502 M.-N. THANG

et elle devient parall61e k celle dans les acides amin6s apr6s une phase de latence. Lorsque les nitrates exog6nes sont 6puis6s, cette incorporation dans les prot6ines continue ~ augmenter pendant que celle dans les acides amin6s diminue. Ceci indique que le marquage au 15N dans les prot6ines a pour origine les acides amin6s marqu6s. Le fait que l'on assiste k une augmentation de prot6ines totales indique qu'il y a synth6se des prot6ines k partir de l'azote des nitrates par l'interm6diaire des acides amin6s.

B I B L I O G R A P H I E

1 M.-N. THANG, Biochim. Biophys. Acta, 52 (1961) 478. 2 A. G. GORNALL, C. J. BARDAWILL ET M. DAVID, J. Biol. Chem., 177 (1949) 751. 3 j . p . ZALTA, communica t ion personnelle. 4 B. LOBOCHINSKY ET M. N. THANG, m6thode non publi6e. 5 B. LUBOCHINSKY ET J. P. ZALTA, Bull. soe. chim. biol., 36 (1954) 1363. e D. B. SPRINSON ET D. RITTENBERG, J. Biol. Chem., 18o (1949) 707. 7 M. N. THANG, Compt. rend., 249 (1959) 2415. 8 V. C. STEWARD, R. G. S. BIDWELL ET E. W. YEMIN, Nature, I78 (1956) 734. 9 A. P. SIMS, darts FOLKER, Utilization o] Nitrogen and its Compounds by Plants, Academic Press,

Inc., New York, 1959. 10 M. CRAMER ET J. MYERS, J. Gen. Physiol., 32 (1948) 93. 11 p. C. SYRETT, Physiol. Plantarum, 9 (1956) 19. l a p . C. SYRETT, Physiol. Plantarum, 9 (1956) 28. 13 H. B. VICKERY, G. W. PUCHER, R. SCHOENHEIMER ET D. RITTENBERG, J. Blot. Chem., 13,

(194 ° ) 531 . 14 R. SCHOENHEIMER, The Dynamic State o] Body Constituents, Harvard Univ. Press, 1942. is H. HALVORSON, Biockim. Biophys. Acta, 27 (1958) 267. is j . MANDELSTAM, Biochem. J., 69 (1958) IiO. 17 H. HALVORSON, Bioehim. Biophys. Acta, 27 (I958) 255.

Biochim. Biophys. Acta, 52 (1961) 495-5o2

ON T H E O R I G I N OF T H E T O X I C I T Y OF T O X O F L A V I N

H. E. LATUASAN AND W. B E R E N D S *

Biochemical and Biophysical Laboratory o] the Technological University o] Del]t (The Netherlands)

(Received March I4th, 1961)

SUMMARY

The mechanism of action of toxoflavin was studied by examining the effect on yeast and some other microorganisms. Toxoflavin appears to act as an electron-cartier which makes possible a by-passing of the cytochrome-system. The final result of this electron-transfer is the production of hydrogen-peroxide.

The peroxide-generating capacity of toxoflavin may be responsible for the strong antibiotic activity and poisonous character of this compound. The inactivity of toxo- flavin under anaerobic conditions is in agreement with this assumption.

* Jul ianalaan 67, Delft (The Netherlands).

Biochim. Biophys. ¢tcta, 52 (1961) 5o2-5o8

TOXICITY OF TOXOFLAVIN 503

INTRODUCTION

Toxoflavin, a poison from Pseudomonas cocovenenans was isolated by VAN VEEN AND MERTENS 1 in 1934. This microorganism, which also produces bongkrekic acid, has been found responsible for several fatal food poisonings occurring amongst the natives in the densely populated parts of Mid-Java 2.

The conclusions from the work on the structure 3 were not accepted by us and after a reinvestigation we have proposed a choice between two structures, one of these seeming the most probable 4. This structure has been confirmed by a recent r rntgenographic investigation 5.

O II

C N HsC- -N/~ ~ ' C ~ ~ C H

I i It O=C\N//C\ N/N J CH s

Apart of its toxicity for man and animals, toxoflavin has a strong antibiotic ac t iv i ty 4.

In 1954 an antibiotic, called xanthothricin, was isolated by MACHLOWITZ et al. 6 from a culture of a member of the genus Streptomyces. Owing to the high toxicity of this antibiotic (intravenous toxicity (LD 50) of 1, 7 mg/kg and oral toxicity (LD 50) of 8.4 mg/kg on mice) no work on the chemical structure was done. According to all available evidence xanthothricine must be identical with toxoflavin. The over- looking of this identi ty by MACHLOWITZ et al. is completely understandable as the data available in the literature on toxoflavin were erroneous.

The occurrence of toxoflavin in two quite different microorganisms suggests a biochemical function of this compound. Although toxoflavin inhibits the growth of several microorganisms at low concentrations, it does not affect the growth of yeast ( Saccharomyces cerevisiae).

In starting work on the mechanism of the antibiotic activity we wanted first to know if toxoflavin has no infuence whatsoever on the metabolism of yeast cells. The results of this s tudy have brought to light a mechanism of action which might explain why this compound is so highly toxic to many cells and almost harmless to yeast.

METHODS

The antibiotic act ivi ty was determined by the dilution method. The effect of toxo- flavin on the growth of yeast was tested in a medium described by WHIFFEN 7 (glucose, 7 %; Difco yeast extract, 0.25 % and KH2P04, o.i %). Sterilization during 30 min at 115 °. Toxoflavin was added before sterilization or afterwards as a solution which had passed through a Seitz filter.

The effect of toxoflavin on the respiration of yeast was determined in a Warburg apparatus. Commercial pressed or dried yeast*, or yeast obtained from a Whiffen

* Products of the "Nether lands Yeast and Fermenta t ion Industr ies", Delft (The Netherlands).

Biochim. Biophys. Acta, 52 (1961) 502-508

504 I-L E. LATUASAN, W. BERENDS

medium in stationary or shake culture, was used. The intensity of the respiration is expressed as the Qo2*.

Homogenates of yeast were prepared by destruction of the cells of pressed yeast in a slightly modified Nossal apparatus 8. Preparations showing the highest respiration were obtained by twice repeated shaking of 3 g pressed yeast with 8 g ballotine beads and 9 ml water for 3o sec at o ° followed by centrifugation at 3ooo × g during 15 min.

In the experiments on oxidative phosphorylation the uptake of inorganic phos- phate was determined by applying the FISKE-SUBBAROW method 9 after precipitation of the proteins with trichloroacetic acid (2o %) and centrifugation at 3ooo × g for IO min.

The oxygen consumption of the cytochrome deficient yeast 1° Saccharomyces cerevisiae Pfaff 3oo in the presence of toxoflavin and glucose was determined after inhibiting the alcohol dehydrogenase with actidion n.

For detection of H20 ~ the method of AEBI ~2 was used (peroxydase catalyzed oxidation of o-dianisidine).

RESULTS

The growth-inhibiting concentrations assembled in Table I are of course dependent on the media and methods used, so the figures given have only a comparative value.

Neither in stationary nor in shake cultures any growth inhibition of yeast cells by toxoflavin to concentrations of IOO/~g/ml could be observed.

T A B L E I

GROWTH INHIBITION BY TOXOFLAVIN

Concentration of coraplele inkibition

Microorganism of growth (~glrm)

E. coli o.5 Shigella 1.2 ~Vlicrococcus pyogenes 1.2 Bacillus subtilis 2.5 Proteus vulgaris 25

The endogenous respiration of yeast cells suspended in a phosphate buffer is considerably increased by toxoflavin. An increase of oxygen uptake of about ioo % was observed after adding 30 ~g toxoflavin/ml (blank: Qo2, 6-1o; with toxoflavin Qo2, 14-2o). Even at a concentration of 0.6 t~g/ml toxoflavin the increase in oxygen uptake was about 4o %. The stimulation of oxygen consumption by toxoflavin was almost the same for commercial pressed yeast, dried yeast or yeast cultivated in a Whiffen medium.

The RQ of the respiration of intact yeast cells (commercial pressed yeast washed twice with distilled water) was reduced from 0.85 to 0.8 upon adding toxoflavin (lO -4 M). According to STIGKLAND 13 the RQ of endogenously respiring yeast is 0.8. Hence it is not only carbohydrate that is utilized in endogenous respiration of yeast.

* Qo~ = / A of 02 /mg dry wt. /h.

Biochim. Biophys. Acta, 52 (I96I) 502-508

TOXICITY OF TOXOFLAVIN 505

A considerable increase (about 60 %) of the endogenous respiration could also be demonstrated with yeast homogenates prepared by destruction of the cells of pressed yeast in a Nossal apparatus (blank: Qo2 about 2; after addition of 30/zg toxoflavin/ml the Qo2 was increased to 3.3).

Toxoflavin (lO -4 M) only caused a negligible increase of oxygen uptake in the presence of 3" lO-2 M glucose. (In these conditions the Qo2 is 4 to 5 times the Qo2 in the absence of glucose.)

Toxoflavin did not show any effect on the anaerobic fermentation of glucose by intact cells or homogenates of pressed yeast.

Toxoflavin did not inhibit, but rather slightly promoted the oxidative phospho- rylation in a Nossal homogenate of yeast at pH 6.3. (The internal pH of yeast cells is 6.3 (see ref. 14).) When intact yeast was incubated with lO -4 M dinitrophenol the initially increased endogenous respiration gradually diminished again within I h. Subsequent addition of lO -4 M toxoflavin restored the oxygen consumption to its original value. This also proved that the effect of toxoflavin must differ from that of dinitrophenol.

Several inhibitors of the respiration of yeast are known. I t seemed interesting to study the effect of toxoflavin on inhibitions brought about by these compounds.

Iodoacetate (lO -4 M) had no effect at all on the endogenous respiration of intact yeast cells. HOLZER, HOLZER AND SCHULTZ 15 have found that the oxygen consumption of yeast in the presence of glucose is not suppressed by iodoacetate, whereas the fermentation is strongly inhibited. An explanation for this remarkable phenomenon has not yet been given.

The increase of the endogenous oxygen uptake by toxoflavin (about 8O-lOO ~o) is not affected by the presence of iodoacetate.

The antibiotic antimycin A intercepts the transfer of electrons between the cytochromes b and c (see refs. 16, 17). Traces of antimycin appeared to block the endogenous respiration of yeast almost completely, whereas the C02 production was stimulated. These effects could be annihilated by the addition of toxoflavin. A concen- tration of lO -4 M was sufficient to restore the respiration completely. (The Qo2 of 8.5 of yeast cells washed twice with distilled water decreased to practically zero after 15 min incubation with antimycin; addition of lO -4 M toxoflavin brought the Qo2 to 11.7.) Similar results were obtained with Nossal homogenates.

From these experiments the conclusion may be drawn that toxoflavin causes a by-passing of the cytochrome system.

Chlorpromazine is known as an inhibitor of flavin-adenine-dinucleotide con- taining enzymes. At low concentrations competition between chlorpromazine and FAD has been assumed 18 whereas at higher concentrations formation of a complex of these compounds has been demonstrated 12, ~o. The endogenous oxygen consumption of a yeast homogenate was found to be inhibited for about 5 ° °/o by lO -3 M chlor- promazine. Toxoflavin (lO -4 M) could counteract this effect completely. However, the inhibition of the respiration of intact cells by chlorpromazine (also about 50 %) was not affected by toxoflavin. An explanation of this result cannot be given as yet.

Potassium cyanide (lO -2 M) was found to suppress the endogenous respiration to about 30 % of the original value. Toxoflavin (lO -4 M) completely abolished this inhibition. This again suggests a bypassing of the cytochrome system.

Biochim. Biophys. Acta, 52 (i96i) 502-508

506 It. E, LATUASAN, W. BERENDS

The presumed by passing of the cytochrome system by toxoflavin could be con- firmed by the following experiment.

The mitochondria-containing fraction of a Nossal homogenate was sedimented by centrifuging at 25 ooo x g for 30 rain. The oxygen uptake of the supernatant was very low (Qo2 = o.II) . Combination of the supernatant and the sediment practically restored the original respiration (Qo, = 2.7), while toxoflavin (IO -4 M) could com- pletely replace the sediment (Qo= = 3.2).

Saccharomyces cerevisiae Pfaff 300 is a cytochrome deficient yeast, that almost exclusively shows fermentation and no respiration 1°. Toxoflavin, however, brought about an oxygen uptake in the absence as well as in the presence of glucose. I f alcohol dehydrogenase is inhibited at the same time by actidion the RQ was found to be I.

Accepting an electron-transferring function of toxoflavin it was essential to have more detailed information available about the red-ox characteristics of this compound. The experiments of VAN VEEN AND BAARS 3 on the hydrogenation of toxoflavin could only par t ly be confirmed by STERN ~1. A reexamination demonstrated tha t toxoflavin absorbs exactly one mole of hydrogen under the influence of a palladium catalyst (IO %) in acetic acid at room temperature. By shaking this reduced solution with air, after removal of the catalyst, toxoflavin is restored by reoxidation very rapidly (the brown solution turns yellow again).

The same reduction of toxoflavin takes place anaerobically by a yeast homo- genate to which D P N H has been added. The rapid aerobic oxidation of D P N H by toxoflavin is illustrated in Fig. 2 (decrease of extinction at 34 ° m/z plotted against time). Experiments using 25000 × g supernatants have given the same results. Heated homogenates, however, were completely inactive, so the reduction of toxo- flavin must be an enzymic process.

From the experiments described it may be deduced that toxoflavin acts as an electron carrier between D P N H and oxygen. This would lead to a production of hydrogen-peroxide.

2~

le

14 1,4 M

X

I

I • I I

/ I

r t |

@-. t i i

2 ' ~" I

250 - - i - - - -~ ~ ;

350 400 4 5 0

Wavelength Fig. I. Absorp t ion spect ra of toxoflavin ( ) and of reduced ( . . . . . . ) and re-oxidized ( - - . - - )

toxoflavin.

. . . . . . . . . . . . . . Blanks O.8 \,.,.,

\ ' ' 0 . 6 ' ~+Ckl)Jg toxoflavin/ml o ',

< 0.4 \~~1,0~ ~ 0.2 ~

oflavinlrnl C 1 2 3 4 5

Fig. 2. E lec t ron t rans fe r D P N H - o x y g e n s t imu- la ted by toxoflavin.

Biochim. Biophys. Acta, 52 (1961) 5o2-5o8

TOXICITY OF TOXOFLAVIN 507

D P N H + Toxoflavin ~ D P N + + Toxof lav in-Hz

Toxof lav in -H 2 + O 3 - ~ Toxoflavin + HzO 2

Indeed the presence of H.202 could easily be demonstrated. Toxoflavin (I mg) and D P N H (I mg) were added to the 25000 × g supernatant of a Nossal homogenate in an evacuated Thunberg tube. After a few minutes the tube was heated in a boiling water bath to destroy the catalase. Hydrogen peroxide production could be detected by the method of AEB112 immediately after opening of the tube. A negative reaction was obtained upon omitting toxoflavin or yeast extract from the reaction mixture.

Hydrogen peroxide is a very strong poison to catalase-deficient cells. The rather high catalase content of yeast may explain the fact tha t the growth of yeast is not inhibited by toxoflavin. If the poisonous effect of toxoflavin is only an indirect one, due to the formation of H20 ~, the latter compound must be detectable in cultures of toxoflavin-sensitive microorganisms to which toxoflavin is added. This was tested with E. coli, a microorganism highly sensitive to toxoflavin.

According to the figures given in Table n toxoflavin strongly inhibits the oxygen consumption of E. coli.

No hydrogen-peroxide could be detected in the blanks, but all the toxoflavin containing vessels gave a strongly positive reaction. Even in suspensions of endo-

T A B L E I I

E. coli cells were obtained f rom a 24-h aerated cul ture in "Difco nut r ient b r o t h " (2 g/25o ml water) by centrifugation. After washing the cells twice with distilled water, 5 ° mg (wet wt.) of cells

were suspended in 20 ml of 0.o5 M phospha te buffer, p H 7, to which subs t ra tes were added.

Subs~ta~e Q02 Blank + zo I~g toxoflavin

o.o 5 M glucose 8o 42 o.o 5 M pyruva t e I52 29 o.o 5 M succinate 245 88

~100 E 0

-Q 8 0

o~ 6 0

¢-

~ 4 ¢

2C

A n a e P o ~ c

i Aerobic t I t I,

\ 0

I I

Tox~flovin t ~lg/ml

Fig. 3. Effect of toxoflavin on growth of Esche- richia coli.

100

8 0

,~ 6o

e -

~ 4o 0 L 19

20

i i Anaerobic

t

~ A e r o ~ c

Toxoflovin, ~g/ml

Fig, 4- Effect of toxoflavin on growth of Strepto- coccus l a c t i s .

BiocMm. Biophys. Acta, 52 (1961) 5o2-5o8

508 H. E. LATUASAN, W. BERENDS

genously respiring cells, to which toxoflavin was added, the presence of H~O, could be demonstrated. In similar experiments with yeast cells no H~0 2 was found.

If toxoflavin acts by producing H~O 2 no effect of this compound was to be expected under anaerobic conditions. From the Figs. 3 and 4 it is clear that the growth of E. coli and Streptococcus lactis is completely insensitive to toxoflavin if oxygen is excluded from the media.

DISCUSSION

According to the results reported in this paper toxoflavin is a very effective electron carrier, which under aerobic conditions ultimately gives rise to H,O 2 production.

It seems propable that the poisonous effect of toxoflavin is always due to the hydrogen peroxide formed. The effects of toxoflavin on animals are in agreement with this assumption. According to STERN 21 toxoflavin stimulates the oxygen uptake of erythrocytes very strongly, while the oxyhaemoglobin is converted with fair velocity to methaemoglobin.

The lack of antibiotic activity of toxoflavin in anaerobic conditions also supports this hypothesis. The comparative high catalase content of yeast might prevent the action of toxoflavin on this microorganism.

R E F E R E N C E S

1 A. G. VAN VEEN AND W. K. MERTENS, Rec. tray. chim., 53 (1934) 257, 398. 2 W. K. MERTENS AND A. G. VAN VEEN, Geneesk. Tijdschr. Ned. Indi~, 73 (1933) 1223, 13o9;

Mededeel. Dienst der Volksgezondheid Ned. Indi~, 22 (1933) 209; Rec. tray. chim., 53 (1934) 257. 3 A. G. VAN VEEN AND J. K. BAARS, Rec. tray. chim., 57 (1938) 248. 4 p. A. VAN DAMME, Miss A. G. JOHANNES, H. C. Cox AND W. BERENDS, Rec. tray. chim., 79

(196o) 255. 5 A. S. HELLENDOORN, R. IV[. TEN CA.TE-DHONT AND A. F. PEERDEMAN, Rec. tray. chim., 80

(1961) 3o 7 . 6 R. A. MACHLOWITZ, W. P. FISHER, B. S. MCKAY, A. A. TYTTEL AND J. CHARNE¥, Antibiotics

and Chemotherapy, 4 (1954) 259. 7 A. J. WHIFFEN, J. Bacteriol., 56 (1948) 283. 8 p. M. NOSSAL, Austral. J. Exptl. Biol., 31 (1953) 583. 9 C. H. FISKE AND Y. SUBBAROW, J. Biol. Chem., 66 (1925) 375.

lO H. J. PHAFF, Science, lO5 (1947) 44. 11 H. E. LATUASAN AND W. BERENDS, Rec. tray. chim., 77 (1958) 416. 12 H . A E B I , A . T E M P E R L I , R . G R E S S L Y , R . OESTREICHER AND A. ZUPPINGER, I-Ielv. Chim. Acta,

43 (196o) 1714. 13 L. H. STICKLAND, Biochem. J., 64 (1956) 498. 14 A. GOTTSCHALK, in J. B. SUMNER AND K. MYRBACK, The t~nzymes, Vol. I, Academic Press, Inc.,

New York, I95O, p. 575. 15 H. HOLZER, E. HOLZER AND G. SCHULTZ, Biochem. Z., 326 (1955) 385 . 16 B. CHANCE, in O. H. GAEBLER, Enzymes: Units o] Biological Structure and Function, Academic

Press, Inc , New York, 1956, p. 447. 17 B. CHANCE, J. Biol. Chem., 233 (1958) 1223. 18 K. YAGI, T. NAGATSU AND T. OZAWA, Nature, 77 (1956) 891- 18 A . SZENT-GY~RGYI, Bioenergetics, Academic Press, Inc., New York, 1957, p. 118. 2o K. YAGI, T. OZAWA AND T. NAGATSU, Nature, 184 (1959) 982. ex K. G. STERN, Biochem. J., 29 (1935) 5oo.

Biochlm. Biophys. Aeta, 52 (1961) 502-508