t s , i n o u r labor a t o r y and in others [51-53], w h e r e a l o w a n d i n c o n s t a n t effect of i r o n in vitro could be observed, is not at p r e s e n t u n d e r s t o o d , although it could result f r o m m i n u t e t e c h n i c a l d i f f e r e n c e s ; in v i e w of the results r e p o r t e d above, h o w e v e r , it seems safe to c o n c l u d e that in o r d e r to a p p r e c i a bly i n c r e a s e f e r r i t i n synthesis, i r o n m u s t act in
TABLE III.
Radioactive amino acid incorporation into ferritin by rat liver microsomes incubated with (Fe) or without (N) iron directly added in vitro.
Iron
CPM into ferritin )er incubation
S.A. ot total protein
CPM into I ferritiu cor-I rected for a I total t protein S.A.or 2000 CPM/MG
Relative increase (p. !00)
Statistical significance of the difference
1
2¥/m1
199 + 67 187 + 43
1640 + 282 1628 + 230
239 -'~ 40 234 -'~ 38
--
NS
2
10y]ml
642 -~- 33 605 T- 18
3220 -4- 144 3950 ~ 157
400 + 6 308 + 19
- - 30
3
-22y/ml
973 ~ 133 735 ___~163
3480 -P 793' 550 + 70 3029 ~ 626 488 1- 19
- - 13
Experiment N°
p :
0,001 NS
Each entry in the table represents the mean (with the standard deviation) of 3 different incubations. 3 separate experiments are presented, each using a different pool of microsomes. A clear-cut d e m o n s t r a t i o n of an i r o n effect in vitro w o u l d h a v e h a d a great t h e o r e t i c a l signi: ficance s i n c e it w o u l d have p r o v i d e d the d i r e c t p r o o f of the p o s t - t r a n s c r i p t i o n a l site of i r o n stimulation, a p r o o f w h i c h is still needed, since, as d e s c r i b e d above, an i n h i b i t o r y effect of Actinom y c i n D in this s t i m u l a t i o n is not c o m p l e t e l y r u l e d out at present. Besides, it w o u l d h a v e greatly simplified the in vitro study of the site and m o d e of i r o n action. T h e r e f o r e , in a c c o u n t of the l o w (20 to 30 p. cent) and i r r e g u l a r effects o b t a i n e d by "the a d d i t i o n of i r o n in vitro in several l a b o r a t o r i e s , it has been f o u n d n e c e s s a r y iv r e e x a m i n e the question. I r o n a d d i t i o n has been f r o m 2 to 22 ~ g / m l of i n c u b a t i o n , these b e i n g the c o n c e n t r a t i o n s o b t a i n e d in sampIes p r e p a r e d f r o m rats i n j e c t e d w i t h 5 to 10 mg i r o n IM, 3 to 4 h r s before death, samples w i t h w h i c h an i m p o r t a n t i n c r e a s e of f e r r i t i n synthesis o v e r c o n t r o l has r e g u l a r l y been found.
BIOCHIMIE, 1974, 56, n ° 5.
vivo : this suggests that the m o d i f i c a t i o n r e s u l t i n g in an i n c r e a s e d c a p a c i t y for a cell to synthesize f e r r i t i n i n v o l v e s a step w h i c h needs a degree of o r g a n i z a t i o n of the cell m a c h i n e r y not a c h i e v e d in cell-free p r e p a r a t i o n s , Next step to inject i r o n in vivo and, by crosse x p e r i m e n t s w i t h p r e p a r a t i o n s o b t a i n e d f r o m normal animals, to locate in cell-free i n c u b a t i o n s the f r a c t i o n (soluble or m i c r o s o m a l ) w h i c h h a d b e e n changed. (2) I r o n acts m a i n l y t h r o u g h a m o d i f i c a t i o n of the m i c r o s o m a l apparatus, and not of the soluble f r a c t i o n of the cell cytoplasm. This a p p e a r s f r o m data p r e s e n t e d in table IV : m i c r o s o i n e s p r e p a r e d f r o m i r o n i n j e c t e d animals 3 hrs before death <> a p p r o x i m a t e l y t r e b l e d the counts i n c o r p o r a t e d into f e r r i t i n by n o r m a l m i c r o s o m e s <>, w h i c h e v e r supernatant was used. H o w e v e r t h e r e exists a weak
R. Saddi, R. Fagard, J. Feingold and M. M. Sabatier.
746
effect of t h e <> a c t i n g p o s i t i v e l y u p o n <> a n d n e g a t i v e l y u p o n <>. T h e i n c r e a s e b r o u g h t a b o u t b y t h e <> w h e n a c t i n g on ¢ N Ms >> c o u l d be d u e to t h e p r e s e n c e of s o m e p a r t i c u l a t e m a t t e r ( m o n o s o m e s o r s u b p a r t i c l e s ) not c o m p l e t e l y s p u n d o w n at t h e c e n t r i f u g e d f o r c e a p p l i e d (4.5,000 r p m X 3/4 hr).
i n j e c t e d w i t h i r o n 3 to 4 h r s b e f o r e d e a t h . It r a i s e s t h e q u e s t i o n of t h e p r e s e n c e of an i n h i b i t o r of f e r r i t i n t r a n s l a t i o n in t h e <>. T h i s i n h i b i t o r is n o t t h e f e r r i t i n s y n t h e s i z e d during these 3 hrs under the influence of iron, since, 14 h r s a f t e r i n j e c t i o n , t h e f e r r i t i n c o n t e n t of t h e s u p e r n a t a n t is m u c h g r e a t e r a n d y e t it does not display any inhibitory action.
TABLE IV. Fraction origin S.A. ofthelast ovalb-antiovalb. precipitate
CPM into Ierri'dn per incubation
S.A. of total protein
CPM into ferritin corrected for a total protein S.A. of 2000 CPM/MG
Relahve increase (p. t00)
Statistical significance of the difference
Microsomes
45.000 RPM × 3/4 h supernatant
N
N
96 ~
7
1957 -+- 93
98 ~___ 4
N
Fe
137 ~
12
1930 -4- 182
140 ~___I0
+
Fe
Fe
294 -t- 12
1940 ~__ 56
303 ~_
6
÷ 2O0
)
Fe
N
330 -~- 19
1992 -+- 102
332 ~
23
+ 240
3
43
I p <
0,01
p <
0,05
TABLE V. Fraction origin S.A. of the last ovalb-autiovalb. precipitate
CPM into ferritiu per incubation
Mierosomes
50.000 RPM × lh30 supernatant
N
N
9
N
Fe
10
334 -t- 30
Fe
Fe
9
1037 ~ 45
Fe
N
7
1237 ~
398 ~
87
172
F r o m t a b l e V w h e r e t h e s u p e r n a t a n t has b e e n r e c e n t r i f u g e d at 50,0.00 r p m / 1 . 3 0 hr, it is c l e a r t h a t t h i s is i n d e e d tile e a s e a n d t h a t t h e <> i t s e l f h a s n o s t i m u l a t o r y action. (3) N a t u r e o f t h e i n h i b i t o r y effect of t h e <>: t h e i n h i b i t o r y a c t i o n disp l a y e d b y t h e <~F e 3 h r S u p e r >> w h e n a c t i n g u p o n homologous microsomes (table ¥ and even t a b l e IV) w h e r e t h e n e g a t i v e e f f e c t is still a p p a rent though partly masked by the positive action of t h e p a r t i c u l a t e m a t t e r t h a t it c o n t a i n s ) , i n h i b i t o r y a c t i o n w h i c h is r e l i e v e d w h e n <> is i n c u b a t e d -with ¢ F e 3 h r Ms >> i n t h e p l a c e of h o m o l o g o u s <> h a s r e g u l a r l y b e e n f o u n d in s u p e r n a t a n t s p r e p a r e d f r o m a n i m a l s
BIOCHIMIE, 1974, 56, n ° 5.
S.A. total protein
CPM into ferritin corrected for a total protein S.A. o[ 2000 CPM/MG
Relative increase (p. 100)
3840 -~- 439
204 ~
27
--
3560 ~
192 ___~ 43
-- 7
3477 ~- 246
590 ~
83
-J- 190
2640 ~
957 ~
176
~- 370
847
616
Statistical significance of the difference
N
p <
0,01
T h e d e x t r i n c o n t a i n e d in t h e i r o n p r e p a r a t i o n used (Jectofer) could, early' after injection account f o r t h i s n e g a t i v e effect : l a t e r i n t i m e t h i s i n h i b i tion would disappear, the dextrin having been metabolized. I r o n c i t r a t e a n d s u l f a t e w e r e t h e r e f o r e u s e d in p l a c e of i r o n d e x t r i n . T a b l e VI s h o w s t h a t u n d e r t h e s e c o n d i t i o n s , no i n h i b i t o r y a c t i o n is a p p a r e n t . A d i r e c t p r o o f of t h e s e e n i n table VII.
dextrin
effect m a y
be
(4) t h e i n c r e a s e i n f e r r i t i n s y n t h e s i s b r o u g h t a b o u t by i r o n i n j e c t i o n affects a l m o s t e x c l u s i v e l y the ferritin spontaneously released into the supern a t a n t (table VIII), at l e a s t i n t h e f e w h o u r s a f t e r
Iron s t i m u l a t i o n of ferritin biosynthesis. s t i m u l a t i o n . T h i s is e v i d e n c e d b y e x p e r i m e n t s i n which after the incubation, the samples were separated into supernatant and pellet by a short (30 r a i n ) u l t r a c e n t r i f u g a t i o n at 40,0.00 r p m a n d t h e ferritin prepared and counted in these two frac-
747
p o l y s o m e s i n v o l v e d i n t h e s y n t h e s i s of t h e p r o t e i n a n d t h u s the actual q u a n t i t y of active f e r r i t i n m e s s e n g e r RNA is of t h e s a m e m a g n i t u d e i n n o r m a l a n d i n i r o n t r e a t e d r a t s (at l e a s t i n t h e f i r s t hours after iron stimulation), the difference being
TABLE V[. Fraction origin
Microsomes
Supernatant
CPM into ferritin per incubation
CPMinto ferritin S.A. of total ]corrected for a protein / total /protein S.A. oi 2ooo CPM/MG
Relative increase (p. t00)
Statistica 1 significance ol the diIlereace
r
N
N
428 -4- 166
6150 ______2616
140 ~____ 6
N
Fe
356 ___+ 141
6010 ______2284
117 ~
Fe
Fe
890 -I-
5150 -t-
354 -~- 52
~
Fe
N
763 -~- 130
294 ~
+ 110
NS 71
654
5150 _____ ~
14
22
--
16
153
NS
TABLE VII. Fraction origin
MicroSOrties
Supernatant
Fe
N
Fe
D
CPM into ferritin per incubation
1659 ~
S.A. ol total protein
CPM into Ierritin Relative corrected for a i n c r e a s e total (p. t00) protein S.A. ol 2000 CPM/MG _ _
1055 13.199 4- 8328
254 -4- 48
--
851 --I- 188 14.014 -+- 5416
165 q- 12
- - 46
Statistical significance of the difference
P <
0.001
1~C lencine incorporation into ferritin by rut liver microsomes prepared from normal (N) or ion injected (FE) animals # hours before sacrifice, incubated with homologous or heterologous supernatant. Each e n t r y in the table represents the m e a n (with the s t a n d a r d deviation) of 3 incubations p r e p a r e d w i t h fractions derived f r o m a pool of the livers of 3 i d e n t i c a l l y treated rats. - - In Table IV and V the iron p r e p a r a t i o n injected was iron d e x t r i n sorbitol (jeetofer) and in Table VI iron sulfate. - - In Table IV mierosomes and s u p e r n a t a n t s were p r e p a r e d by u l t r a e e n t r i f u g a t i o n at 45,000 r p m × 3/4 hr. In Table V, mixed m i e r o s o m e s of 2 u l t r a e e n t r i f u g a t i o n s , the one at 45,000 r p m × 3/4 hr, the o t h e r at 50,000 r p m × I h r 30 of the s u p e r n a t a n t resulting f r o m the first centrifugation were used ; the s u p e r n a t a n t s used for the incubation being the ones resulting f r o m the second u l t r a c e n t r i f u g a t i o n (50,000 r p m × 1 h r 30). -In Table VII, mieros0mes (Fe) were p r e p a r e d f r o m ferric a m m o n i u m citrate injected rats (600 lxg per 100 g of body weight). S u p e r n a t a n t s were f r o m n o r m a l r a t s (N) or d e x t r i n injected a n i m a l s (D). The dextrin, the same as t h a t incorporated into jeetofer, was a gift f r o m Dr Lindvall (Astra S f d e r t a j l e , Sweden) ; the dose used, 0.33 mg per 100 g b o d y weight, was t h e one received by j e e t o f e r injected rats.
l i o n s a f t e r a d d i t i o n of c a r r i e r . T h e f e r r i t i n p o l y peptides artificially detached from the microsom a l pelle~ b y u l t r a s o n i c v i b r a t i o n s h o w a b o u t t h e s a m e a m o u n t of 14C l e u c i n e i n c o r p o r a t i o n w h e t h e r t h e y come f r o m iron t r e a t e d or control rats. This finding could suggest that the number of
BIOCHIMIE, 1974, 56, n ° 5.
i n t h e s p e e d of t r a n s l a t i o n , o r i n t h e n u m b e r o f the initiation events. This interpretation would be in keeping with t h e l a c k of a c t i o n o f a c t i n o m y c i n D i n i n d u c t i o n of fcrritin synthesis.
R. Saddi, R. Fagard, J. Feingold and M. M. Sabatier.
748
T a n L E VIII.
~ C incorporation into ferritin naturally released into the supernatant and inlo [erritin polypeptides artificially detached from microsomes by ultrasonic vibration (U.S.) at the end of the incubation time. Fraction origin CPM into supernatant ferritin
Relative increase
CPM into ferritin polypeptides detached by U.S. from microsomes
Microsomes
Supernatant
N
N
61
90
N
Fe
62
98
Fe
Fe
193
-q- 216
97
Fe
N
327
+ 436
115
(p. 100)
Relative increase
(p. t00)
+9 ÷8 ÷ 2g
Microsomes a n d s u p e r n a t a n t f r a c t i o n s are f r o m n o r m a l (N) or 4 h r p r e v i o u s l y j e e t o f e r injected a n i m a l s (Fe) i n c u b a t e d w i t h h o m o l o g o u s or h e t e r o l o g o u s s u p e r natants. Mixed m i e r o s o m e s of 2 successive u l t r a e e n t r i f u g a t i o n s , t h e first a t 32,500 r p m × 3/4 hr, t h e second a t 50,00D r p m × 1 h r of t h e s u p e r n a t a n t r e s u l t i n g f r o m t h e first one, w e r e used. T h e s u p e r n a t a n t u s e d is t h e one o b t a i n e d a f t e r t h e second u l t r a c e n t r i f u g a t i o n a t 50,0t)0 r p m × 1 hr.
CONCLUSION. The present work clearly establishes that iron action mainly results in the introduction of a m o d i f i c a t i o n of the m i c r o s o m a l p a r t of t h e t r a n s lational machinery. T h e m o l e c u l a r n a t u r e of t h i s i r o n - i n d u c e d m o d i f i c a t i o n a n d t h e c a u s e o f t h e n o n ( o r at l e a s t p o o r ) o p e r a t i o n of t h i s p r o c e s s in vitro m u s t b e now unravelled. T h i s w o r k is b e i n g c u r r e n t l y u n d e r t a k e n laboratory.
in our
Ce t r a v a i l p r 6 s e n t e les p r e m i e r s r 6 s u l t a t s d ' u n e 6tude d ' e n s e m b l e e n t r e p r i s e p o u r t e n t e r de pr6eiser le lieu, et d'dlucider le m o d e d ' a c t i o n du fer d a n s ce syst6me. 1) Le fer agit p a r l ' i n t e r m ~ d i a i r e d ' u n e modification de l ' a p p a r e i l m i e r o s o m a l , et n o n de la f r a c t i o n soluble du s u r n a g e a n t ; 2) Cette modification ne p e u t 6tre apport6e a u x m i c r o s o m e s de faqon apprdciable qu'in vivo ; 3) l ' a e t i v a t i o n m i e r o s o m a l e , u n e fois i n t r o d u i t e in oioo p e u t dtre 6tudi~e in vitro ; elle se t r a d u i t p a r u n e a u g m e n t a t i o n eonsid6rable de la f e r r i t i n e m a r q u 6 e lib6r6e d a n s le s u r n a g e a n t d ' i n e u b a t i o n , alors que les p o l y p e p t i d e s f e r r i t i n i q u e s m a r q u 6 s ddtaeh6s artifie i e l l e m e n t p a r les u l t r a s o n s s o n t p e u modifies, du m o t h s apr6s u n e s t i m u l a t i o n de q u e l q u e s h e u r e s .
Acknowledgements. T h i s w o r k w a s s u p p o r t e d b y g r a n t s of t h e <>, t h e <> a n d t h e <>. R~suML P l u s i e n r s 6tudes, i s s u e s de d i v e r s l a b o r a t o i r e s , ont m o n t r 6 que l ' a u g m e n t a t i o n de s y n t h 6 s e de f e r r i t i n e i n d u i t e p a r le fer n ' e s t p a s s u p p r i m 6 e p a r l'injection, p r d a l a b l e m e n t a u fer, d ' a e t i n o m y c i n e D ~ t a b l i s s a n t a i n s i la n a t u r e p o s t - t r a n s c r i p t i o n n e l l e de cette stimulation. La s y n t h ~ s e de f e r r i t i n e , d ' a u t r e part, a i n s i que l ' a u g m e n t a t i o n de cette s y n t h b s e o b t e n u e avee des f r a c t i o n s a c e l l u l a i r e s pr6par6es h p a r t i r d ' a n i m a u x inject~s de fer, a p u ~tre r6alis6e in vitro. L ' e n s e m b l e de ces d o n n 6 e s p e r m e t de consid~rer la s y n t h ~ s e de f e r r i t i n e et sa s t i m u l a t i o n p a r le f e r c o m m e u n module c o m m o d e p o u r l'~tude a n a l y t i q u e de la t r a d u c t i o n et de sa r 6 g u l a t i o n chez les a n i m a u x .
BIOCHIMIE, 1974, 56, n ° 5.
REFERENCES. 1. L a u f b e r g e r , V. (1937) Bull. Soc. Chim. Biol. (Paris) 19, 1575-1582. Granick, S. ( 1 9 4 2 ) J . Biol. Chem., 146, 451-461. Granick, S. (1951) Physiol. Rev., 31, 489-511. Saddi, R. (1962) Reo. Jr. Etud. Clin. Biol., 7, 408-410. T h e r o n , J. J., H a w t r e y , A. O. & Seherron, (1963) Clin. China. Acta, 8, 165-167. 6. Kopp, R., Vogt, A. & Maass, G. (1963) Nature (London) 198, 892-893. 7. Fine, J. M. & Harris, G. (1963) Clin. Chim. Acta 8, 794-798. 8. Richter, G. W. (1963) Lab. Invest., 12, 1026-1039. 9. Richter, G. W. (1964) Brit. J. Exper. Pathol., 46, 88-94. 10. Carnevoli, F. & Tecce, G. (1964) Arch. Biochem. Biophys., 105, 207. 11. Kopp, R., Vogt, A. & Maass, G. (1964) Nature (London), 202, 1211-1212. 12. S u r a n , A. A. & Tarver, H. (1965) Arch. Biochem. Biophys., 111, 399-406. 13. H a r r i s o n , P. M. & Gregory, D. W. (1965) J. Mol. Biol., 14, 626-629. 14. Richter, G. W. & W a l k e r , G. F. (1967) Biochemistry, 6, 2871-2880. 2. 3. 4. 5.
Iron stimulation
of ferritin
15. Williams, M. A. • Harrison, P. M. (1968) Biochem. J., 110, 265-280. 16. Richter, G. W. (1965) Nature (London), 207, 616618. 17. Richter, G. W. ~ Lee, J. C. K. (1970) Cancer Res., 30, 880-888. 18. Allfrey, C. P. Jr, Lynch, E. C. & Whitley, C. E. (1967) J. Lab. Clin. Med., 70, 419-428. 19. Crichton, R. R., Millar, J. A. & Cumming, R. I. C. (1970) Biochem. J., 117, 35 P. 20. Gabudza, T. G. ~ Gardner, F. H. (1967) Blood, 29, 770-779. 21. Gabudza, T. G. ~ Pearson, J. (1969) Biochem. Biophys. Aeta, 194, 50-54. 22. Harrison, P. M. (1959) J. MoL Biol., 1, 69-80. 23. Saddi, R., Schapira, G. ~ Dreyfus, J. C. (1961) Bull. Soc. Chim. Biol., 43, 409~413. 24. Harrison, P. M. (1963) J. Mol. Biol., 6, 404-422. 25. Harrison, P. M., H o f m a n n , T. ~ Mainwaring, W. I. P. (1962) J. MoL Biol., 4, 251-256. 26. Harrison, P. M. ~ Hofmann, T. (1962) J. Mol. Biol., 4, 239-250. 27. Mainwaring, W. I. P. ~ Hofmann, T. (1968) Arch. Biochem. Biophys., 125, 975-980. 28. Hofmann, T. ~ Harrison, P. M. (1963) J. Mol. Biol., 6, 256-267. 29. Harrison, P. M. ~ Gregory, D. W. (1968) Nature (London), 220, 578-590. 30. Suran, A. A. (1966) Arch. Biochem. Biophys., 113, 1-4. 31. Crichton, R. R. (1969) Biochim. Biophys. Acta, 194, 34-42. 32. Crichton, R. R. ~ Barbiroli, V. (1970) FEBS Letters, 6, 121-124 and 134-136. 33. Crichton, R. R. (1971) Hoppe Seyler's Z. Physiol. Chem., 352, 9-10. 34. Crichton, R. R. (1971) Biochim. Biophys. Acta, 229, 75-82.
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35. Granick, S. (1946) J. Biol. Chem., 164, 737-746. 36. Granick, S. (1949) Bull. N. Y. Acad. Med., 25, 403428. 37. Fineberg, R. A. & Greenberg, D. M. (1955) J. Biol. Chem., 214, 97-113. 38. Millar, J. A., Cumming, R. L. C., Smith, J. A. & Goldberg, A. (1970) Biochem. J., 119, 643-649. 39. Drysdale, J. W. & Munro, H. N. (1966) J. Biol. Chem., 241, 3630-3637. 40. Munro, H. N. ~ Drysdale, J. W. (1970) Fed. Proc., 29, 1469-1473. 41. Saddi, R. a yon der Decken, A. (1965) Biochim. Biophys. Acta, 111, 124-133. 42. Drysdale, J. W. ~ Munro, H. N. (1965) Biochim. Biophys. Acta, 103, 185-188. 43. Greenberg, O. a Acs, G. (1962) Biochim. Biophys. Acta, 61, 652-656. 43 (bis). Yu-Fu-Li a Fineberg, R. A. (1965) J. Biol. Chem., 240, 2083-2087. 44. Yoshino, Y., Morris, J. ~ Schachter, D. (1966) Nature (London), 210, 538-539. 45. Yoshino, Y., Morris, J. a Schachter, D. (1968) J. Biol. Chem., 243, 2911-2917. 46. Fineberg, R. A. personal communication. 47. Cohn, M. (1962) Methods Med. Res., 5, 271. 48. Jackson, R. J., Munro, A. J. & Korner, A. (1964) Biochim. Biophys. Acta, 91, 666-688. 49. Schneider, W. C. (1963) J. Biol. Chem., 238, 35723578. 50. Lowry, O. H., Rosebrough, N. J., Farr, A. L. a R a n dall, R. J. (1951) J. Biol. Chem., 193, 265-275. 51. Pearson, E. J. (1966) P h . D . Thesis, Univ. of Californie. San Francisco. 52. Drysdale, J. W. ~ Munro, H. N. Biochim. Biophys. Acta, in press cited by Chu, L. L. H. & Fineberg, R. A. (1969) J. Biol. Chem., 244, 3847-3854 (Ref. 46). 53. Saddi, R. unpublished observations.
Iron stimulation of ferritin biosynthesis. I. Localization of the iron effect. R. SADDI (*), R. FAGARD (*), J. FEINGOLD (~ *) and M. M. SABATIER (*).
(*) I n s t i t u t de Pathologie Mol~cnlaire ( * ~ ) , 24, rue da faubourg Saint-Jacques, 75014 Paris. (**) Groupe 1NSERM U 12, H6pital des Enfants-Malades, lZ~9, rue de S~vres, 75015 Paris. (11..1-1974). Summary. - - Studies stemming from several laboratories have shown that the increase in synthesis of ferritin brought about by iron is aetimyein D independent, thus establishing its post-transcriptional nature. Ferritin synthesis as well as its increase when tested with fractions prepared from iron injected animaIs have been realized in vitro. Ferritin synthesis thus provides a convenient model for the study of translational control of protein synthesis in mammals. This work presents the first results of a comprehensive study aiming at the analysis of the site and mechanism of iron action : (1) iron acts through a modification of the microsomal apparatus which is not mediated by a change in the soluble fraction of the cell ; (2) this modification can he obtained appreciably only in oivo ; (3) once induced, this mierosomal activation may be studied in cell-free preparations ; it results in an increase of the amount of completed ferritin molecules released into the incubation medium, whereas labelled ferritin polypeptides detached from microsomes by sonication are little if at all increased, at least in the few hours after iron injection.
INTRODUCTION. F e r r i t i n is a w a t e r soluble i r o n storage molecule w i d e l y d i s t r i b u t e d a m o n g m a m m a l s , plants and fungi. It is r e a d i l y purified o w i n g to its h i g h m o l e c u l a r w e i g h t (MW) and density, its r e l a t i v e r e s i s t a n c e to heat d e n a t u r a t i o n and r e a d y crystallization w i t h C a d m i u m ions [1~3]. Its t w o constitutive parts are easily identified in the electron m i c r o s c o p e , d i r e c t l y for the i r o n core ( d i a m e t e r 70 X), after s h a d o w i n g for the p r o t e i n shell (diam e t e r 120 A). A n u m b e r of o l i g o m e r s n a t u r a l l y coexist in solution along w i t h m o n o m e r i c ferritin [4-15]. Several i s o f e r r i t i n s h a v e been c h a r a c t e r i z e d : in a given species in different tissues, in c u l t u r e d cells in the e m b r y o n i c and n e o p l a s t i c state, i n d i c a t i n g the existence of several s t r u c t u r a l genes differently e x p r e s s e d in each cell type [8, 9, 16-19]. Other d i f f e r e n c e s h a v e been described, p r o b a b l y i n v o l v e d in different b i o c h e m i c a l roles [21}, 211. T h e p r o t e i n moiety, a p o f e r r i t i n , h a v i n g in the m o n o m e r i c state a MW of 460,0,00 daltons, is m a d e of 20 to 2,4 p r o b a b l y i d e n t i c a l subunits (**~) Groupe U 15 de l'Institut National de la Sant6 et de la Recherche M6dicale, Laboratoire Associ6 au Centre National de la Recherche Scientifique.
[22-24J. S t r u c t u r a l studies on a p o f e r r i t i n are in p r o g r e s s in s e v e r a l l a b o r a t o r i e s [25-34]. T h e a m o u n t of f e r r i t i n p r o t e i n has long been k n o w n to d e p e n d on the tissues c o n t e n t of i r o n [35, 36]. Studies s t e m m i n g f r o m s e v e r a l laboratories have s h o w n : (1) that the r e l a t i o n l i n k i n g the a m o u n t of ferr i t i n p r o t e i n to the tissue c o n t e n t of i r o n m a i n l y results f r o m an i r o n s t i m u l a t i o n of f e r r i t i n synthesis [37], as e v i d e n c e d by its c o m p l e t e i n h i b i tion by c y c l o h e x i m i d e [38]. T h e c o n t r i b u t i o n of the stabilization of the p r o t e i n m o i e t y by i r o n is still a m a t t e r of c o n t r o v e r s y ; c o n f l i c t i n g results h a v i n g been p u b l i s h e d r e c e n t l y , d e m o n s t r a t i n g [39, 40] or d e n y i n g [38] s u c h an effect of the metal ; (2) that the i r o n i n d u c e d s t i m u l a t i o n of s y n t h e sis is a c t i n o m y c i n i n d e p e n d e n t , i n d i c a t i n g a postt r a n s c r i p t i o n a l m e c h a n i s m , l o c a t e d in the cytoplasm. This p o i n t results f r o m the w o r k of s e v e r a l groups ([41, 42, 39i, p r o v i d e d doses of a c t i n o m y e i n D (8,0 > 150 ~g p e r 100 g of rat) w h i c h specifically depress DNA d e p e n d e n t RNA synthesis [43] w i t h o u t affecting p r o t e i n synthesis w e r e used,
R. Saddi, R. Fagard, J. Feingold and M. M. Sabatier.
742
as i n d i c a t e d in each e x p e r i m e n t [41] b y simultaneous l a b e l l i n g of a l b u m i n as i n t e r n a l control. One must keep in m i n d h o w e v e r that this c r i t i c a l p o i n t is not c o m p l e t e l y settled yet, two groups h a v i n g f o u n d i n h i b i t i o n of the i r o n a c t i o n b y actin o m y c i n D [43 bis-45]. T h e results of one of these have been c r i t i c i z e d as t h e y w e r e i m p r o p e r l y u s i n g the a n t i b i o t i c , in too large doses k n o w n to result in aspecific i n h i b i t i o n of p r o t e i n s y n t h e s i s ; a n d i n d e e d in s u b s e q u e n t e x p e r i m e n t s , this group d i d not confirm t h e i r p r e v i o u s results ; (3) that f e r r i t i n a n d its increase, w h e n tested w i t h cell f r a c t i o n s c o m i n g f r o m i r o n i n j e c t e d animals, m a y be a c h i e v e d in vitro in cell-free systems ; the a m o u n t of l a b e l l e d m a t e r i a l r e c o v e r e d is ulinute but it p r e s e r v e s the c h a r a c t e r i s t i c s of f e r r i t i n : heat stability, e l e c t r o p h o r e t i c a n d salt i n g out p r o p e r t i e s , high m o l e c u l a r weight, antigenicity, specific s t i m u l a t i o n b y i r o n [41]. On the basis of the above d e l i n e a t e d 3 points, it a p p e a r e d that f e r r i t i n s y n t h e s i s p r o v i d e s a conv e n i e n t m o d e l for the s t u d y of t r a n s l a t i o n a l cont r o l of p r o t e i n s y n t h e s i s in m a m m a l s . The e x p e r i m e n t s p r e s e n t e d h e r e are p a r t of a c o m p r e h e n s i v e s t u d y d e v i s e d to analyze the p l a c e and mechanism by which iron enhances ferritin synthesis. T h e y s h o w that i r o n a c t i o n is p r i m a r i l y to i n t r o d u c e a m o d i f i c a t i o n of the m i c r o s o m a l app a r a t u s , a m o d i f i c a t i o n w h i c h c a n n o t be i n d u c e d to a n y a p p r e c i a b l e extent in vitro a n d results in an i n c r e a s e of the amount of c o m p l e t e d f e r r i t i n m o l e c u l e s r e l e a s e d in the i n c u b a t i o n m e d i u m . MATERIALS AND METHODS. CHEMICALS" ATP, GTP, CP a n d CPK (*) w e r e o b t a i n e d from the Sigma Chemical Co., SaintLouis (Miss.) USA, I r o n d e x t r a n (inferon) a n d I r o n s o r b i t o l c i t r a t e (jectofer r e s p e c t i v e l y f r o m L athevet (Paris) a n d from Astra (Sweden), Bovine s e r u m a l b u m i n (BSA) a n d o v a l b u m i n f r o m NBC ('Cleveland, Ohio). I4C l e u c i n e u n i f o r m l y labelled, 270 m C i / m m o l e , a n d 1¢C c h l o r e l l a h y d r o l y s a t e (algal) 30 mCi/14C nl atom, w e r e o b t a i n e d r e s p e c t i v e l y from A m e r s h a m (G.B.) a n d C.E.A. fSaclay, France).
ANIMALS: Albinos male rats of the W i s t a r s t r a i n r e c e i v i n g n o r m a l diet w e r e used t h r o u g hout. RAT
FERRITIN
-"
w e r e p e r f u s e d t h r o u g h the p o r t a l vein, r e m o v e d , h o m o g e n i z e d w i t h 3 volumes of w a t e r , e x t r a c t e d 1/2 h w i t h shaking, c e n t r i f u g e d for 30 mAn at i'0,0,00 g in a Sorvall c e n t r i f u g e ; the s u p e r n a t a n t was h e a t e d for 5 mAn at 76 ± 2°C, f r e e d of coagulated p r o t e i n s b y eentrifugation, p r e c i p i t a t e d t w i c e w i t h a m m o n i u m sulfate b e t w e e n 15 a n d 35 p. 100 c o n c e n t r a t i o n ( w / v ) , finally d i a l y z e d against a p p r o p r i a t e buffer, t i t r a t e d at 3,25,0 nm, a n d s t o r e d frozen in aliquots w h i c h w e r e t h a w e d only once ( f e r r i t i n i n d e e d is p a r t i a l l y d e n a t u r e d on r e p e a t e d freezing a n d thawing)• (2) Ferritin twice crystallized : • C r y s t a l l i z a t i o n w a s a c h i e v e d using the m e t h o d of F i n e b e r g [46] : to c a r r i e r f e r r i t i n d i a l y z e d against NaC1 6.0 raM, 2.6 l u t i d i n e 2.0 mM p H 7.2 buffer (N.L) was a d d e d an equal volume of Cadm i u m sulfate 4 p. cent ( w / v ) d i s s o l v e d in N L a n d a d j u s t e d to p H 7.2 (NIL Cd.). C r y s t a l l i z a t i o n took p l a c e in a few h o u r s a n d w a s c o m p l e t e after a few d a y s at 4°C. It w a s m o n i t o r e d b y m i c r o c o s p i c examination.
• Separation of crystals from amorphous precipitate (AP): s e d i m e n t a b l e m a t e r i a l (crystals + &P) was w a s h e d t w i c e b y c e n t r i f u g a t i o n w i t h NL Cd, r e s u s p e n d e d in the m i n i n u m volume of NL Cd a n d h o m o g e n i z e d w i t h 23 p a r t s of 2.4 M sucrose in NL Cd, w h i c h r e s u l t e d in d i s s o l u t i o n of a m o r p h o u s material, l a y e r e d over 6 ml sucrose 2.4 M in N~L Cd a n d c e n t r i f u g e d for 1 h r at 20,000 r p m in a SW 25 rotor. Crystals w e r e recov e r e d free of AP at the b o t t o m of the tube, w a s h e d in 57L Cd, dissolved in NaC1 c o m p l e x o n buffer p H 7 . 2 (NaC1 0.9 p. cent, c o i n p l e x o n 5 p. cent) a n d d i a l y z e d against the same buffer once, then 3 times against NL buffer and r e c r y s t a l l i z e d a n d r e p u r i f i e d u s i n g the same p r o c e d u r e . T w i c e c r y s t a l l i z e d Cd free r a t f e r r i t i n was finally d i a l y zed against NaCI 0.1 M, p h o s p h a t e 0.025 M p H 7.2, c o n t r o l l e d for p u r i t y b y i m m u n o e l e c t r o p h o r e s i s , using r a b b i t a n t i s e r a d i r e c t e d against r a t l i v e r soluble p r o t e i n s a n d p l a s m a p r o t e i n s (usually not a single p r e c i p i t a t i o n line o t h e r t h a n f e r r i t i n was p r e s e n t ) ; f e r r i t i n w a s finally t i t r a t e d at 3,250 m n a n d s t o r e d frozen in fractions. PREPARATION OF ANTISE'RA : antisera d i r e c t e d against t w i c e c r y s t a l l i z e d rat f e r r i t i n , BSA, a n d o v a l b u m i n w e r e p r e p a r e d in rabbits, u s i n g these p r o t e i n s m i x e d w i t h F r e u n d ' s a d j u v a n t [47].
(1) Carrier ferritin: 250 g rats w e r e i n j e c t e d i n t r a m u s c u l a r l y w i t h i r o n d e x t r a n (130, mg iron) for 15 days. After a lag p e r i o d of 15 days, livers
Preparation of the microsomal and soluble tractions :
(*) Adenosine triphosphate, guanosine triphosphate, Creatine phosphate and creatine phosphokinase.
( 1 ) I R O N S T I M U L A T I O N in v i v o : male rats weighing b e t w e e n 120 a n d 150 g, s t a r v e d overnight,
BIOCHIMIE, 1974, 56, n ° 5.
CE'LL-F~EE
INCUBATIONS
-"
Iron stimulation
w e r e i n j e c t e d w i t h 10 m g p e r 100 g b o d y w e i g h t with iron sorbitol citrate intraperitoneally, usually 3 and o c c a s i o n a l l y 14 hrs before b e i n g killed. Control rats r e c e i v e d saline i n s t e a d of iron. Rats w e r e killed by d e c a p i t a t i o n , bled, t h e i r livers w e r e r e m o v e d , m i n c e d and h o m o g e n i z e d in a loose fitting teflon glass h o m o g e n i z e r in 2.5 times t h e i r w e i g h t of m e d i u m (0.26 M sucrose 0.05 M Tris buffer (pH 7.8 at 20°C), 0.05 M KC1, 0,00,5 M Mg+~C12, 0.005 M glutathione adjusted to p H 7 w i t h KOH). The h o m o g e n a t e s w e r e c e n t r i fuged t w i c e at 10,000 g for 10 m i n ; after r e m o v a l of the fat layer, the p o s t m i t o c h o n d r i a l supernatants w e r e c e n t r i f u g e d in a S p i n c o {model L) TABLE I.
F e r r i t i n stability near the isoeleetrie p o i n t (pH I4.8) at various ionic strengths. pH 7.8 Ionic strengths
0.150 0.100 0.050 0.035 0.025 0.015 0.010
pH 4.8
Ferritin [remaining E 3.250nmlin solution E 3.250 nm {% of inii (tial value . ~ 06 0 0.270 0.250 0.250 0.258 o. 245 0.260
1
100 104 96 100 100 94 100
0.260 0.275 0.215 0.220 0.215 0.145 0.025
Fer.rit.in remaining in solution {% ol initial value 100 105 83 85 83 56 9.6
Rat ferritin samples were dissolved in Tris NaCI buffer at decreasing ionic strengths. Each sample was then divided into 2 halves and placed in an ice bath : one half was acidified to pH 4.8 with diluted acetic acid. After 1/2 hr standing in ice, the samples were centrifuged and the E 3,250 nm absorbancy of the supernatants recorded.
p r e p a r a t i v e u l t r a - c e n t r i f u g e at 32,500, 45,000 rpln for 45 rain in the 40 or 50 rotor. T h e m i d d l e p a r t of the s u p e r n a t a n t w a s r e c o v e r e d a n d used as such, or alternatively, ~n some e x p e r i m e n t s , rec e n t r i f u g e d at 50,000 r p m .for 1 h r 30 in the 50 t i t a n i u m r o t o r to give p a r t i c l e free supernatant. The m i c r o s o m a l (Ms) pellets of the first and, if necessary, of the s e c o n d c e n t r i f u g a t i o n w e r e m i x e d , r e s u s p e n d e d by gentle h o m o g e n i z a t i o n , f r e e d of aggregated m a t e r i a l by a s h o r t l o w speed c e n t r i f u g a t i o n ; the a b s o r b a n c y at 2,6,00, 2,800, 3,250 n m was r e a d to give a m e a s u r e of the conc e n t r a t i o n s of the Ms suspension and the supernatant f r a c t i o n s ; these w e r e adjusted to s t a n d a r d c o n c e n t r a t i o n s ; a l l o w a n c e w a s m a d e f o r the a b s o r b a n c y at 2,60.0 n m due to f e r r i t i n by subs-
BIOCHIMIE, 1974, 56, n ° 5.
743
of ferritin biosynthesis• t r a c t i n g the a b s o r b a n c y at 3,250 nm from the a b s o r b a n c y at 2,60,0 nm.
×
1.6 [48]
(2) IRON ADDED in vitro : f r a c t i o n s w e r e prep a r e d using the same p r o c e d u r e f r o m n o r m a l rats. T h e y w e r e then s e p a r a t e d in t w o h a l v e s : to one was a d d e d i r o n f e r r i c a m m o n i u m c i t r a t e : 2, 10 and 22 .ag p e r ml of i n c u b a t i o n m e d i u m and to the o t h e r was a d d e d the e q u i v a l e n t q u a n t i t y of medium. • The incubation m e d i u m contained in a volume of 6.5 m l : total m i c r o s o m e s f r o m 4 to 5 g of liver, s u s p e n d e d in 2.5 ml of m e d i u m , 2.75 ml of the c o r r e s p o n d i n g s u p e r n a t a n t ( c o n t a i n i n g 100 m g of p r o t e i n ) , 6.5 uM ATP, 1.3 ~M GTP, 6.5 ~M phosp h o c r e a t i n e , 125 eg GPK, and usually 20 ~Ci 1'C leucine. T h e final ion c o n c e n t r a t i o n s w e r e : Mg ÷+ : 0.005 M; K + : 0.043 M. The m i x t u r e was i n c u b a t e d for 3/4 h r at 37°C in air, in a s h a k i n g i n c u b a t o r .
P u r i f i c a t i o n of labelled f e r r i t i n : at the end of the i n c u b a t i o n time, aliquots w e r e set a p a r t to d e t e r m i n e the s p e c i f i c a c t i v i t y (SA) of total protein used as a m e a s u r e of the b i o s y n t h e t i c activity of the i n d i v i d u a l i n c u b a t i o n s . E a c h i n c u b a tion m i x t u r e was t h e n t r e a t e d in one of the following ways : - - in some e x p e r i m e n t s , w h e r e it w a s d e c i d e d to d i s t i n g u i s h the p a r t of the s y n t h e s i z e d ferritin n a t u r a l l y r e l e a s e d in the s u p e r n a t a n t f r o m the one r e m a i n i n g attached to the ribosomes, the incubation m i x t u r e w a s c e n t r i f u g e d for 30 rain at 40,000 r p m (Spinco, Model L c e n t r i f u g e 40 rotor) and m i c r o s o i n e s and s u p e r n a t a n t w e r e t r e a t e d separately ; --in others, the w i t h o u t separation.
whole
sample
was
treated
T h e n each f r a c t i o n , w h e t h e r s e p a r a t e d or not, was h a n d l e d in the same w a y : a d d i t i o n of c a r r i e r f e r r i t i n , e x p o s u r e to US v i b r a t i o n (1.5 m n MSE s o n i c a t o r m o d e l PG 153, p r o b e n ° 25 926 setting : m e d i u m 3), h e a t i n g to 76 ± 2°C d u r i n g 5 rain. The r e s u l t i n g s u p e r n a t a n t was t h e n p r e c i p i t a t e d w i t h a m m o n i u m sulfate b e t w e e n 15 and 35 p. cent conc e n t r a t i o n ( w / v ) ; the p r e c i p i t a t e d f e r r i t i n w a s d i s s o l v e d in 2.5 ml of NaC1 0.2 M, p h o s p h a t e 0.05 M buffer p H 7.3 (*) and t i t r a t e d to p H 4.85 in ice w i t h acetic acid N/3. T h e p r e c i p i t a t e was disc a r d e d and the s u p e r n a t a n t n e u t r a l i z e d a n d cent r i f u g e d free of debris (some f e r r i t i n is lost in this a c i d i f i c a t i o n step but it w a s a s c e r t a i n e d that its SA w a s not different f r o m the one d i s p l a y e d by the acidosoluble ferritin). To each s a m p l e was (*) This high ionic strength ensures stability of the ferritin during the acidification step (table I).
744
R. Saddi, R. Fagard, J. Feingold and M. M. Sabatier.
then a d d e d 0.5 ml of ~2C l e u c i n e s a t u r a t e d saline c o n t a i n i n g 400 gg o v a l b u m i n , and a first p r e c i pitate o v a l b u m i n - a n t i o v a l b u m i n w a s o b t a i n e d a n d s e p a r a t e d by c e n t r i f u g a t i o n ; 2 such p r e c i p i t a t e s w e r e o b t a i n e d b e f o r e the s p e c i f i c f e r r i t i n - a n t i f e r r i t i n p r e c i p i t a t e was p r o d u c e d by a d d i t i o n of r a b b i t anti rat f e r r i t i n in excess (it is w e l l - k n o w n that w i t h r a b b i t a n t i s e r a t h e r e is no solubilization of the antigene a n t i b o d y c o m p l e x e s in the antib o d y excess zone).
on the c l a r i f i e d a n t i f e r r i t i n p r e c i p i t a t e o b t a i n e d p e r ml thereof. In table II an e x a m p l e of the results thus obtained in one e x p e r i m e n t by the 3 t e c h n i q u e s of ferritin e s t i m a t i o n is shown. TABLE II.
Protein content of the ferritin-anti[erritin precipitates as measured by different methods. Lowry
P R O C E S S I N G AND COUNTING OF RADIOACTIVE A N T I GENE ANTIBODY P R E C I P I T A T E S ,
FERRITIN
AND P R O -
TEIN TITRATION :
--The antigene a n t i b o d y p r e c i p i t a t e s w e r e w a s h e d 3 times by c e n t r i f u g a t i o n in p h o s p h a t e buffered saline, s u s p e n d e d in 6 ml distilled water. One d r o p of p e r c h l o r i c acid (PCA) N / 3 w a s t h e n a d d e d r e s u l t i n g in the d i s s o c i a t i o n and c l a r i f i c a tion of the c o m p l e x w h i c h allows d i r e c t absorb a n c y r e a d i n g at 3,2.5.0 n m for f e r r i t i n a n d 2,800 nm for o v a l b u m i n p r e c i p i t a t e s ; --they w e r e t h e r e a f t e r p r e c i p i t a t e d by PCA N / 3 and the p r o t e i n s p r o c e s s e d by a t e c h n i q u e d e r i v e d f r o m that of S c h n e i d e r [49]; the d r y p o w d e r was t h e n dissolved in c o n c e n t r a t e d form i c acid, aliquots w e r e set apart for p r o t e i n assay by the m e t h o d of L o w r y [50], and the r e m a i n d e r w a s c o u n t e d e i t h e r in a l o w b a c k g r o u n d N u c l e a r Chicago c o u n t e r (after e v a p o r a t i n g the f o r m i c acid on steel p l a n c h e t s and w e i g h i n g ) or d i r e c t l y in an I n t e r t e c h n i q u e l i q u i d s c i n t i l l a t i o n counter, each vial c o n t a i n i n g 0.2 ml of the p r o t e i n s dissolved in f o r m i c acid, 0,8 ml of distilled w a t e r a n d 10 ml of Instagel (Packard). - - F e r r i t i n t i t r a t i o n : p r e l i m i n a r y studies h a v e s h o w n that t h e r e exists a good c o r r e l a t i o n (in a n y case sufficient for this k i n d of c o m p a r a t i v e studies) b e t w e e n p r o t e i n e s t i m a t i o n by w e i g h i n g on planchets, L o w r y estimation, and use of extinction coefficient at 3,25.0 nm on a n t i b o d y p r e c i p i tates clari~fied by the above d e s c r i b e d t e c h n i q u e : a c a l i b r a t i o n c u r v e w a s w o r k e d out u s i n g both the r e a d i n g s at 3,250 nm and the p r o t e i n estimation by the m e t h o d of L o w r y : an e x t i n c t i o n coefficient of E 3o._~0L~m nm 1 m g / m l of 9.4 Do units has been established for c r y s t a l l i z e d f e r r i t i n of m e a n i r o n c o n t e n t 20 p. cent ( w / w ) . U s i n g c a r r i e r f e r r i t i n the same coefficient m a y be used p r o v i d e d the a m o u n t of f e r r i t i n p r o t e i n that it c o n t a i n s p e r ml is established for each i n d i v i d u a l pool of c a r r i e r f e r r i t i n , by r e a d i n g the 3,250 nm absorb a n c y d i r e c t l y on the solution as such, and then
BIOCHIMIE, 1974, 56, n ° 5.
Exp 1 Normal.. i -~- F e . . . Exp 2 Normal.. +
Fe...
Weight on E 3,250nm E 3.250 n m planchels absorbancy ×2
2.47 2.85
2.15 2.90
1.21 1.325
2.42 2.65
0.98
1.10 1.25
0.49 0.54
O. 98 1.08
1.145
In this table, the results of the ferritin estimation by E 3,250 nm absorbancy readings are matched with those of the protein assay by the method of Lowry and direct weighing on steel planehets. One must take into account that Lowry titration and weighing measure the total protein (ferritin -b antiferritin) of the precipitate, when extinction at 3,250 nm only estimates ferritin moiety ; bearing this in mind, the results above presented stronly suggest that in the ferritin-antiferritin precipitate there is about the same amount of ferritin and antiferritin protein, which means that for each molecule of ferritin (MW 460,000) there exists a mean of 3 antibody molecules 7 S (MW : 160,000 X 3 -----480,000).
RBSeULTS P R E S E N T A T I O N : Results h a v e been e x p r e s s e d in 2 w a y s listed f r o m left to r i g h t in the tables :
(1) the c p m i n c o r p o r a t e d into f e r r i t i n p e r incubation i.e. by the m i c r o s o m e s of 4 g l i v e r (after d e d u c t i o n of b a c k g r o u n d counts, and t h e ones r e c o v e r e d into f e r r i t i n for the test s a m p l e prep a r e d w i t h o u t e n e r g y and p l a c e d in ice at the b e g i n n i n g of the i n c u b a t i o n p e r i o d i.e. about 15 c p m each). This figure has also been c o r r e c t e d to the i n i t i a l l y a d d e d a m o u n t of c a r r i e r f e r r i t i n , u s i n g the a b s o r b a n c y at 3,250 n m of the c l a r i f i e d p r e c i p i t a t e at the end of the p u r i f i c a t i o n p r o c e d u r e : this c o r r e c t i o n accounts for the losses of material during purification ; (2) the last figure takes into a c c o u n t for the sake of c o m p a r i s o n the b i o s y n t h e t i c a c t i v i t y of the i n d i v i d u a l i n c u b a t i o n (each p r e p a r e d f r o m a d i f f e r e n t rat), m e a s u r e d by the s p e c i f i c activity (SA) of total p r o t e i n s at t h e end of the i n c u b a tion ; this c o r r e c t i o n brings the f e r r i t i n c p m p e r i n c u b a t i o n to the one w h i c h w o u l d h a v e been f o u n d for a h y p o t h e t i c a l SA for total p r o t e i n of 2,000 c p m / m g .
Iron s t i m u l a t i o n of ferritin biosynthesis. (3) the data h a v e been statistically t r e a t e d by analysis of the v a r i a n c e and by the t. test of Student. RESULTS. F o r c l a r i t y each result w i l l be p r e s e n t e d a n d discussed s e p a r a t e l y in a series of tables a n d figures. (1) I r o n s t i m u l a t i o n c a n n o t be a p p r e c i a b l y o b t a i n e d w h e n the metal is d i r e c t l y a d d e d in vitro to a cell-free system p r e p a r e d f r o m a normal rat liver.
745
F r o m results s h o w n in table III, it is clear that no s t i m u l a t i o n w a s o b t a i n e d by the d i r e c t a d d i tion of i r o n in the i n c u b a t i o n m e d i u m . (In some e x p e r i m e n t s , instead, i n h i b i t i o n w a s observed). The d i s c r e p a n c y b e t w e e n these results and those of p r e v i o u s p r e l i m i n a r y e x p e r i m e n t s , i n o u r labor a t o r y and in others [51-53], w h e r e a l o w a n d i n c o n s t a n t effect of i r o n in vitro could be observed, is not at p r e s e n t u n d e r s t o o d , although it could result f r o m m i n u t e t e c h n i c a l d i f f e r e n c e s ; in v i e w of the results r e p o r t e d above, h o w e v e r , it seems safe to c o n c l u d e that in o r d e r to a p p r e c i a bly i n c r e a s e f e r r i t i n synthesis, i r o n m u s t act in
TABLE III.
Radioactive amino acid incorporation into ferritin by rat liver microsomes incubated with (Fe) or without (N) iron directly added in vitro.
Iron
CPM into ferritin )er incubation
S.A. ot total protein
CPM into I ferritiu cor-I rected for a I total t protein S.A.or 2000 CPM/MG
Relative increase (p. !00)
Statistical significance of the difference
1
2¥/m1
199 + 67 187 + 43
1640 + 282 1628 + 230
239 -'~ 40 234 -'~ 38
--
NS
2
10y]ml
642 -~- 33 605 T- 18
3220 -4- 144 3950 ~ 157
400 + 6 308 + 19
- - 30
3
-22y/ml
973 ~ 133 735 ___~163
3480 -P 793' 550 + 70 3029 ~ 626 488 1- 19
- - 13
Experiment N°
p :
0,001 NS
Each entry in the table represents the mean (with the standard deviation) of 3 different incubations. 3 separate experiments are presented, each using a different pool of microsomes. A clear-cut d e m o n s t r a t i o n of an i r o n effect in vitro w o u l d h a v e h a d a great t h e o r e t i c a l signi: ficance s i n c e it w o u l d have p r o v i d e d the d i r e c t p r o o f of the p o s t - t r a n s c r i p t i o n a l site of i r o n stimulation, a p r o o f w h i c h is still needed, since, as d e s c r i b e d above, an i n h i b i t o r y effect of Actinom y c i n D in this s t i m u l a t i o n is not c o m p l e t e l y r u l e d out at present. Besides, it w o u l d h a v e greatly simplified the in vitro study of the site and m o d e of i r o n action. T h e r e f o r e , in a c c o u n t of the l o w (20 to 30 p. cent) and i r r e g u l a r effects o b t a i n e d by "the a d d i t i o n of i r o n in vitro in several l a b o r a t o r i e s , it has been f o u n d n e c e s s a r y iv r e e x a m i n e the question. I r o n a d d i t i o n has been f r o m 2 to 22 ~ g / m l of i n c u b a t i o n , these b e i n g the c o n c e n t r a t i o n s o b t a i n e d in sampIes p r e p a r e d f r o m rats i n j e c t e d w i t h 5 to 10 mg i r o n IM, 3 to 4 h r s before death, samples w i t h w h i c h an i m p o r t a n t i n c r e a s e of f e r r i t i n synthesis o v e r c o n t r o l has r e g u l a r l y been found.
BIOCHIMIE, 1974, 56, n ° 5.
vivo : this suggests that the m o d i f i c a t i o n r e s u l t i n g in an i n c r e a s e d c a p a c i t y for a cell to synthesize f e r r i t i n i n v o l v e s a step w h i c h needs a degree of o r g a n i z a t i o n of the cell m a c h i n e r y not a c h i e v e d in cell-free p r e p a r a t i o n s , Next step to inject i r o n in vivo and, by crosse x p e r i m e n t s w i t h p r e p a r a t i o n s o b t a i n e d f r o m normal animals, to locate in cell-free i n c u b a t i o n s the f r a c t i o n (soluble or m i c r o s o m a l ) w h i c h h a d b e e n changed. (2) I r o n acts m a i n l y t h r o u g h a m o d i f i c a t i o n of the m i c r o s o m a l apparatus, and not of the soluble f r a c t i o n of the cell cytoplasm. This a p p e a r s f r o m data p r e s e n t e d in table IV : m i c r o s o i n e s p r e p a r e d f r o m i r o n i n j e c t e d animals 3 hrs before death <
R. Saddi, R. Fagard, J. Feingold and M. M. Sabatier.
746
effect of t h e <
i n j e c t e d w i t h i r o n 3 to 4 h r s b e f o r e d e a t h . It r a i s e s t h e q u e s t i o n of t h e p r e s e n c e of an i n h i b i t o r of f e r r i t i n t r a n s l a t i o n in t h e <
TABLE IV. Fraction origin S.A. ofthelast ovalb-antiovalb. precipitate
CPM into Ierri'dn per incubation
S.A. of total protein
CPM into ferritin corrected for a total protein S.A. of 2000 CPM/MG
Relahve increase (p. t00)
Statistical significance of the difference
Microsomes
45.000 RPM × 3/4 h supernatant
N
N
96 ~
7
1957 -+- 93
98 ~___ 4
N
Fe
137 ~
12
1930 -4- 182
140 ~___I0
+
Fe
Fe
294 -t- 12
1940 ~__ 56
303 ~_
6
÷ 2O0
)
Fe
N
330 -~- 19
1992 -+- 102
332 ~
23
+ 240
3
43
I p <
0,01
p <
0,05
TABLE V. Fraction origin S.A. of the last ovalb-autiovalb. precipitate
CPM into ferritiu per incubation
Mierosomes
50.000 RPM × lh30 supernatant
N
N
9
N
Fe
10
334 -t- 30
Fe
Fe
9
1037 ~ 45
Fe
N
7
1237 ~
398 ~
87
172
F r o m t a b l e V w h e r e t h e s u p e r n a t a n t has b e e n r e c e n t r i f u g e d at 50,0.00 r p m / 1 . 3 0 hr, it is c l e a r t h a t t h i s is i n d e e d tile e a s e a n d t h a t t h e <
BIOCHIMIE, 1974, 56, n ° 5.
S.A. total protein
CPM into ferritin corrected for a total protein S.A. o[ 2000 CPM/MG
Relative increase (p. 100)
3840 -~- 439
204 ~
27
--
3560 ~
192 ___~ 43
-- 7
3477 ~- 246
590 ~
83
-J- 190
2640 ~
957 ~
176
~- 370
847
616
Statistical significance of the difference
N
p <
0,01
T h e d e x t r i n c o n t a i n e d in t h e i r o n p r e p a r a t i o n used (Jectofer) could, early' after injection account f o r t h i s n e g a t i v e effect : l a t e r i n t i m e t h i s i n h i b i tion would disappear, the dextrin having been metabolized. I r o n c i t r a t e a n d s u l f a t e w e r e t h e r e f o r e u s e d in p l a c e of i r o n d e x t r i n . T a b l e VI s h o w s t h a t u n d e r t h e s e c o n d i t i o n s , no i n h i b i t o r y a c t i o n is a p p a r e n t . A d i r e c t p r o o f of t h e s e e n i n table VII.
dextrin
effect m a y
be
(4) t h e i n c r e a s e i n f e r r i t i n s y n t h e s i s b r o u g h t a b o u t by i r o n i n j e c t i o n affects a l m o s t e x c l u s i v e l y the ferritin spontaneously released into the supern a t a n t (table VIII), at l e a s t i n t h e f e w h o u r s a f t e r
Iron s t i m u l a t i o n of ferritin biosynthesis. s t i m u l a t i o n . T h i s is e v i d e n c e d b y e x p e r i m e n t s i n which after the incubation, the samples were separated into supernatant and pellet by a short (30 r a i n ) u l t r a c e n t r i f u g a t i o n at 40,0.00 r p m a n d t h e ferritin prepared and counted in these two frac-
747
p o l y s o m e s i n v o l v e d i n t h e s y n t h e s i s of t h e p r o t e i n a n d t h u s the actual q u a n t i t y of active f e r r i t i n m e s s e n g e r RNA is of t h e s a m e m a g n i t u d e i n n o r m a l a n d i n i r o n t r e a t e d r a t s (at l e a s t i n t h e f i r s t hours after iron stimulation), the difference being
TABLE V[. Fraction origin
Microsomes
Supernatant
CPM into ferritin per incubation
CPMinto ferritin S.A. of total ]corrected for a protein / total /protein S.A. oi 2ooo CPM/MG
Relative increase (p. t00)
Statistica 1 significance ol the diIlereace
r
N
N
428 -4- 166
6150 ______2616
140 ~____ 6
N
Fe
356 ___+ 141
6010 ______2284
117 ~
Fe
Fe
890 -I-
5150 -t-
354 -~- 52
~
Fe
N
763 -~- 130
294 ~
+ 110
NS 71
654
5150 _____ ~
14
22
--
16
153
NS
TABLE VII. Fraction origin
MicroSOrties
Supernatant
Fe
N
Fe
D
CPM into ferritin per incubation
1659 ~
S.A. ol total protein
CPM into Ierritin Relative corrected for a i n c r e a s e total (p. t00) protein S.A. ol 2000 CPM/MG _ _
1055 13.199 4- 8328
254 -4- 48
--
851 --I- 188 14.014 -+- 5416
165 q- 12
- - 46
Statistical significance of the difference
P <
0.001
1~C lencine incorporation into ferritin by rut liver microsomes prepared from normal (N) or ion injected (FE) animals # hours before sacrifice, incubated with homologous or heterologous supernatant. Each e n t r y in the table represents the m e a n (with the s t a n d a r d deviation) of 3 incubations p r e p a r e d w i t h fractions derived f r o m a pool of the livers of 3 i d e n t i c a l l y treated rats. - - In Table IV and V the iron p r e p a r a t i o n injected was iron d e x t r i n sorbitol (jeetofer) and in Table VI iron sulfate. - - In Table IV mierosomes and s u p e r n a t a n t s were p r e p a r e d by u l t r a e e n t r i f u g a t i o n at 45,000 r p m × 3/4 hr. In Table V, mixed m i e r o s o m e s of 2 u l t r a e e n t r i f u g a t i o n s , the one at 45,000 r p m × 3/4 hr, the o t h e r at 50,000 r p m × I h r 30 of the s u p e r n a t a n t resulting f r o m the first centrifugation were used ; the s u p e r n a t a n t s used for the incubation being the ones resulting f r o m the second u l t r a c e n t r i f u g a t i o n (50,000 r p m × 1 h r 30). -In Table VII, mieros0mes (Fe) were p r e p a r e d f r o m ferric a m m o n i u m citrate injected rats (600 lxg per 100 g of body weight). S u p e r n a t a n t s were f r o m n o r m a l r a t s (N) or d e x t r i n injected a n i m a l s (D). The dextrin, the same as t h a t incorporated into jeetofer, was a gift f r o m Dr Lindvall (Astra S f d e r t a j l e , Sweden) ; the dose used, 0.33 mg per 100 g b o d y weight, was t h e one received by j e e t o f e r injected rats.
l i o n s a f t e r a d d i t i o n of c a r r i e r . T h e f e r r i t i n p o l y peptides artificially detached from the microsom a l pelle~ b y u l t r a s o n i c v i b r a t i o n s h o w a b o u t t h e s a m e a m o u n t of 14C l e u c i n e i n c o r p o r a t i o n w h e t h e r t h e y come f r o m iron t r e a t e d or control rats. This finding could suggest that the number of
BIOCHIMIE, 1974, 56, n ° 5.
i n t h e s p e e d of t r a n s l a t i o n , o r i n t h e n u m b e r o f the initiation events. This interpretation would be in keeping with t h e l a c k of a c t i o n o f a c t i n o m y c i n D i n i n d u c t i o n of fcrritin synthesis.
R. Saddi, R. Fagard, J. Feingold and M. M. Sabatier.
748
T a n L E VIII.
~ C incorporation into ferritin naturally released into the supernatant and inlo [erritin polypeptides artificially detached from microsomes by ultrasonic vibration (U.S.) at the end of the incubation time. Fraction origin CPM into supernatant ferritin
Relative increase
CPM into ferritin polypeptides detached by U.S. from microsomes
Microsomes
Supernatant
N
N
61
90
N
Fe
62
98
Fe
Fe
193
-q- 216
97
Fe
N
327
+ 436
115
(p. 100)
Relative increase
(p. t00)
+9 ÷8 ÷ 2g
Microsomes a n d s u p e r n a t a n t f r a c t i o n s are f r o m n o r m a l (N) or 4 h r p r e v i o u s l y j e e t o f e r injected a n i m a l s (Fe) i n c u b a t e d w i t h h o m o l o g o u s or h e t e r o l o g o u s s u p e r natants. Mixed m i e r o s o m e s of 2 successive u l t r a e e n t r i f u g a t i o n s , t h e first a t 32,500 r p m × 3/4 hr, t h e second a t 50,00D r p m × 1 h r of t h e s u p e r n a t a n t r e s u l t i n g f r o m t h e first one, w e r e used. T h e s u p e r n a t a n t u s e d is t h e one o b t a i n e d a f t e r t h e second u l t r a c e n t r i f u g a t i o n a t 50,0t)0 r p m × 1 hr.
CONCLUSION. The present work clearly establishes that iron action mainly results in the introduction of a m o d i f i c a t i o n of the m i c r o s o m a l p a r t of t h e t r a n s lational machinery. T h e m o l e c u l a r n a t u r e of t h i s i r o n - i n d u c e d m o d i f i c a t i o n a n d t h e c a u s e o f t h e n o n ( o r at l e a s t p o o r ) o p e r a t i o n of t h i s p r o c e s s in vitro m u s t b e now unravelled. T h i s w o r k is b e i n g c u r r e n t l y u n d e r t a k e n laboratory.
in our
Ce t r a v a i l p r 6 s e n t e les p r e m i e r s r 6 s u l t a t s d ' u n e 6tude d ' e n s e m b l e e n t r e p r i s e p o u r t e n t e r de pr6eiser le lieu, et d'dlucider le m o d e d ' a c t i o n du fer d a n s ce syst6me. 1) Le fer agit p a r l ' i n t e r m ~ d i a i r e d ' u n e modification de l ' a p p a r e i l m i e r o s o m a l , et n o n de la f r a c t i o n soluble du s u r n a g e a n t ; 2) Cette modification ne p e u t 6tre apport6e a u x m i c r o s o m e s de faqon apprdciable qu'in vivo ; 3) l ' a e t i v a t i o n m i e r o s o m a l e , u n e fois i n t r o d u i t e in oioo p e u t dtre 6tudi~e in vitro ; elle se t r a d u i t p a r u n e a u g m e n t a t i o n eonsid6rable de la f e r r i t i n e m a r q u 6 e lib6r6e d a n s le s u r n a g e a n t d ' i n e u b a t i o n , alors que les p o l y p e p t i d e s f e r r i t i n i q u e s m a r q u 6 s ddtaeh6s artifie i e l l e m e n t p a r les u l t r a s o n s s o n t p e u modifies, du m o t h s apr6s u n e s t i m u l a t i o n de q u e l q u e s h e u r e s .
Acknowledgements. T h i s w o r k w a s s u p p o r t e d b y g r a n t s of t h e <
BIOCHIMIE, 1974, 56, n ° 5.
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