9 30
NOTIZEN
men stellt das Chloramphenicol die D-threo-Form dar
und ist bei weitem am wirksamsten. Die i.-threo-F orm
hat weniger als 0,5% der W irksamkeit der D-Form.
Die beiden Erythro-Isomeren sind biologisch in a k tiv 6.
Beim PNPG dürfte ein Gemisch zweier oder mehr der
vier möglichen stereoisomeren Formen vorliegen.
Im vorstehenden Zusammenhang interessierte die
Frage, ob die schwärmhemmende Wirkung des PNPG
in ähnlicher Weise an eine bestimmte sterische Kon­
figuration gebunden ist wie die biologische Aktivität
des Chloramphenicols an die D-threo-Form. Für die
Untersuchungen standen die Stereoisomeren des PNPG
nicht zur Verfügung, wohl aber Stereoisomere der
Chloram phenicolbase7, die sich vom PNPG dadurch
unterscheidet, daß eine Hydroxyl-Gruppe am C-Atom 2
durch eine Amino-Gruppe ersetzt ist.
D ie zur Verfügung stehenden Isomeren der Chlor­
amphenicolbase wurden in gleicher Weise wie für an­
dere Zusätze an anderer Stelle 8 beschrieben in abge­
stuften Konzentrationen einem einfachen FleischpeptonAgar ( 1%, pH 7,2) zugesetzt. Geprüft wurde die
schwärmhemmende 8 und die bakteriostatische Wirkung
verschiedener Konzentrationen dieser Zusätze auf einen
aus menschlichem Urin isolierten Stamm von P roteus
m irabilis 9. Die Konzentrationen, die notwendig waren
um einerseits das Schwärmen und andererseits das
Wachstum des untersuchten Proteus-Stammes vollstän­
dig zu hemmen, zeigt die Tab. 1.
Der Versuch zeigt, daß die Stärke der schwärmhemmenden Wirkung des l-p-Nitrophenyl-2-amino-propan1.3-diols maßgeblich von der sterischen Konfiguration
an den beiden asymmetrischen C-Atomen abhängt. Wie
beim Chloramphenicol, so ist auch hier die d-threoVerbindung am wirksamsten. Während beim Chlor­
amphenicol die L-threo-Verbindung nur 0,5% der W irk­
samkeit der D-threo-Form zeigt und die beiden Erythro6 R. E. M a x w e l l u . V. S. N ic k e l , Antibiotics and Chemo­
therapy, 4 , 289 [1954].
7 Freundlicherweise zur Verfügung gestellt von H errn Dr.
E. H a a c k , C. F. Boehringer & Söhne GmbH, M annheim .
The D issociation of R eticulocyte R ibosom es
G eorg
R.
P h il ip p s *
Division of Biology, California Institute of Technology
Pasadena, California
(Z. Naturforschg. 20 b, 930— 932 [1965] ; ein gegan gen am 11. Juni 1965)
It is now generally accepted that ribosomes are the
primary site of the amino acid polymerization during
protein synthesis. In the active state they appear as
polysomes: clusters of single risosomes (monosomes)
joined together by a mRNA molecule. Each monosome
is composed of two subunits (60 s and 40 s) which dif­
fer in size and function. Bacterial ribosomes are easily
dissociated into subunits upon removal of M g2® ions;
but mammalian ribosomes require chelating agents for
* Supported by AEC G rant At (04-3) 51 and U SPH G rant
HE-01624-14.
B akterio- Schw ärm sta tisc h e hem m K onz.
K onz.
[-m.]
[-m.]
D-<Areo-l-p-nitrophenyl-2a m in o -p ro p a n -1.3-diol
L -JA reo-l-p-nitrophenyl-2am ino-propan-1.3-diol
l.-eryth ro -l -j?-nitrophenyl-2a m in o -p ro p a n -1.3-diol
D'L-erythro- 1 -p-nitrophenyl-2a m in o -p ro p an -1.3-diol
1 -^ -N itro p h en y l-p ro p a n 1 .2.3-triol ( P N P G )
D-iAreo-Chloram phenicol
0,005
0,0004
0,01
0,005
0,1
0,005
0,03
0,003
0,05
0,0001
0,0002
Tab. 1. Erforderliche Konzentrationen zur vollständigen
Hem mung des Schwärmens und des Wachstums des ProteusStammes.
Isomeren ganz unwirksam sind, zeigen die L-threoForm und i-erythro-F orm der freien Chloramphenicol­
base noch etwa 10% der schwärmhemmenden Wirkung
des D-£/ireo-Derivats.
Keines der untersuchten Isomeren des 1-p-Nitrophenyl-2-amino-propan-1.3-diols erreicht die Wirksam­
keit des PNPG. Wichtig für die volle schwärmhem­
mende Wirksamkeit scheint die Hydroxylgruppe am
C-Atom 2 zu sein.
Ein Vergleich der W irkungen von PNPG und d i/ireo-Chloramphenicol zeigt, daß PNPG bei hoher
schwärmhemmender Wirksamkeit (0,0002-m.) das
Wachstum nur mäßig hemmt (0,05-m .), während d fAreo-Chloramphenicol bei starker Wachstumshem­
mung ( 0 ,0001 -m.) keine isolierte schwärmhemmende
W irkung erkennen läßt.
8 R.
9 R.
K o pp,
Z. Hyg. Infektionskrankh. 148, 501 [1962].
J. M ü l l e r , Appl. Microbiol., im Druck.
K o pp u .
a comparable dissociation. We wish to report here the
successful dissociation of reticulocyte ribosomes without
chelating agents and the influence of the growing pep­
tide chain (peptidyl-sRNA) on this dissociation. A full
report of this research will be published elsewhere
( P h i l i p p s , in preparation).
The preparation and purification of ribosomes from
reticulocytes, incubated in vitro with radioactive leu­
cine, has been d escrib ed 1. If these ribosomes were
dialysed against 0.01 M tris-HCl (ph 7 .2 ), 0.04 M
KC1, 3 x l O - 5 M M g2® for 16 hours and centrifuged
for 5 hours through a 20 to 5% sucrose gradient in the
same buffer at 25,000 rpm, the sedimentation profile
usually showed a single peak. No differences were
noticed, if M g2® ions were omitted. The same ribo­
somes, dialysed against tris buffer containing 0.1 M
1 G. R.
P h ilip p s ,
Nature [London]
205,
567 [1965].
Unauthenticated
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931
NOTIZEN
NaCl and 3 x l O - 5 M Mg2®, gave a quite different
Sedimentation profile. This is shown in F ig. 1. W hile
the main peak (60 s) is present under both conditions,
two more fractions on the heavier side of the gradient
are seen at 0.04 M KC1 (solid lin e ). The arrow at 80 s
indicates the persistence of undissociated monosomes.
Fig. 2. Sucrose gradient of ribosomes from reticulocytes,
labeled in vitro with leucine-H3. One ml ribosomes dialysed
against solution Gt were centrifuged as described in Fig. 1.
16 and 4 drops were collected alternately. The radioactivity
(16 drops) was determined in a Nuclear Chicago scintilla­
tion counter with 15 ml B r a y ’ s solution, the optical
density (4 drops) was m easured after dilution with 0.4 ml
of distilled water. — • — • — optical density at 260 m [X
— O— O— radioactivity in cts per min.
Fig. 1. Superimposed gradients of ribosomes after dialysis
against 0.01 M tris-HCl (ph 7 . 2 ) , 0.04 M KC1 (solution M0)
and 0.01 M tris-HCl (p h 7 .2 ) , 0.1 M NaCl, 3 -1 0 “5 M Mg2®
(solution G j). One ml ribosomes were layered onto a 28 ml
20 to 5% (w/v) sucrose gradient and centrifuged 5 hours at
25 00 0 rp m in a SW 25 rotor a t 4 °C. Fractions were ob­
tained by drop- counting and the optical density determ ined.
The position of 80 s material was confirmed by com­
parison with a control gradient of ribosomes dialysed
against high Mg2® concentration, in which the mono­
somes are preserved. A second heavier fraction is in­
dicated by the shoulder on the slope of the 60 s p ea k ;
this material ( > 60 s) represents an intermediate of the
dissociation found also in liver ribosomes 2. The sm al­
ler subunit (40 s) is absent in 0.04 M KC1. The dis­
sociation pattern at 0.1 M NaCl (dashed line) de­
monstrates the separation into two subunits, with al­
most no material of a sedimentation constant greater
than 60 s. The same pattern with two clearly separated
subunits was also obtained after dialysis against trisbuffer at 0.04 M KC1 and 1 x 1 0 -4 M versene.
In Fig. 2 the distribution of the radioactivity in
peptidyl-sRNA after dissociation at 0.1 M NaCl is
shown. W hile most of the peptidyl-sRNA is released
from the ribosomes, that remaining is found associated
with both subunits. U sually approximately 50 percent
of the label was found in the 4 s position of sR NA , but
differences were noted with ribosomes from several
batches of reticulocytes. If the same ribosomes were
dialysed in the presence of 0.04 M KC1, less than
2 Y. Tashiro u. P. Siekevitz, J. molecular Biol. 11, 149
[1 9 6 5 ].
20 percent of the counts were released. The radio­
activity bound to the incompletely dissociated ribo­
somes (compare Fig. 1, solid line) was found for the
most part in the heavy material (!> 60 s and 80 s ) . We
compared the sedimentation profiles of several gra­
dients performed with ribosomes from various incuba­
tions. W hile in 3 x 1 0 -5 M Mg2® plus 0.1 M NaCl
(Solution Gx) a complete dissociation of the ribosomes
was always obtained, changing the NaCl concentration
to 0.04 M gave variable results. Some preparations
gave a curve like the one shown for Gx in Fig. 1;
others gave sedimentation profiles intermediate between
the two curves shown in Fig. 1, with very little material
in the 40 s position. In the latter cases a great per­
centage of the labeled peptidyl-sRNA was found in
those fractions denser than 60 s. These results suggest
that the failure to release the growing peptide chain
from the ribosomes is responsible for the incomplete
dissociation of reticulocyte and liver 3 ribosomes. Con­
trol experiments had shown that actually all the label
in purified ribosomes is associated with the growing
peptide chain.
Since the amount of radioactivity does not allow an
estimation of the peptide chain length, these experi­
ments were repeated with ribosomes labeled in the
terminal trinucleotide of the sRNA. By comparing the
radioactivity of the H3-labeled sRNA moiety with the
C14-labeled peptidyl chain, and knowing that 17 leucyl
residues should be present in the complete chain, an
estimation of the length of the peptide chain (ratio
H 3 : C14) should be possible. It is known that the
CpCpA end of the sR N A turns over by an enzymatic
mechanism independent of the polymerase-directed
3 F. O. W e t t s t e i n
[1965].
u.
H.
N o ll,
J. molecular Biol. 11, 35
Unauthenticated
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NOTIZEN
932
R NA synthesis. If ribosomes labeled in the sRNA
moiety were dissociated, most of the radioactivity was
released, while the remaining label was associated with
both subunits. By comparing the ratio of H 3 to C 14
labeled peptidyl-sRNA bound to ribosomal subunits the
conclusion was reached that the longer the peptidyl
chain the more tightly peptidyl-sRNA is bound to the ri­
bosomes. Furthermore, they suggest that the peptidyl
moiety rather than the sRNA molecule is responsible
for the binding of the growing peptide chain to the
ribosomes. This was confirmed by treatment of dis­
sociated and undissociated ribosomes with RNase. In
neither case was an increase in the radioactivity re­
leased from ribosomes obtained.
Using the poly-U directed synthesis of polyphenyl­
alanine in a E. coli cell-free system, G i l b e r t 4 has
shown that the growing peptide chain is exclusively
bound to the major subunit. These results could not be
confirmed by our experiments. The reason (s) for the
differential binding of polyphenylalanyl-sRNA and
the natural intermediate of hemoglobin is (are) not
4
W.
G il b e r t ,
J. m olecular Biol. 6, 389 [1963].
known, either the chain length and/or the composition
of the polypeptide may be responsible for this.
A comparison of bacterial and mammalian ribosomes
shows that the latter are more stable in low Mg2® ion
concentrations. One of the reasons for the stability of
mammalian ribosomes, as shown here, seems to be the
presence of the growing peptide chain. The release of
labeled material after dissociation is far more complete
in bacteria 5 than in reticulocytes. The two species dif­
fer, furthermore, with respect to the stability of the
mRNA-ribosome complex: by removal of Mg2® ions
from bacterial ribosomes, mRNA can be separated
from the subunits 5. This is not possible with reticulo­
cyte ribosomes ( P h i l i p p s , unpublished d ata). Our
studies suggest that after dissociation with chelating
agents, mRNA remains attached to the smaller sub­
unit, while after dialysis in 0.04 M KC1 and low Mg2®
concentration, polysome-like material is still present.
In conclusion, our results indicate that the complex of
mRNA-ribosomes-peptidyl-sRNA in mammalion cells is
more stable than in bacteria, but that the differences
are more quantitative than qualitative.
5 D.
S c h l e s s in g e r
u
.
F r a n c o is e G r o s ,
J. m olecular Biol. 7,
350 [1963].
Nachdruck — auch auszugsw eise — nur m it sch riftlich er G enehm igung des V erlages g esta ttet
Verantwortlich für den In h a lt: H . F r i e d r i c h - F r e k s a
G esam th erstellung: K onrad T riltsch, W ürzburg
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