Identification of neighbouring protein pairs in the rat liver 40-s ribosomal subunits cross-linked with dimethyl suberimidate

72

Biochimiea et Biophysica Acta, 621 (1980)72--82 © Elsevier/North-Holland Biomedical Press

BBA 38333

IDENTIFICATION OF NEIGHBOURING PROTEIN PAIRS IN THE RAT LIVER 40-S RIBOSOMAL SUBUNITS CROSS-LINKED WITH DIMETHYL SUBERIMIDATE

KAZUO TERAO, TOSHIO UCHIUMI, YUKIMI KOBAYASHI and KIKUO OGATA

Department of Biochemistry, Niigata University School of Medicine, Niigata (Japan) (Received February 19th, 1979)

Key words: Protein neighbour; Ribosomal subunit; Dimethyl suberimidate; Cross-linking; (Rat liver)

Summary (1) The 40-S ribosomal subunits of rat liver were treated with a bifunctional cross-linking reagent, dimethyl suberimidate. Cross-linked protein-protein dimers were separated by two-dimensional acrylamide gel electrophoresis. The stained cross-linked complexes within the gel were radioiodinated without the elution of proteins from the gel and were cloven into the original monomeric protein constituents by ammonolysis. The proteins in each dimer were finally identified by two-dimensional acrylamide gel electrophoresis of the cloven monomeric proteins, followed by radioautography of the stained gel. (2) The molecular weights of cross-linked complexes were determined by SDS-polyacrylamide gel electrophoresis and were compared with those of their constituent proteins. (3) The following dimers were proposed from these results: $3-S12 ($3 or S3a-Sll), $4-S12 (S3b-Sll), $5-$7 ($4-$6), $5-$22 ($4-$23 or $24), $6-$8 ($5-$7), $8-S16 ($7-S18), S17-$21 (S16--S19) and S22A-S22B ($23-$24), designated according to our numbering system [ 1 ]. The designations according to the proposed uniform nomenclature [2] are described in parentheses.

Introduction Knowledge of the spatial arrangement of ribosomal proteins in the ribosomes is important for the detailed understanding of the function of ribosomal proteins in protein synthesis. Several reports describe the use of a variety of cleavable and non-cleavable cross-linking agents to obtain information on the Abbreviations:

DMS0dimethyl suberimidate dihydrochloride; SDS, sodium dodecyl sulfate.

73 topography of proteins in Escherichia coli ribosomes [3]. On the other hand, no study on the protein topography of rat liver ribosomes with bifunctional reagents has yet been reported. It has been shown that dimethyl suberimidate can form amidine linkages between lysine e-amino groups in ribosomal proteins and the imidate group of this bifunctional reagent, and the crosslinked proteins are cloven by ammonolysis to form the original monomeric protein constituents [4--9]. In this paper, we describe the isolation of cross-linked complexes formed by the action of DMS on 40-S ribosomal subunits of rat liver and the identification of the protein constituents by using radioiodination of the cross-linked complex in the two-dimensional acrylamide gel. Cross-linked pairs of 40-S ribosomal proteins identified by these methods are proposed. Materials and Methods

Chemicals Na12SI (17 Ci/mg) was obtained from New England Nuclear, Boston. Dimethyl suberimidate dihydrochloride was purchased from Wako Pure Chemicals. Treatment o f ribosomal subunits with DMS Ribosomal 40-S subunits were prepared from rat liver by the methods described previously [1,10]. Immediately before use, DMS was dissolved at a concentration of 10 rag/50/zl of 0.5 M triethanolamine. 4 mg DMS was added to 2 mg RNA of 40-S subunits in 2 ml of buffer I (0.25 M sucrose/50 mM KC1/ 10 mM MgC12/10 mM 2-mercaptoethanol/20 mM Triethanolamine-HC1, pH 7.6) and the mixture was incubated at 37°C for 60 min. After incubation, the mixture was chilled in ice-cold water and was layered on the medium comprising 0.5 M sucrose/50 mM KC1/10 mM MgC1J10 mM 2-mercaptoethanol/50 mM Tris-HC1, pH 7.6, and was centrifuged at 60 000 rev./min for 2 h. Forty-S subunits thus obtained were homogenized in 0.1 M MgC12 and 40-S ribosomal proteins were extracted with 67% acetic acid as described previously [1]. The ribosomal proteins were precipitated by adding acetone, and subjected to twodimensional acrylamide gel electrophoresis by a slight modification of the method of Kaltschmidt and Wittmann [11] as described previously [1]. The 40-S proteins on the gel were numbered according to our numbering system

[1].

Radioiodination o f cross-linked proteins in the gel and ammonolysis The proteins in the gel were radioiodinated according to a modified method of Elder et al. [12]. 40-S ribosomal proteins were separated on a 150 × 150 X 3 mm plate by two-dimensional polyacrylamide gel electrophoresis. After staining with Amido Black 10B the gel disc containing the cross-linked protein complex was cut out carefully with a surgical knife, avoiding contamination with a neighbouring spot, for radioiodination. After the gel disc was dried by lyophilization, cross-linked protein in the gel discs was directly radioiodinated with 12sI by a modification of the chloramine T method [13]. The following reagents were added sequentially to a plastic tube: 30/zl 0.5 M sodium phos-

74 phate buffer, pH 7.5, 160/~Ci Na12SI in 5 pl of redistilled water and 5 pl chloramine T (1 mg/ml). Finally, each gel disc was put into the tube, and after incubation at 30°C for 60 min 1 ml of sodium bisulfide (1 mg/ml) was added to stop the reaction. After 15 min the sodium disulfite solution was removed. The gel disc was wrapped in gauze and dialyzed against water at 0°C overnight. The gel disc was transferred to a plastic tube and crashed with a glass rod, and incubated in 1 ml of SDS solution (1% SDS and 0.1 M phosphate buffer, pH 7.0) at 37°C for 3 h. These extraction procedures were repeated twice. The extract was mixed with 4 vols. 12 M NH4OH containing 1 M acetic acid and 2% SDS, and the mixture was incubated at 30°C for 16 h to cleave the imidoester cross-links. After the addition of 300/~g of 40-S ribosomal proteins as a carrier, the labeled proteins were recovered from the mixture by precipitation with 5 vols. acetone. The precipitated proteins were dissolved in 1 ml of 6 M urea and were diluted with an equal volume of water. The proteins were reprecipitated by the addition of acetone as described above, dissolved in an appropriate volume of the sample solution for two-dimensional gel electrophoresis [1], and subjected to two-dimensional polyacrylamide gel electrophoresis.

Radioautography o f the cross-linked proteins on two-dimensional gel After electrophoresis, the gel slab was stained with Amido black 10B and was sliced into two layers. One half was placed onto a filter paper, dried and exposed onto a Fuji X-ray film. Radioautographs were exposed at --70 ° C. The other was used for the measurement of the radioactivity of labeled protein to determine the expoqure time period. Molecular weigh t measurement o f cross-linked components The molecular weights of cross-linked protein complexes were determined by three-dimenisonal gel electrophoresis as described previously [1]. In brief, the ribosomal proteins extracted from DMS-treated 40-S subunits were subjected to two-dimensional polyacrylamide gel electrophoresis and the gel discs containing the cross-linked complexes were cut out. The gel discs thus obtained were incubated in SDS solution and applied to SDS-gel electrophoresis as described previously [1] except that 7.5% polyacrylamide gel was used. Results Separation o f the cross-linked complexes Total proteins isolated from 40-S ribosomal subunits pretreated with the cross-linking agent were analyzed by two
75

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(B) G' q~m H •.

2

$23

Q S24

's2s

t

~h,,D ~ A E qllbF

-,,,. B

~ C

s3

s26

Fig. 1. T w o - d i m e n s i o n a l p o l y 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 o f DMS-cxoss-linked 40-S r i b o s o m a l p r o t e i n s . 3 0 0 / ~ g of proteins isolated f r o m r a t liver 40-S s u b u n i t s t r e a t e d w i t h DMS were s u b j e c t e d to t w o - d i m e n sional polyacrylamide gel elect~ophoresis. (A) The entire gel p a t t e r n ; (B) t h e p a t t e r n o f the u p p e r left r e g i o n o f t h e gel.

Radioiodination of the protein in the gel It was reported that the amidine bonds of the cross-linked products were cloven by ammonolysis [5,9]. However, the yield of the cross-linked products on the gel slab of two-dimensional electrophoresis was so low that it was impossible to identify the individual proteins cloven by ammonolysis from the staining pattern on two-dimensional gel. Therefore the cross-linked proteins in the gel were labeled in vitro with 12sI [12]. It must be added that it is difficult to obtain in highly labeled ribosomal proteins of rat liver by labeling in vivo. For the identification of cross-linked proteins by the present method, it is important to know whether all 40-S proteins are labeled with 12sI. Therefore, the following experiments were carried out. 40-S proteins were labeled with 12sI as described in the legend of Fig. 2. After two
76

F i g . 2. T w o - d i m e n s i o n a l e l e c t ~ o p h o r e s i s o f i o d i n a t e d 4 0 - S r i b o s o m a l p r o t e i n s . 1 m g 4 0 - S r i b o s o m a l p r o t e i n s in 0 . 0 7 m l 6 M u r e a w a s i o d i n a t e d w i t h 1 0 0 ~Ci 12 S I b y c h l o r a m i n T m e t h o d . A f t e r 1 5 m i n a t 3 0 ° C, the reaction was stopped by the addition of sodium bisulfite. The incubation mixture was dialyzed to 4 M u r e a a n d f o l l o w e d t o 0 . 2 M HCI. L a b e l e d r i b o s o m a l p r o t e i n s w e r e p r e c i p i t a t e d b y a c e t O n e a n d subjected t o 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 s i s . A f t e r s t a i n i n g w i t h A m i d o b l a c k 1 0 B t h e gel slab w a s sliced i n t o t w o layers and dried, and one layer was radioautographed. (A) The staining pattern of iodinated 40-S ribos o m a l p r o t e i n s ; (B) r a d i o a u t o g r a p h o f l o d i n a t e d 4 0 - S r i b o s o m a l p r o t e i n s .

and $22 as described later, was labeled with 12sI in vitro and extracted with SDS solution. The extract was divided into three equal aliquots. Each aliquot was mixed with 4 vols. of (A) 6.5 M NH4OH, (B) 8 M NH4OH or (C) 12 M NH4OH, each containing 1.0 M acetic acid and 2% SDS. The mixture were incubated at 30°C for 16 h to cleave the imidoester cross-link. After the addition of carrier 40-S proteins, proteins were collected with acetone and were subjected to one
Effects of ammonolysis on the electrophoretogram of 40-S proteins As the next step, effects of ammonolysis on the electrophoretogram of 40-S proteins were examined. For this purpose gel discs containing protein spots~on the two-dimensional electrophoresis of 40-S ribosomal proteins were cut o u t and labeled with 12sI in vitro. The labeled protein was extracted b y SDS solution and subjected to ammonolysis. The labeled protein was then subjected to

77 (c)

(B)

(A) A $5

1

$22

A $5

A $5

$22

$22

22 x

u

5

i0

15

20

25

i0 Slice

15

20

25

5

i0

15

20

25

Number

F i g . 3. E f f e c t s o f t h e c o n c e n t r a t i o n o f N H 4 O H o n t h e c l e a v a g e o f t h e i m i d o e s t e r b o n d . C o m p l e x A labeled w i t h 1251 w a s e x t r a c t e d b y S D S and subjected t o o n e - 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 a t p H 4 . 6 under t h e s a m e c o n d i t i o n s o f 2 n d - d i m c n s t o n a l gel e l e c t r o p h o r e s i s f o r 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 s i s [ 9 ] . A f t e r s t a i n i n g , t h e gel w a s sliced i n t o S m m w i d t h , a n d t h e r a d i o a c t i v i t i e s o f t h e slices w e r e m e a s u r e d b y a S b i m a z u g a m m a c o u n t e r . A r r o w s s h o w t h e p o s i t i o n s o f c o m p l e x A , $ 5 and S22 p r o t e i n s .

two~limensional acrylamide gel electrophoresis after the addition of carrier 40-S proteins. Fig. 4 shows the radioautographs of several 12sI labeled 40-S proteins which were cross-linked with DMS as described later. The dotted circles in Fig. 4 represent tracings of staining spots of ribosomal proteins on the radioautograph. The dark spots represent the labeled ribosomal proteins after ammonolysis. The radioactive spots are more diffuse than the staining spots of corresponding proteins under our experimental conditions. As shown in Fig. 4A, the radioactive spot of $5 protein shifts somewhat to the anodic region from the staining spot. This shift may be caused by the modification of $5 protein by ammonolysis, since iodination itself does not shift S5 protein as shown in Fig. 2 and SDS treatment of 40-S proteins followed by acetone precipitation did not change the position of 40-S proteins including $5 protein on the gel (data not shown). The other radioactive spots coincided generally with the stained spots of corresponding ribosomal proteins (Figs. 4B--F).

Protein composition of cross-linked complexes The products of ammonolysis of cross-linked complexes were analysed by two
78 A

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B

C

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C.

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E

F

i27:. :~;]~

Fig. 4. 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 i c analysis o f the 12 S I l a b e l e d proteins after a m m o n o l y s i s . Proteins ( $ 5 , $ 7 , $ 8 , $ 1 3 , S 1 7 a n d $ 2 2 ) labeled w i t h 125I in vitro w e r e subjected to a m m o n o l y s l s and the prod u c t s w e r e a n a l y s e d b y 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 l s . Gel slabs w e r e stained w i t h A m i d o b l a c k 1 0 B , dried and r a d i o a u t o g r a p h e d . In e a c h p a n e l o f the figure the staining spots o f 40-S r i b o s o m a l p r o t e i n s w e r e traced o n the p h o t o g r a p h o f a r a d i o a u t o g ~ a p h o f the s a m e gel slab a n d s h o w n as the d o t t e d circles. A , B, C, D, E a n d F aze r a d i o a u t o g r a p h s o f S5, $ 7 , $ 8 , S 1 3 , S 1 7 a n d $ 2 2 p r o t e i n s o n the slab gel, r e s p e c t i v e l y .

products of ammonolysis of complex B contain only one protein $22 (Fig. 5B). Since it was reported that our $22 protein consists of two protein species designated here as $22 A and B [14,15], it is possible that these two kinds of protein form a pair component. Tentatively, we propose the combination of $22A and $22B which may be contained in the $22 spot. However, the possibility cannot be ruled out that a partner with $22 protein is $19, as described later, which is hardly labeled with 1:sI in vitro. Further experiments are required to elucidate this problem. (3) Ammonolysis of complex C liberates proteins $17 and $21 (Fig. 5C). (4) The products of ammonolysis of complex ]3 are $5 and $7 proteins. As described above, the radioactive spot of $5 protein shifts somewhat to the anodic side (Fig. 5D). (5) Ammonolysis of complex E releases $8 and $16 proteins (Fig. 5E). The radioactive spot in the lower region from complex E cannot be identified. (6) Ammonolysis of complex G shows a distinct radioactive spot of $12 and two faint spots of $3 and $4 on the gel. Proteins $3 and $4 have a same molecular weight and one of them seems to be a modified protein of the other as described later. Taken together

79 •

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Fig. 5. T w o - d l m e n s i o n a l e l e c t r o p h o r e s i s o f t h e p r o d u c t s o f a m m o n o l y s i s o f t h e l a b e l e d c r o S s - l i n k e d c o m p l e x e s . A m m o n o l y s l s p r o d u c t s o f t h e c r o s s - l i n k e d c o m p l e x e s l a b e l e d w i t h 1251 w e r e m i x e d w i t h u n l a b e l e d 40-S r i b o s o m a l p r o t e i n s a n d w e r e a n a l y s e d b y t w o - d i m e n s i o n a l e l e c t ~ o p h o r e s i s . G e l s l a b s w e r e s t a i n e d , d r i e d and radioautographed. I n e a c h p a n e l o f t h e f i g u r e , t h e p o s i t i o n s o f 4 0 - S p r o t e i n s a r c s h o w n b y t h e s a m e w a y as d e s c r i b e d i n Fig. 4. A , B, C, D, E, F , G a n d H a r e r a d i o a u t o g r a m s o f a m m o n o l y s i s p r o d u c t s o f c o m p l e x e s A , B, C, D , E, F , G a n d H s h o w n in Fig. 1, r e s p e c t i v e l y .

these results with the molecular weights of complex G (Table I), the combinations of $3-S12 proteins and $4-S12 proteins are proposed as the possible pairings. (7) Ammonolysis products of complex H may be $6 and $8 proteins, although the radioactive spot of $6 is diffuse and shifts somewhat to the anodic side from the staining position of $6 protein. (8) Ammonolysis products of complex F could not be determined. The radioactive spot in the upper left region from complex F may be aggregated protein due to ammonolysis. It must be added that in the cases of complexes E, F, G and H large parts of radioactivity remained at the original positions of cross-linked complexes, probably due to the incomplete cleavage of the imidoester bond. The molecular weights o f cross-linked complexes and o f their components The molecular weights of the cross-linked complexes were determined by SDS-acrylamide gel electrophoresis of protein spots (A, B, C, D, E, G, H) on two
80 TABLE I MOLECULAR WEIGHTS AND PROTEIN COMPOSITION OF CROSS-LINKED LATED FROM DMS-TREATED 40-S RIBOSOMAL SUBUNITS

COMPLEXES

ISO-

P h o s p h o r y l a s e a f r o m rabbit muscle (M r 9 4 0 0 0 ; Boehringer Mannheim), b o v i n e serum albumin (M r 6 8 0 0 0 ; A r m o u r ) , creatine kinase from rabbit muscle (M r 4 0 0 0 0 ; Sigma) and c h y m o t r y p s i n o g e n from b o v i n e p a n c r e a s (M r 2 5 7 0 0 ; Schwarz-Mann) were used as markers o n S D S gel electrophoresis. Molecular weights o f r i b o s o m a l proteins listed b e l o w are taken from the previous report [ 1 ] . P r o p o s e d u n i f o r m n o m e n c l a t u r e [2] is described in the parentheses. Cornplex

Protein

Mr

Proposed composition of complex

Sum of M r of

Observed M r of

cornplex proteins

complex

A

$5 $22

($4) ($23 or $24)

32 000 21 000

$5-$22

($4-$23 or $24)

53 0 0 0

48 000

B

$22 [S19]

($23 or $24) (S20)

21 000 17 000

S22A-S22B [$19-$22]

(23-$24) ($20-$23 or $24)

42 000 [38 000]

38 000

C

$17 $21

(SlS) (S19)

18 000 20 000

$17-$21 $17-$21

(S16-$19) (S16-$19)

38 000

32 000

D

$5 S7

($4) ($6)

32 000 37 000

$5-$7

(S4-$6)

69 000

62 000

E

$8 S16

(S7) (S18)

23 000 23 000

S8-SlS

($7-S18)

46 000

46 000

G

$3 $4 $12

($3 or S3a) (S3b) ($11)

35 000 35 000 21 000

$3-S12 $4-$12

($3 or S3a-S117 (S3b-Sll)

56 000 56 000

52 000

$6 S8

($5) ($7)

26 000 23 000

$6-$8

($5-$7)

49 000

48 000

H

Discussion The identification of proteins which are cross-linked by protein-specific bifunctional reagents may be one of the experimental approaches to determine the proximity relationship between proteins in the ribosomes. In this experiment, DMS was used, since ammonolysis of the cross-linked complexes by DMS release proteins which can be identified by two~limensional acrylamide gel electrophoresis. It wa~ found that the yields of cross-linking of 40-S proteins by this reagent and of ammonolysis were very low under our experimental conditions. Therefore, it was necessary that cross-linked complexes were highly labeled in vitro with 12sI. Although in the case of bacterial ribosomes the in vivo labeling was used, it is difficult to obtain the highly labeled ribosomal proteins of rat liver by labeling in vivo. Radioiodination of protein in the gel without elution was found to be useful to obtain the highly labeled cross-linked proteins. There is, however, a certain obvious limitation of this type of analysis as follows: The proteins to be analysed must contain amino acid residues radioiodinated by the chloramin T method [13]. When the 40-S ribosomal proteins in urea solution were labeled with I2SI and subjected to two-dimensional gel electrophoresis, it was observed

81 that almost of all 40-S proteins except for two proteins, S19 and $25, were labeled under our experimental conditions. It was reported by Leader that when 40-S ribosomal proteins were subjected to iodination, catalyzed by lactoperoxidase, 19 out of 23 proteins of 40-S subunits were labeled [16]. Their unlabeled two proteins coincided with our S19 and $25 proteins, while the other unlabeled two proteins, $9 and S10, were labeled in our experiment. These differences may be due to the m e t h o d of radioiodination, because they used lactoperoxidase while we used chloramin T. It must be added that since S19 and $25 proteins are~hardly labeled with 12sI in vitro, the possibility may not be ruled out that these two proteins are partners with other proteins in cross-linked complexes. Since the molecular weight of $25 is 11 000, it is unlikely that $25 protein is contained in the cross-linked complexes in these experiments (Table I). However, since the molecular weight of S19 is 17 000, S19 protein may be contained in the cross-linked complexes especially as the partner of $22 in the complex B. Further experiments should be made to elucidate this problem. Ammonolysis products of complex G show a distinct radioactive spot of S12 and two faint spots of $3 and $4. It is noted that darkness of $3, $4 and S12 proteins on the radioautograph is proportional to the staining intensity of the corresponding protein (Fig. 2). Therefore, it is reasonable that there are pairs of $3-S12 and $4-S12, in the case of complex G, since the intensity of S12 protein on the radioautograph was markedly higher than that of $3 or $4 (Fig. 5). Furthermore, the pair of $3 and $4 proteins in complex G is unlikely from the molecular weights of these proteins (Table I). It is noted that the relative staining intensity of $3 protein to $4 protein on two-dimensional gel was variable among the different preparations of rat liver 40-S subunits but the staining intensity of $3 + $4 proteins was quite constant. When rats fasted on water for one week, the $4 spot disappeared on two
82 there may be some limitations on the detection and the identification of crosslinked proteins. After completion of this work, the proposed uniform nomenclature for ribosomal proteins was reported [2]. Since the conditions of two-dimensional polyacrylamide gel were somewhat different, we found that it was difficult to correlate our nomenclature with the proposed one in cases of some ribosomal proteins. Therefore, we added the designation of cross-linked proteins according to the c o m m o n nomenclature in the Summary and Table I which our conclusion concerning the cross-linked 40-S proteins is described. Acknowledgement This work was supported by a Grant-in-Aid for Scientific Research (No. 1 4 7 1 1 9 and No. 3 5 7 0 7 6 ) from the Ministry of Education, Science and Culture of Japan, and by the Naito Research Grant for 1978. References 1 Terao, K. and Ogata, K. (1975) Biochim. Biophys. Acta 402, 2 1 4 - - 2 2 9 2 McConkey, E.H., Bielka, H., Gordon, J., Lastlk, S.H., Lin, A., Ogata, K., Reboud, J.-P., Traugh, J.A., Traut, R.R., Warner, J.R., Welfle, H. and Wool, I.G. (1979) Mol. Gen. Genet. 169, 1---6 3 Traut, R.R., Heomark, R.L., Sun, T.-T., Hershey, J.W.B. and BoHen, A. (1974) in R i b o s o m e s (Nomura, M., Tissi~res, A. and Lengyel, P., eds.), pp. 271--308, Cold Spring Harbor Lab., N e w York 4 Davis, G.E. and Stark, G.R. (1970) Proc. Natl. Acad. Sci. U.S.A. 66, 651--656 5 Bickle, T.A., Hershey, J.W.B. and Traut, R.R. (1972) Proc. Natl. Acad. Sci. U.S.A. 69, 1327--1331 6 Clegg, C. and Hayes, D.H. (1974) Eur. J. Biochem. 42, 2 1 - - 2 8 7 Barritault, D., Expert-Benzancon, A., Milet, M. and Hayes, D.H. (1975) FEBS Lett. 50, 114--120 8 Bode, V., Lutter, L.C. and StUffier, C.G. (1974) FEBS Lett. 45, 2 3 2 - - 2 3 6 9 Expert-Benzan~on, A., Barriault, D., Milet, M., Gu~rin, M.-F. and Hayes, D.H. (1977) J. Mol. Biol. 112, 603---629 10 Terao, K. and Ogata, K. (1970) Biochem. Biophys. Res. C o m m u n . 38, 80--85 11 Kaltschmidt, E. and Wittmann, H.G. (1970) Anal. Biochem. 36, 401--412 12 Elder, J.H., Pickett If, R.A., Hampton, J. and Lerne~, R.A. (1977) J. Biol. Chem. 252, 6510--6515 13 Greenwood, F.C., Hunter, W.M. and Glover, J.S. (1963) Biochem. J. 89, 114--123 14 Welfle, H., Staid, J. and Bielka, H. (1972) FEBS Lett. 26, 2 2 8 - - 2 3 2 15 Sherton, C.C. and Wool, I.G. (1974) J. Biol. Chem. 2 4 9 , 2 2 5 8 - - 2 2 6 7 16 Leader, D.P. (1976) Biochem. J. 162, 373--378