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Effects of intercalating agents on the structure of the ribosome
Vol. 65, No. 2, 1 9 7 5
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
EFFECTS OF INTERCALATING AGENTS ON THE STRUCTURE OF THE RIBOSOME T. Suryanarayana*
and D. P. Burma
M o l e c u l a r Biology Unit, Department of Biochemistry, Institute of Medical Sciences, Banaras Hindu University Varanasi 221005, U.P., India Received
June 3,1975
SUMMARY R i b o s o m e s t r e a t e d w i t h an i n t e r c a l a t i n g dye (ethidium bromide or acridine orange) are degraded m o r e r a p i d l y b y RNase I t h a n u n t r e a t e d ribosomes. Spermidine or spermine counteracts to great extent the effect produced by the intercalating dye. It appears that the intercalation leads to the 'loosening' of the structure of the ribosome which is also reflected in the decreased inhibitory capacity of the treated r i b o s o m e s t o w a r d s RNase I . Intercalation appears to decrease the affinity o f rRNA towards proteins causing the structure of the r i b o s o m e t o become u n f o l d e d .
INTRODUCTION The intercalating
dyes like ethldium bromide and
acridine orange have been extensively used to study the structure
of polynueleotides
to RNA in yeast ribosomes
(1,2).
Acridine
(3); ethldlum bromide binds to
free rRNA as well as to rRNA in ribosomes laboratory with
the mechanism of artificial
30S s u b u n i t
(5)
is being
Because the hydrolytic
attack
d e p e n d s on t h e c o n f o r m a t i o n tion
can be utilised
ribosomes.
to
(4).
investigated
of the the
In this
association in detail
o f RNase I on t h e
study
In the present
orange binds
ribosome structural
communication the
o f RNase I (6).
ribosome
(7) this
associa-
topography effects
of
* Senior Research Fellow under a project financed by the Indian Council of Medical Research.
Copyright © 1975 by Academic Press, Inc. All rights o f reproduction in any form reserved.
708
of
VoI. 65, No. 2, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Intercalating monitored
agents
on t h e
b y RNase I w i l l
structure
of the
ribosomes
as
be described.
MATERIALS AND METHODS P o l y ( A ) was t h e p r o d u c t o f M i l e s L a b o r a t o r i e s , U.S.A. S p e r m i d i n e , s p e r m i n e , b o v i n e s e r u m a l b u m i n and e t h i d i u m bromide were purchased from Sigma Chemical Co., U.S.A. Acridine o r a n g e was t h e p r o d u c t o f B r i t i s h D r u g H o u s e , E n g l a n d . E . e o l i r i b o s o m e s a n d S . t y p h i m u r i u m RNase I w e r e prepared a~ribed earlier (~). Ribosomes were treated with ethidium bromide or acridine orange according to the m e t h o d o f S t e v e n s and P a s c o e ( 4 ) . The degradation of ethidium bromide-or acridine orange-treated and untreated r i b o s o m e s b y RNase I was f o l l o w e d i n Z e i s s PMQ I I s p e c t r o p h o t o m e t e r . The r e a c t i o n was c a r r i e d o u t i n t h e c u v e t t e a t 25°C i n a t o t a l v o l u m e o f 1 ml c o n t a i n i n g 0 . 1 M T r i s - H C 1 pH 7 , 1 A 2 6 0 . u n i t o f t r e a t e d or untreated r i b o s o m e , 4 u n i t s o f RNase I t 6 ) and d e s i r e d a m o u n t o f Mg ++. A b l a n k m i x t u r e w i t h o u t t h e e n z y m e was u s e d t o a d j u s t t h e initial absorhancy of the reaction mixture to zero. The i n h i b i t i o n o f RNase I - e a t a l y s e d hydrolysis of poly(A) by treated and u n t r e a t e d r i b o s o m e s was m e a s u r e d a c c o r d i n g t o t h e m e t h o d d e s c r i b e d b y D a t t a and Burma ( 6 ) . RESULTS AND DISCUSSION Degradation . of ethidium ribosomes
b~ RNase I :
produce a large (Fig.lA).
bromide-and
amount of distortion
are completely
protected
in the
untreated
ribosomes
Similar trations.
degraded
differences Even a t
treated
ribosomes
Similar
results
orange
(Fig.lB).
at
ribosomal
against
treated ribosomes are degraded at ++ When Mg concentration is lowered to are
seems t o structure
o f 1 . 3 5 mM t h e u n t r e a t e d
the
ribosomes
orange-treated
Ethtdtum bromide treatment
At a Mg ++ c o n c e n t r a t i o n
ribosomes
acridine
a much f a s t e r
RNase I ,
whereas
a reasonably
fast
0 . 3 5 mM t h e
treated
rate
whereas
rate.
the
are degraded
s l o w l y and i n c o m p l e t e l y . ++ a r e a l s o o b s e r v e d a t o t h e r Mg concert÷÷ a v e r y h i g h Mg concentration (5 mM) t h e
are not completely
were
obtained
These
results
protected
following suggest
709
against
treatment that
with
RNase I . acridine
the distortion
Vol. 65, No. 2, 1975
I
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
|
I
|
I0
15
A ]
I
I
I
5
I0
15
50
O 40
~A
_Z 30
~ zo
io
5
20
20
MINUTES
Fig.l.Degradation,,of ethidium bromide (A) and acridine orange (B) treated ,rlbosomes by RNase I at different Mg ++ concentrations. The treatment of ribosomes with ethidlum bromide an~cridine o r a n g e and t h e a s s a y w e r e c a r r i e d o u t a s described under 'Materials and M e t h o d s ' Closed symbols and o p e n s y m b o l s r e p r e s e n t u n t r e a t e d a,nd t r e a t e d r i b o s o m e s respectively. Mg ++ c o n c e n t r a t i o n s (mM) a r e A . e O 0 . 3 5 ! A A 0.7|m ~1,355[]~2.35|m ® 5.0 B.e o 0.355A/k 0.7~Br~l.0; m~l.35;B ~ 2.35.
produced by the the
overall
the
attack
structure
of polycationtc
of the
degradation amines:
on t h e
leads
to the unfolding
r i b o s o m e and m a k e s i t
of
amenable to
It
is well
spermidine
and s p e r m i n e
of treate d ribosome in presence
Polycationtc
structure
(4,7-10).
amines have profound
and f u n c t i o n
known t h a t cause the
ribosome to become tightened Spermidine
agent
o f RNase I .
RNase I c a t a l y s e d
influence
intercalating
of the
polycationic overall
ribosome amines such as
structure
of the
and t h u s mimick t h e e f f e c t
o r s p e r m i n e was f o u n d t o d e c r e a s e
710
the
rates
o f Mg of
++
Vol. 65, No. 2, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
degradation of both ethidium bromide-treated and untreated ribosomes (Flg.2).
Similar results were obtained with
ribosomes treated with acridine orange (data not shown). the
presence
produced
in the
Inhibition a~ents:
of a polycationic ribosome
agent
by the
ability
of
a polynucleottde
as catalysed
is
dependent
concentration
capacity
on t h e
which
is
dependent
T - - T - -
the distortion
intercalating
of RNase I by ribosomes
Ribosomes have the
reverses
treated to
agent. with
inhibit
the
intercalatin~
the hydrolysis
by RNase I and this o f Mg + + ( 6 ) .
on t h e
overall
A
Thus
inhibition
This
inhibitory
structure
of the
I
T
f
5
I0
15
B
50 o
"
_z ,,<, :50 Z
20
-
I0
5
I0
15
MINUTES
Fig.2.De~radation .of e t h i d i u m b r o m i d e t r e a t e d ribosomes In p r e s e n c e o f s p e r m i d i n e (A) a n d s p e r m t n e ( B ) . The assay was c a r r i e d out as described under tMaterials and Methods I excepting that indicated amount of spermtdine or spermine was p r e s e n t w h e r e m e n t i o n e d . The concentration o f Mg ++ during assay was invariably 0 . 3 5 raM. C l o s e d s y m b o l s a n d open symbols represent untreated and treated ribosomes respectively. Spermidine concentrations (raM) a r e A.eOntl~A/\0.05~ID 0.251U ~ 0.5. B.• 0 nil|A/\0.01! ID0.05! !1~ 0.125.
711
Vol. 65, No. 2, 1 9 7 5
ribosome,
is
also
intercalating tion
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
profoundly
dyes
(Fig.3).
o f 0 . 8 mM t h e
ribosomes
are
inhibition, observed
untreated
ribosomes
inhibition
ribosomes
with
0.02 I I
0
0
0.04 I
the
orange-treated considerable
of ribosomes, ++ A t 5 mM Mg the
capacities
produced by the
treated
acridine
ribosomes.
becomes smaller
carried
the
concentration
inhibitory
Studies
with
a t a Mg ++ c o n c e n t r a -
o f RNase I w h e r e a s
on t h e
the untreated
in the
less
bromide-or
poor inhibitors
difference
by treatment
For example,
ethidium
dependent with
affected
out
of the
hut
treated
in this
thiol
there
treated is
is
and
definitely
ribosomes. laboratory
reacting
with
reagents
the
like
N-ethyl-
AMOUNT OF RIBOSOME(A26O) .-o-. 0"02 0.04 0.06 0.06 I AI
0"06
0,08
o;2
003
oo4
~
0"01
0'02
0-03 0"04 AMOUNT OF RIBOSOME(Az6O)
Ftg.3.Inhtbitton of RNase I by ribosomes treated with ethtdium b r o m i d e (A) a n d a 0 r i d i u . e o r a n g e ( B ) . The treatment of ribosomes with ethldium bromide and acridine orange and Inhibition assay have been described in the text. Closed s y m b o l s and o p e n s y m b o l s r e p r e s e n t untreated and treated rlbosome respectively.@ O a s s a y e d a t 0 . 8 mM Mg ++ A~assayed a t 5 . 0 mM Mg ÷+.
712
Vol. 65, No. 2, 1 9 7 5
maleimide,
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
parachloromercuribenzoate,
benzoic acid),
indicate
the ribosome.
Apparently the distortion
structure
of either
similar
5,5t-dithiobis(2-nitro-
unfolding
rRNAs o r p r o t e i n s
and t h u s t h e
of the ribosome. monitoring
~loosening'
in t h e r i b o s o m e
between nucleic
of the overall
RNase I a p p e a r s t o be a u s e f u l
such c o n f o r m a t i o n a l
of
produced in the
present
leads to the weakening of the interaction and p r o t e i n s
of the structure
acids
structure
tool for
change.
REFERENCES /l/
(3)
Waring, J . , N a t u r e , 219, 1 3 2 0 ( 1 9 6 8 ) . Carlberg-Bacq~C.M., in Fundamentals of Biochemical Pharmacology (Z.M.Bacq,R.Capek,R.Paoletti, and J . R e n s o n , E d s . ) Pergamon P r e s s , p . 4 7 6 ( 1 9 7 1 ) . M o r g a n , R . S . , and D . G . R h o a d s , B i o c h i m . B i o p h y s . A c t a , 102, 311 ( 1 9 6 5 ) . Neu,H.C. and L . A . H e p p e l , ~ , 1267 ( 1 9 6 4 ) .
Proc.Nat.Acad:Sci.,
U.S.A.,
Suryanarayana,T., Ph.D.Dissertation submit~e'd t o B a n a r a s Hindu U n i v e r s i t y , V a r a n a s i , I n d i a ( 1 9 7 5 ) . (8) B r e t t h a u e r , R . K . , L.Marcus,J.Chaloupka,H.0:IIalvorson, and R.M.Bock, B i o c h e m i s t r y , 2, 1079 ( 1 9 6 3 ) . 9) T a k e d a , Y . , B i o e h i m . B i o l D h y s . A c t a , 182, 2 5 ~ ( 1 9 6 9 ) . 10) S p i r i n , A . S . , in ' M o l e c u l a r mechanism o f a n t i b i o t i c a c t i o n on p r o t e i n s y n t h e s i s and membranes' (E.Munoz, F.Garcia-Ferrandiz, and D . ~ a z q u e z , Eds. ) E l s e v i e r Scientific P u b l i s h i n g C o . , Amsterdam, p . l l ( 1 9 7 2 ) .
713
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
EFFECTS OF INTERCALATING AGENTS ON THE STRUCTURE OF THE RIBOSOME T. Suryanarayana*
and D. P. Burma
M o l e c u l a r Biology Unit, Department of Biochemistry, Institute of Medical Sciences, Banaras Hindu University Varanasi 221005, U.P., India Received
June 3,1975
SUMMARY R i b o s o m e s t r e a t e d w i t h an i n t e r c a l a t i n g dye (ethidium bromide or acridine orange) are degraded m o r e r a p i d l y b y RNase I t h a n u n t r e a t e d ribosomes. Spermidine or spermine counteracts to great extent the effect produced by the intercalating dye. It appears that the intercalation leads to the 'loosening' of the structure of the ribosome which is also reflected in the decreased inhibitory capacity of the treated r i b o s o m e s t o w a r d s RNase I . Intercalation appears to decrease the affinity o f rRNA towards proteins causing the structure of the r i b o s o m e t o become u n f o l d e d .
INTRODUCTION The intercalating
dyes like ethldium bromide and
acridine orange have been extensively used to study the structure
of polynueleotides
to RNA in yeast ribosomes
(1,2).
Acridine
(3); ethldlum bromide binds to
free rRNA as well as to rRNA in ribosomes laboratory with
the mechanism of artificial
30S s u b u n i t
(5)
is being
Because the hydrolytic
attack
d e p e n d s on t h e c o n f o r m a t i o n tion
can be utilised
ribosomes.
to
(4).
investigated
of the the
In this
association in detail
o f RNase I on t h e
study
In the present
orange binds
ribosome structural
communication the
o f RNase I (6).
ribosome
(7) this
associa-
topography effects
of
* Senior Research Fellow under a project financed by the Indian Council of Medical Research.
Copyright © 1975 by Academic Press, Inc. All rights o f reproduction in any form reserved.
708
of
VoI. 65, No. 2, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Intercalating monitored
agents
on t h e
b y RNase I w i l l
structure
of the
ribosomes
as
be described.
MATERIALS AND METHODS P o l y ( A ) was t h e p r o d u c t o f M i l e s L a b o r a t o r i e s , U.S.A. S p e r m i d i n e , s p e r m i n e , b o v i n e s e r u m a l b u m i n and e t h i d i u m bromide were purchased from Sigma Chemical Co., U.S.A. Acridine o r a n g e was t h e p r o d u c t o f B r i t i s h D r u g H o u s e , E n g l a n d . E . e o l i r i b o s o m e s a n d S . t y p h i m u r i u m RNase I w e r e prepared a~ribed earlier (~). Ribosomes were treated with ethidium bromide or acridine orange according to the m e t h o d o f S t e v e n s and P a s c o e ( 4 ) . The degradation of ethidium bromide-or acridine orange-treated and untreated r i b o s o m e s b y RNase I was f o l l o w e d i n Z e i s s PMQ I I s p e c t r o p h o t o m e t e r . The r e a c t i o n was c a r r i e d o u t i n t h e c u v e t t e a t 25°C i n a t o t a l v o l u m e o f 1 ml c o n t a i n i n g 0 . 1 M T r i s - H C 1 pH 7 , 1 A 2 6 0 . u n i t o f t r e a t e d or untreated r i b o s o m e , 4 u n i t s o f RNase I t 6 ) and d e s i r e d a m o u n t o f Mg ++. A b l a n k m i x t u r e w i t h o u t t h e e n z y m e was u s e d t o a d j u s t t h e initial absorhancy of the reaction mixture to zero. The i n h i b i t i o n o f RNase I - e a t a l y s e d hydrolysis of poly(A) by treated and u n t r e a t e d r i b o s o m e s was m e a s u r e d a c c o r d i n g t o t h e m e t h o d d e s c r i b e d b y D a t t a and Burma ( 6 ) . RESULTS AND DISCUSSION Degradation . of ethidium ribosomes
b~ RNase I :
produce a large (Fig.lA).
bromide-and
amount of distortion
are completely
protected
in the
untreated
ribosomes
Similar trations.
degraded
differences Even a t
treated
ribosomes
Similar
results
orange
(Fig.lB).
at
ribosomal
against
treated ribosomes are degraded at ++ When Mg concentration is lowered to are
seems t o structure
o f 1 . 3 5 mM t h e u n t r e a t e d
the
ribosomes
orange-treated
Ethtdtum bromide treatment
At a Mg ++ c o n c e n t r a t i o n
ribosomes
acridine
a much f a s t e r
RNase I ,
whereas
a reasonably
fast
0 . 3 5 mM t h e
treated
rate
whereas
rate.
the
are degraded
s l o w l y and i n c o m p l e t e l y . ++ a r e a l s o o b s e r v e d a t o t h e r Mg concert÷÷ a v e r y h i g h Mg concentration (5 mM) t h e
are not completely
were
obtained
These
results
protected
following suggest
709
against
treatment that
with
RNase I . acridine
the distortion
Vol. 65, No. 2, 1975
I
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
|
I
|
I0
15
A ]
I
I
I
5
I0
15
50
O 40
~A
_Z 30
~ zo
io
5
20
20
MINUTES
Fig.l.Degradation,,of ethidium bromide (A) and acridine orange (B) treated ,rlbosomes by RNase I at different Mg ++ concentrations. The treatment of ribosomes with ethidlum bromide an~cridine o r a n g e and t h e a s s a y w e r e c a r r i e d o u t a s described under 'Materials and M e t h o d s ' Closed symbols and o p e n s y m b o l s r e p r e s e n t u n t r e a t e d a,nd t r e a t e d r i b o s o m e s respectively. Mg ++ c o n c e n t r a t i o n s (mM) a r e A . e O 0 . 3 5 ! A A 0.7|m ~1,355[]~2.35|m ® 5.0 B.e o 0.355A/k 0.7~Br~l.0; m~l.35;B ~ 2.35.
produced by the the
overall
the
attack
structure
of polycationtc
of the
degradation amines:
on t h e
leads
to the unfolding
r i b o s o m e and m a k e s i t
of
amenable to
It
is well
spermidine
and s p e r m i n e
of treate d ribosome in presence
Polycationtc
structure
(4,7-10).
amines have profound
and f u n c t i o n
known t h a t cause the
ribosome to become tightened Spermidine
agent
o f RNase I .
RNase I c a t a l y s e d
influence
intercalating
of the
polycationic overall
ribosome amines such as
structure
of the
and t h u s mimick t h e e f f e c t
o r s p e r m i n e was f o u n d t o d e c r e a s e
710
the
rates
o f Mg of
++
Vol. 65, No. 2, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
degradation of both ethidium bromide-treated and untreated ribosomes (Flg.2).
Similar results were obtained with
ribosomes treated with acridine orange (data not shown). the
presence
produced
in the
Inhibition a~ents:
of a polycationic ribosome
agent
by the
ability
of
a polynucleottde
as catalysed
is
dependent
concentration
capacity
on t h e
which
is
dependent
T - - T - -
the distortion
intercalating
of RNase I by ribosomes
Ribosomes have the
reverses
treated to
agent. with
inhibit
the
intercalatin~
the hydrolysis
by RNase I and this o f Mg + + ( 6 ) .
on t h e
overall
A
Thus
inhibition
This
inhibitory
structure
of the
I
T
f
5
I0
15
B
50 o
"
_z ,,<, :50 Z
20
-
I0
5
I0
15
MINUTES
Fig.2.De~radation .of e t h i d i u m b r o m i d e t r e a t e d ribosomes In p r e s e n c e o f s p e r m i d i n e (A) a n d s p e r m t n e ( B ) . The assay was c a r r i e d out as described under tMaterials and Methods I excepting that indicated amount of spermtdine or spermine was p r e s e n t w h e r e m e n t i o n e d . The concentration o f Mg ++ during assay was invariably 0 . 3 5 raM. C l o s e d s y m b o l s a n d open symbols represent untreated and treated ribosomes respectively. Spermidine concentrations (raM) a r e A.eOntl~A/\0.05~ID 0.251U ~ 0.5. B.• 0 nil|A/\0.01! ID0.05! !1~ 0.125.
711
Vol. 65, No. 2, 1 9 7 5
ribosome,
is
also
intercalating tion
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
profoundly
dyes
(Fig.3).
o f 0 . 8 mM t h e
ribosomes
are
inhibition, observed
untreated
ribosomes
inhibition
ribosomes
with
0.02 I I
0
0
0.04 I
the
orange-treated considerable
of ribosomes, ++ A t 5 mM Mg the
capacities
produced by the
treated
acridine
ribosomes.
becomes smaller
carried
the
concentration
inhibitory
Studies
with
a t a Mg ++ c o n c e n t r a -
o f RNase I w h e r e a s
on t h e
the untreated
in the
less
bromide-or
poor inhibitors
difference
by treatment
For example,
ethidium
dependent with
affected
out
of the
hut
treated
in this
thiol
there
treated is
is
and
definitely
ribosomes. laboratory
reacting
with
reagents
the
like
N-ethyl-
AMOUNT OF RIBOSOME(A26O) .-o-. 0"02 0.04 0.06 0.06 I AI
0"06
0,08
o;2
003
oo4
~
0"01
0'02
0-03 0"04 AMOUNT OF RIBOSOME(Az6O)
Ftg.3.Inhtbitton of RNase I by ribosomes treated with ethtdium b r o m i d e (A) a n d a 0 r i d i u . e o r a n g e ( B ) . The treatment of ribosomes with ethldium bromide and acridine orange and Inhibition assay have been described in the text. Closed s y m b o l s and o p e n s y m b o l s r e p r e s e n t untreated and treated rlbosome respectively.@ O a s s a y e d a t 0 . 8 mM Mg ++ A~assayed a t 5 . 0 mM Mg ÷+.
712
Vol. 65, No. 2, 1 9 7 5
maleimide,
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
parachloromercuribenzoate,
benzoic acid),
indicate
the ribosome.
Apparently the distortion
structure
of either
similar
5,5t-dithiobis(2-nitro-
unfolding
rRNAs o r p r o t e i n s
and t h u s t h e
of the ribosome. monitoring
~loosening'
in t h e r i b o s o m e
between nucleic
of the overall
RNase I a p p e a r s t o be a u s e f u l
such c o n f o r m a t i o n a l
of
produced in the
present
leads to the weakening of the interaction and p r o t e i n s
of the structure
acids
structure
tool for
change.
REFERENCES /l/
(3)
Waring, J . , N a t u r e , 219, 1 3 2 0 ( 1 9 6 8 ) . Carlberg-Bacq~C.M., in Fundamentals of Biochemical Pharmacology (Z.M.Bacq,R.Capek,R.Paoletti, and J . R e n s o n , E d s . ) Pergamon P r e s s , p . 4 7 6 ( 1 9 7 1 ) . M o r g a n , R . S . , and D . G . R h o a d s , B i o c h i m . B i o p h y s . A c t a , 102, 311 ( 1 9 6 5 ) . Neu,H.C. and L . A . H e p p e l , ~ , 1267 ( 1 9 6 4 ) .
Proc.Nat.Acad:Sci.,
U.S.A.,
Suryanarayana,T., Ph.D.Dissertation submit~e'd t o B a n a r a s Hindu U n i v e r s i t y , V a r a n a s i , I n d i a ( 1 9 7 5 ) . (8) B r e t t h a u e r , R . K . , L.Marcus,J.Chaloupka,H.0:IIalvorson, and R.M.Bock, B i o c h e m i s t r y , 2, 1079 ( 1 9 6 3 ) . 9) T a k e d a , Y . , B i o e h i m . B i o l D h y s . A c t a , 182, 2 5 ~ ( 1 9 6 9 ) . 10) S p i r i n , A . S . , in ' M o l e c u l a r mechanism o f a n t i b i o t i c a c t i o n on p r o t e i n s y n t h e s i s and membranes' (E.Munoz, F.Garcia-Ferrandiz, and D . ~ a z q u e z , Eds. ) E l s e v i e r Scientific P u b l i s h i n g C o . , Amsterdam, p . l l ( 1 9 7 2 ) .
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