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Ribosomal proteins
BIOCHIMIE, 1972, 54, 167-175.
Ribosomal proteins. X X X I I : Comparison of several extraction methods for proteins from Escherichia colt ribosomes. E. KALTSCHMIDT (*) a n d H. G. WITTMANN. Extraction methods [or ribosomal proteins. Max-Planck-lnstitut [iir Molekulare Genetik, Berlin-Dahlem, Germany. (7/2/1972).
Summary. - - The following methods were used for the extraction of proteins from E. colt ribosomes.: LiCl-urea, RNase, phenol or acetic acid in presence of mono- and bivalent cations (Li +, Na ÷, K÷, Mg++, Ca ++, Mn+*). After separation in a two-dimensional polyacrylamide gel eleetrophoresis the protein patterns were compared. By treatlnent of ribosomes with LiCl-urea, with RNase or with acetic acid at high salt concentration the highest number of proteins were extracted. Treatment with phenol or acetic acid without addition of salts extracted only a portion of the ribosomal proteins. The rest can be obtained by repeating the extraction with acetic acid at high salt concentration. This offers the possibility to prefraetionate ribosomal proteins by simple partial extraction with acetic acid.
In o r d e r to study the s t r u c t u r e and f u n c t i o n of r i b o s o m a l p r o t e i n s it is n e c e s s a r y to isolate all of the r i b o s o m a l p r o t e i n c o m p o n e n t s by e x t r a c t i o n p r o c e d u r e s w h i c h are c o m p a t i b l e w i t h the requir e m e n t s of subsequent e x p e r i m e n t s . T w o - d i m e n sional a c r y l a m i d e gel e l e c t r o p h o r e s i s p r o v i d e s a c o n v e n i e n t m e t h o d for c o m p a r i s o n of the compositions of r i b o s o m a l p r o t e i n extracts [1, 2]. In this p a p e r w e c o m p a r e the c o m p o s i t i o n s of r i b o s o m a l p r o t e i n m i x t u r e s p r e p a r e d by the methods c o m m o n l y used for isolation of the p r o t e i n m o i e t y of n u c l e o p r o t e i n s : e x t r a c t i o n w i t h acetic acid in the p r e s e n c e of various salts [3-7], extraction w i t h Li,Cl-urea [8-111, digestion w i t h RNase and p h e n o l e x t r a c t i o n [121. It is s h o w n that all r i b o s o m a l p r o t e i n s are e x t r a c t e d by some of these methods whereas other methods yielded incomplete p r o t e i n mixtures. Partial e x t r a c t i o n of ribosomal p r o t e i n s can be useful as a first s e p a r a t i o n step in the p u r i f i c a t i o n of r i b o s o m a l proteins. MATERIALS AND METHODS.
Strains and ribosomes : Ribosomes are o b t a i n e d f r o m E. colt K12 (strain A19) and E. colt B g r o w n to 3/4 log phase. After the cells w e r e g r o u n d w i t h a l u m i n a p o w d e r (Alcoa 305) the r i b o s o m e s w e r e p r e p a r e d by differential c e n t r i f u g a t i o n in 0,02 M tris-HC1, pH 8.0, 0.01 M m a g n e s i u m acetate. After the s e c o n d h i g h s p e e d c e n t r i f u g a t i o n the r i b o s o m e s (') Present address : Institute for Enzyme Research, University of Wisconsin, Madison, Wisconsin, USA.
w e r e w a s h e d in 0.02 M tris-HC'l, 0.5 M NH~ C1, 0.001 M m a g n e s i u m acetate, pH 7.8, pelleted again, r e s u s p e n d e d and stored in 0.02 M tris-HC1, pH 7.8, 0.01 M m a g n e s i u m acetate. Some hatches of ribo. somes w e r e r e p e a t e d l y p r e c i p i t a t e d w i t h (NH4),., SO 4 before differential c e n t r i f u g a t i o n [13].
Extraction o[ proteins : Optimal e x t r a c l i o n of r i b o s o m a l p r o t e i n s w i t h acetic acid was o b t a i n e d by the f o l l o w i n g p r o c e d u r e : MgC12 was a d d e d to a r i b o s o m e suspension c o n t a i n i n g 15.0-300 OD26o/ ml to i n c r e a s e the c o n c e n t r a t i o n Of Mg ÷* to 0.2 Mr. T w o volumes of glacial acetic acid w e r e a d d e d to one v o l u m e of r i b o s o m e s u s p e n s i o n at 0°C w i t h vigorous stirring. This m i x t u r e was kept in ice for 40 m i n u t e s and stirred. The p r e c i p i t a t e d RNA was pelleted by c e n t r i f u g a t i o n at 30 000 g for 20 min. The acetic acid c o n c e n t r a t i o n "was reduced by stepwise dialysis against 5(I p. cent, 40 p. cent, 30 p. cent, 20 p. cent and 10 p. cenl acetic acid. The p r o t e i n s w e r e stored in 10 p. cenl acetic acid at - - 2 0 ° C . The acetic acid e x t r a c t i o n p r o c e d u r e was v a r i e d by the a d d i t i o n of v a r i o u s salts to the ribosome suspension, e.g. MnC12, CaC12, NaC1, KCI or LiCI. The r i b o s o m e s w e r e dialyzed at 4°C firsl against 0.005 M Na2EDTA to reluove b o u n d Mg +~, then against 0.02 M tris-C1, pH 7.8, and finally the desired salt was added. T h e pellet o b t a i n e d by acetic acid e x t r a c t i o n was r e s u s p e n d e d in the same buffer or in a buffer w i t h h i g h e r salt c o n c e n t r a t i o n and e x t r a c t e d for
E. K a l t s c h m i d t and H. G. W i t t m a n n .
168
a s e c o n d time. Difficulties in r e s u s p e n s i o n of the pellet w e r e o v e r c o m e by g r i n d i n g the frozen pellet in a motar.
LiCl-urea extraction of proteins w a s carried out by the f o l l o w i n g p r o c e d u r e : One v o l u m e of 4 M L i C 1 - 8 M urea solution w a s m i x e d w i t h one v o l u m e of r i b o s o m e s u s p e n s i o n c o n t a i n i n g 0.004 M Mg *+ and the resulting s u s p e n s i o n w a s stored 48 hours at 0°C. The R~NA precipitate w a s pelleted by centrifugation at 30 000 g for 20 rain. RNase digestion w a s carried out by the f o l l o w i n g p r o c e d u r e : The r i b o s o m e s u s p e n s i o n w a s d i a l y z e d against 0.0,06 M EDTA. F i v e ~g pancreatic RNase and 0.3 ~g (8,0 units) T l - R N a s e w e r e a d d e d to 0.1- 0.2 m l s u s p e n s i o n c o n t a i n i n g 100 OD26 o r i b o s o m e s and the resulting s u s p e n s i o n w a s i n c u b a t e d at 37°C for 1 hour. Phenol extraction w a s carried out by the f o l l o w i n g p r o c e d u r e : One v o l u m e p h e n o l w a s a d d e d to one v o l u m e r i b o s o m e s u s p e n s i o n containing
0.02 M tris-HC1, p H 7.8, 0.001 Mg ÷÷ and the resulting m i x t u r e w a s shaken for 10 rain at 4°C. The p h e n o l p h a s e w a s extracted three a d d i t i o n a l t i m e s w i t h an equal v o l u m e of buffer (---- first extraction proteins). The t h i c k interphase o b t a i n e d in the first extraction w a s d i s s o l v e d w i t h KOH, neutral i z e d and extracted w i t h an equal v o l u m e of phenol. The extraction of the interphase p r o t e i n s w a s repeated three m o r e times ( = interphase proteins). Proteins w e r e precipitated by a d d i t i o n of 4 v o l u m e s of cold e t h a n o l + ether (1:1) in the presence of 0.01 M s o d i u m acetate. P h e n o l w a s removed b y repeated w a s h i n g s of the precipitate w i t h ethanol.
Oxidation of proteins w i t h p e r f o r m i c a c i d and r e d u c t i o n w i t h ~-mercapto6thanol f o l l o w e d by alkylation w i t h i o d o a c e t a m i d e w a s p e r f o r m e d as d e s c r i b e d by Hirs [14]. Two-dimensional polyacrylamide gel electrophoresis (2-D) w a s carried out as p r e v i o u s l y described [l, 2].
TABLE I.
Extraction of ribosomal proteins bg [our methods. ._=
~+ i
S 1 S 2 S 3 S 4 S 5 S 6 S 7 S 8 S 9 S t0 Sll S12 S13 S14 S15 S16 S17 S18 S19 S 2O $21
+ + + + ÷ + + + + %
+ + + + + + + + + +
+ + + + + + + + +
+ (+) + + + + + + + +
L 1 L 2 L3 L 4 L 5 L 6
+ + + + + +
+ + + + + +
BIOCHIMIE, 1972, 54, n ° 2.
+
++ (+) +
(+)
÷ +
++ +
+
+
+ + + (+)
L 7 L L9 L10
+ + + +
+ + + +
+ +
Lll L12 L13 L14 LI5 L16 L17 L18 L19 L20 L21 L22 L23
+ +: t + + + + + + ÷ + + ÷
+ (+) + + + + + + +
+ ! + + (+) +
L24 L25 L26 L27 L28 L29 L 30 L31 L32 L33
+ + + + + +
+ + + + + + + + + +
+
+i
+ + + +
+ + +
+ + + -~
(-4-)
÷ + + + ÷ ÷ +
Extraction methods for ribosomal proteins. RESULTS.
Acetic acid m e t h o d : T h e p r o t e i n s o b t a i n e d by e x t r a c t i o n of 0,2 M Mg ÷÷ c o n t a i n i n g r i b o s o m e susp e n s i o n s w i t h acetic acid gave the m a x i m u m number of p r o t e i n spots in the 2-D e l e c t r o p h o r e s i s p a t t e r n (Fig. l a ; Table I). A few p r o t e i n s need some c o n s i d e r a t i o n : a) C o m p a r a t i v e studies [151 on p u r i f i e d 3,0S p r o t e i n s have s h o w n the existence
169
pattern given by r e d u c e d p r o t e i n s (Fis. 5b) suggesting that S16A m a y be a r e d u c e d f o r m of S17. d) The spot L12 given in the p a t t e r n of p r o t e i n s e x t r a c t e d f r o m 50S subuniis was often v e r y weak. L12 and L7 h a v e v e r y s i m i l a r chemical, p h y s i c a l and i m m u n o l o g i c a l p r o p e r t i e s suggesting that L12 and J~7 are d e r i v a t i v e s of the same protein, e) The i n t e n s i t y of spot L31 was always v e r y w e a k in the 50S pattern.
Fx6. 1 a. - - Two-dimensional polyacrylamide gel electrophoresis (2-D) pattern of E. colt K12 total 70S ribosomal proteins prepared by acetic acid extraction of a ribosome suspension containing 0,2 M Mg~ as described in Material and Methods. 2-D eleetrophoresis was carried out as previously described [1]. Enumeration of the spots folloxvs the suggestion of Kaltschmidt and Wittmann [2]. FI6. 1 b. - - 2-D pattern of first extraction proteins of 70S ribosomes of E. colt K12 prepared by phenol extraction as described in Material and Methods. FI6. 1 e. - - 2-D pattern of in terphase proteins of 70S ribosomes of E. colt I~.12 prepared by phenol extraction as described in Material and Methods.
of t w o forms of p r o t e i n S l l w h i c h are s e p a r a t e d by 2=1) e l e c t r o p h o r e s i s , b) The i n t e n s i t y of spot S12 v a r i e d i n v e r s e l y w i t h the i n t e n s i t y of spot S12A seen in the p o s i t i o n of spot L20. c) Spot S17 was v e r y w e a k in the 30 S p r o t e i n pattern. H o w ever, a n e w spot, SI~A, on the left h a n d side of S16 was o b s e r v e d in this 2-D pattern. This is the same r e l a t i o n s h i p w h i c h was o b s e r v e d in the
BIOCHIMIE, 1972, 54, n ° 2.
LiCl-urea m e t h o d : E x t r a c t i o n of r i b o s o m e s w i t h 2 M LiCI, 4 M u r e a in p r e s e n c e of 0.0'005 M Mg ++ gave p a t t e r n s v e r y s i m i l a r to the p a t t e r n s o b t a i n e d by e x t r a c t i o n w i t h acetic acid in h i g h salts (Table I). Spot S13 was r e d u c e d in i n t e n s i t y and an a p p a r e n t d e r i v a t i v e was visible near the eenterline. Spot L20 was not o b s e r v e d in the 50S extract.
170
E. Kaltschmidt
a n d H . G. W i t t m a n n .
R N a s e d i g e s t i o n : T h e p a t t e r n g i v e n b y l~Nase d i g e s t of r i b o s o m e s w a s s i m i l a r to t h e a c e t i c a c i d p a t t e r n ( T a b l e I). T h e i n t e n s i t y of s p o t S12 w a s w e a k a n d t h a t of S12A w a s s t r o n g as p r e v i o u s l y d e s c r i b e d . S p o t S17 w a s f a i n t w h i l e s p o t S16A w a s i n t e n s e a n d o v e r l a p p e d s p o t s S15 a n d S16. T h e i n t e n s i t y of s p o t L20 w a s r e d u c e d . H o w e v e r , a n e w s p o t w h i c h m a y b e a d e r i v a t i v e o f L20 a p p e a r e d n e a r t h e p o s i t i o n of s p o t S l l .
p r o t e i n s . M o s t of t h e s e p r o t e i n s w e r e e x t r a c t e d f r o m t h e 50S s u b u n i t . A n a d d i t i o n a l s p o t , 1A, appeared in this pattern which has not previously been observed.
FIG. 2 a. - - 2-D patLern of p r o t e i n s p r e p a r e d b y acetic some suspension c o n t a i n i n g Fro. 2 b. - - 2-D p a t t e r n of p r o t e i n s p r e p a r e d by acetic some suspension c o n t a i n i n g
FIG. 2 C. - - 2-D p a t t e r n of p r o t e i n s p r e p a r e d b y acetic some s u s p e n s i o n c o n t a i n i n g FIG. 2 d. - - 2-D p a t t e r n of p r o t e i n s p r e p a r e d b y acetic some suspension c o n t a i n i n g
E. colt K12 70'S acid extraction 0.01 Mf Mg÷+. E. colt K12 70S acid extraction 0:01 M Mn +÷.
ribosomal of a riboribosomal of a ribo-
Phenol method : The extract obtained from the first p h e n o l e x t r a c t i o n ( F i g . l b ; T a b l e I) c o n t a i n e d a b o u t 25 p. c e n t of t h e t o t a l 70S r i b o s o m a l BIOCHIMIE, 1'972, 54, n ° 2.
The interphase extract (see M a t e r i a l and M e t h o d s ) c o n t a i n e d m a n y of t h e p r o t e i n s m i s s i n g i n t h e f i r s t p h e n o l e x t r a c t (Fig. l c ; T a b l e 1). However, twelve proteins were not found in
E. colt K12 70S acid e x t r a c t i o n 0:01 M Ca ++. E. colt K12 70S acid extraction 0.4 M Na +.
ribosomal of a riboribosomal of a ribo-
either extracts and several proteins were found in both extracts. In the pattern obtained from the i n t e r p h a s e e x t r a c t s p o t s L8 a n d L9 w e r e n e a r l y
~±
-~
~
.
~
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"-~"~-
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±++
-7-+$+
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+
~
~ .
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.
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+
++++
$ ++±
+
~-
++
+
+
..1_._~_
++
-~-
+++
_~_ _~_..~_..~_ ~ .
+
.
~ ~
-7-~-7--7-~+ . .
++++-7-++-7-+ + +
-~-'-~- + + - 4 - + + + + + + +
~+++ ++~
~
.
-?--7--7--?-+ + + +
+
++
.
~ - - ~
+-7-+-7-++++-?-++++~ ~ ~ -7--%7--7--7-+~.~v_ +
.
~+~+~+ + + +. ~ + +. + + ~ +. + + +. . .
"-~-4--~"[- "31-~--~. . .
.
+
.
-~--~-
~+~+ ~ + ~ + ~
"-'"
.
. . . . . . . .
-?--?-+-?-++-~$++-?-+~.~ .~ .~.~ .~
~
~-7~. + ~ +.~ .
±±
~"
~
+
+
_~_
+
"-~-'-~---~- -4-+
..~
+
+
_~
+
-~-'-~--~
.~
++-7-+++++
~-~.
+ + ++++_?__7_+
++-7--++++-?--7-+~ ~
~ w ~ ~ w ~
(0,02 M Na+) Reextraction : 0,~ M Na +
Acetic acid
Acetic acid (0,001 M Ca++) Reextraction : 0,2 M Ca++
Acetic acid (0,001M Mn++) Reextraction : 0,2 M Ma++
0,2 M Mg++
Reextraetion :
Acetic acid (0,0IM Mg~+)
0,005 M EDTA
Acetic water
0~02 M Li+
0~02 M Na+
o,~M~a+
Acetic acid
Aceticacid
0,0t M ca++
o,o~ ~ M~+
Acetic acid
iI0.0t M Mg++
Protein
~-
~,~'~
--o
~¢
O
.~
~
0
t,~° ~ "
O
~°
O
t--L
~'
.¢
172
E. Kallschmidt
a n d H . G. W i t t m a n n .
P r e s e n c e o f salts : T h e i n f l u e n c e of v a r i o u s salts a n d salt c o n c e n t r a t i o n s on the p r o t e i n comp o s i t i o n of t h e a c e t i c a c i d e x t r a c t o f r i b o s o m e s a r e s h o w n i n Fig. 2, 3, 4 a n d T a b l e II. W h e n t h e Mg ++ c o n c e n t r a t i o n i n t h e r i b o s o m e s u s p e n s i o n
extracted proteins. The electrophoretic patterns o b t a i n e d w i t h 0.2 M Mn ++ o r 0.2 M Ca ++ i n t h e r i b o s o m e s u s p e n s i o n w e r e c o m p a r a b l e to t h a t o b t a i n e d w i t h 0.2 M Mg ++. S i m i l a r l y t h e p a t t e r n s o b t a i n e d w i t h 0.01 M Mn ++ o r 0.01 M Ca ++ i n t h e r i b o -
Fro. 3 a. - - 2-D p a t t e r n of E. coli B 7OS ribosomal p r o t e i n s prepared b y acetic acid extraction of a ribosome suspension containing 0.0'2 M Na +. FIa. 3 b. - - 2-D p a t t e r n of E. coli B 70~S ribosomal proteins p r e p a r e d by acetic acid extraction of a ribosome suspension containing 0,0~2 M Li ÷.
Fro. 3 c. - - 2-D p a t t e r n of E. coli B 7'0S ribosomal proteins, p r e p a r e d b y acetic acid extraction of a ribosome suspension w h i c h w a s dialyzed against desqdlled water. FIG. 3 d. - - 2-D p a t t e r n of E. coli B 70 S ribosomal p r o t e i n s p r e p a r e d b y acetic acid extraction of a ribosome suspension w h i c h w a s dialyzed against 0.805 M E DTA.
w a s r e d u c e d t o 0.01 M t h e n u m b e r o f p r o t e i n s e x t r a c t e d f r o m t h e 30~S a n d 50S r i b o s o m e s w a s d e c r e a s e d . The i n t e n s i t y of several o t h e r spots w a s r e d u c e d . T h e s e p r o t e i n s a r e l i s t e d i n T a b l e II.
s o m e s u s p e n s i o n w e r e c o m p a r a b l e to that obtain e d w i t h 0.01 M Mg ~÷ ( F i g . 2a, 2b, 2c a n d T a b l e II).
Mg ++ w a s r e p l a c e d b y o t h e r b i v a l e n t c a t i o n s w i t h o u t s i g n i f i c a n t c h a n g e s i n t h e n u m b e r of
BIOCHIM1E, 1972, 54, n ° 2.
A c e t i c a e i d e x t r a c t i o n of r i b o s o m e s u s p e n s i o n c o n t a i n i n g 0,4 M NaC1 (Fig. 2 d ; T a b l e 2) o r 0,4 M KC1 o r LiC1 r e s u l t e d i n s i m i l a r e l e c t r o p h o r e t i e p a t t e r n s to t h a t o b t a i n e d b y e x t r a c t i o n o f
E x t r a c t i o n m e t h o d s [or r i b o s o m a l p r o t e i n s . r i b o s o m e s u s p e n s i o n c o n t a i n i n g 0,01 M Mg ~+ a l t h o u g h t h e r e w a s a c o n s i d e r a b l e d i f f e r e n c e in the i o n i c s t r e n g t h of these s u s p e n s i o n s c o n t a i n i n g m o n o v a l e n t o r b i v a l e n t c a t i o n s . W h e n 0,02 M NaC1, LiC1 (Fig. 3 a, 3 b ; T a b l e II) o r 0,02 M KCI were present in the ribosomes suspension during acetic acid e x t r a c t i o n , the n u m b e r of the e x t r a c t e d proteins was considerably reduced. This pattern w a s v e r y similar to that o b t a i n e d f r o m r i b o s o m e s
Fit;. 4 a. - - 2-D p a t t e r n of E, colt B 70 S ribosomal proteins extracted f r o m the sediment obtained by acetic acid t r e a t m e n t of a 0,01 M Mg++ containing ribosome suspension w h i c h w a s r e s u s p e n d e d in buffer containing 0.2 M Mg++ and again extracted w i t h acetic acid as described in Material and Methods. FIG. 4 b. - - 2-D p a t t e r n of E. colt K12 70S ribosomal proteins prepared f r o m the sediment obtained by acetic acid extraction of a 0.001 M Mn ++ containing ribosome suspension w h i c h w a s resuspended in buffer containing 0.2 M Mn ÷~ and again extracted w i t h acetic acid as described in Material and Methods.
BIOCHIMIE, 1972, 54, n ° 2.
173
which were dialyzed against destilled water prior to a c e t i c a c i d e x t r a c t i o n (Fig. 3 c ; T a b l e II). R e m o v a l o f t h e r i b o s o m e b o u n d M g ~÷ b y d i a l y sis of t h e r i b o s o m e s u s p e n s i o n a g a i n s t 0,005 M I~DTA s o l u t i o n r e d u c e d t h e n u m b e r o f p r o t e i n s f o u n d in the acetic acid extracts m o r e t h a n dialysis against destilled water. Only seven proteins w e r e f o u n d in this extract. Several of these w e r e p r e s e n t i n r e d u c e d a m o u n t s (Fig. 3 d ; T a b l e II).
Fro. 4 c. - - 2-D p a t t e r n of E. colt K12 70S ribosomal proteins p r e p a r e d f r o m the sediment obtained by acetic acid extraction of a 0.001 M Ca ++ containing ribosome suspension w h i c h was resuspen.ded in buffer conraining 0.2 M Ca ++ and again extracted w i t h acetic acid as described in Material a n d Methods. FIG. 4 d. - - 2-D p a t t e r n of E. colt B 7OS ribosomal proteins prepared f r o m the s e d i m e n t obtained by acetic acid extraction of a 0.02 M Na + containing ribosome suspension ~'hich w a s resuspended in buffer containing 0.4 1~. Na ~ and' again extracted w i t h acetic acid as described Material and Methods.
174
E. K a l t s c h m i d t
a n d H. G. W i t t m a n n .
The R N A - p r o t e i n pellets w h i c h are left after a c e t i c a c i d e x t r a c t i o n s of r i b o s o m e s s u s p e n d e d in 0',01 M Mg +÷, 0;00.1 M Mn+% 0,001 M Ca ++, 0,02 M Na ÷, 0,02 M K + or 0,0,2 M L i ÷ w e r e f u r t h e r e x t r a c t e d w i t h acetic acid after the salt c o n c e n t r a t i o n s w e r e i n c r e a s e d to 0,2 M Mg ÷+, 0,2 M Mn +÷, 0,2 M Ca ++, 0,4 M Na ÷, (Fig. 4 a, 4 b, 4 c, 4 d and Table II), 0,4 M K + and 0,4 M Li ÷ r e s p e c t i v e l y . Almost all of the p r o t e i n s w h i c h w e r e absent f r o m the first extracts w e r e found after e x t r a c t i o n at h i g h e r salt concentrations. DISCUSSION.
teins w i t h b o u n d n u c l e o t i d e s or by c o n t a m i n a t i n g c y t o p l a s m i c proteins. The n u m b e r of p r o t e i n s e x t r a c t e d by the acetic acid m e t h o d d e p e n d s s t r o n g l y on the com-entration and c o m p o s i t i o n of salt in w h i c h the ribosomes are r e s u s p e n d e d suggesting that electrostatic forces are also i m p o r t a n t in p r o t e i n - R N A interactions. Bivalent cations are m o r e effective in this r e s p e c t than the m o n o v a l e n t cations tested. We o b s e r v e d no significant differences b e t w e e n the t h r e e b i v a l e n t cations or b e t w e e n the t h r e e m o n o v a l e n t cations tested.
The results p r e s e n t e d in this p a p e r s h o w that acetic a c i d e x t r a c t i o n in p r e s e n c e of h i g h salt c o n c e n t r a t i o n , LiC1 u r e a e x t r a c t i o n and RNase digestion give r i b o s o m a l p r o t e i n extracts of similar c o m p o s i t i o n . The c h o i c e of one of these methods w i l l d e p e n d on the necessity of a v o i d i n g c o n t a c t of the r i b o s o m a l p r o t e i n s w i t h u r e a o r
The group of p r o t e i n s w h i c h are only e x t r a c t e d by acetic acid in p r e s e n c e of a h i g h c o n c e n t r a t i o n of salts c o n t a i n almost all of the p r o t e i n s w h i c h b i n d to the 16S [16-21] or the 2~S RNA [18, 22]. T h e r e f o r e it is likely that differential e x t r a c t i o n by acetic acid in absence of salt is p a r t l y based on i n t e r a c t i o n of p r o t e i n s w i t h other c o m p o n e n t s of the ribosome.
FIG. 5 a. - - 2-D pattern of oxidized proteins of E. colt K12 70 S ribosomes. The proteins were extracted as described in Fig. 1 a and oxidized according to Hirs [14].
Fro,. 5 b. - - 2-D pattern of reduced and alkylated proteins of E. colt K12 7'0 S ribosomes. The proteins were extracted as described in Fig. 1 a and reduced and alkytared according to Hirs [14].
c o n t a m i n a t i o n of the p r o t e i n e x t r a c t w i t h RNase. The s i m i l a r i t y of the p r o t e i n p a t t e r n s o b t a i n e d by several different e x t r a c t i o n p r o c e d u r e s suggests that these m e t h o d s e x t r a c t all r i b o s o m a l proteins.
Because of the h i g h n u m b e r of p r o t e i n s in E. colt ribosomes, s e p a r a t i o n of subunits in zonal r o t o r s and f u r t h e r f r a c t i o n a t i o n steos are necessary before the p r o t e i n m i x t u r e s are sufficiently s i m p l e for s e p a r a t i o n by c o l u m n c h r o m a t o g r a p h y and gel filtration. A c o n v e n i e n t w a y of p r e f r a c t i o n a t i o n consists in e x t r a c t i o n of r i b o s o m a l proteins by acetic acid in p r e s e n c e of low c o n c e n t r a tion of salt. This results in two almost equal groups of different p r o t e i n s w h i c h can t h e n be subjected to f u r t h e r fractionation.
P r o t e i n s f o u n d in a t y p i c a l positions are probably o x i d i z e d or r e d u c e d d e r i v a t i v e s s i m i l a r to those o b t a i n e d by o x i d a t i o n of r e d u c t i o n of total p r o t e i n m i x t u r e s (Fig. 5 a and 5 b). The p r o t e i n s w h i c h give spots in the u p p e r p a r t of the gel t o w a r d s the anode h a v e not yet been identified. These spots could be caused by ribosomal proBIOCH1MIE, 1972, 54, n ° 2,
Extraction
methods
Acknoa~ledgement. W e t h a n k Mrs. J. K u h l m e y e r , M i s s J. H u f , Miss I. Hindennach for their excellent technical assistance and Dr. Lawren,ee K a h a n f o r c r i t i c a l r e a d i n g of t h e m a n u s cript. T h e ~vork w a s s u p p o r t e d b y t h e D e u t s c h e Forsehungsgemeinsehaft.
for
ribosomal
proteins.
175
E x t r a k t i o n m i t Essigs~iure bei h o h e r S a l z k o n z e n t r a t i o n g e w o n n e n w e r d e n . D a d u r c h b i e t e t s i e h die M 6 g l i c h k e i t , eine Praefraktionierung der ribosomalen Proteine d u r c h e i n f a c h e p a r t i e l ] e E x t r a k t i o n m i t Essigs~iure durchzuffihren. REFERENCES.
R~suM~.
L e s m 6 t h o d e s s u i v a n t e s son¢ u t i l i s 6 e s p o u r l ' o b t e n tion d e s p r o t 6 i n e s des r i b o s o m e s d'E. coli : a c t i o n d ' u n e s o l u t i o n de L i C l - n r 6 e , de la R N a s e , d u p h 6 n o l o u de l ' a c i d e a c 6 t i q u e e n p r 6 s e n e e de c a t i o n s m o n o o u d i v a l e n t s (Li +, Na +, K +, Mg ++, Ca *+, Mn++). A pr6s s 6 p a r a t i o n p a r d l e e t r o p h o r ~ s e en d e u x d i m e n s i o n s s u r gel de polyacrylamide les positions relatives des prot6ines sont compar6es. E n t r a i t a n t les r i b o s o m e s p a r u n e s o l u t i o n de LiC1ur6e, p a r de la R N a s e ou p a r de / ' a c i d e a c 6 t i q u e d a n s u n m i l i e u de c o n c e n t r a t i o n s a l i n e s ~lev6es le p l u s g r a n d n o m b r e de p r o t 6 i n e s e s t e x t r a i t . Le t r a i t e m e n t p a r le p h 6 n o l ou l ' a c i d e a c 6 t i q u e s a n s a d d i t i o n de sels n e p e r met d'extraire qu'une partie des prot6ines ribosomales. L e s p r o t 6 i n e s r e s t a n t e s p e u v e n t 6tre o b t e n u e s e n r e e x t r a y a n t p a r de l ' a c i d e a c ~ t i q u e d a n s u n m i l i e u de c o n c e n t r a t i o n ~ s a l i n e s 61ev6es. Il est d o n c p o s s i b l e d~op~rer u n p r ~ f r a c t i o n n e m e n t d e s p r o t ~ i n e s r i b o s o m a l e s p a r s i m p l e e x t r a c t i o n p a r t i e l l e a u m o y e n de l'acide ae6tique. ZUSAMMENFASSUNG. Folgende Methoden wurden zur Extraktion der Prot e i n e a u s E. coli R i h o s o m e n a n g e w e n d e t : L i C 1 - H a r n stoff, R N a s e , P h e n o l o d e r Essigs~iure in G e g e n w a r t v o n ein- u n d z w e i w e r t i g e n K a t i o n e n (Li ÷, Na t , K +, Mg *÷, Ca ++, Mn++). N a e h d e r A u f t r e n n u n g in e i n e r z w e i d i m e n s i o n a l e n P o l y a c r y l a m i d g e l - E l e k t r o p h o r e s e w u r d e n die Proteinmuster verg|ichen. Dureh Behandlung der Ribosomen mit LiCl-Harnstoff, ]nit R N a s e o d e r m i t I~ssigsiiure bei h o h e r S a l z k o n z e n t r a t i o n veurde die g r 6 s s t e Z a h l a n P r o t e i n e n e x t r a h i e r t . Behandlung mit Phenol oder Essigsiiure ohne Zusatz v o n Sal,zen e x t r a h i e r t e n u r e i n e n Teil d e r r i b o s o m a l e n Proteine. Der Rest kann durch Wiederholung der
BIOCHIMIE, 1972, 54, n ° 2.
1. K a l t s c h m i d t , E. ~ W i t t m a n n , H. G. (1970) Analyt. Biochem., 36, 401. 2. K a l t s c h m i d t , E. ~ W i t t m a n n , H. G. (1970) Proc. Nat. Acad. Sci. ( W a s h ) , 67, 1276. 3. F r a e n k e l - C o n r a t , H. (1957) Viroloyy, 4, 1. 4. W a l l e r , J. P. ~ H a r r i s , J. I. (19.61) Proc. Nat. Acud. Sci. ( W a s h . ) , 47, 18 5. K a l t s c h m i d t , E., D z i o n a r a , M., D o n n e r , D. ~ W i t t m a n n , H. G. (1967) Molec. Gen. Genetics, 100, 364. 6. Moore, P. B., T r a u t , R. R., NoLler, H., P e a r s o n , P. D e l i u s , H. (1968) J. Mol. Biol., 31, 441. 7. H a r d y , S. J. S., K u r l a n d , C. G., V o y n o w , P. ~ Mora, G. (1969) Biochemistry, 8, 2897. 8. S p i t n i k - E q s o n , P. (1965) Biochem. Biophys. Res. Com., 19, 557. 9 M a r c o t - Q u e i r g z , J. • M o n i e r , R. (1966) Bull. Soc. Chim. Biol., 48, 446. 10. Itoh, T., O t a k a , E. ~ O s a w a , S. (1968) J. Mol. Biol., 33, 109. 11. L e r m a n , M. I., S p i r i n , A. S., G a v r i l o v a , L. P. ~ Colov, V. F. (1966) J. Mol. Biol., 15, 268. 12. S c h u s t e r , H., S c h r a m m , G. ,~ Zillig, W . (1956) Z. Natur[., 116, 339. 13. K u r l a n d , C. G. (1988) J. Mol. Biol., 18, 90. 14. Hirs, C. H. W . (1967) Methods in Enzymology, 11, 195. 15. W i t t m a n n , H. G., StSffler, G., H i n d e n n a c h , I., K u r l a n d , C. G., R a n d a l l - H a z e l b a u e r , L., Birge, E. A. N o m u r a , M., K a l t s c h m i d t , E., M i z u s h i m a , S., T r a u t , R. R. ,~ Bickle, T. A. (1971) Molec. Gen. Genetics, 111, 327. 16. M i z n s h i m a , S. ,~ N o m u r a , M. (1970) Nature, 226, 1214. 17. S c h a u p , H. W., Green, M. ~ K u r l a n d , C. G. (1970) Molec. Gen. Genetics, 109, 193. 18. St6ffler, G., D a y a , L., R a k , K. H. • G a r r e t t , R. (1971) J. Mol. BioL, 62, 411. 19. S c h a u p , H. W., Green, M. & K u r l a n d , C. G. (1971) Molec. Gen. Genetics, 112, 1. 20. G a r r e t t , R. A., R a k , K. H., D a y a , L. ~ St6ffler, G. (1971) Molec. Gen. Genetics, 114, 112. 21. N a s h i m o t o , H., Held, W., K a l t s e h m i d t , E. t, N o m u r a , M. (1971) J. Mol. Biol., 62, 121. 22. St6ffler, G., D a y a , L., R a k , K. H. ~ G a r r e t t , R. A. (1971) Molec. Gen. Genetics, 112, 125.
Ribosomal proteins. X X X I I : Comparison of several extraction methods for proteins from Escherichia colt ribosomes. E. KALTSCHMIDT (*) a n d H. G. WITTMANN. Extraction methods [or ribosomal proteins. Max-Planck-lnstitut [iir Molekulare Genetik, Berlin-Dahlem, Germany. (7/2/1972).
Summary. - - The following methods were used for the extraction of proteins from E. colt ribosomes.: LiCl-urea, RNase, phenol or acetic acid in presence of mono- and bivalent cations (Li +, Na ÷, K÷, Mg++, Ca ++, Mn+*). After separation in a two-dimensional polyacrylamide gel eleetrophoresis the protein patterns were compared. By treatlnent of ribosomes with LiCl-urea, with RNase or with acetic acid at high salt concentration the highest number of proteins were extracted. Treatment with phenol or acetic acid without addition of salts extracted only a portion of the ribosomal proteins. The rest can be obtained by repeating the extraction with acetic acid at high salt concentration. This offers the possibility to prefraetionate ribosomal proteins by simple partial extraction with acetic acid.
In o r d e r to study the s t r u c t u r e and f u n c t i o n of r i b o s o m a l p r o t e i n s it is n e c e s s a r y to isolate all of the r i b o s o m a l p r o t e i n c o m p o n e n t s by e x t r a c t i o n p r o c e d u r e s w h i c h are c o m p a t i b l e w i t h the requir e m e n t s of subsequent e x p e r i m e n t s . T w o - d i m e n sional a c r y l a m i d e gel e l e c t r o p h o r e s i s p r o v i d e s a c o n v e n i e n t m e t h o d for c o m p a r i s o n of the compositions of r i b o s o m a l p r o t e i n extracts [1, 2]. In this p a p e r w e c o m p a r e the c o m p o s i t i o n s of r i b o s o m a l p r o t e i n m i x t u r e s p r e p a r e d by the methods c o m m o n l y used for isolation of the p r o t e i n m o i e t y of n u c l e o p r o t e i n s : e x t r a c t i o n w i t h acetic acid in the p r e s e n c e of various salts [3-7], extraction w i t h Li,Cl-urea [8-111, digestion w i t h RNase and p h e n o l e x t r a c t i o n [121. It is s h o w n that all r i b o s o m a l p r o t e i n s are e x t r a c t e d by some of these methods whereas other methods yielded incomplete p r o t e i n mixtures. Partial e x t r a c t i o n of ribosomal p r o t e i n s can be useful as a first s e p a r a t i o n step in the p u r i f i c a t i o n of r i b o s o m a l proteins. MATERIALS AND METHODS.
Strains and ribosomes : Ribosomes are o b t a i n e d f r o m E. colt K12 (strain A19) and E. colt B g r o w n to 3/4 log phase. After the cells w e r e g r o u n d w i t h a l u m i n a p o w d e r (Alcoa 305) the r i b o s o m e s w e r e p r e p a r e d by differential c e n t r i f u g a t i o n in 0,02 M tris-HC1, pH 8.0, 0.01 M m a g n e s i u m acetate. After the s e c o n d h i g h s p e e d c e n t r i f u g a t i o n the r i b o s o m e s (') Present address : Institute for Enzyme Research, University of Wisconsin, Madison, Wisconsin, USA.
w e r e w a s h e d in 0.02 M tris-HC'l, 0.5 M NH~ C1, 0.001 M m a g n e s i u m acetate, pH 7.8, pelleted again, r e s u s p e n d e d and stored in 0.02 M tris-HC1, pH 7.8, 0.01 M m a g n e s i u m acetate. Some hatches of ribo. somes w e r e r e p e a t e d l y p r e c i p i t a t e d w i t h (NH4),., SO 4 before differential c e n t r i f u g a t i o n [13].
Extraction o[ proteins : Optimal e x t r a c l i o n of r i b o s o m a l p r o t e i n s w i t h acetic acid was o b t a i n e d by the f o l l o w i n g p r o c e d u r e : MgC12 was a d d e d to a r i b o s o m e suspension c o n t a i n i n g 15.0-300 OD26o/ ml to i n c r e a s e the c o n c e n t r a t i o n Of Mg ÷* to 0.2 Mr. T w o volumes of glacial acetic acid w e r e a d d e d to one v o l u m e of r i b o s o m e s u s p e n s i o n at 0°C w i t h vigorous stirring. This m i x t u r e was kept in ice for 40 m i n u t e s and stirred. The p r e c i p i t a t e d RNA was pelleted by c e n t r i f u g a t i o n at 30 000 g for 20 min. The acetic acid c o n c e n t r a t i o n "was reduced by stepwise dialysis against 5(I p. cent, 40 p. cent, 30 p. cent, 20 p. cent and 10 p. cenl acetic acid. The p r o t e i n s w e r e stored in 10 p. cenl acetic acid at - - 2 0 ° C . The acetic acid e x t r a c t i o n p r o c e d u r e was v a r i e d by the a d d i t i o n of v a r i o u s salts to the ribosome suspension, e.g. MnC12, CaC12, NaC1, KCI or LiCI. The r i b o s o m e s w e r e dialyzed at 4°C firsl against 0.005 M Na2EDTA to reluove b o u n d Mg +~, then against 0.02 M tris-C1, pH 7.8, and finally the desired salt was added. T h e pellet o b t a i n e d by acetic acid e x t r a c t i o n was r e s u s p e n d e d in the same buffer or in a buffer w i t h h i g h e r salt c o n c e n t r a t i o n and e x t r a c t e d for
E. K a l t s c h m i d t and H. G. W i t t m a n n .
168
a s e c o n d time. Difficulties in r e s u s p e n s i o n of the pellet w e r e o v e r c o m e by g r i n d i n g the frozen pellet in a motar.
LiCl-urea extraction of proteins w a s carried out by the f o l l o w i n g p r o c e d u r e : One v o l u m e of 4 M L i C 1 - 8 M urea solution w a s m i x e d w i t h one v o l u m e of r i b o s o m e s u s p e n s i o n c o n t a i n i n g 0.004 M Mg *+ and the resulting s u s p e n s i o n w a s stored 48 hours at 0°C. The R~NA precipitate w a s pelleted by centrifugation at 30 000 g for 20 rain. RNase digestion w a s carried out by the f o l l o w i n g p r o c e d u r e : The r i b o s o m e s u s p e n s i o n w a s d i a l y z e d against 0.0,06 M EDTA. F i v e ~g pancreatic RNase and 0.3 ~g (8,0 units) T l - R N a s e w e r e a d d e d to 0.1- 0.2 m l s u s p e n s i o n c o n t a i n i n g 100 OD26 o r i b o s o m e s and the resulting s u s p e n s i o n w a s i n c u b a t e d at 37°C for 1 hour. Phenol extraction w a s carried out by the f o l l o w i n g p r o c e d u r e : One v o l u m e p h e n o l w a s a d d e d to one v o l u m e r i b o s o m e s u s p e n s i o n containing
0.02 M tris-HC1, p H 7.8, 0.001 Mg ÷÷ and the resulting m i x t u r e w a s shaken for 10 rain at 4°C. The p h e n o l p h a s e w a s extracted three a d d i t i o n a l t i m e s w i t h an equal v o l u m e of buffer (---- first extraction proteins). The t h i c k interphase o b t a i n e d in the first extraction w a s d i s s o l v e d w i t h KOH, neutral i z e d and extracted w i t h an equal v o l u m e of phenol. The extraction of the interphase p r o t e i n s w a s repeated three m o r e times ( = interphase proteins). Proteins w e r e precipitated by a d d i t i o n of 4 v o l u m e s of cold e t h a n o l + ether (1:1) in the presence of 0.01 M s o d i u m acetate. P h e n o l w a s removed b y repeated w a s h i n g s of the precipitate w i t h ethanol.
Oxidation of proteins w i t h p e r f o r m i c a c i d and r e d u c t i o n w i t h ~-mercapto6thanol f o l l o w e d by alkylation w i t h i o d o a c e t a m i d e w a s p e r f o r m e d as d e s c r i b e d by Hirs [14]. Two-dimensional polyacrylamide gel electrophoresis (2-D) w a s carried out as p r e v i o u s l y described [l, 2].
TABLE I.
Extraction of ribosomal proteins bg [our methods. ._=
~+ i
S 1 S 2 S 3 S 4 S 5 S 6 S 7 S 8 S 9 S t0 Sll S12 S13 S14 S15 S16 S17 S18 S19 S 2O $21
+ + + + ÷ + + + + %
+ + + + + + + + + +
+ + + + + + + + +
+ (+) + + + + + + + +
L 1 L 2 L3 L 4 L 5 L 6
+ + + + + +
+ + + + + +
BIOCHIMIE, 1972, 54, n ° 2.
+
++ (+) +
(+)
÷ +
++ +
+
+
+ + + (+)
L 7 L L9 L10
+ + + +
+ + + +
+ +
Lll L12 L13 L14 LI5 L16 L17 L18 L19 L20 L21 L22 L23
+ +: t + + + + + + ÷ + + ÷
+ (+) + + + + + + +
+ ! + + (+) +
L24 L25 L26 L27 L28 L29 L 30 L31 L32 L33
+ + + + + +
+ + + + + + + + + +
+
+i
+ + + +
+ + +
+ + + -~
(-4-)
÷ + + + ÷ ÷ +
Extraction methods for ribosomal proteins. RESULTS.
Acetic acid m e t h o d : T h e p r o t e i n s o b t a i n e d by e x t r a c t i o n of 0,2 M Mg ÷÷ c o n t a i n i n g r i b o s o m e susp e n s i o n s w i t h acetic acid gave the m a x i m u m number of p r o t e i n spots in the 2-D e l e c t r o p h o r e s i s p a t t e r n (Fig. l a ; Table I). A few p r o t e i n s need some c o n s i d e r a t i o n : a) C o m p a r a t i v e studies [151 on p u r i f i e d 3,0S p r o t e i n s have s h o w n the existence
169
pattern given by r e d u c e d p r o t e i n s (Fis. 5b) suggesting that S16A m a y be a r e d u c e d f o r m of S17. d) The spot L12 given in the p a t t e r n of p r o t e i n s e x t r a c t e d f r o m 50S subuniis was often v e r y weak. L12 and L7 h a v e v e r y s i m i l a r chemical, p h y s i c a l and i m m u n o l o g i c a l p r o p e r t i e s suggesting that L12 and J~7 are d e r i v a t i v e s of the same protein, e) The i n t e n s i t y of spot L31 was always v e r y w e a k in the 50S pattern.
Fx6. 1 a. - - Two-dimensional polyacrylamide gel electrophoresis (2-D) pattern of E. colt K12 total 70S ribosomal proteins prepared by acetic acid extraction of a ribosome suspension containing 0,2 M Mg~ as described in Material and Methods. 2-D eleetrophoresis was carried out as previously described [1]. Enumeration of the spots folloxvs the suggestion of Kaltschmidt and Wittmann [2]. FI6. 1 b. - - 2-D pattern of first extraction proteins of 70S ribosomes of E. colt K12 prepared by phenol extraction as described in Material and Methods. FI6. 1 e. - - 2-D pattern of in terphase proteins of 70S ribosomes of E. colt I~.12 prepared by phenol extraction as described in Material and Methods.
of t w o forms of p r o t e i n S l l w h i c h are s e p a r a t e d by 2=1) e l e c t r o p h o r e s i s , b) The i n t e n s i t y of spot S12 v a r i e d i n v e r s e l y w i t h the i n t e n s i t y of spot S12A seen in the p o s i t i o n of spot L20. c) Spot S17 was v e r y w e a k in the 30 S p r o t e i n pattern. H o w ever, a n e w spot, SI~A, on the left h a n d side of S16 was o b s e r v e d in this 2-D pattern. This is the same r e l a t i o n s h i p w h i c h was o b s e r v e d in the
BIOCHIMIE, 1972, 54, n ° 2.
LiCl-urea m e t h o d : E x t r a c t i o n of r i b o s o m e s w i t h 2 M LiCI, 4 M u r e a in p r e s e n c e of 0.0'005 M Mg ++ gave p a t t e r n s v e r y s i m i l a r to the p a t t e r n s o b t a i n e d by e x t r a c t i o n w i t h acetic acid in h i g h salts (Table I). Spot S13 was r e d u c e d in i n t e n s i t y and an a p p a r e n t d e r i v a t i v e was visible near the eenterline. Spot L20 was not o b s e r v e d in the 50S extract.
170
E. Kaltschmidt
a n d H . G. W i t t m a n n .
R N a s e d i g e s t i o n : T h e p a t t e r n g i v e n b y l~Nase d i g e s t of r i b o s o m e s w a s s i m i l a r to t h e a c e t i c a c i d p a t t e r n ( T a b l e I). T h e i n t e n s i t y of s p o t S12 w a s w e a k a n d t h a t of S12A w a s s t r o n g as p r e v i o u s l y d e s c r i b e d . S p o t S17 w a s f a i n t w h i l e s p o t S16A w a s i n t e n s e a n d o v e r l a p p e d s p o t s S15 a n d S16. T h e i n t e n s i t y of s p o t L20 w a s r e d u c e d . H o w e v e r , a n e w s p o t w h i c h m a y b e a d e r i v a t i v e o f L20 a p p e a r e d n e a r t h e p o s i t i o n of s p o t S l l .
p r o t e i n s . M o s t of t h e s e p r o t e i n s w e r e e x t r a c t e d f r o m t h e 50S s u b u n i t . A n a d d i t i o n a l s p o t , 1A, appeared in this pattern which has not previously been observed.
FIG. 2 a. - - 2-D patLern of p r o t e i n s p r e p a r e d b y acetic some suspension c o n t a i n i n g Fro. 2 b. - - 2-D p a t t e r n of p r o t e i n s p r e p a r e d by acetic some suspension c o n t a i n i n g
FIG. 2 C. - - 2-D p a t t e r n of p r o t e i n s p r e p a r e d b y acetic some s u s p e n s i o n c o n t a i n i n g FIG. 2 d. - - 2-D p a t t e r n of p r o t e i n s p r e p a r e d b y acetic some suspension c o n t a i n i n g
E. colt K12 70'S acid extraction 0.01 Mf Mg÷+. E. colt K12 70S acid extraction 0:01 M Mn +÷.
ribosomal of a riboribosomal of a ribo-
Phenol method : The extract obtained from the first p h e n o l e x t r a c t i o n ( F i g . l b ; T a b l e I) c o n t a i n e d a b o u t 25 p. c e n t of t h e t o t a l 70S r i b o s o m a l BIOCHIMIE, 1'972, 54, n ° 2.
The interphase extract (see M a t e r i a l and M e t h o d s ) c o n t a i n e d m a n y of t h e p r o t e i n s m i s s i n g i n t h e f i r s t p h e n o l e x t r a c t (Fig. l c ; T a b l e 1). However, twelve proteins were not found in
E. colt K12 70S acid e x t r a c t i o n 0:01 M Ca ++. E. colt K12 70S acid extraction 0.4 M Na +.
ribosomal of a riboribosomal of a ribo-
either extracts and several proteins were found in both extracts. In the pattern obtained from the i n t e r p h a s e e x t r a c t s p o t s L8 a n d L9 w e r e n e a r l y
~±
-~
~
.
~
.
.
"-~"~-
.
.
±++
-7-+$+
.
_~
.
....
.
+
~
~ .
.
.
.
.
+
++++
$ ++±
+
~-
++
+
+
..1_._~_
++
-~-
+++
_~_ _~_..~_..~_ ~ .
+
.
~ ~
-7-~-7--7-~+ . .
++++-7-++-7-+ + +
-~-'-~- + + - 4 - + + + + + + +
~+++ ++~
~
.
-?--7--7--?-+ + + +
+
++
.
~ - - ~
+-7-+-7-++++-?-++++~ ~ ~ -7--%7--7--7-+~.~v_ +
.
~+~+~+ + + +. ~ + +. + + ~ +. + + +. . .
"-~-4--~"[- "31-~--~. . .
.
+
.
-~--~-
~+~+ ~ + ~ + ~
"-'"
.
. . . . . . . .
-?--?-+-?-++-~$++-?-+~.~ .~ .~.~ .~
~
~-7~. + ~ +.~ .
±±
~"
~
+
+
_~_
+
"-~-'-~---~- -4-+
..~
+
+
_~
+
-~-'-~--~
.~
++-7-+++++
~-~.
+ + ++++_?__7_+
++-7--++++-?--7-+~ ~
~ w ~ ~ w ~
(0,02 M Na+) Reextraction : 0,~ M Na +
Acetic acid
Acetic acid (0,001 M Ca++) Reextraction : 0,2 M Ca++
Acetic acid (0,001M Mn++) Reextraction : 0,2 M Ma++
0,2 M Mg++
Reextraetion :
Acetic acid (0,0IM Mg~+)
0,005 M EDTA
Acetic water
0~02 M Li+
0~02 M Na+
o,~M~a+
Acetic acid
Aceticacid
0,0t M ca++
o,o~ ~ M~+
Acetic acid
iI0.0t M Mg++
Protein
~-
~,~'~
--o
~¢
O
.~
~
0
t,~° ~ "
O
~°
O
t--L
~'
.¢
172
E. Kallschmidt
a n d H . G. W i t t m a n n .
P r e s e n c e o f salts : T h e i n f l u e n c e of v a r i o u s salts a n d salt c o n c e n t r a t i o n s on the p r o t e i n comp o s i t i o n of t h e a c e t i c a c i d e x t r a c t o f r i b o s o m e s a r e s h o w n i n Fig. 2, 3, 4 a n d T a b l e II. W h e n t h e Mg ++ c o n c e n t r a t i o n i n t h e r i b o s o m e s u s p e n s i o n
extracted proteins. The electrophoretic patterns o b t a i n e d w i t h 0.2 M Mn ++ o r 0.2 M Ca ++ i n t h e r i b o s o m e s u s p e n s i o n w e r e c o m p a r a b l e to t h a t o b t a i n e d w i t h 0.2 M Mg ++. S i m i l a r l y t h e p a t t e r n s o b t a i n e d w i t h 0.01 M Mn ++ o r 0.01 M Ca ++ i n t h e r i b o -
Fro. 3 a. - - 2-D p a t t e r n of E. coli B 7OS ribosomal p r o t e i n s prepared b y acetic acid extraction of a ribosome suspension containing 0.0'2 M Na +. FIa. 3 b. - - 2-D p a t t e r n of E. coli B 70~S ribosomal proteins p r e p a r e d by acetic acid extraction of a ribosome suspension containing 0,0~2 M Li ÷.
Fro. 3 c. - - 2-D p a t t e r n of E. coli B 7'0S ribosomal proteins, p r e p a r e d b y acetic acid extraction of a ribosome suspension w h i c h w a s dialyzed against desqdlled water. FIG. 3 d. - - 2-D p a t t e r n of E. coli B 70 S ribosomal p r o t e i n s p r e p a r e d b y acetic acid extraction of a ribosome suspension w h i c h w a s dialyzed against 0.805 M E DTA.
w a s r e d u c e d t o 0.01 M t h e n u m b e r o f p r o t e i n s e x t r a c t e d f r o m t h e 30~S a n d 50S r i b o s o m e s w a s d e c r e a s e d . The i n t e n s i t y of several o t h e r spots w a s r e d u c e d . T h e s e p r o t e i n s a r e l i s t e d i n T a b l e II.
s o m e s u s p e n s i o n w e r e c o m p a r a b l e to that obtain e d w i t h 0.01 M Mg ~÷ ( F i g . 2a, 2b, 2c a n d T a b l e II).
Mg ++ w a s r e p l a c e d b y o t h e r b i v a l e n t c a t i o n s w i t h o u t s i g n i f i c a n t c h a n g e s i n t h e n u m b e r of
BIOCHIM1E, 1972, 54, n ° 2.
A c e t i c a e i d e x t r a c t i o n of r i b o s o m e s u s p e n s i o n c o n t a i n i n g 0,4 M NaC1 (Fig. 2 d ; T a b l e 2) o r 0,4 M KC1 o r LiC1 r e s u l t e d i n s i m i l a r e l e c t r o p h o r e t i e p a t t e r n s to t h a t o b t a i n e d b y e x t r a c t i o n o f
E x t r a c t i o n m e t h o d s [or r i b o s o m a l p r o t e i n s . r i b o s o m e s u s p e n s i o n c o n t a i n i n g 0,01 M Mg ~+ a l t h o u g h t h e r e w a s a c o n s i d e r a b l e d i f f e r e n c e in the i o n i c s t r e n g t h of these s u s p e n s i o n s c o n t a i n i n g m o n o v a l e n t o r b i v a l e n t c a t i o n s . W h e n 0,02 M NaC1, LiC1 (Fig. 3 a, 3 b ; T a b l e II) o r 0,02 M KCI were present in the ribosomes suspension during acetic acid e x t r a c t i o n , the n u m b e r of the e x t r a c t e d proteins was considerably reduced. This pattern w a s v e r y similar to that o b t a i n e d f r o m r i b o s o m e s
Fit;. 4 a. - - 2-D p a t t e r n of E, colt B 70 S ribosomal proteins extracted f r o m the sediment obtained by acetic acid t r e a t m e n t of a 0,01 M Mg++ containing ribosome suspension w h i c h w a s r e s u s p e n d e d in buffer containing 0.2 M Mg++ and again extracted w i t h acetic acid as described in Material and Methods. FIG. 4 b. - - 2-D p a t t e r n of E. colt K12 70S ribosomal proteins prepared f r o m the sediment obtained by acetic acid extraction of a 0.001 M Mn ++ containing ribosome suspension w h i c h w a s resuspended in buffer containing 0.2 M Mn ÷~ and again extracted w i t h acetic acid as described in Material and Methods.
BIOCHIMIE, 1972, 54, n ° 2.
173
which were dialyzed against destilled water prior to a c e t i c a c i d e x t r a c t i o n (Fig. 3 c ; T a b l e II). R e m o v a l o f t h e r i b o s o m e b o u n d M g ~÷ b y d i a l y sis of t h e r i b o s o m e s u s p e n s i o n a g a i n s t 0,005 M I~DTA s o l u t i o n r e d u c e d t h e n u m b e r o f p r o t e i n s f o u n d in the acetic acid extracts m o r e t h a n dialysis against destilled water. Only seven proteins w e r e f o u n d in this extract. Several of these w e r e p r e s e n t i n r e d u c e d a m o u n t s (Fig. 3 d ; T a b l e II).
Fro. 4 c. - - 2-D p a t t e r n of E. colt K12 70S ribosomal proteins p r e p a r e d f r o m the sediment obtained by acetic acid extraction of a 0.001 M Ca ++ containing ribosome suspension w h i c h was resuspen.ded in buffer conraining 0.2 M Ca ++ and again extracted w i t h acetic acid as described in Material a n d Methods. FIG. 4 d. - - 2-D p a t t e r n of E. colt B 7OS ribosomal proteins prepared f r o m the s e d i m e n t obtained by acetic acid extraction of a 0.02 M Na + containing ribosome suspension ~'hich w a s resuspended in buffer containing 0.4 1~. Na ~ and' again extracted w i t h acetic acid as described Material and Methods.
174
E. K a l t s c h m i d t
a n d H. G. W i t t m a n n .
The R N A - p r o t e i n pellets w h i c h are left after a c e t i c a c i d e x t r a c t i o n s of r i b o s o m e s s u s p e n d e d in 0',01 M Mg +÷, 0;00.1 M Mn+% 0,001 M Ca ++, 0,02 M Na ÷, 0,02 M K + or 0,0,2 M L i ÷ w e r e f u r t h e r e x t r a c t e d w i t h acetic acid after the salt c o n c e n t r a t i o n s w e r e i n c r e a s e d to 0,2 M Mg ÷+, 0,2 M Mn +÷, 0,2 M Ca ++, 0,4 M Na ÷, (Fig. 4 a, 4 b, 4 c, 4 d and Table II), 0,4 M K + and 0,4 M Li ÷ r e s p e c t i v e l y . Almost all of the p r o t e i n s w h i c h w e r e absent f r o m the first extracts w e r e found after e x t r a c t i o n at h i g h e r salt concentrations. DISCUSSION.
teins w i t h b o u n d n u c l e o t i d e s or by c o n t a m i n a t i n g c y t o p l a s m i c proteins. The n u m b e r of p r o t e i n s e x t r a c t e d by the acetic acid m e t h o d d e p e n d s s t r o n g l y on the com-entration and c o m p o s i t i o n of salt in w h i c h the ribosomes are r e s u s p e n d e d suggesting that electrostatic forces are also i m p o r t a n t in p r o t e i n - R N A interactions. Bivalent cations are m o r e effective in this r e s p e c t than the m o n o v a l e n t cations tested. We o b s e r v e d no significant differences b e t w e e n the t h r e e b i v a l e n t cations or b e t w e e n the t h r e e m o n o v a l e n t cations tested.
The results p r e s e n t e d in this p a p e r s h o w that acetic a c i d e x t r a c t i o n in p r e s e n c e of h i g h salt c o n c e n t r a t i o n , LiC1 u r e a e x t r a c t i o n and RNase digestion give r i b o s o m a l p r o t e i n extracts of similar c o m p o s i t i o n . The c h o i c e of one of these methods w i l l d e p e n d on the necessity of a v o i d i n g c o n t a c t of the r i b o s o m a l p r o t e i n s w i t h u r e a o r
The group of p r o t e i n s w h i c h are only e x t r a c t e d by acetic acid in p r e s e n c e of a h i g h c o n c e n t r a t i o n of salts c o n t a i n almost all of the p r o t e i n s w h i c h b i n d to the 16S [16-21] or the 2~S RNA [18, 22]. T h e r e f o r e it is likely that differential e x t r a c t i o n by acetic acid in absence of salt is p a r t l y based on i n t e r a c t i o n of p r o t e i n s w i t h other c o m p o n e n t s of the ribosome.
FIG. 5 a. - - 2-D pattern of oxidized proteins of E. colt K12 70 S ribosomes. The proteins were extracted as described in Fig. 1 a and oxidized according to Hirs [14].
Fro,. 5 b. - - 2-D pattern of reduced and alkylated proteins of E. colt K12 7'0 S ribosomes. The proteins were extracted as described in Fig. 1 a and reduced and alkytared according to Hirs [14].
c o n t a m i n a t i o n of the p r o t e i n e x t r a c t w i t h RNase. The s i m i l a r i t y of the p r o t e i n p a t t e r n s o b t a i n e d by several different e x t r a c t i o n p r o c e d u r e s suggests that these m e t h o d s e x t r a c t all r i b o s o m a l proteins.
Because of the h i g h n u m b e r of p r o t e i n s in E. colt ribosomes, s e p a r a t i o n of subunits in zonal r o t o r s and f u r t h e r f r a c t i o n a t i o n steos are necessary before the p r o t e i n m i x t u r e s are sufficiently s i m p l e for s e p a r a t i o n by c o l u m n c h r o m a t o g r a p h y and gel filtration. A c o n v e n i e n t w a y of p r e f r a c t i o n a t i o n consists in e x t r a c t i o n of r i b o s o m a l proteins by acetic acid in p r e s e n c e of low c o n c e n t r a tion of salt. This results in two almost equal groups of different p r o t e i n s w h i c h can t h e n be subjected to f u r t h e r fractionation.
P r o t e i n s f o u n d in a t y p i c a l positions are probably o x i d i z e d or r e d u c e d d e r i v a t i v e s s i m i l a r to those o b t a i n e d by o x i d a t i o n of r e d u c t i o n of total p r o t e i n m i x t u r e s (Fig. 5 a and 5 b). The p r o t e i n s w h i c h give spots in the u p p e r p a r t of the gel t o w a r d s the anode h a v e not yet been identified. These spots could be caused by ribosomal proBIOCH1MIE, 1972, 54, n ° 2,
Extraction
methods
Acknoa~ledgement. W e t h a n k Mrs. J. K u h l m e y e r , M i s s J. H u f , Miss I. Hindennach for their excellent technical assistance and Dr. Lawren,ee K a h a n f o r c r i t i c a l r e a d i n g of t h e m a n u s cript. T h e ~vork w a s s u p p o r t e d b y t h e D e u t s c h e Forsehungsgemeinsehaft.
for
ribosomal
proteins.
175
E x t r a k t i o n m i t Essigs~iure bei h o h e r S a l z k o n z e n t r a t i o n g e w o n n e n w e r d e n . D a d u r c h b i e t e t s i e h die M 6 g l i c h k e i t , eine Praefraktionierung der ribosomalen Proteine d u r c h e i n f a c h e p a r t i e l ] e E x t r a k t i o n m i t Essigs~iure durchzuffihren. REFERENCES.
R~suM~.
L e s m 6 t h o d e s s u i v a n t e s son¢ u t i l i s 6 e s p o u r l ' o b t e n tion d e s p r o t 6 i n e s des r i b o s o m e s d'E. coli : a c t i o n d ' u n e s o l u t i o n de L i C l - n r 6 e , de la R N a s e , d u p h 6 n o l o u de l ' a c i d e a c 6 t i q u e e n p r 6 s e n e e de c a t i o n s m o n o o u d i v a l e n t s (Li +, Na +, K +, Mg ++, Ca *+, Mn++). A pr6s s 6 p a r a t i o n p a r d l e e t r o p h o r ~ s e en d e u x d i m e n s i o n s s u r gel de polyacrylamide les positions relatives des prot6ines sont compar6es. E n t r a i t a n t les r i b o s o m e s p a r u n e s o l u t i o n de LiC1ur6e, p a r de la R N a s e ou p a r de / ' a c i d e a c 6 t i q u e d a n s u n m i l i e u de c o n c e n t r a t i o n s a l i n e s ~lev6es le p l u s g r a n d n o m b r e de p r o t 6 i n e s e s t e x t r a i t . Le t r a i t e m e n t p a r le p h 6 n o l ou l ' a c i d e a c 6 t i q u e s a n s a d d i t i o n de sels n e p e r met d'extraire qu'une partie des prot6ines ribosomales. L e s p r o t 6 i n e s r e s t a n t e s p e u v e n t 6tre o b t e n u e s e n r e e x t r a y a n t p a r de l ' a c i d e a c ~ t i q u e d a n s u n m i l i e u de c o n c e n t r a t i o n ~ s a l i n e s 61ev6es. Il est d o n c p o s s i b l e d~op~rer u n p r ~ f r a c t i o n n e m e n t d e s p r o t ~ i n e s r i b o s o m a l e s p a r s i m p l e e x t r a c t i o n p a r t i e l l e a u m o y e n de l'acide ae6tique. ZUSAMMENFASSUNG. Folgende Methoden wurden zur Extraktion der Prot e i n e a u s E. coli R i h o s o m e n a n g e w e n d e t : L i C 1 - H a r n stoff, R N a s e , P h e n o l o d e r Essigs~iure in G e g e n w a r t v o n ein- u n d z w e i w e r t i g e n K a t i o n e n (Li ÷, Na t , K +, Mg *÷, Ca ++, Mn++). N a e h d e r A u f t r e n n u n g in e i n e r z w e i d i m e n s i o n a l e n P o l y a c r y l a m i d g e l - E l e k t r o p h o r e s e w u r d e n die Proteinmuster verg|ichen. Dureh Behandlung der Ribosomen mit LiCl-Harnstoff, ]nit R N a s e o d e r m i t I~ssigsiiure bei h o h e r S a l z k o n z e n t r a t i o n veurde die g r 6 s s t e Z a h l a n P r o t e i n e n e x t r a h i e r t . Behandlung mit Phenol oder Essigsiiure ohne Zusatz v o n Sal,zen e x t r a h i e r t e n u r e i n e n Teil d e r r i b o s o m a l e n Proteine. Der Rest kann durch Wiederholung der
BIOCHIMIE, 1972, 54, n ° 2.
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