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TRANSCRIPT
INHIBITION OF RNA SYNTHESIS BY ct-AMANITIN 1119
ner waren. Lediglich der Gehalt an Chlorophyll b war auch prozentual erheblich geringer.
Elektronenmikroskopische Arbeiten haben ergeben, daß das Lamellarsystem der Chloropiasten z. T. aus den Plastidenzentren der Etioplasten hervorgeht. Andererseits können unter bestimmten Bedingungen
29 M. W r i s c h e r , Z. Pflanzenphysiol. 55, 296 [1966].
Teile des Lamellarsystems der Chloropiasten wieder in Plastidenzentren umgewandelt werden 29. Die chemischen Analysen haben nun gezeigt, daß in den Plastidenzentren der Etioplasten und im Lamellarsystem der Chloroplasten die gleichen farblosen Lipide Vorkommen. Ihr Mengenverhältnis ist jedoch in den beiden Formen des Membransystems verschieden.
Inhibition of RNA Synthesis by a-Amanitin in VivoJ. N i e s s i n g , B. S c h n i e d e r s , W. K u n z , K. H. S e i f a r t , and C. E. S e k e r i s
Departments of Physiological Chemistry and Pharmacology, University of Marburg, Marburg/Lahnberge, W. Germany
(Z. Naturforsch. 25 b, 1119— 1125 [1970] ; eingegangen am 12. Juni 1970)
Injection of a-amanitin inhibits both the synthesis of ribosomal and DNA-like RNA as measured by the incorporation of radioactively labelled orotic acid and oriAo-phosphate into RNA extracted by phenol and subsequently fractionated by density gradient centrifugation and MAK column chromatography.
a-amanitin, a very potent and toxic cyclic peptide isolated from the toadstool amanita phalloides 1, inhibits RNA synthesis either after injection into m ice2 or after addition to isolated mouse or rat liver nuclei 2’ 3 and it has been recently demonstrated that the toxin exerts its effect by acting directly on the enzyme RNA polymerase3_6. Enzymes from mammals and yeast7 are susceptible to the toxin whereas bacterial polymerase is not affected at all 3-6. In addition it could be shown that neither the binding of the enzyme to the DNA template nor initiation is effected, but that the chain elongation step is probably completely arrested. K e d i n g e r et al. 6 recently isolated two species of RNA polymerase from calf thymus, only one of which is susceptible to amanitin. Similary R o e d e r and R u t
t e r 8 separated RNA polymerase from rat liver into two main components one of which they attributed to a nucleolar RNA polymerase 9 and the other to
Reprints request to Dr. J. N ie s s in g , Dept, of Physiological Chemistry, Univ. Marburg, D-3550 M arburgjLahn, Lahnberge.
1 T h . W i e l a n d , Science [Washington] 159, 946 [1968].2 F . S t i r p e and L. F iu m e , Biochem. J. 105, 776 [1967].3 K. H. S e i f a r t and C. E. S e k e r i s , Z. Naturforsch. 24 b.
1538 [1969].4 F . N o v e l l o and F . S t i r p e , Biochem. J. 112, 721 [1969].5 S . T. J a c o b , E. M . S a j d e l , and H. N. M u n r o , Nature
[London] 225,60 [1970],
the polymerase of the nucleoplasm. According to J a c o b et al. 10 only the latter polymerase is inhibited by amanitin whereas the nucleolar enzyme is fully resistent to the toxin. On the basis of these findings, the above mentioned authors concluded that amanitin does not impair the formation of ribosomal RNA.
To gain more insight into the nature of the RNA(s) inhibited after administration of amanitin in vivo we have analysed ribonucleic acid synthesized in rat liver by phenol extraction at different temperatures, by density gradient centrifugation and MAK column chromatography. The results of these experiments are presented below.
M aterials and M ethods
a) Animals and ChemicalsMale Wistar BR II rats (130 — 200 g) were ob
tained commercially. 6-14C-orotic acid (44.5 mCi/
6 C . K e d i n g e r , M . G n ia z d o w s k i , J . L. M a n d e l , F . G is s in - g e r , and P. C h a m b o n , Biochem. biophysic. Res. Commun. 38, 165 [1970],
7 S. D e z e l e e , A. S e n t e n a c , and P. F r o m a g e o t , FEBS letters 77, 220 [1970].
8 R . G . R o e d e r and W. J . R u t t e r , Nature [London] 224, 234 [1969].
9 R . G . R o e d e r and W. J . R u t t e r , Proc. nat. Acad. Sei. USA 65, 675 [1970].
10 S . T. J a c o b , E. M . S a j d e l , and H. N. M u n r o , Biochem. biophysic. Res. Commun. 38, 765 [1970].
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1120 J. NIESSING, B. SCHNIEDERS, W. KUNZ, K. H. SEIF ART, AND C. E. SEKERIS
mmole), 5-3H-orotic acid (14.3 Ci/mmole) and carrier- free 32P-or£/io-phosphate were purchased from the Radiochemical Center (Amersham). Bentonite, sodium- dodecylsulfate and potassium-polyvinylsulfate were products of Serva, Heidelberg. a-Aminitin was generously supplied by Prof. T h . W i e l a n d (Max-Planck-Institut, Heidelberg).
b) Radioactive labelling and preparation of RNA
If not stated otherwise, a-amanitin was injected intra- peritoneally 3 hrs prior to sacrifice at a dose of 1 /ug/g body weight; control rats received an injection of 0.065 m Tris-HCl pH 7.9. 3H-orotic acid (lOOjitCi/ 100 g body weight), 14C-orotic acid (30 /zCi/100 g) or 32P-oriAo-phosphate (1 or 2 mCi/rat) were administered intraperitoneally at the times appropriately indicated for each experiment. Rats were killed by cervical dislocation, the livers were homogenized in 5 vol. of buffer (0.05 M Tris-HCl, pH 7.55 containing 0.25 M sucrose 0.025 M KC1, 0.01 M MgCl2) and a cytoplasmic supernatant fraction was obtained after centrifugation of the homogenate at 800 x g. The nuclear pellet was resuspended in extraction buffer (0.05 sodium acetate pH 5.0, 0.14 M NaCl, 150 /ug polyvinylsulfate/ml, 4 mg bentonite/ml, 5 mg sodium dodecylsulfate/ml). The RNA was extracted at 65° after the addition of an equal volume of water saturated phenol as previously described n . If a thermal fractionation 12 was required, the livers were homogenized directly in extraction buffer without prior separation of cytoplasmic supernatant and nuclear pellet and phenol extraction was subsequently conducted at two temperatures (4°; 65°). The final aqueous layers were washed with ether, then the RNA was precipitated by the addition of 2 vol. of ethanol and stored at —20 °C for at least 3 hours.
c) Sucrose density gradient analysis of RNA
The RNA, dissolved in distilled water (2 —3 mg per sample), was layered on 28ml of 15 — 30% sucrose gradient (prepared in 0.05 M sodiumacetate pH 5.0 containing 0.14 M NaCl and 1 mM EDTA) and ultracentrifuged in a SW25.1 rotor. Fractionation of the sucrose gradient and measurement of radioactivity was performed as previously described 13.
d) MAK-column chromatography of RNA
This was accomplished as described earlier14. Equal amounts of RNA from control and amanitin- treated animals were applied to the columns which were then eluted with a linear 0.1 —2.0 m NaCl gradient in 0.05 M phosphate buffer followed by a wash with 1 M ammonium hydroxide.
11 G . S c h ü t z , D. G a l l w i t z , and C. E. S e k e r i s , Europ. J.Biochem. 4, 149 [1968].
12 G . P. G e o r g ie v and V. L. M a n t i e v a , Biochim. biophysicaActa [Amsterdam] 61,153 [1962].
13 J. N ie s s in g and C. E. S e k e r i s , Biochim. biophysica Acta[Amsterdam] 209,484 [1970].
g) General assays
Protein was determined according to L o w r y et al. 15 The concentration of RNA was estimated spectrophoto- metrically at 260 nm (25 A2eo units correspond to 1 mg RNA/ml). Radioactivity was measured as follows: an aliquot of each RNA-containing sample was precipitated on paper discs (Schleicher and Schuell, 2043 b) in ice cold 5% HC104 , washed several times with 5% HC104 , then with ethanol and ether. If the RNA had been labelled with 32P-orMo-phosphate, the paper discs were washed with 5% HC104 contaning 20 mM H3P 0 4 . The radioactivity of the dried paper discs was counted in toluene (5 g PPO, 200 mg POPOP//) with a Nuclear Chicago liquid scintillation counter.
R esults
In a first series of experiments, rats treated with amanitin for a total of 3 hrs ante mortem were administered 14C- and 3H-orotic acid 120 min and 10 min before sacrifice. Subsequently, total RNA was prepared from the liver by phenol extraction at 65° in the presence of 0.5% SDS from control and amanitin treated animals followed by measure-
4-5S 285 18S
Fractions -----■-
Fig. 1. Effect of amanitin on the sedimentation profile of the RNA extracted from a whole rat liver homogenate at 65°. RNAfrom untreated animal: 3H-labelled RNA (10 min pulse) • —• , 14C-labelled RNA (120 min labelling time ) o — o. RNA from amanitin treated animal: 3H-labelled RNA (10 min pulse) ■ — ■. 14C-labelled RNA (120 min labelling time) □ - - - Density gradient centrifugation was carried out on a 15 — 30% sucrose gradient in a SW 25,1 rotor of a Spinco
L II model at 23000 rev./min for 13 hours.
14 W. K u n z , J. N ie s s in g , B. S c h n i e d e r s , and C. E. S e k e r is , Biochem. J. 116. 563 [1970].
15 O. H. L o w r y , N . J. R o s e b r o u g h , R . L. F a r r , and R . J. R a n d a l l , J. biol. Chemistry 193, 265 [1951].
INHIBITION OF RNA SYNTHESIS BY a-AMANITIN 1121
ment of its specific activity. In the short pulse a 10% inhibition of incorporation of 3H-orotic acid into RNA of the amanitin treated animals was observed whereas in the long pulse, the inhibition amounted to 80 percent. The RNA was then submitted to sucrose gradient centrifugation. The results (Fig. 1) of the short pulse demonstrate a slight inhibition of labelling in the 45 — 10 S part of the gradient whereas in the long pulse, the RNA is inhibited throughout the gradient.
Rats treated with amanitin for 3 hrs as above were pulsed for 15 and 90 min with 3H- and 14C-
orotic acid respectively. RNA was subsequently extracted from a crude nuclear preparation and from the 800 g cytoplasmic supernatant with phenol at 65° in the presence of SDS and submitted to density gradient centrifugation. Measurement of the specific activity showed that the inhibition of RNA labelling by amanitin was 15% and 65 — 70% for the short and long pulse respectively both for the nuclear and the cytoplasmic fractions. The results of a typical centrifugation analysis of the RNA extracted from the nuclear pellet is depicted in Fig.2 a and demonstrates, that RNA synthesis after a
4 5 S 26S IBS1 1 1 Fig. 2 a
F ra c tio n No.
Fig. 2 b
Fig. 2. Effect of amanitin on the sedimentation pattern of the RNA extracted from rat liver nuclear sediment (a) and cytoplasm (b). RNA from control animals: 3H-labelled RNA (15 min pulse) • — 14C-labelled RNA (120 min labelling time) o - - - O . RNA from amanitin-treated animals: 3H-labelled RNA (15 min pulse) ■ 14C-labelled RNA (120 min label
ling time) □ — □ .
6Q.o
Fract ions
1122 [.N IESSING , B. SCHNIEDERS, W. KUNZ, K. H. SEIFART. AND C. E. SEK.ERIS
long pulse is significantly depressed by amanitin treatment especially in the 45, 28 and 18 S region of the gradient, whereas the 4 — 10 S RNA is not inhibited to the same extent. In contrast, RNA synthesized within the short pulse is effected to a lesser degree. A larger series of experiments, of which only one is shown in Fig. 2 a, has revealed (see also Fig. 1) that the short-pulse RNA with a sedimentation coefficient of over 50 S is only slightly inhibited, whereas the region between 45 and 10 S is clearly depressed. As for the long pulse, the4 — 10 S RNA is least effected by the toxin.
From the results of the gradients containing the cytoplasmic RNA it is clear (Fig. 2 b) that admini- . stration of amanitin resulted in almost no inhibi- | tion of the appearance of label in the 5 — 10 S re- £ gion during the short pulse. However, a completely Y different picture emerges from the results of theit long pulse (Fig. 2 b). In this case labelling of the cytoplasmic 28 and 18 S RNA is completely abolished after amanitin treatment and the 4 — 10 S region, although inhibited, is effected to a lesser extent. It is therefore clear that amanitin administration completely blocks the appearance of 18 and 28 S ribosomal RNA in the cytoplasm.
In order to establish whether the lesser degree of inhibition mediated by amanitin during the short orotate pulses is due to the long time interval between injection of amanitin and injection of the precursor (180 — 15 = 165 min) or whether this is due to a preferential effect on the ribosomal RNA, rats were pretreated for only 90 min with amanitin and then pulsed for 15 min with 3H-orotic acid. In this case a 50% inhibition of cytoplasmic RNA could be observed, indicating that the amanitin inhibition was more pronounced with a shorter time interval between application of the toxin and the orotic acid pulse. To elucidate this point more clearly and to investigate whether the formation of ribosomal RNA was less effected if longer periods of time were allowed to elapse after amanitin treatment, the toxin was administered to rats 7 hrs ante mortem. 3H- and 14C-orotic acid was then pulsed at 30 min and 120 min before sacrifice respectively and total liver RNA was then extracted with phenol at 4° to preferentially analyse ribosomal and transfer RNA. Measurement of the specific activities showed inhibition figs. of 16% and 37% for the 30 min 3H- and 120 min 14C-pulse respectively. Comparable figs. for an amanitin treatment period
of 3 hrs (experiments of Fig. 3) are 10% and 80% respectively, which suggest a time-dependence of the effect as pointed out before. However, the results of the centrifugation analysis (Fig. 3) show, that even after the very long period of 7 hrs, no labelling of ribosomal RNA occurs, whereas the synthesis of transfer RNA is much less impaired.
F ra c tio n s —►
Fig. 3. Effect of amanitin treatment for 7 hrs on the sedimentation pattern of the RNA extracted from a whole rat liver homogenate at 4°. RNA from control animals: 3H-labelled RNA (30 min pulse) • —• , 14C-labelled RNA (120 min pulse) O - - - o. RNA from amanitin treated animals: 3H-labelled RNA (30 min pulse) ■ —■, 14C-labelled RNA (120 min
pulse) □ — □ .
In another series of experiments 32P-oriAo-phos- phate was injected to animals pretreated with amanitin for 90 minutes. Ninety min thereafter, the rats were sacrificed, RNA was isolated from a crude nuclear and a 800 g liver supernatant fraction and analyzed by density gradient centrifugation and MAK-column chromatography. Both the labelling of the nuclear (59% inbilition) and of the cytoplasmif RNA (28% inhibition) is impaired. Sucrose gradient centrifugation of the nuclear RNA (Fig. 4 a) reveals that the 45 and 28 S RNA is inhibited more in comparison to the RNA heavier than 50 S Avhereas the 18 and 10 S regions of the gradient are effected to a lesser extent. The cytoplasmic 28, 18 and 4 — 10 S RNA (see Fig. 4 b) are inhibited by 85, 55 and 35% respectively. MAK column chromatography (Fig. 5 a) reveals an almost total inhibition of the nuclear fractions 45 — 61 corresponding to the 18 — 30 S RNA with a concomitant inhibition of the RNA eluting with the 1 M ammonia fraction, which is composed mostly of DNA-like
- cp
m
INHIBITION OF RNA SYNTHESIS BY a-AMANITIN 1123
75 30 45 60 75 90 705 120 F ra c tio n s
—-----------0.7- 2.0M - A/o C /----------------- -M-AWy—
Fig. 5. MAK-column chromatography of nuclear (a) and cytoplasmic RNA (b) from untreated and amanitin-treated rats after labelling with 32P-ort/io-phosphate (2 mCi per animal) for 90 minutes. RNA from control rats: • — RNA from
amanitin-treated rats: ö —Q .
65° was inhibited to 75% whereas the 4° fraction
was depressed to approximately 39 percent. As is
seen from the gradient centrifugation of the 4° RNA
(Fig. 6 a), most of the label sediments in the region
between 4 and 18 S with only a small proportion
of 28 S RNA being present. Inhibition by amanitin
is 63, 15 and 30% for the 28, 18 and 4 — 10 S part
of the gradient respectively. In the 65° fraction
(Fig. 6 b) all the RNA species are almost totally
inhibited after amanitin treatment with the excep
tion of the 4 — 10 S RNA.
3000
2400
1200
J---- 1---- !_____ I_____ I_____ I_5 10 15 20 25 30
F ra c tio n s —►
Fig. 4 a
220C
170015001400
Fig. 4. Sedimentation profile of nuclear RNA (a) and cytoplasmic RNA (b) from control and amanitin-treated animals after labelling with 32P-or/Ao-phosphate (2 mCi per animal) for 90 minutes. RNA from control rats • — • , RNA from ama-
tin-treated rats EH — CH-
RN A 14. The MAK column chromatography (Fig.
5 b) of cytoplasmic RNA demonstrates an almost
complete inhibition of ribosomal RNA (Fractions
54 — 6 6 ) with the 1 M ammonia fraction being de
pressed to 75% whereas the synthesis of transfer
RNA is only inhibited to 35 percent.
Finally, a 60 min 32P-orJAo-phosphate pulse was
given to animals treated with amanitin for a total
of 3 hours. The RNA was then extracted from the
liver by phenol extraction first at 4° and then at
65°. As is well documented, extraction at 4° yields
transfer and ribosomal RNA whereas extraction at
high temperatures yields the DNA-like RNA and the
ribosomal precursor RNA. The RNA extracted at
1200
t1 10006cl« 600 ia.C\J
<3 600
400
200
Fig. 5 a
30 45 60 75 F ra c tio n s
- 0.1♦ 2.0 M -N a C l-----
-1
90 105 120
-- M -N H3- »
5 10 15 20 25 30 F ra c tio n s —►
t' N
ECL
10.000 6 000
6 000 4000 2000
1124 J. NIESSING, B. SCHNIEDERS, \K. KUNZ, K. H. SEIFART. AND C. E. SEKERIS
4-5S 28S IBS
1 1 I4-500 - i \
/ io 13500 - I o
5 10 15 20 25 30Fractions — ►
Fig. 6 a
45 S 28S 18S
Fractions -- »-242/70 N ie s s m g r ig . 6b
Fig. 6. Effect of amanitin on the sedimentation profile of the RNA extracted from rat liver homogenate after labelling with 32P-orf/>o-phosphate (1 mCi per rat) for 60 minutes. RNA was extracted at either 4° ((Fig. 6 a) from control (o — o) and amanitin treated rats (Q — □) or at 65° (Fig. 6 b)
from control ( • — • ) and amanitin treated rats (■ — ■).
Discussion
From the experiments presented above it is evi
dent that a-amanitin inhibits the synthesis of both
ribosomal as well as DNA-like RNA as characterized
by sucrose density centrifugation and MAK column
chromatography. The synthesis of transfer RNA is
effected only to a much lesser extent. In the long
orotic acid pulses an almost complete inhibition of
nuclear RNA synthesis is seen involving all RNA
species. No labelled ribosomal RNA whatever finds
its Avay into the cytoplasm and it is therefore justi
fied to conclude that the synthesis of this RNA
species is impeded by a-amanitin in vivo.
The degree of inhibition of RNA synthesis de
pends on the period of pretreatment of the animals
with amanitin. The appearance of labelled RNA
after a 15 min orotic acid pulse is inhibited up to
50% if administered to animals treated for 90 min
with amanitin whereas in animals treated with the
toxin for 165 min the depression is only about
10 — 15 percent. The same tendency is observed in
the 120 min orotate pulses, where amanitin treat
ment periods for a total of 3 and 7 hrs results in
80% and 35% depression of RNA synthesis. These
results suggest that the animals can probably in
activate the toxin with a resulting escape-synthesis
occuring. However, even after a 7 hr treatment period
no labelled ribosomal RNA is found in the cyto
plasm, indicating that the recovery synthesis does
not selectively involve the ribosomal species.
The results reported here after in vivo administra
tion of a-amanitin are at variance with the in vitro
findings of other authors 6-10 who reported that the
RNA polymerase from nucleoli is insensitive to
a-amanitin in vitro. The differentiation of the two
enzymes which are supposedly associated with
either the nucleolus9 (Form I) or extranucleolar
region (Form II) was initially achieved6,9 by their
different chromatographic behaviour on DEAE-
Sephadex (or cellulose) and their different response
to Mg20 (Form I) or Mn23 (Form II) as well as
their optima to synthesize RNA in vitro under con
ditions of low (Form I) or high (Form II) ionic
strength 9.
If these in vitro findings could be transferred to
the in vivo situation, they would imply, that the syn
thesis of ribosomal RNA should remain unimpaired
after amanitin treatment. However, initial microsco
pic observations by F iu m e and L a s c h i 16 and elec
tron-microscopic investigations conducted in this
laboratory17, revealed that the morphological ar
chitecture of the nucleolus tends to disintegrate com
pletely after in vivo application of a-amanitin, sug
gesting that synthesis of ribosomal RNA is like
wise impeded. The discrepancy between our in vivo
findings and the in vitro experiments reported by
others warrants further consideration. One possible
explanation could be a requirement for coordinated
16 L . F ium e and R. L a s c h i, Sperimentale 113, 288 [1965],17 Unpublished observations.
BIOSYNTHESE DER PENICILLINE 1125
synthesis of DNA-like RNA and ribosomal RNA.
Depression of the former would then lead to a
secondary inhibition of ribosomal RNA synthesis,
thus overshadowing the a-amanitin resistance of
the enzyme responsible for its synthesis. There is
however, little experimental evidence available at
present, supporting this hypothesis. Another alter
native, which encompasses an in vivo transforma
tion of a-amanitin to a metabolite capable of ef
fecting the nucleolar RNA polymerase, should also
be considered and cannot be resolved at present.
The availability of radioactively labelled amanitin
should render such studies feasible.
During transcription in vivo, it is conceivable that
the enzyme could be modified by dissociation of, or
association with, an enzyme component responsible
for the altered amanitin susceptibility of the
enzyme. At the present state of our understanding
of the transcriptional process and its control in
higher organisms, it is premature to ascribe a cor
rect explanation to the observed phenomena. How
ever, the reported experiments exclude the use of
a-amanitin in the intact organism to eliminate spe
cifically the synthesis of DNA-like RNA and may be
interesting and useful to investigators in other labo
ratories attempting to study the posttranscriptional
processing of RNA synthesized in vivo.
We wish to thank Professor P. K a r l s o n and W. S c h m id for their continued interest and encouragement and Miss H . R a d l e r , Mrs. U. P a n k o v , Mrs. C h .
H a e n is c h and Miss G. F r ö h l i c h for competent technical assistance. The Deutsche Forschungsgemeinschaft has provided financial support.
Zur Biosynthese der Penicilline: Bildung von 5-(2-Aminoadipyl)- cysteinyl-valin in Extrakten von Penicillium chrysogenum
Formation of 5- (2-Aminoadipyl) -cysteinyl-valin by Extracts of Penicillium
chrysogenum
K a r l B a u e r
Isotopenlaboratorium der Universität Stuttgart
(Z. Naturforsch. 25 b, 1125— 1129 [1970] ; eingegangen am 25. April 1970)
Mycel-extracts of P. chrysogenum catalyse the formation of 5-(2-aminoadipoyl)-cysteinyl-valin out of its aminoacid components.
For the synthetical preparation of this compound the solid-phase method is particularly suited. The synthesis can be carried out even with very small quantities of substance and thereby allows the preparation of radioactive labelled L-5-(2-aminoadipoyl)-L-cysteinyl-L-valin of relatively high specific activity.
Die Penicilline gehören zur Gruppe der Peptid-
antibiotica. Sie werden von P. chrysogenum aus den
Aminosäuren Cystein und Valin, die das 3-Lactam-
thiazolidin-Ringsystem (6-Aminopenicillansäure)
bilden, und einem Seitenketten-Precursor aufge
baut 1.
Der Mechanismus der Biosynthese der Penicil
line ist noch weitgehend unbekannt, obwohl er seit
langem Gegenstand umfangreicher Untersuchungen
ist2. Von besonderem Interesse war dabei die Ent
deckung des 5-(2-Aminoadipyl)-cysteinyl-valin (1)
im Mycel von P. chrysogenum3, da es in enger
struktureller Beziehung steht zu dem bereits früher
aus dem Fermentationsmedium von Cephalosporium
isolierten Penicillin N 4 und zu dem später in P.
chrysogenum entdeckten Isopenicillin N 3 (2), das
Sonderdruckanforderungen an Dr. K a r l B a u e r , Isotopenlaboratorium d. Chem. Institute d. T.H., D-7000 Stuttgart 1, Azenbergstr. 14 — 16.
1 H . R. V. A r n s te in , Annu. Rep. Chem. Soc. 54, 339 [1957].
2 S. Reviews: A. L. D em a in , in: Biosynthesis of Antibiotics, Vol. I, S. 30, I. F. S n e l l , Academic Press, New York, London 1966; E. P. A b rah a m , G. G. F. N e w to n , S. C. W a r
re n , in: Z. V ane k , Z. H o s ta le k , Biogenesis of Anti
biotic Substances, Czech. Acad. Sei., (distr. Academic Press, New York, London), Prag 1965.
3 H. R. V. A r n s te in and D. M o r r is , Biochim. biophysica Acta [Amsterdam] 35,561 [1959].
4 G. G. F. N e w to n , E. P. A b ra h am , and C. W. H a le , Biochem. J. 58,94 [1954].
5 E. H. F ly n n , M . H. M c C o rm ic k , M . C. S tam p e r, H. D e V a le r a , and C. W . G odzesk i, J. Amer. chem. Soc. 84, 4594 [1962].