Reductive alkylation of mammalian ribosomes

578

Blochzm~ca el B~ophyswa Acta, 474 (1977) 578--587

© Elsevier/North-Holland Biomedical Press

BBA 98833 REDUCTIVE ALKYLATION OF MAMMALIAN RIBOSOMES

ANNE-MARIE REBOUD, MONIQUE BUISSON, MONIQUE ARPIN and JEAN-PAUL REBOUD Laboratotre de Blochimw Mdd~cale, UER Lyon Nord, 43, Boulevard du 11 Novembre 1918, 69621 Vdleurbanne (France)

(Received July 20th, 1976)

Summary 40- and 60-S ribosomal subunits and 80-S ribosomes from rat liver were highly labelled by reductive methylation using formaldehyde and sodium boro[3H] hydride, under conditions which did not decrease their activity m poly-Udirected polyphenylalanine synthesis. Dissociation of the monosomes, subunit dimers, and polysomes into free subunits was observed after methylation. Free proteins labelled after extraction from the ribosomal subunits incorporated 7 times more radioactivity than when labelled in the subunits. Proteins extracted from methylated subunits and ribosomes were analyzed by two-dimensional gel electrophoresis, and the radioactivity of each protein was compared to that of the same free protein. A classification of the proteins was established according to their accessibility to the reagents in the subunits and the ribosomes.

Introduction Moore et al. [1] reported that active bacterial ribosomes with very high specific radioactivity could be prepared by reductive methylation of their lysine residues. Under these conditions all the ribosomal proteins were labelled. In the following we show that ribosomal subunits and ribosomes from rat liver could also be labelled using a m e t h o d with mild conditions, adapted from that of Means et al. [2]. Reductive methylation, which does not affect the charge of the proteins appreciably, did not change the two-dimensional electrophoretic pattern of the ribosomal proteins, as this had been observed using bacterial ribosomes [1]. Since the biological activity of the labelled ribosomes and subunits was n o t decreased, information was obtained on the accessibility of the lysine residues of proteins in native eucaryotic ribosomes and subunits.

579 Material and Method

Ribosomes and subunits. Ribosomes from rat liver were isolated according to a method adapted from that of Moldave [3]. Active 40- and 60-S subunits were prepared using 0.3 M KC1 as previously described [4] and stored in buffer A (1 mM potassium phosphate pH 7.3, 30 mM KC1, 1.5 mM MgC12, 20 mM ~mercaptoethanol). The A~60 of a 1 mg/ml solution of either subunits or ribosomes was considered to be equal to 14.0. Reductive methylation. The reaction was carried o u t in a fume cupboard as follows: 37 pl of 0.040 M formaldehyde were added to 40-, 60- or 80-S ribosomes (12 A260 units) in 0.37 ml of buffer A, containing 20 mM sodium borate at 0°C, the pH of this buffer being equal to 8.6. This addition was followed in 30 s by four sequential additions of 7.4 pl of a 5 mg/ml solution of sodium boro [3H] hydride. After 1 min the treatment with formaldehyde and sodium borohydride was repeated once more. The reaction mixture was left at 0°C for 10 min and then dialyzed against buffer A which was changed several times. In other experiments the proteins extracted from ribosomes or subunits by acetic acid [5] were dialyzed against buffer A containing in addition 20 mM sodium borate and 4 M guanidinium chloride, and reductive methylation was performed as described above. The solution was dialyzed against 7% acetic acid, 6 mM ~-mercaptoethanol and lyophilized. The number of methyl groups incorporated into the sample was calculated on the assumption that one hydrogen atom derived from sodium borohydride is incorporated per methyl group per lysine residue. Two-dimensional polyacrylamide gel electrophoresis. UnlabeUed ribosomes or subunits (20 A260 units) were added to each labelled sample. After extraction of the RNA, the proteins were separated by two-dimensional gel electrophoresis and the gel was stained with amido black. The conditions of electrophoresis and the code for numering the proteins have already been described [6,7] and are very similar to those described by Sherton and Wool [8]. Spots were cut o u t and the proteins eluted with 1% sodium dodecyl sulfate/6 M urea at 37°C. The extract was chilled and then filtered through 0.45-pm Millipore filters according to a method described elsewhere [9]. The filters were dried and counted in a scintillation mixture prepared with toluene. The yield of the counting was estimated to be 12.2%. We noticed, as did Moore et al. [ 1 ] , that the positions of the spots on the plates were unaffected by reductive methylation. Assay of amino-acid incorporation activity. The activity of methylated ribosomes or subunits was assayed under conditions of limiting ribosomes, by using a poly(U)-dependent phenylalanine incorporation system [4]. Treated subunits were tested in conjunction with the untreated complementary ones. Material. Sodium boro[3H]hydride (5 Ci/mmol) was obtained from the Commissariat fi l'Energie Atomique. It was diluted with unlabelled sodium borohydride, so that the final solution contained 5 mg/ml of this c o m p o u n d , with a specific activity of 150 mCi/mmol.

580

Results

Radioactivity incorporation into ribosomal subunits by reductive methylation 40- and 60-S subunits treated with formaldehyde and sodium boro[3H] hydride under the conditions described in Material and Method were labelled with a high specific activity: 4 • 106 and 3 • 106 d p m / m g of subunits respectively. When free ribosomal proteins were labelled, after extraction from the subunits, they incorporated 7 times more radioactivity than when they were included in the subunits. Omission of formaldehyde during the reaction reduced the radioactivity incorporated to a low level (10% of the incorporation with a complete mixture). This confirms that the main radioactive derivate is methylated (e-N-dimethyllysine) [2], but does not exclude any side reaction of small extent. Assuming that the 40- and 60-S subunits have molecular weights of 1.2 • 106 and 3.1 • 106 [10] and contain 462 and 975 lysine residues respectively [11], one can calculate that about 6% of the total lysines were methylated in treated subunits, and that this percentage increased to 41--43% when free proteins were allowed to react. When formaldehyde was replaced by another aldehyde c o m p o u n d having a much bigger molecular size, such as 3,4,5-trim e t h o x y b e n z a l d e h y d e , the number of reactive lysines in ribosomal subunits was only slightly decreased. Activity and sedimentation o f treated ribosomes The activity of methylated 40-, 60- and 80-S particles in poly(U)-directed polyphenylalanine synthesis was entirely preserved. Using the m e t h o d of Crichton et al. [1] who employed 4 times more formaldehyde and sodium borohydride per mg of protein, only the activity of the treated 60-S subunits was slightly diminished (75% of the control) (Table I). The sedimentation profiles on sucrose gradient of methylated 40-, 60- and 80-S particles and free polysomes are shown in Fig. 1. Control preparations kept under the same conditions at pH 7.4 and pH 8.6 (the pH at which the methylation reaction is carried out) were also analyzed (Fig. l a and lb). In buffer A, the two subunits and the monosomes were present mainly as dimers.

TABLE

I

ACTIVITY

OF METHYLATED

SUBUNITS

In thin typical experiment, 4.5 pg of 40-S and 11.25 #g of 60-S subumts were used for poly(U)-dlrected polyphenylalanme synthesis [4]. M means methylated subunits, M* means methylated under Cnehton's conditaons [1]. Subumts

Polyphenylalamne

40S 60S 40S 40S 60S 40S 40S 40S 40S

37 402 2346 24 422 2105 2464 2100 1759

+ M M M + M +

60S

+ 60S 60S M * + 60S 60S M *

(counts/ram)

581 a 0.2

b

c 40S

40 S

65S

Small subun,t

545

J< ,J\

0.1

Large subumt

84S

6~)S7BS

ji

60S 78S

t

0.'

0

40S

"i

blonosomes 0.2

"°$ 6I0 S

725 62S 805

t

t ~ 86, ~t

0.1

0

~olysomes 98S

0.2

145s

40S

l

63 S

80S 90~

t

* It

2

3

8os9o$

i

I

~

3

4

5

I

Effluent

4

5

! 1

2

3

4

v o l u m e (rnl )

Fig. 1. S e d i m e n t a t * o n analys*s o f m e t h y l a t e d r i b o s o m e s a n d s u b u n i t s . I s o l a t e d 400, 6 0 ° a n d 80-S p a r t , cles a n d free p o l y s o m e s m b u f f e r ( A ) p H 7.4 (see Material a n d M e t h o d ) w e r e e a c h f r a c t i o n a t e d i n t o 3 e q u a l p o r t i o n s . S a m p l e s a w e r e k e p t in b u f f e r A. 1 / 1 0 v o l u m e o f 0.2 M s o d m m b o r a t e p H 8.6 was a d d e d t o s a m p l e s b a n d c0 in o r d e r to give a 2 0 m M s o d i u m b o r a t e c o n c e n t r a t i o n a n d a p H of 8.6. Only s a m p l e s c w e r e m e t h y l a t e d as d e s c r i b e d in t h e t e x t . A t t h e e n d o f t h e i n c u b a t i o n , s a m p l e s a, b a n d c (25 Dg of r i b o s o m e s ) w e r e f i x e d b y 6% f o r m a l d e h y d e t o a v o i d a n y c h a n g e in t h e s e d i m e n t a t i o n p r o f i l e d u r i n g z o n a l c e n t r i f u g a t i o n . S e d i m e n t a t i o n analysis was c a r r i e d o u t on a 1 5 - - 5 0 % s u c r o s e g r a d i e n t p r e p a r e d in b u f f e r (A). C e n t r i f u g a t i o n was d o n e in a s p i n c o SW 50.1 r o t o r a t 50 0 0 0 r e v . / m i n f o r 3 h (400, 600 a n d 800S, p o l y s o m e s ) . H o w e v e r , s a m p l e a o f p o l y s o m e s w a s c e n t r i f u g e d f o r o n l y 2 h 1 0 rain in o r d e r t o h a v e a b e t t e r s e p a r a t i o n o f t h e h e a v y m a t e r i a l p r e s e n t in this s a m p l e . T h e u l t r a v i o l e t a b s o r b a n c e n e a r t h e t o p o f t h e g r a d i e n t s w a s d u e to t h e f o r m a l d e h y d e .

As could be expected the incubation itself at basic pH had two effects: a partial dissociation of ribosomes, polysomes and subunit dimers and a decrease in the sedimentation coefficients of the resulting subunits. This decrease was particularly apparent in the case of the small subunit (from 52 to 40 S) which probably has a more flexible structure than the large one, (whose sedimentation coefficient varied from 67 to 60 S). In the case of the monosomes, a new 72-S component appeared, which should constitute swollen monosomes in which subunits were weakly bound. The effect of methylation was a significant increase in the dissociation which yielded free subunits.However, this dissociation was not irreversible, since methylated particles dialyzed against buffer A showed the same sedimentation pattern as the untreated ones. Although the

582

F i g , 2. T w o - d i m e n s i o n a l e l e c t r o p h o r e t l c p a t t e r n s o f p r o t e i n s e x t r a c t e d f r o m m e t h y l a t e d subunl~s. Prot e i n s e x t r a c t e d f r o m m e t h y l a t e d 40-S ( u p p e r ) , 60-S ( m i d d l e ) s u b u n t t s a n d m o n o s o m e s ( l o w e r ) w e r e a n a l y z e d as d e s c r i b e d in M a t e r i a l a n d M e t h o d . I d e n t i c a l p a t t e r n s w e r e o b t a i n e d u s i n g r a d i o a c t i v e m e t h y l a t e d p a r t i c l e s m i x e d w i t h u n m e t h y l a t e d m a t e r i a l as c a r n e r , o r o n l y m e t h y l a t e d p a r t i c l e s . S e v e r a l s p o t s , w i n c h w e r e a p p a r e n t a n d well r e s o l v e d in t h e e l e c t r o p h o r e t i c p l a t e s , are n o t v i s i b l e o n t h e p h o t o g r a p h s . L 7 w a s f o u n d r e p r o d u c i b l y n o t e x a c t l y a t t h e s a m e p l a c e , i n t h e 60-S s u b u n l t s a n d in t h e m o n o somes.

4.78 3.41 4.56 8.13 2.71 11.02 3.34 3.05 1.36 4.19 6.04 2.04 2.00 1.84 6.42 5.74 8.17 4.31 4.51 3.07 1.00 5.11 0.75

5.30 3.76 7.55 9.95 2.92 10.47 1.24 1.61 1.15 8.96 6.17 1.04 0.36 1.48 8.33 6.20 9.20 3.17 4.36 1.00 0.19 4.23 0.45

0.287 0.205 0.274 0.488 0.163 0.161 0.200 0.183 0.112 0.251 0.362 0.122 0.120 0.110 0.385 0.344 0.490 0.259 0.271 0.184 0.060 0.307 0.045

2.28 1.61 3.23 4.26 1.25 4.48 0.53 0.69 0.49 3.83 2.64 0.44 0.37 0.63 3.57 2.66 3.94 1.36 1.87 0.43 0.08 1.81 0.19

0.126 0.127 0.085 0.114 0.130 0.147 0.376 0.265 0.227 0.065 0.137 0.274 0.326 0.174 0.108 0.129 0.124 0.190 0.145 0.429 0.737 0.170 0.233

(5)

Free proteins

Intact subumts

+ + + + + + +++ ++ ++ + + ++ ++ + + + + + + +++ +++ + ++

(6)

P r o t e i n rea c t i v i t y in subunits

1.2 1.4 1.2 2.1 3.8

I.i 1.3 1.7 0.6 1.4 0.8 1.6 1.8 2.8 1.4 0.2 0.6 3.5

0.119 0.137 0.273 0.559

0.251 0.085 0.233 0.166 0.454 0.139 0.173 0.232 0.532 0.203 0.257 0.149 0.595

(8)

Subunits

Ribosomes

0.152

(7)

Free proteins

Ribosomes

As w e a l r e a d y m e n t i o n e d [ 6 ] , t h e s e p r o t e i n s , w h i c h w e r e q u i t e d i s t i n c t o n o u r d m g r a m s , w e r e n o t n u m b e r e d b y S h e r t o n e t al. [ 8 ] .

$2 $3 34 PX * $6 $7 $8 39 SI0 $11 S13 814 $15 PY * $16 $17 $18 819 820 822--23 $24 $25 $28

Free proteins (4)

Intact subunits (3)

Intact subumts (1)

Free p r o t e i n s (2)

P e r c e n t a g e of t o t a l p r o t e i n lysine-methylated

Percentage of total incorporation

Proteins e x t r a c t e d f r o m r i b o s o m a l s u b u n i t s or r i b o s o m e s labelled b y r e d u c t i v e m e t h y l a t i o n using s o d i u m b o r o [ 3 H ] h y d r l d e , o r free p r o t e i n s d i r e c t l y labelled, w e r e s e p a r a t e d b y t w o - d i m e n s i o n a l ge] e l e c t r o p h o r e s i s a n d t h e i r r a d i o a c t i v i t y was c o u n t e d as d e s c r i b e d m Material a n d M e t h o d . I t was a s s u m e d t h a t t h e p r o t e l n s r e c o v ered in t h e d i f f e r e n t s p o t s w e r e in t h e s a m e p r o p o r t i o n s as in t h e u n f r a c t i o n a t e d p r o t e i n m i x t u r e . F o r e a c h p r o t e i n , t h e p e r c e n t a g e o f t h e t o t a l p r o t e i n i y s i n e m e t h y l a t e d was c a l c u l a t e d f r o m t h e p e r c e n t a g e o f r a d i o a c t i v i t y r e c o v e r e d in e a c h p r o t e i n s p o t ( c o l u m n s 1 a n d 2), a n d f r o m t h e t o t a l p e r c e n t a g e of lysine m e t h y l a t e d in s u b u n i t s o r free p r o t e i n s . (6% a n d 5.7% o f t h e l y s i n e was m e t h y l a t e d in 40- a n d 60-S s u b u m t s w i n l e m free p r o t e l n s d e n v e d f r o m t h e s e s u b u n i t s , t h e perc e n t a g e s w e r e 43% a n d 41%, r e s p e c t i v e l y . )

R E D U C T I V E M E T H Y L A T I O N O F 40-S S U B U N I T P R O T E I N S

T A B L E II

L3 L4 L5 L6 L7 L8 L9 LIO Lll L12 L13 L14 L15 L16 L17 L18 L19 L21--23 L26 L27 L28 L29 L31 L33 L34 L35 L36 L37

1.36 0.82 1.74 4.58 3.80 1.80 4.62 1.20 4.51 9.57 1.87 4.06 1.80 2.94 1.55 2.15 3.02 8.23 15.39 4.43 1.05 0.76 2.78 1.54 2.53 8.05 1.38 1.11

Intact subunlts (1)

3.63 0.85 3.46 8.77 6.91 2.30 7.20 1.64 7.63 6.19 2.13 3.54 2.52 3.16 1.37 1.76 1.96 6.36 9.80 2.09 1.16 1.01 2.84 1.57 2.74 4.79 0.75 0.33

Free proteins (2)

Percentage of total incorporation

See t h e c a p t i o n o f T a b l e I.

0.078 0.047 0.099 0.261 0.217 0.103 0.263 0.068 0.257 0.545 0.107 0.231 0.103 0.168 0.088 0.123 0.172 0.469 0.877 0.253 0.060 0.043 0.158 0.088 0.144 0.459 0.079 0.063

Intact subumts (3) 1.49 0.35 1.42 3.60 2.83 0.94 2.95 0.67 3.13 2.54 0.87 1.45 1.03 1.30 0.56 0.72 0.80 2.61 4.02 0.86 0.48 0.41 1.16 0.64 1.12 1.96 0.31 0.13

(4)

F r e e protelns

Percentage of total protein lysme-methylated

R E D U C T I V E M E T H Y L A T I O N O F 60-S S U B U N I T P R O T E I N S

T A B L E III

0.052 0.135 0.071 0.073 0.077 0.110 0.090 0.102 0.083 0.217 0.123 0.161 0.100 0.130 0.158 0.171 0.216 0.181 0.220 0.297 0.127 0.105 0.138 0.137 0.128 0.235 0 258 0.470

(5)

Free proteins

Intact subumts

1 0.6 29 1.5 3.3 03 15 0.8 09 1.5 0.7 0.5 1.1 0.8 14 0.5 1.7 09

1.4

0.052 0.043 0.212 0.116 0.363 0.306 0.125 0.174 0 145 0.195 0.111 0.108 0.199 0.176 0 416 0.064 0.179 0 124

0 329

+ + + + + + + + + ++ + + + + + + ++ + ++ +++ + + + + + ++ ++ +++

(8)

Subumts

Ribosomes

(7)

Free proteins

Ribosomes

(6)

P r o t e i n rea c t i v i t y in subumts

00

585 40-S subunits contained an endogenous nuclease, which is very active at pH 8.6 [ 6 ] , we observed that methylation at 0°C (10 min) did n o t cause any degradation of the rRNAs, which were examined on sucrose gradients.

Reactivity o f individual proteins in ribosomes and subunits Proteins extracted from methylated subunits and ribosomes were analyzed b y two-dimensional gel electrophoresis (Fig. 2) and their radioactivity measured (Tables II and III). In column 1, results for each protein are expressed in percent of the total radioactivity recovered from the plates, and in column 3 the values represent the percent of the total protein lysine which was methylated. These results were compared with those obtained after methylation of free unfolded ribosomal proteins in guanidinim chloride (columns 2 and 4). In both subunits, all proteins were labelled b u t the degree of reactivity varied considerably from one protein to another. As could be expected, free extracted proteins were more labelled than those included in the subunits. The ratios in column 5 were obtained by dividing the values of column 3 by the values of column 4. These expressions, which are independent of the electrophoretic yields and of the protein properties, such as molecular weight, amino acid composition and fractionality, should depend only on the relative exposure of each protein in the ribosomal subunit. A schematic representation of the protein reactivity by crosses is given in column 6. The values in column 7 were calculated in the same way as were the values in column 5, except that the proteins came from labelled 80-S ribosomes instead of from ribosomal subunits, and therefore these values reflect protein reactivity in the ribosomes. A comparison between the reactivity of the proteins when included in the ribosomes and in the subunits is given by the values of column 8 which were obtained by dividing the values of column 5 by the values of column 7. Since identification of proteins of each subunit is often difficult on the 80-S ribosome plates, only the values corresponding to the most easily identified proteins are given in columns 7 and 8. Keeping in mind the experimental errors which probably affect the values of column 8 (which are ratios of 2 ratios), it is striking that several proteins were more labelled when included in ribosomes than in subunits (particularly $6, ST, S19, S25, L6 and Ls) while several others were less labelled. Discussion

These experiments first show that mammalian ribosomes and ribosomal proteins can be easily labelled, with a high specific radioactivity, using reductive methylation, and, in the case of ribosomes and subunits, without modification of their biological activity. Very often, this can be useful, since in-vivo ribosome labelling raises many more problems with mammalian tissues than with bacteria. Moreover some information a b o u t protein function and topography can be obtained. It is clear that the several amino groups of lysine residues which reacted in ribosomes and subunits do n o t play any direct role in the different steps of polyphenylalanine synthesis. On the other hand, these same groups should be implicated in the association of the subunits in the formation of monosomes or dimers. However, our results concerning the reassociation of

586 methylated 40- and 60-S subunits in a high-Mg 2÷ buffer at pH 7.4 suggest an indirect participation of the lysine residues in the association process. Blocking lysine NH: groups could render labile other linkages and therefore facilitate ribosomal dissociation *. Others have shown the role of SH groups in 40-S and 60-S subunit association [ 13 ]. Among the information concerning protein topography, the fact should be stressed that all proteins reacted in the ribosomal subunits, although a small percentage of the total e-NH: lysine groups was methylated. Thus, all proteins are exposed to the reagent, b u t the accessibility is much lower than with free proteins. The observation that the number of reactive lysines considerably increases when ribosomal subunits, b u t n o t free proteins, are progressively heated ( R e b o u d , A.M., unpublished observation) confirms that the shielding of the reactive groups is due to the ribosomal structure. As the t w o reagent molecules used in the reaction have a small size, their ability to act as surface protein reagent can be doubted. However, as we mentioned, the use of a much larger aldehydic c o m p o u n d , such as 3,4,5-trimethoxybenzaldehyde which should n o t penetrate the ribosomal structure did not significantly decrease the total number of reactive lysines. Also Crichton et al. have shown with Escherichia coli ribosomes that formaldehyde and 3,4,5-trimethoxybenzaldehyde give the same pattern of protein alkylation [ 1 4 ] . It is interesting to compare the protein reactivity determined by reductive methylation to that determined by other reagents, as m e t h o x y n i t r o t r o p o n e , glutaraldehyde, salts and trypsin [7,11,15]. It must be emphasized that methylation of proteins did not modify their properties, particularly during the two-dimensional gel electrophoresls, whereas when using the other reagents mentioned, reacted proteins were n o t visible in the gel plates. Therefore, reductive methylation should give more precise quantitative data. There is often a good agreement between these data and those obtained with the other protein chemical reagents. Thus, L3, L4, Ls, L13, Lls, L21--L23, L2s, L33, $2, Ss, $7, Sll, Sl6 and $17 were always found to be resistant, while L19, L3v and Ss were found to be reactive. Discrepancies which are frequently noticed with the results obtained with trypsin can probably be explained by the differences in the experimental conditions, particularly the temperature during the incubation. The surprising fact that several proteins were found to be more exposed in ribosomes than in subunits suggests that significant conformational changes occur when subunits associate.

Acknowledgments This study has been supported by the Centre National de la Recherche Scientifique (ERA No. 399), the Institut National de la Sant6 et de la Recherche Mddicale (Grant No. 75.1.080.3), the Commissariat ~ l'Energie Atomique and the Fondation pour la Recherche Medicale.

* F o r i n s t a n c e , it has b e e n s u p p o s e d that t h e s e groups, o n thetr c a t i o m c f o r m , m i g h t p r o v i d e c a t l o m c

shielding o f i n t e r n u c l e o s i d e p h o s p h a t e s and t h e r e f o r e s t a b i l i z a t i o n o f h y d r o g e n - b o n d base-prating between RNA molecules of both subumts [12].

587

References 1 Moore, G. and Crichton, R.R. (1973) FEBS Lett. 37, 74--78 2 Means, G.E. and Feeney, R.E. (1968) Biochemistry 7, 2192--2201 3 Moldave, K. and Skogerson, L. (1967) in Methods in Enz ymol ogy, (Colowick, S.P. and Kaplan, N.O., eds.), Vol. 12, pp. 478--481, Academic Press, New York 4 Reb oud, A.M., ArDin, M. and Reboud, J.P. (1972) Eur. J. Biochem. 26, 347--353 5 Hardy, S.J.S., Kurland, C.G., Voynow, P. and Mora, G. (1969) Biochemistry 8, 2897--2905 6 Reboud, A.M., Buisson, M., Marion, M.J. and Reboud, J.P. (1976) Biochim. Biophys. Acta 432, 176-184 7 Arpin, M., Reboud, J.P. and Reboud, A.M. (1975) Blochim~e 57, 1177--1184 8 Sherton, C.C. and Wool, I.G. (1972) J. Biol. Chem. 247, 4460--4467 9 Bmsson, M., Reboud, A.M. and Reboud, J.P. (1976) Anal. Biochcm., m the press I 0 Hamilto n, M.G. and Ruth, M. (1969) Biochemistry 8, 851--856 11 Reboud, A.M., Madjar, J.-J., Buisson, M. and Reboud, J.P. (1975) Blochlmle 57, 285--293 12 Azad, A.A. and Lane, B.G. (1973) Can. J. Biochem. 51, 1669--1672 13 Tamaokl, T. and Miyazawa,- F. (1967) J. Mol. Biol. 23, 35--46 14 Moore, G. and Crichton, R.R. (1974) Biochem. J. 143, 607--612 15 Reboud, A.M., Bulsson, M., Madjar, J.J. and Reboud, J.P. (1975) B1ochirme 57, 295--302