Plant Physiol. (1975) 56, 39-43

Studies on Pea Ribosomal ProteinsCONFORMATIONAL AND BIOLOGICAL ACTIVITY CHANGES OF RIBOSOMAL SUBUNITS DERIVED BYNH4C1 DISSOCIATION' . 2

Received for publication October 11, 1974 and in revised form December 11, 1974

CHU-YUNG LIN, SUBRINA Li-Li CHIA, ROBERT L. TRAVIS, AND JOE L. KEYDepartment of Botany, University of Georgia, Athens, Georgia 30602

ABSTRACT

Ribosomal subunits prepared by NH4Cl dissociation (0.5 M)of the monomeric ribosomes were much less active in in vitroprotein synthesis than those prepared by KCI dissociation. Thedecrease in activity correlated with a detachment of some pro-teins (L2 and LI as shown by gel electrophoresis) within the60S ribosomal subunits. Subunits prepared with 0.3 M NH4Clretained L and L, but the activity remained low. Incubation ofthese 60S subunits in TKM buffer (50 mM tris [pH 7.5], 20mM KCI, and 5 mM MgC12) for 20 min at 37 C restored theactivity almost to the level of those obtained by KCI dissocia-tion. Treatment of the 0.3 M NH4C-derived 60S subunits witha protein reagent, Procion brilliant blue, prior to extractionof the ribosomal proteins resulted in the loss of L2 and L9,showing that these proteins were made accessible for dyebinding. These observations suggest that a considerable degreeof unfolding of the 60S subunit occurs at 0.3 M NH4Cl (thisapparently leads to a preferential detachment of L and L, at0.5 M NH4C1) and that the activity of the purified subunitsdepends not only on the presence of L2 and L but also on theorganization of these proteins within the 60S subunits.

Ribosomal proteins have been studied in bacterial (14, 21)and mammalian (3, 25) systems. Bacterial ribosomes havebeen extensively characterized relative to the total number ofproteins associated with each subunit (13), molecular weightsof individual proteins (4), order of assembly in formation ofactive subunits (22), and function of certain proteins (2, 14).Much less is known about plant ribosomal proteins. Studiesconcerning ribosomal proteins of plants have generally dealtwith a comparison, by one-dimensional acrylamide gel chroma-tography, of the protein complement of cytoplasmic versuschloroplast Pr mitochondrial ribosomes (5-7, 10). Two-dimen-sional acrylamide gel fractionation has been utilized in thestudy of cytoplasmic and chloroplast ribosomes of wheat (12).

In a previous report, we noted that free or run-off 80Sribosomes (N2 gas derived) could be dissociated into biologi-

'This research was supported by a contract from the AtomicEnergy Commission, AT (38-1)-643.

2Part of the work was taken from the thesis submitted byS.L. Chia to the graduate school of the University of Georgia inpartial fulfillment of the requirements for the Master of Sciencedegree.

cally active subunits by treatment with KCl (17, 18). Dissocia-tion with NH4Cl, produced 60S subunits which were essentiallyinactive in in vitro protein synthesis. In the present communica-tion, we have characterized KCl- and NH,Cl-derived subunitsrelative to their complement of ribosomal protein. The resultsindicate that the loss of activity associated with NH,Cl-derivedsubunits relates to the loss of a specific ribosomal protein (L9)and perhaps a portion of another protein (L2). By varying thesalt concentration, it was further demonstrated that thedissociation of protein L. begins as a conformation change inthe subunit followed by selective detachment of the protein.

MATERIALS AND METHODSGrowth of Plant Material and Preparation of Ribosomal

Subunits. Peas (Pisum sativum var. Alaska) were grown for 3days in rolls of moist paper (15). Free or "run-off" 80S mono-ribosomes were prepared from root tips (terminal 0.5-cmsegment) of anaerobically treated seedlings (16). Details ofsalt washing of monoribosomes, monoribosome dissociation,and preparation of ribosomal subunits were as previouslydescribed (17).

Poly(U)-directed Phenylalanine Incorporation Studies. The0.5-ml reaction mixture for in vitro amino acid incorporationcontained 50 mM HEPES buffer, pH 7.5, 80 mm KCl, 80 mMNH,C1, 10 mm MgCl2, 0.3 mm GTP, 40 to 50 ,ug of partiallypurified enzyme fraction from the postribosomal supernatant(18), and 50 jug of poly(U). The level of ribosomal subunitsand `4C-phenylalanyl-tRNA (New England Nuclear) were asnoted in the table legends. The reaction mixture was incubatedat 37 C for 10 or 20 min. At the end of the incubation period,the samples were precipitated with an equal volume of 10%trichloroacetic acid followed by heating to 90 C for 15 min.Hot trichloroacetic acid-insoluble radioactivity retained onWhatman GFA glass fiber disks was determined in a Packardliquid scintillation spectrometer.

Extraction of Ribosomal Proteins. Ribosomal proteins wereextracted from free 80S monoribosomes or ribosomal subunitsby the method of Hardy et al. (9). To one volume of ribosomalsuspension (17) one-tenth volume of 1 M MgCl, and twovolumes of glacial acetic acid were added. The preparation wasstirred for 45 min at 0 to 3 C followed by centrifugation at20,000g for 10 min. The pellet which contained ribosomalRNA was discarded. Ribosomal proteins were precipitatedfrom the supernatant with trichloroacetic acid (final concentra-tion, 10%), dissolved in a small volume of 8 M urea containing1 mM dithiothreitol), and analyzed electrophoretically. Finalconcentration of the protein solution was 2 to 4 mg/ml.

Disc Gel Electrophoresis of Ribosomal Proteins. Acrylamidegel electrophoresis of ribosomal proteins was done by the

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LIN, CHIA, TRAVIS, AND KEY

Table I. In Vitro Pheniylalanzi,ie Inicorporationz by VariousCombiniationis of Ribosomal Suibun7its

Subunits were derived by 0.5 M KCl or 0.5 NI NH4Cl dissociationin the presence of 5 mM MgCl2. The reaction mixture was incubatedfor 20 min at 37 C; 1.0 A260 unit of 40S, 2.0 A260 units of 60S, and19,450 cpm of '4C-phenylalanyl-tRNA per assay. K40S and K60S:KCI-derived subunits; N40S and N60S: NH4CI-derived subunits.

Ribosomes 14C-Phenylalanine Incorporated

cpfrK40S 96K60S 524N40S 57N60 348K40S + K60S 10,027N40S + N60S 1,032K40S + N60S 811N40S + K60S 8,136

method of Panyim and Chalkley (23) modified as follows. Thegel mixture contained 15% (w/v) polyacrylamide, 0.098%N, N'-methylene bisacrylamide, 0.5% tetramethylethylene-diamine, 0.125% freshly prepared ammonium persulfate, 6.25M urea, and 5.4% (w/v) acetic acid. Gels were cast in 0.45 x9 cm Plexiglas tubes. After 1 hr all gels were pre-electro-phoresed for 4 hr at 2 mamp per tube. Electrophoresis ofribosomal protein (40-60 t,g) was done at room temperaturefor 4.75 hr at 1 mamp per gel. Following electrophoresis, thegels were removed from the tubes, stained for 2 hr with 0.1 %amidoblack, destained in 7% acetic acid, and scanned at 600nm with a Gilford model 2400 spectrophotometer.

Reaction with Procion Brilliant Blue. A 0.5-ml volume of60S ribosomal subunit suspension was incubated with 25 1,Iof freshly prepared Procion brilliant blue solution (5 mg/mlin methanol) for 2 min at 0 to 3 C (11). The reaction wasterminated by dilution with 10 volumes of ice-cold 150 mmsucrose containing 35 mM tris buffer, pH 7.5, 20 mm KCI,10 mM MgCl2, and 1 mm dithiothreitol. The diluted reactionmixture was centrifuged immediately at 229,400g for 4 hr.Ribosomal subunits were resuspended (17) for protein extrac-tion.

RESULTSIn Vitro Activity of KCl- and NHICi-derived Subunits. The

in vitro poly(U)-directed incorporation of phenylalanine byribosomal subunits prepared by dissociation of free 80S ribo-somes with 0.5 M KCl or 0.5 M NH4Cl, in the presence of Mg2+,is shown in Table I. Small (K40S, N40S) and large (K60S,N60S) subunits prepared by KCl or NH4Cl dissociation wereessentially inactive at poly(U)-directed phenylalanine incor-poration when incubated separately. Recombination of theKCl-dissociated subunits (K40S + K60S) produced mono-meric ribosomes 10-fold more active than those prepared byrecombination of NH4Cl-dissociated subunits (N40S + N60S).A comparison of the activities of hybrid ribosomes (K40S +N60S and N40S + K60S) suggests that the much lower activityof recombined NH4Cl-derived subunits (N40S + N60S) relatesdirectly to the 60S subunit. The K40S and N40S subunits wereabout equally active in combination with K60S subunits.Monomeric ribosomes derived from recombined subunits thatwere dissociated in the absence of Mg"5 were inactive in in vitropoly(U)-directed phenylalanine incorporation. Recombinationexperiments further indicated that both subunits are adverselyaffected by the absence of Mg"5 (Table II).Ribosomal Proteins of KCl- and NH4CI-derived Subunits.

The relationship of ribosome activity relative to specific

ribosomal proteins in bacterial systems is well established (2,14). Since individual ribosomal proteins have specific func-tions, at least in bacterial systems, possible regulation of ribo-some activity through alteration in the ribosomal proteins wasconsidered. To determine whether the decline in ribosomeactivity associated with NH,C1 dissociation or the absence ofMg2+ from the dissociation medium was related to changes inspecific ribosomal proteins, the distribution of ribosomal pro-teins was studied in subunits derived under varying salt andMg2+ conditions. Proteins were extracted with 67% acetic acid(9) or 4 M LiCl-8 M urea (6) and fractionated on 15%acrylamide gels. Since acetic acid extraction produced betterresolution (data not shown) all subsequent studies utilized thismethod. Populations of ribosomal proteins from KCl-washed80S monomeric ribosomes, K40S subunits, and K60S subunitswith or without Mg2+ in the dissociation buffer are shown inFigure 1 and Figure 2 (A, C, and E). Proteins from 80S

Table II. In Vitro Phentylalaniline Isicorporationl byCornbi,iationi of Pea Ribosomal Subunlits

Subunits were derived by 0.5 M KCl dissociation in the presenceand absence of 5 mM MgCl2. The reaction mixture was incubatedat 37 C for 10 min; 0.6 A260 unit of 40S, 1.2 A260 unit of 60S and10,600 cpm of '4C-phenylalanyl-tRNA were used per assay.K+40S and K+60S: KCl-derived subunits in the presence ofMgCI2; K-40S and K-60S: KCl-derived subunits in the absenceof MgCI2.

Ribosomes '4C-Phenylalanine Incorp)orated

cpm71K+40S + Ki 60S 4748K-40S + K-60S 8K-40S + K+60S 235K+40S + K-60S 48

aI.

A B

a.--a.ta is

C D

.

_--_z. _

.,I....

am

Ei F G

FIG. 1. One-dimensional electrophoretograms of pea ribosomalproteins. Electrophoresis was at pH 4.5 in 15% polyacrylamide gelscontaining 8 M urea. The anode is at the bottom. Ribosomal pro-teins extracted from the 80S monomeric ribosomes prewashed with500 mM KCI + 5 mM MgCl2 (K80S) (A) and prewashed with 500mM NH4Cl + 5 mm MgCI2 (N80S) (B); ribosomal proteins ex-tracted from the small ribosomal subunits by 500 mM KCl + 5mM MgCl2 dissociation (K40S) (C), and by 500 mM NH4C1 +5 mM MgCI2 dissociation (N40S) (D); ribosomal pioteins extractedfrom the large ribosomal subunits by 500 mm KCI + 5 mM MgCI2dissociation (K60S) (E), by 500 ms NH4C1 + 5 mM MgCI2 dis-sociation (N60S) (F), and by 500 mm KCl (in the absence ofMgCl2) dissociation (K-g" 60S) (G).

40 Plant Physiol. Vol. 56, 1975

---* -ow-

m

a

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RIBOSOMAL PROTEINS OF PEA

EC00CD

DISTANCE (CM)

(+) I

E00CD

0 2 4 6 8

DISTANCE (CM)

(-) (4-)

FIG. 2. Densitometric traces of Figure I gels of pea ribosomalproteins. Ribosomal proteins from K80S (A) and N80S (B);ribosomal proteins from K40S (C) and N40S (D); ribosomal pro-

teins from K60S (E), N60S (F), and K-g'+ 60S (G).

0 2 4 6 8

DISTANCE (CM)(t) > (-)

ribosomes were resolved into more than 30 distinct bands,whereas proteins extracted from K40S and K60S subunits were

resolved into 17 and 26 bands, respectively. Dissociation ofmonoribosomes in the absence of Mg2` results in 40S "subunits"practically devoid of protein complement. In a previous report(17), 40S subunits prepared in the absence of Mg` were shownto sediment significantly slower on sucrose gradients. Thedetachment of the ribosomal proteins is sufficient to accountfor both the lowered sedimentation rate and the loss in in vitropoly(U)-directed phenylalanine incorporation. Since maximumin vitro activity was obtained with subunits derived by 0.5 MKCl-dissociation (in the presence of Mg2+), these profiles wereused as a basis of comparison in the following studies.

Electrophoretic patterns of proteins extracted from 0.5 M

NH4Cl-washed monomeric ribosomes and 0.5 M NH4Cl-

derived subunits are shown in Figure 1 and Figure 2 (B, D,and F). NH4Cl-washed 80S monoribosomes lacked one specificprotein and showed decreased amounts of two others (asindicated by arrows). Dissociation of the monoribosomes intosubunits further showed that the proteins in question are

components of the large subunits; L9 and a portion of L, andL" are selectively detached from the large subunit by NHCI(0.5 M). No significant differences were noted in gel patternsbetween 40S subunits prepared by KCI and NH,Cl dissociation(Fig. 1 and Fig. 2, C vs. D).NILCI-induced Conformational Change of 60S Subunit. In

a previous communication (17), we reported that free 80Sribosomes could be dissociated into subunits, in the presenceof Mg"+, with 0.3 M NH4Cl. Subunits prepared by 0.3 M

NH4Cl dissociation were quite low in in vitro protein synthetic

0 ~~~~~~~D

1~

(-)

EC:00co<C

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LIN, CHIA, TRAVIS, AND KEY

Table III. In Vitro Phenzylalaninte Inicorporationz byCombiniationis of Pea Ribosomal Stubuniiits

Subunits were derived by 0.5 M KCl or 0.3 M and 0.5 M NH4Cldissociation in the presence of 5 mM MgCI2. The reaction mixturewas incubated at 37 C for 10 min The 0.6 A260 unit of 40S, 1.2 A260unit of 60S and 10,144 cpm of 14C-phenylalanyl-tRNA per assaywere used for the 0.3 M NH4Cl-derived subunits. The same amountof the ribosomal subunits and 9,754 cpm of 14C-phenylalanyl-tRNA per assay were used for the 0.5 M NH4C1-derived subunits.K40S and K60S: KCI (0.5 M) derived subunits; N40S and N60S:NH4Cl (0.3 M) derived subunits; 0.5 N40S and 0.5 N60S: NH4Cl(0.5 M) derived subunits.

Ribosomes '4c-PhenylalanineIncorporated

cpmt

NH4Cl dissociation 0.3 MK40S 100K60S 193N40S I 54N60S 174K40S + K60S 5813N40S + N60S 1190K40S + N60S 539N40S + K60S 4430N40S + (N60S)' 4837K40S + (K60S)l 5752K40S + (N60S)1 4671

NH4Cl dissociation 0.5 M0.5 N40S + 0.5 N60S 3160.5 N40S + 0.5 N6OS' 1056K40S + 0.5 N 60S' 893

1 60S subunits in the TKM buffer were heated at 37 C for 20min before combination with the 40S subunits for assay.

activity relative to KCI-dissociated subunits (Table III). Therecombination experiments with hybrid ribosomes (K40S +N60S and N40S + K60S) showed that the defect resided inthe N60S ribosomal subunits as the findings with higher NH4Cl-dissociated subunits (0.5 M). Electrophoretic analyses of thecomplement of ribosomal proteins extracted from 0.3 MNH4Cl-dissociated 60S subunits (Fig. 3C versus Fig. 2F)showed that the proteins selectively detached at the highersalt concentration (0.5 M NH4CI), i.e. L9 and a portion of L2and L3 were still present in normal amounts. No major differ-ences were detected in low and high salt-dissociated 40S sub-units (data not shown).

Heating of 0.3 M NH4Cl-derived 60S subunits (37 C for20 min) prior to assay completely reversed the low salt inacti-vation (Table III). Incubation at 37 C did not restore in vitroactivity of the 0.5 M NH4Cl-derived 60S subunits (Table III).These results, taken together with the differential effect of lowand high salt (0.3 M NH4Cl) vs. 0.5 M NH4CI) on the com-plement of 60S subunit proteins suggest that a conformationalchange of the large subunit occurs in response to low salt dis-sociation, followed by eventual detachment of specific proteinsas the salt concentration is increased.To test further for the possibility of a salt-induced conforma-

tional change, 60S subunits were incubated with Procionbrilliant blue according to the method of Hultin (11). If a con-formational change does occur in response to NH4CJ, then onewould expect specific proteins to become accessible for dyebinding. Results of these experiments are presented in Figure3. Electrophoretograms of ribosomal proteins from 0.5 M KCl-derived 60S subunits are identical whether or not the subunits

were incubated with dye (Fig. 3, A versus B), indicatingthat KC1 dissociation does not effect a conformational change(i.e. no proteins were rendered accessible to dye binding).However, when 0.3 M NH4Cl-derived subunits were reactedwith Procion brilliant blue followed by protein extraction, pro-tein L" (and a portion of L2) was not present in the acetic acidextract showing that they were accessible to dye (Fig. 3, Cversus D). These results suggest that the inactivation of 60Ssubunits induced by NH4Cl-dissociation is the consequence ofa conformational change involving these proteins followed by

0'

A

AL/AI 'u 0

L2 B

I)

ilj,;'i !I4

0 2 4 6 8DISTANCE (CM)

(±) (-)

E

L2

.. 01 \ .,_~~~~~~~,i2 ll D

,A

1ID

0 2

(+) -*4 6

DISTANCE (CM)8

(-)FIG. 3. Densitometric traces of gel profiles of ribosomal 60S

subunit proteins: effect of Procion brilliant blue on protein comple-ment. Fractionation was at pH 4.5 in 15% polyacrylamide gelscontaining 8 M urea. Ribosomal subunits for A and B were ob-tained by 500 mm KCl + 5 mM MgCl2 dissociation and for C andD were obtained by 300 mm NH4Cl + 5 mM MgCl2 dissociation.A and C: (Control) ribosomal proteins from the nontreated 60Ssubunits; B and D: ribosomal proteins from the dye-treated 60Ssubunits.

I

42 Plant Physiol. Vol. 56, 1975

CD

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RIBOSOMAL PROTEINS OF PEA

their selective detachment from the subunit as salt concentra-

tion is increased.

DISCUSSION

There have been several attempts to characterize plantribosomal proteins (5-7, 10, 12). These have dealt primarilywith comparative studies of whole protein complements fromcytoplasmic and either chloroplast or mitochondrial ribosomes.Studies concerning the regulation of plant ribosome activity byspecific ribosomal proteins have not been reported. This com-

munication considers the role of at least one specific ribosomalprotein in regulating ribosome activity.

In mammalian systems ribosomal subunits active in in vitroprotein synthesis are prepared in most instances by KCI dis-sociation of polyribosomes (8) or "run-off' monoribosomes(19). The former requires the presence of Mg2` in the dissocia-tion medium, while the latter does not. In procaryotic systemsactive subunits are typically prepared either by NHCl dissocia-tion or by lowering the Mg2` concentration in the dissociationmedium.

Ribosome subunits prepared by KCI dissociation of free or

"run-off" pea monoribosomes were active in in vitro poly(U)-directed phenylalanine incorporation only when Mg2` was

included in the dissociation buffer. A comparison of one-

dimensional acrylamide gel profiles of ribosomal proteins ex-

tracted from KCl-derived subunits showed that the loss ofbiological activity resulting from the absence of Mg2` in thedissociation medium related to the loss of a substantial portionof the ribosomal protein complement.Ammonium chloride dissociation of plant monoribosomes

produced ribosomal subunits which upon recombination were

inactive in in vitro protein synthesis; however, the N40S sub-unit was active in aminoacyl-tRNA binding (17, 18) and was

active in poly(U)-directed phenylalanine incorporation incombination with the K60S subunits. The loss in activity of theN60S subunit related directly to the selective loss of a singleribosomal protein, L5. In addition it appears that a small frac-tion of at least two other proteins of the large subunit (L2 andL3) may be removed during NH4Cl-dissociation. The reversibleloss of protein synthetic activity in low concentrations (0.3M) of NH4Cl apparently relates to a conformational changein the subunit. Dye binding experiments utilizing Procionbrilliant blue show that the protein,L5, is unexposed to thedye in K60S subunits dissociated in Mg2+, but becomes sus-

ceptible to dye binding in the presence of 0.3M NH4Cl, andfinally at higher NH,Cl concentrations (0.5M)L9 is selectivelydetached and loss in protein synthetic activity is no longerreversible. Thus it appears that for the large subunit to befunctional in protein synthesis in a cell-free system proteinL,must not only be present but must be in the correct conforma-tion. Conformational changes in mammalian ribosomal subunitshave been induced by Cs and Rb ions (1), and by Li ion (24).Neither the mechanism of selective removal of protein I, fromthe large subunit, nor the function of protein L in ribosomeactivity is understood. Experiments designed to elucidate boththe mechanism of removal of L. and its role in ribosomeactivity are in progress.

LITERATURE CITED

1. ARPIN, M., A. M. REBOUND, AND J. P. REBOUND. 1972. Conformational changesof large ribosomal subunits of rat liver, induced by some monovalent cations.Biochim. Biophys. Acta 277: 134-139.

2. BROT, N., R. MARCEL, E. YAMASAK, AND H. WEISSBACH. 1973. Further studieson the role of 50S ribosomal proteins in protein synthesis. J. Biol. Chem.248: 6952-6956.

3. DELAUNAY, J., C. MATHIEU, AND G. SCHAPIRA. 1972. Eukaryotic ribosomalproteins. Interspecific and intraspecific comparisons by two-dimensionalpolyacrylamide-gel electrophoresis. Eur. J. Biochem. 31: 561-564.

4. DZIONARA, M., E. KALTSCHMIDT, AND H. G. WIrrmT. 1970. Ribosomal pro-teins. XIII. Molecular weights of isolated ribosomal proteins of Escherichiacoli. Proc. Nat. Acad. Sci. U.S.A. 67: 1909-1913.

5. FREYSSINET, G. AND J. A. SCHIFF. 1974. The chloroplast and cytoplasmicribosomes of Euglena. II. Characterization of ribosomal proteins. PlantPhysiol. 53: 543-554.

6. GlALERZI, C. AND P. CAMMARANO. 1969. Comparative electrophoretic studieson the proteins of chloroplast and cytoplasmic ribosomes of spinachleaves. Biochim. Biophys. Acta 190: 170-186.

7. GuALERZI, C. AND P. CAMMARANO. 1970. Species specificity of ribosomal pro-teins from chloroplast and cytoplasmic ribosomes of higher plants electro-phoresis studies. Biochim. Biophys. Acta 199: 203-213.

8. HAMADA, K., P. YANG, R. HEINZ, AND R. SCHWEET. 1968. Some propertiesof reticulocyte ribosomal subunits. Arch. Biochem. Biophys. 125: 598-603.

9. HARDY, S. J. S., C. G. KURLAND, P. VOYNOW, AND G. M%ORA. 1969. Theribosomal proteins of Escherichia coli. 1. Purification of the 30S ribosomalproteins. Biochemistry 8: 2897-2905.

10. HOOBER, J. K. AND G. BLOBEL. 1969. Characterization of the chloroplasticand cytoplasmic ribosomes of Chlamydomonas reinhardi. J. 'Mol. Biol. 41:121-138.

11. HULTIN, H. 1969. The use of procion blue as a molecular probe in the study ofribosomal structure. Eur. J. Biochem. 9: 579-584.

12. JONEs, B. L., N. NAGALHUSHAM, A. GULYAS, AND S. ZALIK. 192. Two-dimen-sional acrylamide gel electrophoresis of wheat leaf cytoplasmic and chloro-plast ribosomal proteins. FEBS Lett. 23:167-170.

13. KALTSCHMIDT, E. AND H. G. WITTMAN. 1970. Ribosomal proteins. XII. Numberof proteins in small and large ribosomal subunits of Escherichia coli asdetermined by two-dimensional gel electrophoresis. Proc. 'Nat. Acad. Sci.U.S.A. 67: 1276-1282.

14. KUTRLAND, C. G. 1972. Structure and function of the bacterial ribosome. Annu.Rev. Biochem. 41: 377-408.

15. Lin, C. Y.,J. L. KEY, AND C. E. BRACKER. 1966. Association of D-RNAwith polyribosomes in the soybean root. Plant Physiol. 41: 976-982.

16. LIN, C. Y. AND J. L. KEY. 1967. Dissociation and reassembly of poly-ribosomes in relation to protein synthesis in the soybean root. J.Mlol. Biol.26: 237-247.

17. LIN, C. Y. AND J. L. KEY. 1971. Dissociation of N2 gas-induced monomericribosomes and functioning of the derived subunits in protein synthesis inpea. Plant Physiol. 48: 547-552.

18. LIN, C. Y. AND J. L. KEY. 1973. Enzymatic binding of aminoacyl-tRNA andpeptide chain elongation by ribosomes and ribosome subunits in pea.Phytochemistry 12: 43-53.

19. MARTIN, T. E., F. S. ROLLESTON, R. B. Low, AND I. G. WOOL. 1969. Dissocia-tion and reassociation of skeletal muscle ribsomes. J. Mol. Biol. 43: 135-149.

20. NISHIZUKA, Y. AND F. LIPmANY. 1966. Comparison of guanosine triphosphatesplit and polypeptide synthesis with a purified Escherichia coli system.Proc. Nat. Acad. Sci. U.S.A. 55: 212-219.

21. NOM.URA, M. 1970. Bacterial ribosome. Bact. Rev. 34: 228-277.22. NOMURA, M. 1972. Assembly of bacterial ribosomes. Fed. Proc. 31:18-20.23. PAN,YIM, S. AND R. CHALELEY. 1969. High resolution acrylamide gel electro-

phoresis of histones. Arch. Biochem. Biophys. 130: 337-346.24. REBOUND, A. M., M. BUissoN, AND J. P. REBOUND. 1972. Ribosomal subunits

from ratliver. 2. Effect of cation on isolated subunits. Eur.J. Biochem.26: 354-359.

25. SHERTON, C. C. AND I. G. WOOL. 1972. Determination of the number of pro-teins in liver ribosomes and ribosomal subunits by two-dimensional poly-acrylamide gel electrophoresis. J. Biol. Chem. 247: 4460-4467.

26. VASCON-CELOS, A. C. L. AND L. BOGOaAD. 1971. Proteins of cytoplasmic,chloro-plast and mitochondrial ribosomes of some plants. Biochim. Biophys. Acta228: 492-502.

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