BIOCHIMIE, 1975, 57, 285-293.

Study of mammalian ribosomal protein reactivity in situ. I. - Effect o f 2 - m e t h o x y - 5 - n i t r o t r o p o n e

on 4 0 S and 60S s u b u n i t s .

Anne-Marie REBOUD, Jean-Jacques MADJAR, Monique BuissoN a n d J e a n - P a u l REBt)UD. Laboratoire de Biochimie M~dicale, U.E.R. Lyon Nord, 43, bd du 11 N o v e m b r e 1918, 69621 Villeurbanne.

(12-2-1975). Summary. - - Liver ribosomes and subunits were reacted with increasing concentrations of 2-methoxy-5-nitrotropone. At low reagent concentrations (0.3 raM), the molar uptake by 60S stLbunits was more efficient than the uptake by 40S subunits, and the amount of reagent bound to 80S ribosomes was less than that bound to both free subunits considered together. At higher reagent concentrations, the molar uptake of both subunits was equivalent. Subunits and ribosomes remained fully active w~hen reacted with up to 0.3 mM and 1 mM of the reagent, respectively. With 2 mM of the reagent, both subunits were half inactivated, although their sedimentation characteristics were unaltered. The reactivity of each ribosomal protein was assessed by two-dimensional gel electrop~boresis and quantitative measurement of the unmodified proteins. From these results, considered together with the uptake eharacteristies and the inactivation curves, a number of tentative conclusions about ribosome topography can be drawn. The over-all sensitivity of the 60S subunits to the reagent is higher than that of the 40S subunits. Both subunits undergo a conformational change when they combine to form 80S ribosomes. Proteins S~s, S~ S~ and L~, Lg, L=, L~, Lz~, L.~, L~, L~o, L~, L~, La7 have NH.~ groups exposed in native subunits. These groups are not essential for subunit function.

INTRODUCTION. P r o t e i n r e a c t i v i t y in b a c t e r i a l r i b o s o m e s has been studied by the use of s e v e r a l c h e m i c a l and e n z y m a t i c reagents and, m o r e r e c e n t l y , specific antibodies. I n f o r m a t i o n was obtained on the spatial a r r a n g e m e n t of the p r o t e i n s w i t h i n lhe subunits and also, on the f u n c t i o n s of several ribosomal proteins. Until now, f e w such e x p e r i m e n t s have been c a r r i e d out w i t h M a m m a l i a n ribosomes, because the n u m e r o u s p r o t e i n s of 40S and 60S subunits we.re only recen.tly identified w i t h two d i m e n s i o n a l gel e l e c t r o p h o r e s i s . We studied the r e a c t i v i t y of i n d i v i d u a l proteins in rat l i v e r r i b o s o m e s and subunits, using d i f f e r e n t c h e m i c a l an,d e n z y m a t i c reagents. The results w h i c h follow w e r e o b t a i n e d using 2-met h o x y - 5 - n i t r o t r o p o n e , a c h e m i c a l reagent w h i c h is specific for p r i m a r y a m i n o groups [1, 2, 3, 4]. An essential f u n c t i o n of these groups in p o l y p e p tide synthesis w a s e m p h a s i z e d by Moore [5]. In an a t t e m p t to localize the effect of 2-methoxy-5n i t r o t r o p o n e , the r e a c t e d p r o t e i n s w i t h i n both subunits w e r e identified and quantified, and the 0

To whom all correspondence should he addressed.

a c t i v i t y of the t r e a t e d subunits was tested. The p o s s i b i l i t y that a c h e m i c a l r e a g e n t i n d u c e s a s t r u c t u r a l p e r t u r b a t i o n c a n n o t be r u l e d out unless a c t i v i t y is m a i n t a i n e d . As rat l i v e r r i b o s o m e s and r i b o s o m a l subunits kept a large p a r t of t h e i r activity in p r o t e i n synthesis after n i t r o t r o p o n y l a t i o n , the p o s s i b i l i t y that a m a j o r c o n f o r m a t i o n a l change had been i n d u c e d could be excluded. T h e r e f o r e , this r e a g e n t a p p e a r e d to be p a r t i c u l a r l y suitable for s t r u c t u r a l studies of M a m m a l i a n ribosomes. MATERIAL AND METHODS.

Ribosomes and snbunits. R i b o s o m e s w e r e p r e p a r e d by the m e t h o d of Moldave [6] w i t h slight modifications. R i b o s o m e s r e c o v e r e d after p r e c i p i t a t i o n by 50 mM MgC1,_, w e r e dialyzed against buffer N (1 mM p o t a s s i u m p h o s p h a t e p H 7.3, 0.2 mM MgC12, 3 p. cent sucrose). Active r i b o s o m a l subunits w e r e p r e p a r e d w i t h 0.3 M KC1 and isolated as p r e v i o u s l y described [7]. Unfolded subunits w e r e p r e p a r e d by dialysis for 20 hrs against buffer N, c o n t a i n i n g 0.2 mM EDTA, instead of MgC12. T h e y h a d a S.20,~. of 50S and 30S as m e a s u r e d on a sucrose gradient.

286

A.-M. Reboud, J.-J. M a d jar, M. Buisson and J.-P. Reboud.

2-methoxg-5-nitrotropone treatment. R e a c t i o n of subunits or r ib o s o m e s w i t h 2-met h o x y - 5 - n i t r o t r o p o n e w a s c a r r i e d out as d e s c r i b e d by Suzuka [3] w i t h slightly modified conditions. To a set of samples of 40S or 60S subunits or r i b o s o m e s at a c o n c e n t r a t i o n of 1, 2.5 or 3.5 m g / ml, r e s p e c t i v e l y , in buffer A (0.050 M t r i e t h a n o l a m i n e buffer at pH 7.4, c o n t a i n i n g 0.2 M KCI, 4.5 mM MgCI~ and 6 mM ~-mercaptoethanol) was a dd ed a solution of 2 - m e t h o x y - 5 - n i t r o t r o p o n e (27.6 mM in f o r m a m i d e ) . The final c o n c e n t r a t i o n s of the reagent w e r e 0, 0.03, 0.06, 0.15, 0.3, 0.6, 1 and 2 mM. Th e samples w e r e i n c u b a t e d at 29°C for 6~ hrs, t r a n s f e r r e d to dialysis bags, and then d i a l y z e d against buffer A, w h i c h w a s r e p l a c e d several times. Th e n i t r o t r o p o n y l subunits or ribosomes w e r e p e l l e t e d by c e n t r if u g a ti o n . T h e pellets, dissolved in an equal v o l u m e of buffer A, w e r e clarified before i n e a s u r e m e n t of f u n c t i o n a l activity. In addition, p a r t of the suspension was d i r e c t l y e x t r a c t e d by acetic acid w i t h o u t clarification. To estimate the n u m b e r of r e a g e n t molecules p e r 40,S, 60S, or 80S particle, the following criteria were employed : 1 °) the m o l a r a b s o r p t i o n coefficient of N ~ a c et y l - N- : -. n i t ro t r o p o n y l- l y s i n e is 2.07 × 104 M-a c m 1 at 420 nm [!1]. 2 ° ) the m:olecular w e i g h t s of the 40S and 60S subunits are equal to 1.2 X 106 and 3.1 X 106 daltons, r e s p e c t i v e l y [8], and the p e r c e n t a g e s of protein in each subunit 52 and 42..5 p. cent respectively [7]. 3 °) a 1 m g / m l solution of either subunit has an A260of 14.0.

Assay of amino-acid incorporation activity. Th e a c t i v i t y of n i t r o t r o p o n y l r i b o s o m e s or subunits was assayed u n d e r c o n d i t i o n s of l i m i t i n g ribosomes, by using a poly ( U ) - d e p e n d e n t p h e n y l a l an i n e i n c o r p o r a t i o n system [7]. T r e a t e d subunits w e r e tested in c o n j u n c t i o n w i t h the untreated c o m p l e m e n t a r y ones.

Analysis of reaction products. Acetic e x t r a c t i o n of p r o t e i n s c o n t a i n e d in ribosomes was c a r r i e d out as d e s c r i b e d by H a r d y et al. [9]. After e x t r a c t i o n of the p r o t e i n s f r o m the tre at ed particles, the r e s i d u e of r i b o s o m a l RNA was ahnost colorless, suggesting that the r e a c t i o n of 2-methoxy-5-nitro.tropone w i t h n u c l e i c acid was m i n i m a l . T w o - d i m e n s i o n a l a c r y l a m i d e gel e l e c t r o p h o r e s i s w a s p e r f o r m e d as d e s c r i b e d by T r a u t et al. El0] and the gel w a s stained w i t h a m i d o b l ack . W i t h this t e c h n i q u e , a s m a ll e r quantity of pro.teins could be used than w i t h the original p r o c e d u r e of K a l t s c h m i d t and W i t t m a n n [11].

BIOCHIMIE, 1975, 57, n ° 3.

Th e e l e c t r o p h o r e t i c p a t t e r n s w e r e v e r y s i m i l a r to those observed by S h er t o n and W o o l [122, and, therefore, w e used t h e i r n o m e n c l a t u r e . H o w e v e r , because of the slight d i f f er en ces in the t e c h n i q u e s used for subunit p r e p a r a t i o n and for e l e c t r o p h o resis, w e c a n n o t be absolutely sure that all the p r o t e i n s identified on our p a t t e r n s c o r r e s p o n d to the same p r o t e i n s in theirs.

Determination of the intensity of the spots in the electrophoretic pattern. E a c h spot and its s u r r o u n d i n g area w as cut out and i n c u b a t e d o v e r - n i g h t at r o o m t e m p e r a t u r e w i t h 0.25 ml of 25 p. cent p y r i d i n e [13]. Th e ~bsorbanee was m e a s u r e d at 635 nm w i t h a Gilford s p e c t r o p h o t o m e t e r . Extractio.n of the color a t i o n by this m e t h o d w a s f o u n d to be quantitative. Th e c o l o r a t i o n eluted f r o m the spots w a s p r o p o r t i o n a l to the a m o u n t of protein, up to 25 ~,g of proteins. The p e r c e n t a g e of a b s o r b a n c e of each spot r e l a t i v e to the total a b s o r b a n e e of all spots was r e p r o d u c i b l e f o r different gel plates prep a r e d f r o m c o n t r o l r i b o s o m a l proteins. Th e m a i n q u a n t i t a t i v e cause of e r r o r of this method resulted f r o m the difficulty in cu t t i n g a r o u n d the co l o r ed area s u r r o u n d i n g each spot.

Materials. We used male W i s t a r rats w e i g h i n g 200-250 g. 2 - m e t h o x y - 5 - n i t r o t r o p o n e w as p u r c h a s e d from the San:kyo Co., Japan. RESULTS.

Uptake o[ 2-methoxy-5-nitrotropone somes and ribosomal snbanits.

bg ribo-

T h e n i t r o t r o p o n y l a t i o n of r i b o s o m e s can be easily followed by m e a s u r i n g the a b s o r b a n c e at

3 O2

!

|ncub&tion

|

t i m e (hrs)

Fro. 1. - - Ribosomal nitrotroponylation kinelics. One aliquot of ribosomes treated with 2 mM of reagent was dr~wn at each interval of time, as indicated in the figure, and dialyzed (see Methods). The extent of reactions is expressed in absorbance at 420 nm for a concentration of 2.54 m g / m l of ribosomes.

P r o t e i n r e a c t i v i t y in m a m m a l i a n 420 nm. F i g u r e 1 shows t h e uptake of the reagenl as a f u n c t i o n of the i n c u b a t i o n time. After -1 hrs, the g r a p h b e c a m e a plateau. In the f o l l o w i n g

~

287

trated in figure 2. T h e f o l l o w i n g points are w o r t h noting : Firstly, the r i b o s o m e s and the subunits w e r e not saturated w i t h the reagent, in the range of c o n c e n t r a t i o n s used. This failure could be due to the e x i s t e n c e of several classes of free a m i n o groups w i t h d i f f e r e n t r e a c t i v i t y .

&

200

r i b o s o m e s (1).

Secondly, at low c o n c e n t r a t i o n s of reagent, the 80S ribosomes are less r e a c t i v e titan free 40S and 60S subunits c o n s i d e r e d together. This m i g h t be due to s h i e l d i n g of some p r o t e i n s in tile 80S ribosomes. Tile results at high c o n c e n t r a t i o n s are p r o b a b l y ~ess significant, as will he discussed later.

/

,a

T h i r d l y , the uptake by 60S subunits was c o n s i d e r a b l y greater than that by 40S subunits at low c o n c e n t r a t i o n s of the reagent. This and the p r e c e d i n g o b s e r v a t i o n were c o n f i r m e d by studies of isulated proteins.

~100

o

1

2

Reagent c o n c e n t r a t i o n

(mM)

FIG. 2. - - Uptake of 2-melhoxg-5-nilrolropone

by ribosomes and ribosomal subanits.

Riboson~es were treated with 2-methoxv-5-nitrotropone as described in Methods. Ribosome-l~ound 2-methoxy-5-nitrotropone (moles/mole of particles) is plotted against the initial reagent concentration in the reaction mixture. Uptake ljy 80S ribosomes (A) bv 60S subunits ( e ) , by 40S subunits (©) and ~ ; ' 6 0 S plus 40S subunits (A).

e x p e r i m e n t s , the r i b o s o m e s and t r e a t e d by the r e a g e n t for 6 hrs.

subunits

were

The uptake of 2 - m e t h o x y - 5 - n i t r o t r o p o n e by r i b o s o m e s and by 40S and 60S subunits t r e a t e d w i t h i n c r e a s i n g c o n c e n t r a t i o n s of r e a g e n t is illus-

Lastly, the n u m b e r of r e a g e n t nmlecules bound to each subunit or to 80S r i b o s o m e s can be c o m p a r e d to the total n u m b e r of p o t e n t i a l l y r e a c t i v e NH 2 groups. These groups are m a i n l y found in lysine residues. T h e n u m b e r of lysine residues can he a p p r o x i m a t e l y d e t e r m i n e d as follows : the m o l e c u l a r w e i g h t of a mole of 40S or 60S subunit p r o t e i n can be d e d u c e d f r o m the m o l e c u l a r w e i g h t s . o f the r e s p e c t i v e subunits and f r o m the "weight p e r c e n t a g e of p r o t e i n in each subunit, w h i c h have been cited in Methods. Molecular w e i g h t s of 624 × 10 .3 and of 1317 X 10 a for the total p r o t e i n s of the 40S and 60S subunits are found, respectively. Assuming that the m o l a r p e r c e n t a g e of lysine is i d e n t i c a l in both subunits, and equal to 9.63 moles p e r 100 moles of aminoacid [1,1!, and c o n s i d e r i n g the average m o l e c u l a r w e i g h t of the a m i n o a e i d s in both subunits to be 130, the values of 46,2 and 975 residues of lysine p e r 40S and 60S subunits, r e s p e c t i v e l y , are FIG. 3. - - Ribosomal inaclioalion

by 2-methoxg-5-nitrotropone.

A

A) ttibosomes (A) and 40-S ( o ) and 60-S subunits (O), treated with increasing concentrations of 2-methoxy-5-nitrotropone, were assayed for polyphenylalanine-synthesizing activity (see Methods). With treated subunits, the reaction of polymerization was initiated by adding equimolecular amounts of the untreated complementary snbnnit. The quantity of p(~lyphenylalanine synthesized by untreated ribosomes or subunits was considered to be equal to 100. BI Loss of polvphenvlalanine-synthesizing ability of ribosomes ( ~ ) and 40-S ( e ) and 60-S subunits (©) as a function of the proportion of lysine which had reacted with 2-methoxy-5-nitrotropone.

loc

t~

o~ 5O

5C

g ii

o

1

Reagent

concentration

BIOCHIMIE, 1975, 57, n ° 3.

2 (mM)

I0

Reacted

lysine

15 (°//O)

s~

i::~:i:i s,3

~°

i

'1

)0

:iiiii:i:i)~:~:i):~i,~:!i::i i¸•: • :~:•::::::

.....

•"

~

$1L

,:: ;L

0

~

L

~¸ : :

)

::~::i~ i : ¸

::::i:i:iiii~iii,:ii~i:ii~

• 5 :: : i ):: .:)J

.

~IG. 4. I T~oo-dimensional gel eleclrophorcsis patterns of control and nitrotroponyl subunits. 1.5 m g of active 60S s u b u m t s (A) a n d 40S s u b u n i t s (B) w e r e r e a c t e d w i t h (a), 0, (b) 0.3, (c) 1 a n d (d) mM of 2 - m e t h o x y - 5 - n i t r o t r o p o n e . T h e r e a c t i o n w a s t e r i n i n a t e d b y d i a l y s i s a g a i n s t huffer A, a n d the proe i n s w e r e e x t r a c t e d w i t h acetic acid. T w o - d i m e n s i o n a l gel e l e c t r o p h o r e s i s w a s carried out as d e s c r i b e d in 4ethods. N u m e r o u s spots, a l t h o u g h h a r d l y v i s i b l e on the p h o t o g r a p h s , w e r e quite d i s t i n c t l y o b s e r v e d on the :el plates.

: i :: ::•ii•i:i:!:i~:~:~

0

i

c

~o

290

A.-M. Reboud, J.-J. M a d jar, M. B u i s s o n a n d J.-P. Reboud.

obtained. Thus, about 10 p. cent of the total lysine residues in both s u b u n i t s reacted with 2-methoxy-5-nitrotropone at a c o n c e n t r a t i o n of 2 raM. A s i m i l a r low percentage of reacted lysine was calculated from the data of Suzuka [3] conc e r n i n g uptake of reagent by E. coli 50-S s u b u n i t s relative to the total n u m b e r of lysine residues per subunit. It was not possible to measure the uptake o,f the reagent b y the free p r o t e i n s or b y the proteins i n s u b u n i t s u n f o l d e d by EDTA, because of aggregation in both cases.

Activity somes.

and sedilnentation

of treated ribo-

Ribosomes and s u b n n i t s were i n c u b a t e d w i t h v a r y i n g c o n c e n t r a t i o n s of 2-metho.xy-5-nitrotrop o n e in the usual w a y and then assayed for poly (U)-directed p o l y p h e n y l a l a n i n e synthesis. I n figure 3A, activity is plotted against reagent concentration. Ribosomes were able to b e exposed to h i g h e r reagent c o n c e n t r a t i o n s t h a n free su,bunits, w i t h o u t losing a n y activity. The inactivation o£ 40'S an,el 6,0.S s n b u n i t s in the 80S ribosomes, follo~wed by h y b r i d i z a t i o n of the nitrotro~p.onyl ribosomes w i t h s t a n d a r d 60S or 40S snbunits, appeared to be i d e n t i c a l to that of whole ribosomes. The fact that the damage i n c u r r e d was the same for both s u b u n i t s ~,as confirmed for free treated ribosomal subunits. In this case, the activity o.f each s u b u n i t was r e d u c e d to 40-50 p. cent of its n o r m a l level w i t h 2 mM reagent concentration, a n d i n a c t i v a t i o n was n e v e r complete. I n figure 3B, r i b o s o m a l and s u b u n i t activity is plolted against the percentage of reacted lysine. The diagrams show that some lysine residues reacted w i t h o u t causing a n y i n a c t i v a t i o n , and thereafter a l i n e a r r e l a t i o n s h i p between the n u m b e r of reacted lysine and ribosomes a n d subu n i t i n a c t i v a t i o n was observed. A 50 p. cent r e d u c t i o n of activity resulted from the uptake of reagent by only 8 4 5 p. cent of the total Iysine residues. The i n i t i a l rate of p o l y p h e n y l a l a n i n e synthesis was also f o u n d to be lower w i t h treated snbunits. T h e p a r t i a l i n a c t i v a t i o n of the reacted partieles was i n d e p e n d e n t of Mg+÷ c o n c e n t r a t i o n i n the i n c u b a t i o n mixt~we. The s e d i m e n t a t i o n profile on sucrose g r a d i e n t of treated ribosomes a n d s u b u n i t s was i d e n t i c a l to that of control particles.

Reactivity of individual proteins in ribosomal subunits. P r o t e i n s extracted from compact s u b u n i t s treated w i t h 2-methoxy-5-nitrotropone w e r e analyzed by t w o - d i m e n s i o n a l gel electrophoresis. Different p a t t e r n s were o b t a i n e d a c c o r d i n g to the c o n c e n t r a t i o n s of reagent. C o m p a r i s o n w i t h

BIOCHIMIE, 1975, 57, n ° 3.

the p a t t e r n of control s u b u n i t s showed that some spots were absent and some other spots were m u c h less i n t e n s e i n the p a t t e r n s of treated subu n i t s (fig. 4). Most of the yellow coloration o.f the nitrotrop o n y l p r o t e i n s stayed near the origin in the first d i m e n s i o n , a n d no yellow spot was visible in the second d i m e n s i o n . This suggests that the reagent i n d u c e d a modification (probably in the charge) of the proteins, w h i c h p r e v e n t e d their m i g r a t i o n in the t w o - d i m e n s i o n a l electrophoretic system. This a s s u m p t i o n 'was confirmed by the results obtained after n i t r o t r o p o n y l a t i o n of free ribosomal p r o t e i n s (see below) a n d has been s i m i l a r l y d e m o n s t r a t e d in e x p e r i m e n t s w i t h E. colt ribosomes [4]. To q u a n t i t y the v a r i a t i o n in the a m o u n t of each protein, the coloration eluted from each spot was m e a s u r e d (see method). The first observation is that all the p r o t e i n s reacted in both subunits, w i t h o u t exception, but not for the same reagent c o n c e n t r a t i o n and not to the same extent. W h e n the a b s o r b a n c e values were plotted as a f u n c t i o n of 2-methoxy-5-nitrotropone c o n c e n t r a t i o n , three sets of graphs were obtained. To each graph c o r r e s p o n d s a set of p r o t e i n s w h i c h had approximately the same reactivity (fig. 5). The first group c o n t a i n s the p r o t e i n s w h i c h completely reacted w i t h 0.3 mM reagent, that is, Sis, Szo, S._,s a n d Ls, Lg, L,11, L.I~, LI,~, L-_5, L29, Lao, Lal, La4, L.~7. At that c o n c e n t r a t i o n , the activity of the s u b u n i t s was p r a c t i c a l l y u n r e d u e e d . Therefore, it m a y be assumed that these p r o t e i n s had their free a m i n o groups exposed in the native c o n f o r m a t i o n of the subunits. The group II i n c l u d e s p r o t e i n s w h i c h d i s a p p e a r e d from the electrophoretie p a t t e r n w h e n higher c o n c e n t r a t i o n s of reagent (up to 2 raM) were used. The p r o t e i n s i n group III were still visible in the p a t t e r n c o r r e s p o n d i n g to the highest re'agent c o n c e n t r a t i o n used (2 raM). One of these proteins, L~, appears to be significantly less reactive t h a n the others at low reagent concentrations. It should be noted that several p r o t e i n s a m o n g the most reactive (group I) gave faint spots on the control eleetrophoretic pattern. U n d e r such conditions, r e a c t i o n m a y be more easily observed. Ho,wever, the a b s o r b a n c e of each p r o t e i n i n control s u b u n i t s ~ a s easily measurable. I n addition, even a m o n g faint spots of equal i n t e n s i t y , there was clear differences in reactivity. P r o t e i n s from treated 80S ribosomes were also analyzed. Due to the n u m b e r of p r o t e i n s w h i c h were not resolved into i n d i v i d u a l spots, it was not possible to follow the v a r i a t i o n of every ribosomal protein. However, at a c o n c e n t r a t i o n of 1 mM of r e a g e n t ( w h i c h did n o t i n a c t i v a t e the

P r o t e i n r e a c t i v i t y in m a m m a l i a n

ribosomes), several p r o t e i n s were more resistant in 80S ribosomes t h a n in isolated s u b u n i t s : $6, S~ and L4, L~. A few other p r o t e i n s appeared to be more reactive in 80S ribosomes than i n free s u b u n i t s : $13, $19 a n d Sz6.

ribosomes

(I).

291

a b s o r b a n c e at 420 nm. The reacted p r o t e i n s did not migrate in t w o - d i m e n s i o n a l gel electrophorests. Thus, t h e i r modification can be m e a s u r e d from the loss of p r o t e i n s in the electrophoretic pattern. F i n a l l y , modified ribosomes a n d s u b u n i t s

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Fro. 5. - - Extent of reaction of ,60S and -~OS subunit proteins as a function function of 2-methoxy-5-nitrotropone concentration. Intensity of each spot in figure 4 was measured and compared with that of the corresponding spot in control subunits, considered 1o be equal to 100. A : 60S subunits - - B : 40S subunits.

As several p r o t e i n s in the compact s u b u n i t s u n c o m p l e t e l y reacted even at the highest reagent c o n c e n t r a t i o n , e x p e r i m e n t s were p e r f o r m e d w i t h free p r o t e i n s and s u b u n i t s unfolded b y EDTA. With such experiments, it should be possible to d e t e r m i n e 'whether the decrease of r e a c t i v i t y of some p r o t e i n s in the s u b u n i t s was due to their i n c l u s i o n in the particles or to an i n t r i n s i c properry of the proteins. I n u n f o l d e d subunits, all proteins completely reacted ~,ith 0.6 mM 2-methoxy-5-nitrotropone. The results were essentially the same w i t h free proteins. F r o m these experiments, one can conclude that the reactive groups in several ribosomal p r o t e i n s become less accessible to the reagent w h e n these proteins are w i t h i n the compact ribosomal structure than w h e n they are free. DISCUSSION. The results presented above show that there are several advantages in using 2-methoxy-5-nitrot r o p o n e for chemical modification studies of Mammalian ribosomes. The uptake of this reagent by the ribosomes was easily m e a s u r e d from the BIOCHIMIE, 1975, 57, n ° 3.

kept most of their biological activity, and, therefore, extensive s t r u c t u r a l p e r t u r b a t i 9 n could be excluded. This observation differs from the results o b t a i n e d w i t h E. colt large subunits, w h i c h were completely i n a c t i v e in p o l y p h e n y l a l a n i n e synthesis after modification b y the same reagent. No p r o t e i n was completely resistant to 2-methoxy-5-nitrotropone, even though only a small p e r c e n t a g e of all the p o t e n t i a l l y reactive lysine did react. After t r e a t m e n t of both s u b u n i t s w i t h different c o n c e n t r a t i o n s of the reagent, all the p r o t e i n spots were less i n t e n s e than i n the controls, and some completely disappeared. By the q u a n t i t a t i v e m e a s u r e m e n t of the unmodified proteins, it was possible to classify these p r o t e i n s into several groups. The p r o t e i n s w h i c h completely reacted with low c o n c e n t r a t i o n s of reagent (group I) can be c o n s i d e r e d to have free a m i n o groups exposed in the native structure of the subunits, because full activity was m a i n t a i n e d u n d e r these conditions. These free a m i n o groups are, therefore, not i n v o l v e d in the active sites of the subunits, n o r in

2.9,2

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the reassociation process, at least under the cond i t i o n s of p o l y p h e n y l a l a n i n e s y n t h e s i s . M o r e p r o t e i n s of g r o u p e I w e r e f o u n d i n 60S s u b u n i t s t h a n i n 40S subu~nits (11 v e r s u s 3). T h i s c o u l d b e r e l a t e d to a d i f f e r e n c e i n t h e g e n e r a l o r g a n i z a t i o n of t h e t w o s u b u n i t s . H o w e v e r , b e c a u s e 2 - m e f l m x y 5-nitrotropone has a relatively low molecular w e i g h t (181), t h e e x p o s e d p r o t e i n s a r e n o t n e c e s s a r i l y l o c a t e d a t t h e s u r f a c e o.f t h e s u b u n i t s . The proteins in group II reacted with reagent concentrations higher than those which produced t h e c o m p l e t e d i s a p p e a r e n c e of t h e f r e e p r o t e i n s , and the proteins in group III were still present o n t h e p a t t e r n s , e v e n w h e n u s i n g 2 m M of r e a g e n t . All t h e s e p r o t e i n s m u s t b e m o r e o r less s h i e l d e d w h e n i n c l u d e d i n t h e s u b u n i t s . T h i s s h i e l d i n g is p r o b a b l y r e l a t e d t o t h e o v e r - a l l f o l d i n g of t h e r i b o n u c l e o p r o t e i n c h a i n , o r to t h e p r o t e i n - R N A o r p r o t e i n - p r o t e i n i n t e r a c t i o n s . A c t i v i t y of b o t h s u b units was only partly conserved under these conditions. For this reason, one cannot completely exclude the hypothesis of a confomnational m o d i f i c a t i o n of t h e s u b u n i t b y t h e r e a g e n t i n t h i s case. On t h e o t h e r h a n d , t h e s m a l l d e c r e a s e w h i c h w a s o b s e r v e d i n t h e s u b n n i t a c t i v i t y a n d also i n t h e i n t e n s i t y of s e v e r a l p r o t e i n s , w h e n t h e c o n c e n t r a t i o n of r e a g e n t i n c r e a s e d f r o m 1 m M to 2 raM, s u g g e s t s t h e p o s s i b i l i t y of r i b o s o m e h e t e r o g e n e i t y . I t is p o s s i b l e t h a t t h e r e m a i n i n g p r o t e i n s w e r e s h i e l d e d i n a p o p u l a t i o n of s u b u n i t s w h i c h remained completely active. However, the reagent must have reacted with each subunit because several proteins totally disappeared from the e l e c t r o p h o r e t i c p a t t e r n of t r e a t e d s u b u n i t s . I n addition, we did not observe a strict parallelism b e t w e e n t h e u p t a k e of r e a g e n t , ~ - h i c h w a s i d e n tical in several ribosomal preparations, and the a c t i v i t y of t h e s e p r e p a r a t i o n s , w h i c h v a r i e d f r o m 32 t o 75 p. c e n t of t h e c o n t r o l w i t h 2 m M of reagent. R i b o s o m e s w e r e f o u n d to b e m o r e r e s i s t a n t to t h e a c t i o n of t h e r e a g e n t t h a n i s o l a t e d s u b n n i t s . Their activity was maintained with higher conc e n t r a t i o n s of r e a g e n t , a n d t h e r e w e r e less r e a g e n t molecules bound by ribosomes than by subunits, at l e a s t ' w h e n u s i n g l o w c o n c e n t r a t i o n s of r e a g e n t . I n a d d i t i o n , t h e r e a c t i v i t y of s e v e r a l p r o t e i n s w a s not identical in ribosomes and free subunits. T h e s e o b s e r v a t i o n s a r e p r o b a b l y r e l a t e d to differ e n c e s i n t h e c o n f o r m a t i o n s of f r e e a n d c o m b i n e d subunits. A c o m p a r i s o n b e t w e e n t h e e x t e n t of r e a c t i o n of t h e 60S p r o t e i n s a n d t h e i n a c t i v a t i o n of t h e s u b u n i t s s h o w e d t h a n o n l y t h e d e c r e a s e of p r o t e i n L~ a p p r o x i m a t e l y p a r a l l e l e d t h e d e c r e a s e of

BIOCHIMIE, 1975, 57, n ° 3.

s u b u n i t a c t i v i t y . H o w e v e r , d u e to p o s s i b l e s u b u n i t c o n f o r m a t i o n al m o d i f i c a t i o n s , s u c h a n o b s e r v a t i o n is i n s u f f i c i e n t to p o s t u l a t e t h a t a p a r t i c u l a r p r o t e i n is i n v o l v e d i n s u b u n i t a c t i v i t y . W e w i l l compare the above results concerning ribosomal modification by 2-methoxy-5-nitrotropone with t h o s e o b t a i n e d u s i n g o t h e r r e a g e n t s , s u c h as, glutaraldehyde, salts and a macromolecular probe ( s u c h as, t r y p s i n e ) i n o t h e r r e p o r t s .

Acknoimtedgments. This study has been supported b y the ¢ Centre Nat i o n a l de la Recherche Scientifique >> (ERA n ° 399) and the ¢ I n s t i t n t N a t i o n a l de la Sant6 et de la Recherche Mddieale >> (grant n ° 72-1-010-1). R~SUM~. Des ribosomes de foie, et des sous-unit6s ribosomiques ont dt6 trait6s p a r des c o n c e n t r a t i o n s t r o i s sautes de 2-methoxy-5-nitrotropone, u n rc~actif sp6cifique des fonctions amin6es primaires. A basse concent r a t i o n de r6aetif (0.3 raM), il se fixe p r o p o r t i o n n e l l e m e n t plus de moldcules de r6actif sur les so us-unit6s 60S que sur les sons-unit6s 40S (compte-tenu de la diff6rence de poids miol6eulaire), et m o t h s snr les ribosomes 80S que sur les deux sous-nnit~,s prises ensemble. A des c o n c e n t r a t i o n s plus 61ev6es, la quarttit6 de r6actif fixd sur les deux sous-nn.itds est 6quivalente. Les sous-nnit6s et ]es ribosomes conservent toute leur activit6 au cours de la synth6se de polyph(~nylalanine, apr6s t r a i t e m e n t p a r des c o n c e n t r a t i o n s de rdactif a l l a n t r e s p e c t i v e m e n t j n s q u ' h 0.3 et 1 mM. A la c o n c e n t r a t i o n de 2 raM, les deux sous-unit6s out p e r d u la moiti6 de l e n t activit6, mais leur s6dimenration n'est p as modifide. La r6activit6 de chaque prot6ine r i b o s m i q n e a 6t6 d6termin6e, au m o y e n d'dlectrophor6se b i d i m e n s i o n helle sur gel de polyacrylamide. Les mol6eules de prot6ine qui n ' o n t pas rdagi sont dos6es q u a n t i t a t i v e m e n t aprbs dlution des taches. Ces r6sultats obtenus, ainsi que les donn6es relatives h la fixation du r6aetif sur les so us-unit6s et h l ' i n a c t i v a t i o n de celles-ci, p e r m e t t e n t de t i r e r u n c e r t a i n h o m b r e de conclusions concern a n t la t o p o g r a p h i e des ribosomes. La susceptibilit6 globale des sous-unit6s 60S au r6actif est plus 61ev6e que celle des sous-unitds 40S. Les deux types de sonsunit6s c h a n g e n t de c o n f o r m a t i o n s lorsqu'elles se c o m b i n e m p o u r f o r m e r des ribosomes 80S. Les prot~ines S~, S~o, S_~, et L~, Lo, L~, L~, L~¢, L~, L~,, L~,,, L~, L34, L,~7 out des groupelnents amin6s exposds au rdactif clans les sous-unitds n.atives. Ces g r o u p e m e n t s ne sont pas essentiels a n f o n c t i o n n e m e n t des sous-unitds. REFERENCES. 1. Tamaoki, H., Murase, Y., Minato, S. ~ Nakanishi, K. (1967) ,1. Biochem. (Tokyo), 62, 7-11. 2. Craven, G. R. & Gupta, V. (1970) Proe. Nat. Acad. Sci. U.S.A., 67, 1329-1336. 3. Suzuka, I. (1971) Eur. J. Biochem., 23, 61-68. 4. Ballesta, J. P. G., Montejo, V., Hernandez, F. & Vasquez, D. (1974) Eur. J. Biochem., 42, 167-175.

Protein reactivity in m a m m a l i a n ribosomes (I). 5. Moore, P. B. (1966) J. Mol. Biol., 22, 145-163. 6. Moldave, K. ~ Skogerson, L. (1967) in Methods in Enzymology, eds. Colo~wick, S. P. ~ Kaplan, N. O. (Academic Press, New Yor]~), Vol. 12, p. 478481. 7. Reboud, A. M., Arpin, M. ~ Reboud, J. P. (1972) Eur. J. Biochem., 26, 347-353. 8. Hamilton, M. G. ~ Ruth, M. (1969) Biochemistry, 8, 851-856. 9. Hardy, S. J. S., Kurland, C. G., Voynow, P. Mora, G. (1969) Biochemistry, 8, 2897-2905.

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10. Howard, G. A. & Traut, R. R. (1974) Methods in Enzymology, eds. Colo wick, S. P. a Kaplan, N. O. (Academic Press, New York), Vol. ,30, p. 526539. 11. Kaltschmidt, E . . ~ W i t t m a n n , H. G. (1970) Anal. Biochem., 36, 401-412. 12. Sherton, C. C. ~ ~'ool, I. G. (1972) J. Biol. Chem., 247, 4460-4467. 13. Cozzone, A., personal communication. 14. P e t e r m a n n , M. L. (1964) The physical and chemical properties of ribosomes, (Elsevier, Amsterdam), p. 82.