ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 169, 522-528 (1975)

Purification and Partial Characterization of the Antiviral Protein from

Phytolacca americana Which Inhibits Eukaryotic Protein Synthesis

JAMES D. IRVIN

Department of Chemistry, Southwest Texas State University, San Marcos, Texas 78666
Received February 27, 1975

The antiviral protein from the pokeweed plant (Phytolacca americana) which inhibits
eukaryotic protein synthesis has been purified to homogeneity and its molecular weight has
been determined by two physical methods. The protein consists of a single polypeptide
chain of an approximate molecular weight of 27,000. The inhibitory effect of this protein on
the synthesis of polyphenylalanine in a cell-free system from Artemia salina suggests that
this protein acts in an enzymatic manner on eukaryotic ribosomes. It is also demonstrated
that polyphenylalan:ne synthesis on A. salina ribosomes is more sensitive to inhibition by
this protein than on rabbit reticulocyte ribosomes.

A protein from the plant Phytolacca polyphenylalanine synthesis on A. salina

americana has been shown to inhibit viral ribosomes is demonstrated to be more
transmission in plants (1, 2) and the repli- sensitive to PAP inhibition than that re-
cation of polio virus (3) and influenza virus ported for reticulocyte (4) and wheat em-
(2) in tissue culture. This same protein, brvo (5) ribosomes.
referred to as PAP, 1has also been shown to
MATERIALS AND METHODS
inhibit eukarvotic protein synthesis in cell-
free systems derived from rabbit reticulo- Materials
cytes (4) and certain plants (5). The inhibi- The brine shrimp cysts, reticulocyte ribosomes and
tion of protein synthesis by PAP has been supernatant enzyme fraction were the kind gift of Dr.
demonstrated to involve an effect on the Boyd Hardesty, University of Texas at Austin. Brew-
large ribosomal subunit which influences ers yeast transfer RNA was purchased from Boeh-
the interactions of the elongation factors ringer Mannheim Corporation; [‘“Clphenylalanine
EF-1 and EF-2 with the ribosome (4). Also was from Amersham-Searle; polyuridylic acid,
these studies have suggested that PAP may DEAE-cellulose, G-100 Sephadex, bovine serum al-
bumin, ovalbumin, glyceraldehyde-s-phosphate de-
act in an enzymatic manner in its effect on
hydrogenase, trypsin, myoglobin, cytochrome c, blue
the large ribosomal subunit.
dextran and cellulose phosphate were purchased from
PAP has previously been partially puri- Sigma,
fied and reported to be a basic protein
having a minimum molecular weight of Methods
approximately 13,000 (1). Ribosomes and supernatant enzyme fraction were
This communication reports the purifi- prepared from the cysts of A. salina according to the
cation of PAP to homogeneity and the method of Zasloff and Ochoa (6). The supernatant
determination of its molecular weight by enzyme fraction was prepared by precipitation of the
sodium dodecyl sulfate-polyacrylamide gel postribosomal supernatant fluid between the levels of
electrophoresis and gel filtration. Also, 30 and 7Oc7r saturation of ammonium sulfate, dis-
solved in Solution A (10 rnM Tris-HCI, pH 7.5, 0.1 mM
‘Abbreviations used are: PAP, the Ph.vtolacca p-mercaptoethanol, 0.2 rnM EDTA), and dialyzed
americana protein described in these studies. PPO, against 2 liters of Solution A 12-14 h with one change
2,5-diphenyloxazole; POPOP, 1,4-bis [2-(5-phenyl- of dialysis solution. Washed ribosomes were prepared
oxazolyl)]-benzene; and DTT, dithiothreitol. by dissolving the crude ribosomal pellets in a solution
522
Copyright 0 1975 by Academic Press, Inc.
AlI rights of reproduction in any form reserved.
 PAP PURIFICATION AND PROPERTIES 523

containing 50 mM Tris-HCl, pH 7.5, 500 mM KCl, and nine synthesis on A. salina ribosomes
20 mM fi-mercaptoethanol. The ribosomes were iso- which is a faster and more convenient
lated by centrifugation for 2 h at 40,000 rpm in a technique to test activity at all stages of
Spinco 40 Ti fixed-angle rotor. [“ClPhenylalanyl-
purification and in individual fractions
tRNA was prepared according to the procedure of
Hardesty et al. (7) from tRNA from brewers yeast and obtained from column chromatography.
[“C lphenylalanine (100 Ci/mol). Sodium dodecyl All operations were performed at O-4%
sulfate gel electrophoresis was performed in 10% unless otherwise specified.
polyacrylamide gels as described by Weber and Os-
born (8). Gel filtration analysis of PAP on G-100 was
Crude Extract
performed according to the method of Andrews (9). Phytolacca americana leaves were ob-
Polyphenyylalanine synthesis. Reaction mixtures for
tained in early spring from young plants
poly(U) directed polyphenylalanine synthesis con-
tained the following components in a volume of 0.5
growing wild in the immediate area. The
ml: 20 mM Tris-HCI, pH 7.5, 120 mM KCI, 4 mM leaves were frozen and stored prior to use at
MgCI,, 2.5 mM dithiothreitol, 0.2 mM GTP, 200 fig -20°C. Standard preparations utilized 500
washed ribosomes, 0.5 mg 30-70% ammonium sulfate g of leaves which were homogenized in 500
fraction of the postribosomal supernatant, 100 fig ml of deionized water using a Waring-type
poly(U), and 80 pmol [“Clphenylalanyl-tRNA. Incu- blender followed by filtration through
bations were for 5 min at 37°C. The reaction was cheesecloth with mild suction to avoid
terminated by the addition of 5 ml 5% trichloroacetic foaming. Approximately 560 ml of dark
acid. The mixtures were then heated at 90°C for 15 green extract was obtained.
min, cooled to room temperature, collected on Mil-
lipore type HA nitrocellulose filters (0.45 grn pore Ammonium Sulfate Fractionation
size), and washed twice with 5 ml 5% trichloroacetic
acid. The filters were dried and radioactivity was The crude extract was adjusted to 40%
determined by liquid scintillation in 0.5% PPO, 0.05% saturation by addition of solid ammonium
POPOP-toluene using an ambient temperature sulfate with stirring. The resulting precipi-
counter (Beckman LS-10X). tate was removed by centrifugation in an
PAP assa.v. Fractions containing PAP were tested 872 rotor (IEC) in an IEC model B-20
for inhibition of polyphenylalanine synthesis by add- refrigerated centrifuge for 10 min at 9OOOg.
ing various amounts of the fractions diluted with
The brownish-yellow supernatant was re-
water (1/103-1/104) as the first component of the
reaction mixtures. Varying amounts of the diluted
moved and adjusted to 100% saturation by
fractions were added to determine the amount re- addition of solid ammonium sulfate with
quired for 50% inhibition of polyphenylalanine syn- stirring. The solution was centrifuged as
thesis which is defined in this paper as one PAP before,- the precipitate was dissolved in a
activity unit. minimum volume of Solution A and then
dialyzed against 2 liters of Solution A 12-14
RESULTS h with one change of buffer. This fraction
Outline of PAP Purification yields a volume of approximately 160 ml
and may be stored frozen at -90°C with no
The previously reported purification of
PAP by Wyatt and Shepherd (1) utilized appreciable loss of activity being observed
ethanol precipitation and stepwise elution for at least 4 months.
from a cation exchange column. In this
laboratory it was found that the large DEAE-Cellulose Chromatography
volumes resulting from ethanol precipita- The dialyzed 40-100% ammonium sul-
tion were inconvenient to process and that fate fraction was applied to a 2.5 x 61 cm
step elution from various cation exchangers DEAE-cellulose column equilibrated with
did not produce a homogeneous prepara- Solution A and nonabsorbed protein was
tion. Therefore a procedure was devised to eluted with the same buffer. Five-minute
circumvent these problems. The previous fractions, containing approximately 22 ml
procedure employed the assay of inhibition each, were collected and the elution of
of viral replication in uiuo. In this proce- protein was monitored at 280 nm. A broad
dure PAP activity was determined by its peak of protein was eluted from the column
ability to inhibit in vitro polyphenylala- which contained all the PAP activity in its
524 JAMES D. IRVIN

front two-thirds (data not shown). This General Remarks

fraction of 350 ml was concentrated tenfold
The purification of PAP is summarized
by ultrafiltration over a PM-10 filter in an
in Table I. As shown PAP was purified over
Amicon model 402 ultrafiltration cell.
150-fold from the water extract with 25?
Phosphocellulose Chromotography yield. The major loss of activity occurred in
The DEAE-cellulose fraction was ad- the ammonium sulfate fractionation step.
justed to 0.02 M potassium phosphate, pH The yield in this step has at times been as
6.0, by addition of the appropriate volume high as 80%, but more often it has been
of 1 M potassium phosphate, pH 6.0, and near the reported value. Similar recoveries
applied to a 2.0 x 36 cm column of were also observed with ethanol precipita-
cellulose phosphate previously equilibrated tion at this stage of purification. It is
with 0.02 M potassium phosphate, pH 6.0, possible that this may be due to incom-
5 mM P-mercaptoethanol. The column was plete precipitation of PAP or that the
washed with 200 ml of the same buffer, inhibitory activity in the crude extract is
then protein was eluted with a 600 ml linear due to other components (i.e., ribonu-
gradient from 0 to 0.5 M KC1 in the phos- cleases) besides PAP which are removed in
phate buffer. Fractions of 10 ml were col- this step.
lected every 20 min and analyzed for ab-
sorbance at 280 nm and inhibition of poly- Physical Characterization
phenylalanine synthesis. The results are Purified PAP was tested for purity by gel
shown in Fig. 1. The majority of the PAP filtration on Sephadex G-100. As shown in
activity elutes as a sharp peak at 0.12 M Fig. 2A, the PAP preparation yields a
KC1 followed by a minor peak at 0.2 M KCl. symmetrical peak of protein upon gel fil-
The PAP fraction was taken as the three tration. The G-100 column uas previously
highest activity tubes (fractions 23-25) and calibrated with various protein standards
dialyzed versus 2 liters of water for three and a calibration curve was determined for
days with three changes of water. The light the column as shown in Fig. 2B. This
precipitate which appeared on dialysis was analysis shows PAP to have a molecular
removed by low speed centrifugation and weight of 26,500 in solution.
the solution was lyophilized to dryness. The The purified PAP was also analyzed for
prepared PAP was stored as a solid at purity and subunit composition by sodium
-20°C. dodecyl sulfate-polyacrylamide gel electro-

80 -

I I I
IO 30 40
FRA:;ION NUMBER

FIG. 1. Cellulose phosphate chromatography of the DEAE-cellulose fraction. The DEAE-cel-

lulose fraction was applied to a cellulose phosphate column, washed with buffer, and eluted with a
linear KC1 gradient as described in the text. Uninhibited polyphenylalanine synthesis corre-
sponded to 23 pmol of phenylalanine. Absorbance at 280 nm (-0-O-l. Inhibition of poly-
phenylalanine (-O-C-). Kc1 concentration (-----).
 PAP PURIFICATION AND PROPERTIES 525

phoresis. Gel electrophoresis of 25 kg of Both methods of analysis are in good

PAP in a sodium dodecyl sulfate gel results agreement that PAP is a single polypeptide
in one distinct and homogeneous band chain of an approximate molecular weight
stained with Coomassie blue as shown in of 27,000.
the inset of Fig. 3. Comparison of the
mobility of 10 pg PAP versus various pro- Effect of PAP on
tein standards in sodium dodecyl sulfate Polyphenvlalanine Synthesis
gels is shown graphically in Fig. 3. Interpo- The effect of PAP on polyphenylalanine
lation of the PAP mobility on the calibra- synthesis on A. salina ribosomes was tested
tion line yields a molecular weight of and is presented in Fig. 4. In this experi-
27,000. The same results were obtained ment the indicated amounts of PAP based
after pretreatment of PAP with N-ethyl- on the determined molecular weight of
maleimide or upon electrophoresis of PAP 27,000 were preincubated with A. salina
in sodium dodecyl sulfate gels containing 8 ribosomes for 10 min at 37°C under the
M urea (data not shown). same ionic conditions used for poly-
phenylalanine synthesis. The remaining
TABLE I components were added and polymeriza-
PAP PURIFICATION tion assayed for as indicated in the
Fraction Protein” Total Speck % Methods. As previously reported for poly-
(mg) units* fit Yield phenylalanine synthesis on reticulocyte (4)
x lo-’ activ- and wheat embryo (5) ribosomes, PAP
ity’
x10-3 causes efficient inhibition at extremely low
concentrations. As shown, polyphenylala-
Extract 36,800 4.87 1.8 - nine synthesis on A. salina ribosomes can
Ammonium sulfate 3,840 2.52 6.6 52 be inhibited 98% whereas the maximum
DEAE-cellulose 118 2.00 180 41 inhibitions previously reported for reticulo-
Phosphocellulose 45.Bd 1.27 278 25 cyte and wheat embryo ribosomes were 75
n Determined by A280-AZB0 absorbancies. (4) and 60% (5), respectively. Also in the A.
b An activity unit is defined as 50% inhibition of salina system there is little difference be-
polyphenylalanine synthesis. tween the extent of inhibition of poly-
c Activity units/mg. phenylalanine synthesis on ribosomes pre-
d By weight. incubated and not preincubated with PAP.

FRACTION NUMBER

FIG. 2. Sephadex G-100 gel filtration of purified PAP. A. Purified PAP, 2.5 mg in 2.0 ml
column buffer was applied to a 2.5 x 55 cm column equilibrated with 50 mM Tris-HCI, pH 7.5, 100
mM KCl. Fractions containing 2.2 ml were collected every 5 min and absorbance at 230 nm
determined (-O-O-). B. In the same manner the elution volumes of standard proteins bovine
serum albumin (68,000), ovalbumin (45,000), trypsin (24,000). myoglobin (17,200). and cyto-
chrome c (11,700) were determined and plotted versus the logarithms of their molecular weights
(-O-O-). The exclusion volume was determined to be 88 ml with blue dextran. From the elution
volume of PAP the molecular weight was estimated to be 26,500 from the calibration curve (-----).
526 JAMES D. IRVIN

effect previously observed with reticulocyte

ribosomes (4).
These assays contain 200 pg of washed A.
salina ribosomes which constitutes approx-
imately 45 pmol of ribosomes based on a
molecular weight of 4.5 x lo6 as deter-
mined for rat liver ribosomes (10). Thus
the ratios of PAP to ribosomes at 50% and
96% inhibition are l/150 and l/15, respec-
tively. Such inhibition stoichiometry
I
strongly supports the previous suggestions
t
.2 4 .6 6 IO that PAP acts in an enzymatic manner.
MOBILITY The effect of PAP on polyphenylalanine
FIG. 3. Sodium dodecyl sulfateepolyacrylamide
synthesis in a cell-free system from rabbit
gel electrophoresis of purified PAP. The mobility of reticulocytes was tested to compare the
PAP in sodium dodecyl sulfate gels was determined effect to the inhibition obtained in the A.
and compared to the standard proteins bovine serum salina system. As shown in Table II poly-
albumin (S&000), ovalbumin (45,000), glyceraldehyde phenylalanine synthesis is efficiently in-
3-phosphate dehydrogenase (35,500), trypsin (24,006), hibited by PAP but the reticulocyte system
and cytochrome c (11,700). The molecular weight of is not nearly as sensitive to PAP as the one
PAP determined from its mobility is 27,000 (---). The derived from A. salina. Comparison of the
inset shows a gel containing 25 Fg of purified PAP. data in Fig. 4 and Table II shows that
approximately ten times as much PAP is
required to produce the same extent of
inhibition of peptide synthesis with reticu-
locyte ribosomes versus that obtained with
A. salina ribosomes.
TABLE II
THE EFFECT OF PAP ON POLYPHENYLALANINE
SYNTHESIS ON RETICULOCYTE RIBOSOMES

PAP (pmol) Polyphenylalanine synthesis”

No preincubation Preincubatio+

pm01 % pm01 %
1. .a .03 3 10 10
- 34.7 100.0 24.2 100.0
PAP I pmol*sI
0.3 30.7 88.5 21.6 89.3
FIG. 4. Inhibition of polyphenylalanine synthesis 1.0 24.0 69.2 17.3 71.5
by PAP. A. salina ribosomes were preincubated in 3.0 15.4 44.4 10.0 41.3
one-half reaction volumes (0.25 ml) containing 20 mM 10.0 10.2 29.4 6.1 25.2
Tris-HCl, pH 7.5, 120 mM KCl, 4 mM MgCl,, and 2.5 30.0 6.5 18.7 4.0 16.5
mM D’IT with the indicated amounts of PAP for 10
min at 37°C prior to polyphenylalanine synthesis. The a Polyphenylalanine synthesis was performed as
previously described (4) in 0.5 ml reaction volumes
rate of polymerization was measured at 5 minutes
with uninhibited polyphenylalanine synthesis of 25.2 containing 0.25 mg of reticulocyte ribosomes washed
pmoles of phenylalanine. with deoxycholate followed by 0.5 M KC1 and 0.4 mg of
protein from the 40-70s ammonium sulfate fraction
of the postribosomal supernatant fluid both prepared
The only enhancement of inhibition by
according to the methods of Hardesty et al. (7).
preincubation of the ribosomes with PAP is Reactions were carried out for 5 min at 37°C and
observed at low concentrations of PAP in polyphenylalanine formation determined as described
the range 0.01-0.1 pmol PAP in 0.5 ml in Methods.
reaction volume. The extent of increased b Preincubation was carried out in one-half reac-
inhibition is only 10% (data not shown) in tion mixture volumes under the ionic conditions used
marked contrast to the large preincubation for synthesis for 10 min at 37°C.
 PAP PURIFICATION AND PROPERTIES 527

Table II also shows that there is no has a higher molecular weight lends greater
significant enhancement of the inhibitory support to previous results which suggest
effect of PAP upon preincubation of the an enzymatic modification of the ribosome.
inhibitor with the reticulocyte ribosomes For example, it was previously reported (4)
prior to polyphenylalanine synthesis. This that globin synthesis was maximally inhib-
is in marked contrast to the results previ- ited by a PAP/ribosome ratio of one to ten
ously obtained (4) but consistent with the based on the molecular weight of PAP
observation in this study that preincuba- being 13,000; on a molecular weight of
tion of PAP with A. salina ribosomes pro- 27,000 the ratio of PAP to ribosomes be-
duces no significant increase in inhibition. comes less than one to twenty. In the A.
It is of interest to note that on comparison salina system maximum inhibition of poly-
of the data obtained in Table II with the phenylalanine synthesis occurs at a PAP/
previously reported effect of PAP on poly- ribosome ratio of one to fifteen. In the other
phenylalanine synthesis on reticulocyte ri- systems studied (4, 5) polyphenylalanine
bosomes (4), the PAP used in this study is synthesis was shown to be less sensitive to
approximately ten times as effective in the PAP inhibition than synthesis directed by
absence of preincubation. Comparison of natural messengers. This suggests that
the extent inhibition obtained after prein- PAP may be more effective inhibiting nat-
cubation in the previous study (4) to the ural messenger directed protein synthesis
inhibition reported here shows a similar on A. salina ribosomes. Such a stoichiome-
response to PAP in inhibiting poly- try of inhibition lends strong credence to
phenylalanine synthesis, correcting the the possibility of an enzymatic inactivation
previously reported amounts of PAP to the of the ribosome in lieu of direct demonstra-
molecular weight of 27,000 determined in tion of a ribosomal modification. One piece
this study. of indirect evidence to support the concept
The antiviral activity of the purified of PAP acting enzymatically is the stoichi-
PAP was tested on poliovirus replication in ometry in the in u&-o effect of colicin E3 on
HeLa cell cultures and was found to be a bacterial protein synthesis. Colicin E3 has
potent inhibitor of virus replication as been demonstrated to inactivate bacterial
previously described (3). Preparations of ribosomes enzymatically by cleaving a
PAP used in this study were found to be fragment from the 3’ end of 1% RNA (11.
approximately ten times as effective for 12). The inhibition of f2 RNA directed
inhibition of viral replication as the PAP protein synthesis on E. coli ribosomes by
preparations previously tested (Boyd colicin E3 follows the same general concen-
Hardesty and Michael Ussery, personal tration effect as does the action of PAP. It
communication). has been shown that 0.02 pg of’ added
colicin E3 results in 50%~ inhibition of
DISCUSSION protein synthesis whereas maximal inhibi-
tion (99%) requires 0.2 pugof colicin E3 (13).
A convenient method for the isolation of This type of inhibition response is very
homogeneous PAP has been reported and similar to the response produced by PAP in
its molecular weight has been demon- that 10 times as much PAP is required to
strated by two methods to be approxi- produce maximal inhibition compared to
mately 27,000, slightly double the previous 50% inhibition.
reported minimum molecular weight of The inhibition of polyphenylalanine syn-
13,000 (1). The previously reported molec- thesis on A. salina ribosomes by PAP has
ular weight of PAP was calculated from the been repeatedly observed to be biphasic in
amino acid composition based on the as- nature as shown in Fig. 4; the slope of
sumption that PAP contained only one inhibition being less at lower amounts of
histidine residue (1). The results reported added PAP than that observed at higher
here are consistent with about double that concentrations. Possibly such a response
value, thus indicating that the previous could be caused by partial inactivation of
assumption of one histidine residue per PAP in the more dilute solutions or some
molecule is incorrect. The fact that PAP concentration dependent interaction of
528 JAMES D. IRVIN

PAP with the ribosomes limiting the turn- site of such a PAP induced modification
over of PAP with the ribosomes. may lead to the elucidiation of the struc-
The observed difference in susceptibility tural nature of the site(s) of elongation
to PAP inhibition of polyphenylalanine factor interaction with the ribosome.
synthesis on A. salina and reticulocyte
ribosomes may reflect differences in the ACKNOWLEDGMENTS
ribosomes themselves or the ribosomal in- The author is indebted to Dr. Boyd Hardesty and
teractions with their respective elongation Dr. J. M. Cimadevilla for helpful discussions and
factors. The determination of the nature of suggestions. The author is grateful to Miss Shelley
these differences may provide relevant in- Key for her excellent technical assistance and to Mrs.
formation on the action of PAP and are Sandra Posey for her help in preparing the typescript.
currently under investigation in this labo- This work was supported in part by grants from the
ratory. This observed difference in inhibi- Research Corporation and for organized research from
the State of Texas.
tion by PAP cannot be attributed to the
larger amount of reticulocyte ribosomes REFERENCES
(0.25 mg) used for polyphenylalanine syn-
1. Wu~rr, S., AND SHEPHERD, R. (1969) Phytopa-
thesis. This constitutes only 20% more
tho1og.v 59, 178771794.
ribosomes than used in the A. salina sys-
2. TOMLINSON, J. A., WALKER, V. M., FLEWETT, T.
tem, not nearly enough to account for the H., ANDBARCLAY, G. R. (1974) J. Gen. Viral. 22,
observed tenfold differential in inhibition 225-232.
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an enzymatic effect on the eukaryotic ribo- (1973) Arch. Biochem. Riophys. 155, 278-289.
some but merely suggest the possibility of 5. OWENS, R. A., BRUENING, G., AND SHEPHERD, R. J.
(1973) Virology 56, 390-393.
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6. ZASLOFF, M., AND OCHOA. S. (1971) Proc. Nat.
of these studies may be the existence of
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12. BOWMAN, C. M., DAHLBERG, J. E., IKEMURA, T.,
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KONISK~, J., AND NOMURA. M. (1971) Proc. Nat.
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