303

Biochimica et Biophysica Acta, 652 ( 1 9 8 1 ) 3 0 3 - - 3 1 3 © Elsev ie r /Nor th -Hol land Biomedica l Press

BBA 99815

FACTOR-DEPENDENT DISSOCIATION OF WHEAT GERM RIBOSOMES

LESLIE A. G O L D S T E I N and WILLIAM G. R O B I N S O N *

Department of Biochemistry, New York University School of Medicine, 550 First Avenue, New York, N Y 10016 (U.S.A.)

(Received O c t o b e r 1st, 1980)

Key words: Ribosome; Dissociation factor; (Wheat germ)

Summary

Ribosome dissociation factor has been found in wheat germ acetone powder extracts. Further purification of the crude extract by pH and ammonium sul- fate fractionations, DEAE-cellulose and CM-Sephadex Column chromatography has resulted in the separation of two active fractions. The possibility that ribo- some dissociation activity exhibited by either fraction is due to a protease or nuclease is considered unlikely, based on results of experiments involving ribo- some dissociation kinetics, subunit structural integrity, and t reatment with a serine protease inhibitor. Wheat germ ribosome dissociation factor is not species-specific. Dissociation factor from both fractions will p romote the disso- ciation of Escherichia coli 70-S as well as Artemia salina 80-S ribosomes. Although both dissociation factor activities show the same dependence on K ÷ and Mg 2÷ for optimal activity, the two activities exhibit significant differ- ences in their sensitivity to sulfhydryl reagents and heat, and in their depen- dence on incubation temperature for activity. Certain properties of both factors suggest that neither factor is initiation factor eIF-3; however, the pos- sibility that one or both factors are subunits of initiation factor eIF-3 remains to be determined.

Introduction

In eucaryotic protein synthesis, 80-S ribosomes are constantly undergoing dissociation and subunit exchange through repeated rounds of translation, par-

* To w h o m correspondence should be addressed. Abbreviations: PMSF, pheny lmethy l su l fony l fluoride; PCMB, p-chloromercuxobenzoic acid: MalNEt, N-ethylm aleimide.

304

ticipating in a r ibosome cycle similar to that for 70-S ribosomes in procaryotes [1--13]. In the eucaryotic r ibosome cycle, r ibosome dissociation factor must function not only as an anti-association factor preventing the reassociation of subunits, bu t must also act as a dissociation factor promoting the dissociation of 80-S ribosomes which are temporarily sidetracked from the polysome-sub- unit cycle [14,15] . Ribosome dissociation factor activities have been reported for yeast [16], rat liver [17--19] , rabbit ret iculocytes [11,20--23] , bean leaves [24], ascites cells [25,26] , Xenopus [27], and wheat germ [28,29] . Although dissociation factor has been reported from all these sources, it has not been shown to stimulate protein synthesis except in one unconfirmed instance [29]. Because of the uncertainties involved in obtaining a protein-synthesizing system from wheat germ which is depleted only of dissociation factor, we have not reported in this paper data on the role of dissociation factor on the stimulation of protein synthesis in a cell free system.

It has been reported that initiation factor eIF-3 promotes the dissociation of 80-S r ibosomes [30--33] . In addition, Thompson et al. [19] reported that an initiation factor eIF-3-1ike dissociation factor from rat liver promotes the disso- ciation of 80-S ribosomes and prevents the reassociation of ribosomal subunits. However, the existence of native 40-S subunits containing a total of approxi- mately 90 000 daltons of extra ribosomal protein [25,34--39] as well as several reports of r ibosome dissociation factors which appear to be distinct from initiation factor eIF-3 [17,26,29] suggest that in the eucaryotic r ibosome cycle it is unlikely that initiation factor eIF-3 is solely responsible for r ibosome disso- ciation and/or subunit anti-reassociation activity. In this paper we describe the partial purification and properties of a supernatant r ibosome dissociation acti- vity that may consist of at least two distinct dissociation factors, neither of which appear to be initiation factor eIF-3.

Experimental procedures

Materials. Escherichia coli W tRNA, [14C]methionine (260 mCi/mmol; 500 cpm/pmol) , special enzyme grade ammonium sulfate, MalNEt and ribonuclease- free sucrose were obtained from Schwarz/Mann. ATP, PMSF, PCMB and dithio- threitol were from Sigma. Alpha chymotrypsin, trypsin and ribonuclease A from Worthington; proteinase K from EM Laboratories; puromycin dihydro- chloride from Calbiochem; DEAE-cellulose (DE-23) from Whatman; CM-Sepha- dex (C-50) from Pharmacia Fine Chemicals; Long-life brine shrimp eggs (Artemia salina) from Aquarium Supplies (New York, NY) and raw wheat germ from Dixie Portland Company (Arkansas City, KS). All chemicals were reagent grade. Protein concentrations were routinely determined by the method of Warburg and Christian [40].

Artemia salina 80-S ribosomes were prepared by the method of Zasloff and Ochoa [41] and were stored in buffer C at --20°C. Wheat germ 80-S ribosomes [42] and derived 40-S and 60-S subunits [43] were obtained by published methods and stored in buffer C at --20°C.

[14C]Met-tRNA was prepared from unfractionated E. coli W tRNA and un- diluted [~4C]methionine as described by Zasloff and Ochoa [41]. N-acetylation of the [~4C]Met-tRNA was obtained by the method of Haenni and Chapeville [44].

305

Buffer A contained 40 mM KC1, 0.1 mM magnesium acetate, 2 mM Tris-HC1, pH 7.8 at 25 ° C, and 1.25 mM dithiothreitol. Buffer B was 80 mM KC1, 8 mM magnesium acetate, 4 mM Tris-HC1, pH 7.8 at 25°C, and 10 mM dithiothreitol. Buffer C was 40 mM KC1, 4 mM magnesium acetate, 2 mM Tris-HC1, pH 7.8 at 25°C, 5 mM dithiothreitol and 50% (v/v) glycerol. Buffer D contained 50 mM Tris-HC1, pH 7.8 at 25°C, 5% (v/v) glycerol and 0.25 mM dithiothreitol. The KC1 concentration (mM) in buffer D is indicated by the number in Daren- theses. Buffer E was 100 mM sodium acetate, pH 5.5 at 25°C.

Preparation of supernatant ribosomal dissociation factor (Table I) All operations were carried out at 0--4°C unless otherwise stated. Step 1. Preparation of acetone powder extract. Acetone chilled to --20°C

was added to 175-g portions of commercial wheat germ so that the final volume of the acetone-wheat germ suspension was approximately I 1. The sus- pension was homogenized in a Waring blendor for 45 s and the homogenate was quickly filtered by suction. The precipitate was washed twice with chilled ace- tone, spread in a thin layer on paper, and allowed to dry at room temperature for 3 h. No loss in activity was observed after prolonged storage of the acetone powder at --20°C. Wheat germ acetone powder (350 g) was suspended in 2 1 of buffer D(100). After the mixture was stirred for 1 h with a magnetic stirrer, it was centrifuged for 1 h at 13 200 X g. The precipitate was discarded and the supernatant used without further t reatment in the next step.

Step 2. pH 5 fractionation. To the supernatant from step 1 glacial acetic acid was added dropwise with continuous stirring until the pH of the suspension reached 5.0. The suspension was stirred for an additional 15 min and then cen- trifuged for 1 h at 13 200 X g. The supernatant was discarded and the precipi- tate dissolved in 2.5 1 of buffer D(100) and stirred overnight.

Step 3. Ammonium sulfate fractionation. To the solution from step 2, am- monium sulfate was added to give 40% saturation [55]. The resulting suspen- sion was stirred for 1 h, and centrifuged for 45 min at 13 200 X g. The precipi- tate was discarded, and the clear green-yellow supernatant was brought to 65% saturation with ammonium sulfate. The preparation was stirred and centrifuged as before, and the precipitate dissolved in buffer D(100). The solution was dialyzed overnight against 4 1 of buffer D(100).

Step 4. DEAE-cellulose column chromatography. A 4.0 X 12.5 cm column of DE-23-cellulose was equilibrated with buffer D(100). The dialyzed 4 0 - 6 5 % ammonium sulfate fraction was cleared of precipitate by centrifugation and applied to the DE-23 column which was subsequently washed with buffer D(100) at a f low rate of 90 ml/h. A total of 1.6 1 buffer D(100) was allowed to pass through the column before the eluant was changed to buffer D(200). After 4 1 of buffer D(200) had passed through the column, the absorbance at 280 nm of the effluent was 0.02. The final eluting buffer was D(300) which eluted a single peak. All fractions in the peak (1955 ml, 94 mg) which eluted with buffer D(300) were pooled and concentrated by precipitation with ammonium sulfate to 80% saturation. After centrifugation, the precipitate was dissolved in buffer D(100) and dialyzed against 4 1 of the same buffer overnight.

Step 5. Carboxymethyl-Sephadex column chromatography. The dialyzed step 4 solution which had been previously diluted with buffer D(100) to a final

306

concentration of 0.1 mg protein/ml was applied to a 2.0 × 30 cm column of CM-Sephadex (C-50) equilibrated with buffer D(100). The column was washed with buffer D(100) at a flow rate of 36 ml/h. All fractions constituting a single broad peak were pooled and concentrated to 1.7 ml by ultrafiltration in an Amicon apparatus using a PM-10 membrane. This fraction is termed the 0.1 M dissociation factor. The column was washed with buffer D(200) until the absorbance at 280 nm of the effluent was virtually zero. The eluant was changed to buffer D(300) and a single peak of protein which had dissociation activity was eluted. All fractions in the peak were pooled and concentrated by ultrafiltration as before, and dialyzed against 500 ml of buffer D(100) for 1 h. This fraction is termed the 0.3 M dissociation factor. Both the 0.1 M and 0.3 M dissociation factors were divided into aliquots and stored at --80 ° C.

Assays for ribosome dissociation factor activity Ribosome dissociation factor activity was determined essentially by a

coupled ribosome dissociation-peptidyl transferase assay described by Thomp- son et al. [18].

Qualitative measurement of ribosome dissociation factor activity was deter- mined using absorbance profiles from linear 10--30% sucrose gradients.

All ribosome dissociation factor assays contained derived 40-S and 60-S sub- units unless otherwise stated.

Results

Using the coupled ribosome dissociation-peptidyl transferase assay we have detected ribosome dissociation factor activity in wheat germ acetone powder extract. An assay system containing step 3 dissociation factor (920 ttg) and 80-S ribosomes (0.4 A260 40 S + 0.9 A260 60 S) yielded 2900 cpm, but in the absence of dissociation factor 1300 cpm were obtained. Also, the step 3 factor was not a source of exogenous peptidyl transferase activity (60-S ribosomes). In the absence of 80-S ribosomes, the step 3 factor gave 470 cpm, whereas 550 cpm were obtained in the combined absence of factor and 80-S ribosomes.

The crude extract ribosome dissociation activity was partially purified through the five-step procedure shown in Table I, which yielded two dissocia- tion factor activities. As can be seen in Fig. 1, both activities can bring about complete ribosomal dissociation. Absorbance profiles of linear 10--30% sucrose gradients confirm the results obtained by the coupled ribosome dissociation- peptidyl transferase assay as can be seen in Fig. 2. The difference in specific activity of the two dissociation factors is quite apparent. Also, the dissociated 40-S and 60-S peaks co-sediment with the 40-S and 60-S markers.

Trypsin arid pancreatic ribonuclease have been reported to dissociate eucary- otic 80-S ribosomes [16]. We found that wheat germ 80-S ribosomes were dis- sociated by proteases and ribonuclease A using the coupled ribosome dissocia- tion-peptidyl transferase assay system. To test the possibility that the dissocia- t ion factors were in fact non-specific proteases and/or nucleases, three proteases (chymotrypsin, trypsin and proteinase K), ribonuclease A and the 0.1 M and 0.3 M dissociation factors were individually incubated with 80-S

3 0 7

T A B L E I

P U R I F I C A T I O N O F R I B O S O M A L D I S S O C I A T I O N F A C T O R F R O M W H E A T G E R M

D a t a in t h e t ab l e r e p r e s e n t t h e a m o u n t s o f p r o t e i n o b t a i n e d f r o m the in i t i a l 3 5 0 g o f a c e t o n e p o w d e r . All p u r i f i c a t i o n procedtuces we re c a r r i e d o u t as d e s c r i b e d in E x p e r i m e n t a l p r o c e d u r e s . C o u p l e d r i b o s o m e d i s s o c i a t i o n - p e p t i d y l t r a n s f e r a s e assay w a s u s e d t o measuxe d i s s o c i a t i o n f a c t o r a c t i v l t y . The r i b o s o m e dis- s o c i a t i o n r e a c t i o n m i x t u r e ( 5 0 #1) c o n t a i n e d 4 5 m M Tris -HCl , p H 7 .8 a t 2 5 ° C , 1 2 2 m M KC1, 4 .4 m M MgC12, 1 0 0 m M d i t h i o t h r e i t o l , 0 . 2 0 A 2 6 0 40-S r i b o s o m e s , 0 . 4 8 A 2 6 0 60-S r i b o s o m e s , 7% (v/v) g l y c e r o l , 0 - - 3 1 4 # g 0 .1 M d i s s o c i a t i o n f a c t o r o r 0 - - 1 0 0 # g 0 .3 M d i s s o c i a t i o n f a c t o r . A m i x t u r e ( 9 5 #1) c o n t a i n i n g 4 2 2 m M KC1, 4 . 2 2 m M MgCI 2 , 4 2 . 2 m M Tris-HC1, p H 7 .8 a t 2 5 ° C , 5 2 . 7 % (v/v) e t h a n o l a n d 1 0 0 # g p u r o m y c i n was a d d e d t o t h e r e a c t i o n m i x t u r e f r o m t h e r i b o s o m e d i s s o c i a t i o n s t ep . N A c [ 1 4 C ] M e t - t R N A (5 #1, 25 p m o l ) w a s a d d e d a n d the t u b e s we re i n c u b a t e d fo r 6 0 ra in . The p e p t i d y l t r a n s f e r a s e r e a c t i o n w a s t e r m i n a t e d b y a d d i n g 1 m l o f ch i l l ed b u f f e r E (0°C) . 1 .5 m l e t h y l a c e t a t e w a s a d d e d , e a c h t u b e w a s v igor - o u s l y s h a k e n a n d b r i e f l y c e n t r i f u g e d . A 0 .9 m l a l i q u o t o f t h e u p p e r e t h y l a c e t a t e p h a s e w a s r e m o v e d a n d c o u n t e d in 1 0 m l o f B r a y ' s s o l u t i o n . All o t h e r assay c o n d i t i o n s w e r e c a r r i e d o u t as d e s c r i b e d [ 1 8 ] . 1 u n i t o f ac t i v i t y w a s d e f i n e d as t h a t a m o u n t o f d i s s o c i a t i o n f a c t o r r e q u i r e d f o r t h e f o r m a t i o n o f 1 p m o l N A c M e t - p u r o m y c i n in 1 h a t 2 0 ° C in the c o u p l e d assay s y s t e m .

S t e p V o l u m e T o t a l T o t a l Spec i f i c Yie ld (ml) p r o t e i n u n i t s a c t i v i t y (%)

( rag) ( p m o l / h ) ( u n i t s / r a g )

I . A c e t o n e p o w d e r e x t r a c t 1 7 0 0 4 4 5 4 0 53 9 0 0 1 .21 1 0 0 . 0 0 2. p H 5 p r e c i p i t a t e 2 5 0 0 3 0 7 5 0 38 5 0 0 1 . 2 5 7 1 . 5 0 3. A m m o n i u m su l f a t e ( 4 0 - - 6 5 % ) 9 0 5 9 4 0 2 5 5 0 0 4 . 3 0 4 7 . 4 0 4 . D E A E - C e l l u l o s e 6 8 4 0 . 8 1 4 8 5 3 6 . 4 0 2 . 8 0 5. C M - S e p h a d e x

a . 0 .1 M KCI 1 .7 2 9 . 6 5 8 7 1 9 . 8 0 1 . 1 0 b . 0 .3 M KC1 1 .0 1 .4 2 1 2 1 5 1 . 0 0 0 . 3 9

ribosomes. The reaction mixtures were layered on sucrose gradients and centri- fuged. Fig. 3 shows that the profile for either dissociation factor is clearly distinct from those obtained with hydrolytic enzymes. Also, both dissociation factors, alpha chymotrypsin and proteinase K were treated with 5 mM PMSF

0 40 80 120 160 200 240 280 :320

PROTEIN (,ug)

O~-g

O.,M OFI~

O,~g

0.3 M DF

A

164/.~g

20u.g

A

260/~g

0 .75 c

0 . 5 0

0 . 2 5

\ bJ o

,n<:) u.g z

0 .75 m

0 .50 m °

0.25 ~

Fig. 1. F a c t o r - p r o m o t e d (0 .1 M d i s s o c i a t i o n f a c t o r (e ) a n d 0 .3 M d i s s o c i a t i o n f a c t o r (o) ) r i b o s o m e disso- c i a t i o n as a f u n c t i o n o f p r o t e i n c o n c e n t r a t i o n . R i b o s o m e d i s s o c i a t i o n was m e a s u r e d b y the c o u p l e d r ibo - s o m e d i s s 0 c i a t i o n - p e p t i d y l t r a n s f e r a s e assay as d e s c r i b e d in t he l egend t o T a b l e I.

Fig. 2. A b s o r b a n c e p ro f i l e s o f a n a l y t i c a l suc ro se g r a d i e n t s s h o w i n g f a c t o r - p r o m o t e d r i b o s o m a l d issocia- t i o n . V a r y i n g a m o u n t s o f t he 0 .1 M a n d 0 .3 M d i s s o c i a t i o n f a c t o r s ( D F ) we re used . R i b o s o m e d i s s o c i a t i o n assay was c a r r i e d o u t as d e s c r i b e d in t h e l egend to T a b l e I. R e a c t i o n m i x t u r e s ( 5 0 #1) w e r e l a y e r e d o n c o o l e d , 5 m l l inea r 1 0 - - 3 0 % suc rose g r a d i e n t s w h i c h c o n t a i n e d 1 2 6 m M KC1, 4 .8 m M m a g n e s i u m a c e t a t e a n d 4 5 m M Tris-HC1, p H 7 .8 a t 2 5 ° C . T h e g r a d i e n t s were c e n t r i f u g e d a t 4 °C f o r 18 h a t 2 3 0 0 0 X g. A b s o r - b a n c e p ro f i l e s w e r e m o n i t o r e d a t a w a v e l e n g t h o f 2 5 4 n m us ing a n Isco Mode l D g r a d i e n t a n a l y z e r .

308

E 0 . 7 5

0 . 5 0

,~ 0 . 2 5

bJ L) Z

m 0 . 7 5

0 0 5 0

0.25

CHYN~OTRYPSIN

8060 40

RIBONUCLEASE h

TRYPSIN PROTEINASE K

01~ DF 03M DF

Fig. 3. A b s o r b a n c e p rof i l e s o f ana ly t i ca l suc rose g rad i en t s s h o w i n g pro tease - , r i bonuc lease A- and dissocia-

t i on f a c t o r ( D F ) - p r o m o t e d r i b o s o m a l d i s soc ia t ion . 4 .2 #g c h y m o t r y p s i n , 1.2 pg t ryps in , 0.9 pg p r o t e i n a s e K, 0 .16 pg r i bonuc lease A and the 0.1 M and 0.3 M d i s soc i a t i on f ac to r s at 320 #g and 102 •g, respec- t ive ly , were used in th i s e x p e r i m e n t . All assay c o n d i t i o n s and p r o c e d u r e s are as desc r ibed in the l egend to Fig. 2, e x c e p t t h a t the i n c u b a t i o n p e r i o d for r i b o s o m e d i s soc ia t ion was 1 h.

[45,46]. The r ibosome dissociation activities of both proteases were com- pletely inhibited; however, both dissociation factors were unaffected. The kinetics of r ibosome dissociation using a limiting amount of either dissociation factor is shown in Fig. 5. The results suggest that neither dissociation factor activity acts catalytically. As a result of these experiments, we conclude that it is unlikely that either dissociation factor activity was due to a non-specific protease or nuclease.

To determine if the 0.1 M and 0.3 M dissociation factors were two distinct proteins, the r ibosome dissociation activities of both fractions were compared in several different experiments involving sulfhydryl blocking reagents, heat inactivation, react ion-temperature-dependence, etc. As can be seen in Table II, the 0.1 M dissociation factor showed a significantly greater reactivity than the 0.3 M dissociation factor with either sulfhydryl reagent, but most noticeably toward the PCMB, which apparently had no effect on the 0.3 M dissociation factor at the concentrat ions utilized.

The sensitivity of the two dissociation factors to heat is shown in Table III. The most noticeable difference between the two activities in response to heat was in the 5 min incubation at 55 ° C. The 0.1 M dissociation factor retained 32% of its original activity, the 0.3 M dissociation factor, 76%.

Kinetics of the factor p romoted r ibosome dissociation reaction as a funct ion of temperature was also examined. The data in Fig. 4 show a significant differ- ence in the dissociation activity of the two factors when the tempera ture was raised from 25 to 37°C. Kinetics of the fac tor-promoted r ibosome dissociation reaction as a funct ion of time is shown in Fig. 5. No significant difference in factor activity was indicated.

The effect of different concentrat ions of Mg 2÷ and K ÷ on fac tor-promoted ribosome dissociation was also determined. In the magnesium opt imum experi- ments (Fig. 6}, both factors showed similar behavior over the entire magnesium acetate concentra t ion range (1.4--12.4 mM) utilized. The opt imum magnesium acetate concentra t ion for either factor was 4.4 mM. The effect of different KC1

309

T A B L E II

I N H I B I T I O N OF D I S S O C I A T I O N F A C T O R A C T I V I T Y BY S U L F H Y D R Y L B L O C K I N G R E A G E N T S

MalNEt was i n c u b a t e d wi th dissociat ion fac tor (s ) for 30 min a t 25°C in a 40 pl r eac t i on m i x t u r e t ha t c o n t a i n e d 13 or 26 mM M a i N E t / 1 4 8 m M KCI/5 m M MGC12/56 m M Tris-HCl, p H 7.8 a t 25°C. Af t e r 30 rain all t ubes were p laced in ice. Di th io th re i to l (5 #1 of 0.5 M or 1.0 M solu t ions) was a d d e d to each t u b e , giving a final d i t h io th t e i t o l c o n c e n t r a t i o n of 55 or 110 raM. All t ubes were i n c u b a t e d for an add i t iona l 10 rain at 25°C and then p laced in ice. All f u r t h e r process ing was as descr ibed in the legend to Table I. The PCMB e x p e r i m e n t s were car r ied ou t as previous ly descr ibed for the MaiNEt assay, h o w e v e r , d i th io t i~e i to l was o m i t t e d f r o m all buf fe rs . The PCMB-dissociat ion fac to r r eac t i on m i x t u r e has a vol of 43/~1, a PCMB c o n c e n t r a t i o n of 0 .18 or 0 .36 mM and was i n c u b a t e d for 15 rain a t 25°C. Relat ive ac t iv i ty was de te r - m i n e d by t ak ing the ra t io of the ne t c p m ( (80 S + dissociat ion fac to r ) - - 80 S) due to d issocia t ion fac to r ac t iv i ty in t he p resence of su l fhydry l reagen ts to t h a t o b t a i n e d in the absence of su l fhyd ry l reagents .

Dissociat ion fac to r A m o u n t of Inh ib i to r Inh ib i to r Relat ive p r e p a r a t i o n dissociat ion c o n c e n t r a t i o n ac t iv i ty (M) fac to r (raM) (%)

(Dg)

0.1 154 .0 MaiNEt 13 50.8 0.3 61.2 MalNEt 13 99 .0 0.1 154 .0 MaiNEt 26 50.3 0.3 61 .2 MaiNEt 26 71.6 0.1 196 .0 PCMB 0 .18 87.8 0.3 30 .2 PCMB 0 .18 100 .0 0.1 196 . 0 PCMB 0 .36 47.9 0.3 42 .0 PCMB 0.36 100 .0

concentrations (55, 75, 105, 125, 1 5 5 and 185 mM) on dissociation factor activity was also tested. The dissociation activities of the two factors increased as the KC1 concentration was increased from 55 to 185 mM, with both factors showing greatest activity at the latter concentration.

The ribosome specificity of each dissociation factor using native 70-S and

T A B L E n I

I N A C T I V A T I O N OF D I S S O C I A T I O N F A C T O R BY H E A T

Tubes con ta in ing e i ther the 0.1 or 0.3 M dissocia t ion fac to r in a v o l u m e of 45 #1 were i n c u b a t e d a t 45 , 55 or 60°C for the des i red t i m e (1, 3, 5 or 10 min) , t h e n p laced in ice. Con t ro l t ubes for 0.1 an d 0 .3 M disso- c ia t ion f ac to r were k e p t in ice dux~xg this per iod . All o t h e r ope ra t ions were car r ied o u t as desc r ibed previ- ously in the legend to Table I. Each tube rep resen t ing the 0.1 M dissocia t ion fac to r c o n t a i n e d 196 pg of t ha t fac tor , those tubes r ep resen t ing the 0.3 M dissocia t ion fac to r rece ived 30 /~g of the co r r e spond ing fac tor .

I n c u b a t i o n T e m p e r a t u r e 0.1 M Dissociat ion 0 .3 M Dissociat ion t ime (o C) F a c t o r ac t iv i ty f ac to r ac t iv i ty (mfn) r e ma in ing r ema in ing

(%) (%)

0 55 100 .0 100 .0 1 55 89.5 96 .5 3 55 68.5 80 .5 5 45 92 .0 100 .0 5 55 32 .0 76 .0 5 60 27 .5 44 .6

10 55 29 .0 48 .4

3 1 0

6O z 0

5O

40 0 ~ 3o C21

20,

10 u

I I I

100

8O 5 - - 60

40

20

_ I I I I _ t 0 10 20 5 0 4 0 0 4 8 12 16 20

T E M P E R A T U R E TIME (min)

F i g . 4 . F a c t o r - p r o m o t e d r i b o s o m e d i s s o c i a t i o n as a f u n c t i o n o f r e a c t i o n t e m p e r a t u r e . Al l t u b e s w e r e f i r s t

i n c u b a t e d a t 3 7 ° C f o r 15 m i n i n t h e a b s e n c e o f d i s s o c i a t i o n f a c t o r . A f t e r t h e f i r s t i n c u b a t i o n , all t u b e s w e r e p l a c e d in ice f o r a m i n i m u m o f 5 m i n . T o i n i t i a t e t h e r i b o s o m e d i s s o c i a t i o n r e a c t i o n , d i s s o c i a t i o n

f a c t o r (0 .1 ( 1 2 2 p g ) o r 0 . 3 M ( 1 1 # g ) ) w a s a d d e d a n d t h e r e a c t i o n m i x t u r e w a s i n c u b a t e d a t t h e d e s i r e d t e m p e r a t u r e (0 , 1 5 , 25 , 3 0 o r 3 7 ° C ) f o r 15 r a in . A t t h e c o m p l e t i o n o f t h e i n c u b a t i o n , e a c h t u b e w a s

p l a c e d i n i ce f o r 1 m i n a n d t h e r i b o s o m e d i s s o c i a t i o n r e a c t i o n w a s t e r m i n a t e d b y t h e a d d i t i o n o f t h e e t h a n o l - c o n t a i n i n g p e p t i d y l t r a n s f e r a s e a s s a y p r e - m i x . Al l o t h e r c o n d i t i o n s a n d f u r t h e r p r o c e s s i n g a re as d e s c r i b e d in t h e l e g e n d to T a b l e I.

F ig . 5. F a c t o r - p r o m o t e d r i b o s o m e d i s s o c i a t i o n as a f u n c t i o n o f t i m e at 3 7 ° C . R e a c t i o n m i x t u r e s w e r e

i n c u b a t e d a t 3 7 ° C f o r 1 m i n b e f o r e t h e a d d i t i o n o f d i s s o c i a t i o n f a c t o r . D i s s o c i a t i o n f a c t o r (0 .1 ( e ) o r 0 .3 M ( a ) ) w a s a d d e d ( f i n a l v o l u m e o f r e a c t i o n m i x t u r e , 50 p l ) a n d t h e t u b e s i n c u b a t e d f o r t h e t i m e s i n d i - c a t e d . T e r m i n a t i o n a n d f u r t h e r p r o c e s s i n g w e r e c a r r i e d o u t as d e s c r i b e d in t h e l e g e n d to F ig . 4.

100

z o 80 I.--

o 60 O3 123

UJ n-

13 . . . . . .1:3~

E, coli

Artemio salino

WHEAT GERM

70 50 30

806O40

NO FAC IOR

I l I I I I I 2 4 6 8 10 12 14

[_Mg2+~ mM O.1M

A

0.3M

0 75

0.50

0.25 E

t.- 0 7 5 ,~

0.50 z

0.25 < cD

o cD GD

0 75

0.50

0 2 5

Fig . 6 . E f f e c t o f M g 2+ c o n c e n t r a t i o n o n d i s s o c i a t i o n f a c t o r - d e p e n d e n t (e~ e , G . . . . . . ~ ) a n d d i s s o c i a -

t i o n f a c t o r - i n d e p e n d e n t (A ~ ) r i b o s o m a l d i s s o c i a t i o n . D i f f e r e n c e s in t h e M g 2+ c o n c e n t r a t i o n e x i s t e d o n l y i n t h e r i b o s o m e d i s s o c i a t i o n P o r t i o n o f t h e a s s a y . F i n a l i o n i c c o n d i t i o n s f o r all t u b e s w e r e i d e n t i c a l

i n t h e p e P t i d y l t r a n s f e r a s e p o r t i o n o f t h e a s s a y . Al l o t h e r a s s a y c o n d i t i o n s a n d p r o c e d u r e s a r e d e s c r i b e d i n t h e l e g e n d t o T a b l e I . E a c h t u b e r e p r e s e n t i n g t h e 0 .1 M d i s s o c i a t i o n f a c t o r ( e ) r e c e i v e d 1 2 2 # g o f f a c t o r a n d t h o s e t u b e s r e p r e s e n t i n g t h e 0 . 3 M d i s s o c i a t i o n f a c t o r ( a ) r e c e i v e d 13 p g e a c h .

F ig . 7. S p e c i e s - s p e c i f i c i t y o f w h e a t g e r m d i s s o c i a t i o n f a c t o r a c t i v i t i e s w a s d e t e r m i n e d w i t h 70-S r i b o s o m e s f r o m E. coli ( 0 . 7 3 A 2 6 0 ) a n d 80-S r i b o s o m e s f r o m A r t e m i a salina ( 0 . 5 5 A 2 6 0 ) a n d w h e a t g e r m ( 0 . 5 3

A 2 6 0 ) . W h e n E.co l i 70-S r i b o s o m e s w e r e u s e d , t h e r i b o s o m e d i s s o c i a t i o n a s s a y a n d s u c r o s e g r a d i e n t s w e r e 84 m M in KC1 a n d 8 .6 m M in m a g n e s i u m a c e t a t e . A l s o 3 2 0 p g o f t h e 0 .1 M d i s s o c i a t i o n f a c t o r a n d 4 2 p g o f t h e 0 . 3 M d i s s o c i a t i o n f a c t o r w e r e u s e d w i t h t h e 70-S r i b o s o m e s . W i t h e i t h e r t h e A r t e m i a salina o r w h e a t g e r m 80-S r i b o s o m e s , 3 1 4 p g o f t h e 0 .1 M d i s s o c i a t i o n f a c t o r a n d 3 5 ~g o f t h e 0 . 3 M d i s s o c i a t i o n

f a c t o r w e r e u s e d , Al l o t h e r a s s a y c o n d i t i o n s a n d p r o c e d u r e s a re d e s c r i b e d in t h e l e g e n d t o F ig . 2.

311

80-S ribosomes is shown in Fig. 7. Both factors showed similar specificities, although the 0.3 M dissociation factor was more active with eucaryotic ribo- somes.

Preliminary molecular weight determinations using Sephadex G-200 did not indicate any size difference, since both factors eluted at the void volume.

Polyacrylamide disc gel electrophoresis was carried out for both dissociation factors under native (alkaline, 6%) and denaturating conditions [56,57]. The cruder 0.1 M dissociation factor exhibited many major and minor bands on native and denaturing gels. However, the 0.3 M dissociation factor exhibited a minimum of three major bands on native gels (20 pg) and a maximum of six major bands on denaturating gels (40 pg) with molecular weights of 29 000, 53 000, 58 000, 68 000, 73 000 and 84 000. Also, because both activities were only partially purified, comparisons of both dissociation factors on native and denaturating gels gave no definite results concerning the structural relationship of the two factors.

Discussion

Eucaryotic dissociation factor activity has recently been associated with ini- tiation factor eIF-3, a heterogenous, multi-subunit complex consisting of seven to twelve polypeptides which has a reported molecular weight that ranges from about 400 000--700 000 [30--32,35,47--54] and with a high molecular weight (500 000--700 000) initiation factor eIF-3-1ike factor from rat liver [19]. How- ever, certain properties of both wheat germ dissociation factors were not char- acteristic of this protein. It has been reported that initiation factor eIF-3 [29-- 32,50,54] and the initiation factor eIF-3-1ike dissociation factor from rat liver [19] were precipitated virtually completely at 40% ammonium sulfate satur- ation, whereas the majority of the wheat germ dissociation factor activity was precipitated between 40 and 65% ammonium sulfate saturation. Also, the sub- unit peaks produced by the dissociation of 80-S ribosomes by either wheat germ dissociation factor co-sediment with the 40-S and 60-S markers. Benne and Hershey [49] reported that a 40-S-initiation factor eIF-3 complex (50-S), and Thompson et al. [19] reported that complexes formed between the initia- tion factor eIF-3-1ike rat liver dissociation factor and derived 40-S (46 S) and 60-S (69 S) subunits sediment faster than their corresponding subunit markers. In addition, gel electrophoresis data of 0.3 M dissociation factor indicated that this activity did not have an initiation factor eIF-3-1ike quaternary structure. On denaturating gels the partially purified 0.3 M factor exhibited less major bands than homogeneous initiation factor eIF-3 and no major bands with molecular weights of at least 100 000, which are characteristic of this protein.

The possibility that both dissociation factors represent different states of purification of the same factor seems unlikely. If this were the case, it would be expected that the cruder 0.1 M dissociation factor would exhibit the same or an increased level of stability than the 0.3 M dissociation factor when incu- bated at elevated temperatures, and exhibit the same or a decreased reactivity towards sulfhydryl reagents. The simplest explanation of our results is that there are two dissociation factors in wheat germ, neither of which are initiation factor eIF-3, that have different sensitivities to heat, sulfhydryl reagents, etc.,

3 1 2

but have similar behavior with respect to time kinetics, cation concentration, and species-specificity. However, because both dissociation factors were only partially purified, the relationship of each factor to one or more subunits of initiation factor eIF-3 remains to be determined.

References

1 K a e m p f e r , R. , Mese l son , M. a n d Raskas , H . J . ( 1 9 6 8 ) J . Mol . Biol . 31, 2 7 7 - - 2 8 9 2 K a e m p f e r , R . ( 1 9 6 8 ) P r o c . Nat l . A c a d . Sci. U .S .A . 61 , 1 0 6 - - 1 1 3 3 C o l o m b o , B., Vesco , C. a n d Bag l ion i , C. ( 1 9 6 8 ) Proc . Na t l . A c a d . Sci. U .S .A. 61 , 6 5 1 - - 6 5 8 4 H o g a n , B .L .M. a n d K o r n e r , A. ( 1 9 6 8 ) B i o c h i m . B i o p h y s . A c t a 169 , 1 3 9 - - 1 4 9 5 K a e m p f e r , R . ( 1 9 6 9 ) N a t u r e 222 , 9 5 0 - - 9 5 3 6 K a b a t , D. a n d R i c h , A, ( 1 9 6 9 ) B i o c h e m i s t r y 8, 3 7 4 2 - - 3 7 4 9 7 A d a m s o n , S .D. , H o w a r d , G .A . a n d H e r b e r t , E. ( 1 9 6 9 ) Co ld S p r i n g H a r b o r Sy rup . Q u a n t . Biol . 34 ,

5 4 7 - - 5 5 4 8 K a e m p f e r , R . a n d Mese l son , M. ( 1 9 6 9 ) Co ld S p r i n g H a r b o r S y m p . Q u a n t . Biol . 3 4 , 2 0 9 - - 2 2 0 9 S t a e h e l i n , T. a n d F a l v e y , A .K . ( 1 9 7 0 ) J . Mol . Biol . 53 , 2 1 - - 3 4

10 K a e m p f e r , R . a n d K a u f m a n , J . ( 1 9 7 2 ) P roe . Na t l . A c a d . Sci. U .S .A. 69 , 3 3 1 7 - - 3 3 2 1 11 M i z u n o , S. a n d R a b i n o v i t z , M. ( 1 9 7 3 ) Proe . Nat l . A c a d . Sci. U .S .A. 70 , 7 8 7 - - 7 9 1 12 H e n s h a w , E .C. , G u i n e y , D .G. a n d H i r s ch , C .A. ( 1 9 7 3 ) J . Biol . C h e m . 248 , 4 3 6 7 - - 4 3 7 6 13 K a e m p f e r , R . ( 1 9 7 4 ) in R i b o s o m e s ( N o m u r a , M., Tissieres, A. a n d L e n g y e l , P., eds .) , pp . 6 7 9 - - 7 0 4 ,

Co ld Sp r ing H a r b o r L a b o r a t o r y , NY 14 H o g a n , B .L .M. a n d K o r n e r , A . ( 1 9 6 8 ) B i o c h i m . B i o p h y s . A c t a 169 , 1 2 9 - - 1 3 8 15 Bag l ion i , C., Vesco , C. a n d J a c o b s - L o r e n a , M. ( 1 9 6 9 ) Co ld S p r i n g H a r b o r S y r u p Q u a n t . Biol . 34 , 5 5 5 - -

5 6 5 16 Pe t r e , J . ( 1 9 7 0 ) Eur . J . B i o c h e m . 1 4 , 3 9 9 - - 4 0 5 17 L a w f o r d , G . R . , Kaise r , J a n d H e y , W.D. ( 1 9 7 1 ) Can . J . B i o c h e m . 49 , 1 3 0 1 - - 1 3 0 6 18 T h o m p s o n , H . A . , S a d n i k , I. a n d Moldave , K . ( 1 9 7 6 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 73, 5 3 2 - - 5 3 8 19 T h o m p s o n , H .A . , S a d n i k , I., S c h e i n b u k s , J . a n d Moldave , K. ( 1 9 7 7 ) B i o c h e m i s t r y 16, 2 2 2 1 - - 2 2 2 9 20 L u b s e n , N .H . a n d Davis , B .D. ( 1 9 7 2 ) Proc , Nat l . A c a d . Sci. U .S .A. 6 9 3 5 3 - - 3 5 7 21 Mer r i ck , W.C. , L u b s e n , N .H . a n d A n d e r s o n , W.F. ( 1 9 7 3 ) P r o c . Nat l . A c a d . Sci. U .S .A. 70 , 2 2 2 0 - - 2 2 2 3 22 C h e n , Y.C. , W o o d l e y , C .L . , C h a t t e r j e e , B., M a j u m d a r , A. , M f l b r a n d t , J. a n d G u p t a , N .K. ( 1 9 7 4 ) Fed .

P r o c . 33 , 1 2 6 2 23 L u b s e n , N .H . a n d Davis , B.D. ( 1 9 7 4 ) B i o c h i m . B i o p h y s . A c t a 3 3 5 , 1 9 6 - - 2 0 0 24 Wil l iams, G . R . , a n d Maser , J .A . ( 1 9 7 3 ) B i o c h e m . B i o p h y s . Res . C o m m u n . 53, 5 2 - - 5 8 25 H i r s ch , C .A. , C o x , M.A. , van V e n r o o i j , W.J .W. a n d H e n s h a w , E.C. ( 1 9 7 3 ) J . Biol . C h e m . 2 4 8 , 4 3 7 7 - -

4 3 8 5 26 N a k a y a , K. , R a n u , R .S . , a n d Wool , I .G. ( 1 9 7 3 ) B i o c h e m . B i o p h y s . Res . C o m m u n . 54, 2 4 6 - - 2 5 5 27 D e c r o l y , M. a n d G o l d f i n g e r , M. ( 1 9 7 5 ) B i o c h i m . B i o p h y s A c t a 3 9 0 , 8 2 - - 9 3 28 G o l d s t e i n , L. , R o b i n s o n , W. a n d Mause r , L. ( 1 9 7 8 ) Fed . P roc . 37 , 1 4 0 6 29 Russe l l , D.W. a n d S p r e m u l l i , L .L . ( 1 9 7 9 ) J . Biol . C h e m . 254 , 8 7 9 6 - - 8 8 0 0 30 S u n d k v i s t , I .C. a n d S t aehe l i n , T. ( 1 9 7 5 ) J . Mol . Biol . 9 9 , 4 0 1 - - 4 1 8 31 I lan , J . a n d I l an , J . ( 1 9 7 6 ) J . Biol. C h e m . 251 , 5 7 1 8 - - 5 7 2 5 3 2 Sch re i e r , M.H. , E rn i , B. a n d S t aehe l i n , T. ( 1 9 7 7 ) J . Mol. Biol . 116 , 7 2 7 - - 7 5 3 33 T rachse l , H. , Ern i , B., Sch re i e r , M.H. a n d S t aehe l i n , T. ( 1 9 7 7 ) J . Mol. Biol . 116 , 7 5 5 - - 7 6 7 3 4 Ayuso -Pa r i l l a , M., H e n s h a w , E .C. a n d H i r s ch , C .A. ( 1 9 7 3 ) J . Biol . C h e m . 248 , 4 3 8 6 - - 4 3 9 3 35 F r e i e n s t e i n , C. a n d Blobe l , G. ( 1 9 7 5 ) P r o c . Nat l . A c a d . Sci. U .S .A. 72 , 3 3 9 2 - - 3 3 9 6 36 Pa in , V.M. a n d H e n s h a w , E.C. ( 1 9 7 5 ) Eu r . J . B i o c h e m . 5 7 , 3 3 5 - - 3 4 2 37 S m i t h , K .E . a n d H e n s h a w , E.C. ( 1 9 7 5 ) J . Biol . C h e m . 2 5 0 , 6 8 8 0 - - 6 8 8 4 38 S a m e s h i m a , M. a n d I z a w a , M. ( 1 9 7 5 ) B i o c h i m . B i o p h y s . A c t a 3 7 8 , 4 0 5 - - 4 1 4 39 V a n V e n r o o i j , W.J . , J a n s s e n , A .P .M. , H o e y m a k e r s , J .H . a n d D e M a n , B.M. ( 1 9 7 6 ) Eur . J . B i o c h e m . 64 ,

4 2 9 - - 4 3 5 4 0 W a r b u r g , W. a n d Chr i s t i an , W. ( 1 9 4 1 ) B i o c h e m . Z. 3 1 0 , 3 8 4 - - 4 2 1 41 Zas lo f f , M. a n d O c h o a , S. ( 1 9 7 1 ) P roc . Nat l . A c a d . Sci. U .S .A. 68 , 3 0 5 9 - - 3 0 6 3 4 2 Marcus , A. ( 1 9 7 2 ) in M e t h o d s in M o l e c u l a r B i o l o g y (Lask in , A. I . , a n d L a s t , J . A . , eds .) , Vol . 2, pp ,

1 2 7 - - 1 4 5 , Marce l D e k k e r , Inc . , N e w Y o r k 43 Weeks , D.P . , V e r m a , D.P .S . , Seal , S.N. a n d M a r c u s , A. ( 1 9 7 2 ) N a t u r e 236 , 1 6 7 - - 1 6 8 4 4 H a e n n i , A .L . a n d Chapev i l l e , F. ( 1 9 6 6 ) B ioch i rn . B i o p h y s . A c t a 114 , 1 3 5 - - 1 4 8 4 5 Myer s , D .K . ( 1 9 6 0 ) T h e E n z y m e s 4 , 4 7 6 - - 4 7 7 4 6 F a h r n e y , D.E. a n d G o l d , A.M. ( 1 9 6 3 ) J . A m . C h e m . Soc . 85 , 9 9 7 - - 1 0 0 0 47 H e y w o o d , S .M. , K e n n e d y , D.S. a n d Bes te r , A . J . ( 1 9 7 4 ) P roc . Na t l . A c a d . Sci. U .S .A. 71, 2 4 2 8 - - 2 4 3 1

313

48 Safer, B., Adams, S.L., Kemper, W.M., Berry, K.W., Lloyd, M. and Merrick, W.C. (1976) Proe. Natl. Acad. Sci. U.S.A. 73, 2584--2588

49 Benne. R. and Hershey, J.W.B. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 3005--3009 50 Kennedy, D.S. and Heywood, S.M. (1976) FEBS Lett . 72, 314--318 51 Emanuilov, I., Sabatini, D.D,, Lake, J.A. and Freienstein, C. (1978) Proc. Natl. Acad. Sci. U.S.A. 75,

1389--1393 52 Benne, R. and Hershey, J.W.B. (1978) J. Biol. Chem. 253, 3078--3087 53 Spremulli, L.L., Walthan, B.J., Lax, S.R. and Ravel, J.M. (1979) J. Biol. Chem. 254, 143--148 54 Traehsel, H., Erni, B., Schreier, M.H., Braun, L. and Staehelin, T. (1979) Biochim. Biophys. Aeta 561,

484--490 55 Robinson, W.G. (1966) in Methods in Enzymology (Colowick, S.P., and Kaplan, N.O., eds.), Vol. IX,

pp. 332--336, Academic Press, New York 56 Disc Electrophoresis (1972) Canaleo, Rockville, MD 57 Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244, 4406--4412