S. RAMACHANDRAN, D. GOTTLIEB
of phosphate uptake of yeasts in the presence of ascosin is probably the result of the antibiotic blocking the coupled electron transport mechanism. ACKNOWLEDGEMENT
These studies were supported in part by a grant (E-618) from the National Institutes of Health. REFERENCES 1 R.
HICKEY, C. J. CORUM, P. H. HIDY, I. R. COHEN, U. F. B. NAGER AND E. KROPP, Anti-
biotics and Chemotherapy, 2 (1952) 472. 2 W. W. UMBREIT, R. H. BURRIS AND I" F. STAUFFER, Manometric Techniques, Burgess Publishing Company, 1957. 3 K. AHMAD, H. G. SCHIEDER AND F. M. STRONG, Arch. Biochem., 28 (195o) 281. 4 M. M. NACHLAS, S. I. MARGULIES AND A. M. SELIGMAN, dr. Biol. Chem., 235 (196o) 27395 M. B. THORN, Biochem. J., 63 (1956) 420. e F. L. CRANE AND J. L. GLENN, Biochim. Biophys. Acta., 24 (1957) lO7. 7 M. DIXON AND E. WEBB, Enzymes, Academic Press Inc., Publishers, New York, 1958. 8 D. E. GREEN, Advances in Enzymol. Vol. 21 Interscience Publishers Inc., N e w Y ork, 1959, p. 73. o y . HENIS AND N. GROSSOWlCZ, J. Gen. Microbiol., 23 (196o) 345. 10 H. A. KREBS AND H. L. KORNBERG, Energy Trans/ormation in Living Matter, Springer-Verlag Berlin, 1957 .
Biochim. Biophys. Acta, 53 (1961) 391-396
M O D E OF A C T I O N OF A N T I B I O T I C S II. S P E C I F I C I T Y OF ACTION OF ANTIMYCIN A AND ASCOSIN S. R A M A C H A N D R A N
AND D. G O T T L I E B
Department o/Plant Pathology, University o/Illinois, Urbana, Ill. (U.S.A.) (Received March 3oth, 1961)
In a previous report it has been shown that the antibiotic ascosin inhibits the cytochrome linked electron transport system at the same site as does antimycin. This antimycin sensitive site lies between coenzyme Q or cytochrome b and cytochrome Cl. Both antimycin and ascosin exhibit no antibacterial activity. This inactivity of antimycin and ascosin against bacteria is due to the absence of the antimycin sensitive site from their electron transport systems. The possibilities of other factors such as impermeability and inactivation of the antibiotic by the bacteria have been eliminated. The present investigation also shows that the electron transport systems of fungi, higher plants (corn) and mammalian tissues are similar. Abbreviations : D I P, 2 , 6 - d i c h l o r o p h e n o l i n d o p h e n o l , TTC, 2,3,5 -triph enyltetrazoliu m chloride ; Fp, f l a v o p r o t e i n ; CoQ, C o e n z y m e Q; AS, antimycin sensitive site.
Biochim. Biophys. Acta, 53 (I96I) 396 4o2
OF A N T I M Y C 1 N
Differences in sensitivity of organisms to antibiotics might be the result of one or more factors. Some of these factors are: (a) differences of permeability, (b) di,~erences in structure and composition, (c) presence of alternate pathways for a metabolic function, (d) the absence of the sensitive enzyme site, (e) appearance of new pathways by adaptation, (f) the production of agents or enzymes that inactivate the antibiotic (e.g., Penicillinase). The antibiotic, antimycin A, has been shown to inhibit the electron transport mechanism at a site usually designated as AS 1. The heptaene antibiotic has been shown to block the coupled electron transport system at the same site as antimycin 2. Even though these two antibiotics block the most common of the coupled electron transport system, viz., the cytochrome linked electron transport, neither of them exhibit any antibacterial activity3, 4. SMIT~5 has suggested that the insensitivity of bacteria to antimycin A might be due to impermeability. The nature of the specificity of action of antimycin A and ascosin will be discussed in this paper. METHODS
Ascosin and antimycin A were gifts from Commercial Solvents Corporation, Terre Haute, Indiana, and Professor F. M. STRONG, Department of Biochemistry, University of Wisconsin, respectively. Cytochrome c was obtained from Sigma Chemical Company, and D P N H from Pabst Laboratories. Cell-free extracts from filamentous fungi: five grams wet wt. of mycelia from Sclerotinia fructicola, Gibberella zeae, Glomerella cingulata, and Phycomyces nitens which were grown separately as shake-cultures in glucose-yeast medium at 26 ° , were suspended individually in 12.o ml of o.66M sucrose-o.o5M Tris buffer, pH 8, and blended in a Waring blendor for 30 sec. The suspensions were then forced through a "French" pressure vessel at 15 ooo pounds/in. 2 and the resulting homogenates were centrifuged at 800 × g for 20 min. All these extracts were made at 4 °. Cell-free extracts from bacteria. Escherichia coli and Pseudomonas fluorescens were grown as a shake-culture for 24 h at 26 °. The cells were harvested by centrifugation, suspended in o.o2M potassium phosphate buffer, pH 7.0, and sonicated in a IO kc. Raytheon Sonic Oscillator for 5 min. These suspensions were then centrifuged at 18oo × g for 20 rain and the supernatant liquid used in the studies. Cell-flee extracts from rat liver and beef heart: fresh heart and liver tissues were ground individually with one time their weight of acid washed sand at 4 ° for 2 min. The macerated tissues were suspended in o.66M sucrose-o.o5 M, pH 6.8, Tris buffer and centrifuged at 12oo x g for 20 min. The supernatant liquid was used in the experiments. The D P N H cytochromec reductase and succinic-cytochromec reductase systems were assayed in a Beckman D.U. Spectrophotometer. Protein was estimated by the standard Biuret method. Respiratory studies were done by the standard Warburg technique s at 29.6 ° . The corn root mitochondrial preparation was kindly supplied by Dr. J. B. HANSEN of the Agronomy Department of the University of Illinois. The preparation was made following the method described by LUND, VATTER AND HANSEN7. Biochim. Biophys. Acta, 53 (1961) 3 9 6 - 4 o 2
s. RAMACHANDRAN, D. GOTTLIEB RESULTS
The effect of ascosin and antimycin A on yeast glycolosis and respiration are reported in paper I of the series 2. It will be noted that the antibiotics inhibited the oxygen uptake of the yeast cells in the presence of glucose and succinate. They inhibited the succinic-cytochrome c reductase system of such extracts. No inhibition was noted either on the reduction of D I P in the presence of succinate or on the cytochrome c oxidase system. Fig. I and Table I show that the D P N H - cytochrome c reductase of the four filamentous fungi are inhibited by ascosin and antimycin A.
~ 25 / ~"
Fig. i. Effect of a n t i m y c i n a n d ascosin on t h e D P N H c y t o c h r o m e c r e d u c t a s e s y s t e m in t h e cell-free e x t r a c t s of S c l e r o t i n i a /ructicola. The a s s a y s y s t e m is t h e s a m e as described u n d e r T a b l e I. S . / r u c t i c o l a e x t r a c t , 0. 3 rag. 0 - - 0 , c o n t r o l ; [ ] - - o n , + Ascosin; A - - - A , + A n t i mycin.
Time (sec) TABLE I EFFECT OF ASCOSIN A N D ANTIMYCIN ON THE D P N H - c Y T O C H R O M E C REDUCTASE SYSTEM IN THE CELL-FREE EXTRACT OF Gibberella zeae, P h y c o m y c e s n i t e n s , G l o m e r e l l a c i n g u l a t a AND IN CORN ROOT MITOCHONDRIA
The a s s a y s y s t e m c o n s i s t e d of t h e following: c y t o c h r o m e c, 15o m / t l n o l e s ; D P N H , I # m o l e ; p o t a s s i u m p h o s p h a t e buffer, p H 8.o, 6 0 / z m o l e s ; s o d i u m azide, i o # m o l e s ; ascosin, 7.5 # g ; a n t i m ycin , 0. 3 fig; G. zeae cell free e x t r a c t , 1.2 m g p r o t e i n ; P . n i t e n s , 1. 5 m g p r o t e i n ; G. cingulata, 0. 7 m g p r o t e i n ; corn root m i t o c h o n d r i a , 0.6 m g p r o t e i n ; a n d d i s t i l l e d w a t e r to m a k e up to 3.0 ml. mpmoles cytochrome c reduced/min Expt.
I 2 3 4 5
DPNH D P N H + Ascosin % i n h i b i t i o n b y Aseosin DPNH + Antimycin % Inhibition by Antimycin
45 .6 12.3 73 ii 76
45.3 8.3 81 II 76
Cor~ root mitochondria
32 .9 6.9 81 8.9 75
54 20 63 25 54
The oxygen uptake by the whole cells and cell-free extracts of the two bacteria are completely unaffected by either ascosin or antimycin A (Table II). The absence of inhibition of the cell-free extracts proves that the insensitivity of the bacteria to these antibiotics is not a question of permeability. E. coli is known to contain no detectable cytochrome c in its coupled electron transport 5. However, its respiration is cyanide sensitive, thus indicating the participation of a cytochrome oxidase (Table n). The cell-free extract also reduced TTC, which accept electrons from cytochrome oxidase s and D I P which accepts electrons from DPNH, Fp and CoQ (Table III). Mammalian cytochrome c, which served as electron acceptor in P. fluorescens and fungi (Tables I and III), was not reduced by E. coli in any assay system containing DPNH, buffer, cytochrome c, and enzyme preparation. From these data, it is conB i o c h i m . B i o p h y s . A c t a , 53 (1961) 396-402
OF A C T I O N
OF A N T I M Y C I N TABLE
A N D ASCOSIN
EFFECT OF ASCOSIN AND ANTIMYCIN ON THE RESPIRATION OF WHOLE CELLS AND CELL-FREE EXTRACTS OF 2~. coli AND P . f l u o r e s c e n s T h e 0 2 u p t a k e w a s m e a s u r e d m a n o m e t r i c a l l y a t 2 9 . 6 °. T h e f o l l o w i n g a m o u n t s of t h e a d d i t i v e s w e r e a d d e d p e r f l a s k : g l u c o s e , 2 0 / , m o l e s ; s u c c i n a t e , IOO # m o l e s ; a s c o s i n , 12. 3 # g ; a n t i m y c i n , 0. 3 # g ; K C N , t o g i v e a f i n a l c o n c e n t r a t i o n of i . lO -3 M ; a n d 0.02 M p o t a s s i u m p h o s p h a t e b u f f e r t o m a k e u p t o t h e f i n a l v o l u m e of 3 . o m l . #moles 02 uptake/ h Expt.
E. coli Whole cells
i 2 3 4 5 6 7 8
None Glucose Expt. 2 + Expt. 2 + Succinate Expt. 5 + Expt. 5 + Expt. 5 +
P. fluorescens Extracts
o.8 13.6 15.o 13.6 i .8 2.6 1.8 .-
Ascosin Antimycin Ascosin Antimycin IKCN
1.65 4.o 4 .1 4 -1 6.8 6.6 6.9 ..
3.6 7.5 7.o 7.1 I i .6 I 1.6 11.8 1.o
o.9 3.3 3.5 3.3 IO.O lO.5 IO.O 1.2
EFFECT OF ASCOSIN AND ANTIMYCIN ON T T C , D I P AND CYTOCHROME-C REDUCTION BY CELL-FREE EXTRACT OF E . coli AND P . f l u o r e s c e n s The assay systems consisted of the following: succinate, ioo #moles; malate, io #moles; DPNH, I # m o l e ; K C N , 3 # m o l e s ; T T C , 4 # m o l e s ; DIP, 0.4 # m o l e ; c y t o c h r o m e c, o . 1 5 # m o l e ; a s c o s i n , 12. 3 # g ; a n t i m y c i n , 0. 3 # g ; E . coli, c e l l - f r e e e x t r a c t c o n t a i n i n g 4 ° m g p r o t e i n / m l , o . i m l ; P . f l u o rescens cell-free extract containing 90 mg protein/ml, o.I ml for TTC and DIP experiments and o . o i m l f o r c y t o c h r o m e c e x p e r i m e n t ; a n d 0 . 0 2 M p o t a s s i u m p h o s p h a t e b u f f e r , p H 7.0, t o m a k e u p t o f i n a l v o l u m e of 3 . 0 m l .
T T C reduction s + or-E. coli
I 2 3 4 5 6 7 8 9
Endogenous Succinate Malate 2 or 3 + Ascosin 2 or 3 + Antimycir. 2 or 3 + KCN DPNH 7 + Ascosin 7 + Antimscin
-+ + + + . . . .
mpmoles D I P reduction/rain
-+ + + + . . . .
. . . .
. . .
. . . .
33 133 133 i33 133
o 133 133 133 133 .
. . . .
re#moles cytochrome c reduction/min
. . . .
o . . . . . . . . . . . . . . . . .
. . .
. o o o
39.3 39.3 39-3
cluded that in the cytochrome-linked electron transport system of E. coli the steps AS and cytochrome c are absent. In the case of the respiration of whole cells or extracts of P. fluorescens, again ascosin and antimycin exhibit no inhibitory effect. This respiration is cyanide sensitive (Table II), indicating a cytochrome-linked respiration. Table III shows that ascosin and antimycin A did not inhibit this D P N H - c y t o c h r o m e c reductase system, assayed spectrophotometrically. The cell-free extracts reduced D I P and TTC and this reduction is not inhibited by the antibiotics. From these data it seems likely that P. fluorescens, while having a typical cytochrome-linked electron transport system, does not possess the AS site in its electron transport chain. Biochim.
53 (1961) 3 9 6 - 4 o 2
S. R A M A C H A N D R A N ,
. \ s s a y s y s t e m same as described under Table I. Beef-heart preparation: crude extract, o . 2 7 m g protein; 4 I O O O × g p e l l e t , o . o 3 5 m g p r o t e i n ; lO5OOO x g p~Alet, o . o 4 m g protein; 1o5ooo × g supernatant l i q u i d , o 3 2 m g p r o t e i n . R a t - l i v e r preparation: crude extract, o . I m g protein; 4 1 o o o x g p e l l e t , o . o 3 m g p r o t e i n ; 4 i o o o × g supernatant liquid, o . o S m g p r o t e i n . mtwnol:'s cytoch~ome c reduce:l/min Expt.
Fresh crude extract °Jo I n h i b i t i o n
i on storage a t 4 'J o r - - 2 o ~ for 12 h
75 '" 59
63 ~6 6 t o
60 2o 60 o
°/o I n h i b i t i o n 3
E x p t . ~ centrifuged at 4 I o o o f o r 45 r a i n (a) resuspended pellet ° 'o I n h i b i t i o n (b) s u p e r n a t a n t liquid ° 'o I n h i b i t i o n
x g 33 '" . . . .
3.6 90 . . . . . . . .
3 (b) centrifuged at lO5OOO x g f o r 45 min
(a) r e s u s p e n d e d pellet o/ / o Inhibition (b) supernatant liquid o/ /o I n h i b i t i o n
24 '' 2o --
17 29 20 0
17 29 2o 0
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
Experiments with beef heart extracts (Table IV) show the presence of two systems of electron transport, (a) an antimvcin-ascosin sensitive respiration that can be spun down at 4 l o o o × g for 45 rain, and (b) a system that is insensitive to these two antibiotics. This latter system remains largely in the supernatant liquid from the 4 i o o o )< g centrifugation. Even centrifugation at 1o5ooo X g for 45 min does not completely sediment this insensitive system from the supernatant liquid. Such a cytochrome b5 linked, antimycin insensitive system from beef-liver microsomes has been reported by PnYN AND MACKLER 9, and in rat-liver mitochondria by DE DUVE et al. 1° and POTTER AND REIF 11. The respiration of crude extracts from rat liver also exhibits partial sensitivity to ascosin and antimycin (Table IV). The antimycin, ascosin sensitive system, however, was very labile, the activity disappearing completely on a I2-h storage at either 4 ° or - - 2 0 ° while the insensitive system was stable for at least 3 weeks when stored at - - 2 0 °. When the preparation is spun at 40 ooo × g for 45 rain, the antimycin insensitive cytochrome c reductase system distributes itself in both the pellet and supernatant liquid (Table IV). The mitochondria from corn roots were tested for their sensitivity to antimycin and ascosin. Table I shows that the DPNH-eytochrome c reductase system of corn root mitochondria is inhibited to about 60 °/o by the antibiotics. Nothing can be said at this time about the nature of this antimycin-ascosin insensitive system. BiocMm. Biophys..4eta,
S P E C I F I C I T Y OF A C T I O N OF A N T I M Y C 1 N
POTTER AND R E I F 11 have shown that antimycin A was inactivated to some extent by host protein. It occurred to us that the insensitivity of the cytochrome linked electron transport system of bacteria to antimycin and ascosin could be due to inactivation of the antibiotics by some agent present in the bacteria. To verify this hypothesis, inhibitory concentrations of the antibiotics were incubated with extracts from E. coli and this incubated mixture was tested for active antibiotic on the yeast cell-free extracts ( D P N H - - ~ cytochrolne c reductase system). Table V shows that no inactivation of either antimycin or ascosin had taken place on incubation with E. coli extracts. TABLE
THE EFFECT OF ASCOSIN AND ANTIMYCIN, INCUBATED IN CELL-FREE EXTRACT OF E . t o l l , o N THE D P N H - c Y T O C H R O M E C REDUCTASE SYSTEM OF S a c c h a v o m y c e s c e r e v i s e a e CELL-FREE EXTRACT T h e a s s a y s y s t e m is t h e s a m e as d e s c r i b e d u n d e r T a b l e I. A s e o s i n a d d e d , 8. 3 / ~ g ; a n t i m y c i n , 0. 3 /~g; y e a s t c e l l - f r e e e x t r a c t , 1. 3 m g p r o t e i n ; a n d E . coli c e l l - f r e e e x t r a c t , I . o m g p r o t e i n .
m/~moles eytochrome c reduced/Inin
Control + Ascosin
Control + A~timvcin "
-t- E. coli
Control + E. coli Extract + Ascosin
* Control contained the yeast DPNH
Control + E. coli Extract + Antimyci~*
- + c y t o c h r o m e c r e d u c t a s e s y s t e m alone.
Some antibiotics inhibit only specific organisms or groups of organisms, while others inhibit a wide range of cells. Thus, penicillin at low concentrations inhibits gram positive but not gram negative bacteria, and filipin inhibits most fungi but not the bacteria. The cause for this specificity is linked to the variations in the physiological activities amongst the different organisms. Very little information is available on the nature of such differential activity. Antimycin and ascosin have been shown to block the cytochrome linked electron transport system at a site commonly denoted as AS site s,2. Since such an electron transport system is the most common one yet observed among aerobic organisms, one would be led to assume that antimycin and ascosin would have a very broad action spectrum. However, while exhibiting a marked specificity for being toxic to fungi and mammalian cells, neither of these antibiotics are antibacterial at normal physiologic concentrations. The fact that antimycin and ascosin do not inhibit cytochrome-linked respiration of cell free extracts of E. coli and P. fluorescens shows that the inactivity against bacteria is not a question of lack of permeability. As far as can be ascertained, P. fuorcscens has a cvtochrome-linked electron transport mechanism. Our data on TTC, DIP, cytochrome c reduction with succinate, malate, glucose and D P N H as well as the KCN sensitive respiration support the existence of this electron transport system. Various cytochromes and other co-factors of the coupled electron transport system are also known to be present in P. fl,!torescens ~. Nevertheless the D P N H cytochrome c reductase of this organism is insensitive to antimycin and ascosin. Probably the AS site is absent in this microbe and since antimycin and ascosin inhibit at this site th%; are ineffective in preventing electron transport. Biocldm.
A c t a , 53 (1961) 3 9 6 - 4 0 2
s. RAMACHANDRAN, D. GOTTLIEB
The case of E. coli is slightly different. There are reports in the literature t h a t , while cytochromes b a n d a are present, cytochrome c is absent ~. I n our studies the E. coli extracts did not reduce exogenous cytochrome c in the presence of D P N H indicating the absence of a cytochrome c reducing mechanism. However, the E . coli extracts reduced the electron accepting dyes D I P a n d TTC, a n d took up oxygen in the presence of glucose, succinate, m a l a t e a n d D P N H . This oxygen u p t a k e was inhibited b y KCN. I n E . coli also none of the electron t r a n s p o r t activities m e n t i o n e d above were a n t i m y c i n or ascosin sensitive. F u r t h e r m o r e , it is clear from Table V t h a t the antibiotics were n o t i n a c t i v a t e d b y the active bacterial extracts. A n t i m y c i n a n d ascosin, even after i n c u b a t i o n with E. coli extracts, inhibited the sensitive yeast extracts at n o r m a l concentrations. These facts indicate t h a t E. coli has an operative electron t r a n s p o r t system with all the components of the cytochrome linked electron t r a n s p o r t system except AS site a n d cytochrome c. At this time n o t h i n g can be said a b o u t the m e c h a n i s m b y which this a n t i m y c i n - a s c o s i n insensitive site is bypassed in the bacterial electron t r a n s p o r t system. We would also like to draw a t t e n t i o n to the fact t h a t the m a m m a l i a n , fungal a n d higher p l a n t (corn) electron t r a n s p o r t systems appear to have m a n y similarities. All of these have antimycin-ascosin sensitive a n d insensitive systems. I n general this sensitive system is p r e d o m i n a n t l y contained in the particulate fraction t h a t can be sedimented at centrifugal forces of a b o u t 40000 x g for 45 rain. The insensitive system remains in the "40000 / g" s u p e r n a t a n t liquid*. I n conclusion, we find t h a t fungi, higher p l a n t s and m a m m a l i a n cells, all possess cytochrome linked electron t r a n s p o r t systems which c o n t a i n the AS site. On the other hand, the bacteria t h a t were studied had a cytochrome linked electron transport system which contained no AS site. Consequently, a n t i m y c i n a n d ascosin could n o t i n h i b i t the growth a n d respiration of these bacteria b y blocking the electron t r a n s p o r t system. ACKNOWLEDGEMENT
These studies were supported in part b y a g r a n t ( E - 6 1 8 ) f r o m t h e N a t i o n a l I n s t i t u t e s of Health. REFERENCES 1 F. L. CRANEAND J. L. GLENN, Bioehim. Biophys. dcta, 24 (1957) lO7. 2 D. GOTTLIEBAND S. RAMACHANDRAN,Biochim. Biophys. Acta, 53 (1961) 39I. 3 R. J. HICKE¥, C. J. CORUM, P. H. HIDY, I. R. COHEN, U. F. B. NAGERAND E. I~ROPP, Antibiotics and Chemotherapy, 2 (1952) 472. 4 K. AHMAD, H. G. SCHEIDERAND F. M. STRONG,Arch. Biochem., 28 (I95o) 281. 5 LUCILE SMITH, Arch. Bioehem. Biophys., 50 (1954) 299. 0 "~V."vV.UMBREIT,R. H. BURRISAND J. F. STAUFFER, Manometric Techniques, Burgess Publishing Company, 1957. 7 H. A. LUND, A. E. VATTERAND J. B. HANSON, J. Biophys. Bioehem. Cytol., 4 (1957) 87. 8 M. M. NACHLAS,S. I. MARGULIESAND A. M. SELIGMAN,J. Biol. Chem., 235 (196o) 2739. 9 N. W. PENN AND B. MACKLER,Biochim. Biophys. Aela, 27 (1958) 539. 10 C. DE DUVE, B. C. PRESSMAN, R. GIANETTO, R. WATTIAUXAND E. APPELMANS,Biochem. J., 6o (1955) 604. 11 V. R. POTTERANn A. E. REIF, J. Biol. Chem., 194 (1952) 287 . * The information on Selerotinia [ructicola was kindly given to us by Dr. P. D. SHA'~VOf our Department from his unpublished data. Bioehim. Biophys. Acta, 53 (1961) 396-402