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].