Further studies on the hemolytic action of filipin and derivatives

Further studies on the hemolytic action of filipin and derivatives

835 BIOCHIMICA ET BIOPHYSICA ACTA BBA 75O81 F U R T H E R STUDIES ON T H E HEMOLYTIC ACTION OF F I L I P I N AND DERIVATIVES STEPHEN C. K I N S K ...

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835

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 75O81 F U R T H E R STUDIES ON T H E HEMOLYTIC ACTION OF F I L I P I N AND DERIVATIVES

STEPHEN

C. K I N S K Y , R. A. D E M E L AND L. L. M. V A N D E E N E N

Department of Pharmacology, Washington Umversity School of Medicine, St. Louis, Mo. (U.S.A.) and Organisch Chemisch Laboratorium, R~iksuniversiteil, Utrecht (The Netherlands) (Received April I9th, I967)

SUMMARY

The interaction of filipin with model membrane systems, such as lipid monomolecular layers and bilayer films, has been described previously. To find additional criteria by which to evaluate various membrane models, several methods for producing derivatives of filipin, which have little or no biological activity, have been examined. Two methods have proved particularly useful: (a) reduction of the conjugated double bond sequence and (b) irradiation with visible light in the presence of FMN. Perhydrofilipin has approx, i/IOO the hemolytic activity of the parent antibiotic, whereas irradiated filipin has no biological activity (either hemolytic or antifungal). The effect of these derivatives on model membrane systems is described in the following papers. We have extended our observations that also the antibiotic/erythrocyte ratio, and not only the absolute concentration of antibiotic, determines the rate and extent of hemolysis. This finding provides an additional criterion which must be met by an appropriate membrane model.

INTRODUCTION

The availability of agents (mainly, antibiotics) which interfere specifically with the biosynthesis or structure of macromolecules has played an important role in the elucidation of the structure of nucleic acids, proteins, and bacterial cell walls. For the past several years, we have been studying the mode of polyene antibiotic action on the assumption that these compounds might similarly prove useful tools for investigating the lipid architecture of plasma and cytoplasmic membranes. Considerable evidence now indicates that the polyene antibiotics cause permeability alterations in sensitive organisms resulting in the leakage of essential cytoplasmic components from the cell (reviewed in refs. 1-3). Only organisms (e.g. fungi, protozoa, erythrocytes) which contain sterols are affected by the polyenes. It has, in fact, been possible to convert some normally insensitive organisms which lack sterols (MycoBicchim. Biophys. Acta, 135 (I967) 835-843

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S. C. KINSKY, R. A. DEMEL, L. L. M. VAN DEENEN

plasma laidlawii, Pythium ultimum) to polyene sensitivity by cultivating them in media supplemented with cholesterol 4 G. These observations have led to the contention that the selective toxicity of the polyene antibiotics is due to interaction with a unique component (sterol) Nund only in the cell membrane of sensitive organisms. The exact mechanism by which the polyenes affect cell membrane structure, after combining with sterol, is not yet known. Our approach to this problem has been to study interaction of the polyenes with different membrane models such as lipid monomolecular layers and bilayer filmsL8. Obviously, the most suitable membrane model is the one that can account for all the effects which these antibiotics have on natural cell membrane systems. For example, an appropriate membrane model must not only be consistent with the observation that only cells with sterols are apparently affected by the polyenes; it should also explain why (as indicated by numerous studies with yeast cells, fungal protoplasts, or mammalian erythrocytes) filipin causes the most extensive cell membrane damage with etruscomycin, amphotericin B, pimaricin, and nystatin following in that order2, a.

CH3.

OH

CH 3

Fig. I. S t r u c t u r e of filipin proposed b y CEDER AND RYHAGE.

Filipin has been extensively investigated with both natural and model membrane systems for 2 reasons: (a) as noted above, it causes the greatest cell membrane damage of all the polyenes thus far examined and (b) it is one of the few polyenes whose structure has been determined (Fig. 1) 9. To find some additional criteria b y which to evaluate the various membrane models, we have investigated several methods for producing derivatives of filipin which have little or no biological activity. As demonstrated in this paper, 2 methods have proved particularly useful: (a) reduction of the conjugated double bond sequence and (b) irradiation with visible light in the presence of flavin nucleotides. The effect of these derivatives on various model membranes is described in subsequent papers 1°-12. We have also extended our earlier observations TM that the extent of hemolysis induced by the polyenes is also dependent on the antibiotic/cell ratio and not only on the absolute concentration of the antibiotic. This finding provides an additional criterion which must be met b y an appropriate membrane model. MATERIALS AND METHODS

Chemicals The filipin used in this investigation was generously provided by Dr. G . B . Biochim. Bioph),s. Acta, 135 (1967) 835 843

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WHIZFIELD of The Upjotm Company, Kalamazoo, Mich.*. This preparation was approx. 95% pure on the basis of ultraviolet absorption and 80% pure as determined by bioassay. Perhydrofilipin was prepared essentially as described by WHIZFIELD et al. a4. The antibiotic was suspended in a mixture of dimethylformamidemethanol (I :3, v/v) with either palladium on charcoal or platinum oxide as catalyst. Incubation was carried out under a Hz atmosphere at room temperature in the dark for periods as long as 18 h. H 2 uptake was usually complete, however, after 3 h at which time 5 moles per mole of filipin had been consumed. After removal of the catalyst b y filtration or centrifugation, the reaction mixture was used directly or the perhydrofilipin was further purified by thin-layer chromatography on silica-gel plates with CHC13-CH3OH-H20 (65:35:4, v/v/v) as solvent. The majority of the experiments described below were performed with a crystalline sample of perhydrofilipin that had been kindly prepared for us by Dr. J. OUTCHER of the Squibb Institute for Medical Research, New Brunswick, N.J. Saponified filipin was prepared b y dissolving the antibiotic in a small volume of dimethylformamide (approx. I ml) followed by dilution with 15 ml of o.I M NaOH. After incubation at 70-80 ° for 3 h under N~ in the dark, unreacted antibiotic was removed by filtration. The filtrate was acidified with 60 ml of 0.05 M H2SO 4 and the resulting precipitate was washed successively with water and CHC13. The dried material was dissolved in dimethylformamide before use. The preparation of irradiated filipin is described in the following section; illumination was carried out in the apparatus previously employed 15. Absorbance changes and spectra were determined in cuvettes with a I-cm light path.

Measurement of hemolysis R a t or rabbit blood was defibrinated b y shaking with glass beads and the erythrocytes extensively washed with isotonic saline (o.154 M NaC1), before the lysis experiments. Hemolysis was determined b y either of 2 methods. In the first method, tubes containing the indicated amounts of antibiotic, isotonic saline, and erythrocytes were incubated at 37 °. At various times, the tubes were centrifuged at 3000 × g for io min to remove ally intact cells and the hemoglobin released was determined b y measuring the absorbance of the supernatant solution at 550 m#. The reaction was started by addition of the erythrocytes ; an amount of erythrocytes was employed which, upon complete hemolysis in I ml of water, would produce an absorbance between 0.6 to 0. 9. Control tubes containing the appropriate quantity of dimethylformamide were always included in each experiment. The percent hemolysis, relative to water, was calculated after correction for this control value. The preceding method, however, could not be used conveniently to follow the kinetics of hemolysis because, under some conditions, the reaction was complete in less than 2 min. For this purpose, hemolysis was determined spectrophotometrically by measuring the increase in light transmission at 625 m/~ at 20 °. In these experiments absorbance (actually, light scatter) was not a linear function of the number of re* It must be emphasized t h a t Filipin is a registered trade m a r k of the U p j o h n Company, Kalamazoo, Mich. The generic n a m e for this antibiotic is filimarisin. 13ecause filipin has been e m p l o y e d a l m o s t exclusively in the extensive literature on this antibiotic, we prefer to continue use of this n a m e in order to avoid the u n n e c e s s a r y confusion which would result if the generic n a m e were s u b s t i t u t e d . The trade n a m e s for n y s t a t i n and a m p h o t e r i c i n t3 (Squibb and Sons, New York, N.Y.) are Mycostatin and Fungizone, respectively. Biochim. Biophys. dcta, 135 (i967) 835-843

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S . C . KINSKY, R. A. DEMEL, L. L. M. VAN DEENEN

maining intact cells and a calibration curve, prepared by mixing appropriate amounts of non-lysed with lysed cells, was used to calculate the percent hemolysis from the transmission data. Additional experimental details are provided below. For the determination of an approximate value of the molar ratio, filipin added to membrane lipid, a lipid content of 5.I5 mg/ml of packed erythrocytes was assumed (see also P R A N K E R D l l ) . For this calculation a molecular weight of 654 was used for the antibiotic and an average value of 580 for the lipid based on the lipid composition of rabbit erythrocytes is.

Effect of filipin and derivatives on growth of Neurospora This was determined by methods previously described 16. RESULTS AND DISCUSSION

P erhydrofilipin Fig. 2 compares the ability of filipin and perhydrofilipin to lyse rat erythrocytes. Reduction of the conjugated double bond sequence produces a derivative which is far less potent than the parent antibiotic. Thus, with the amount of erythrocytes employed ill this experiment, the critical threshold concentration (i.e. the lowest concentration of antibiotic necessary to induce hemolysis) was approx. 0. 7 #g/ml for filipin and 7° #g/ml for perhydrofilipin. It is important to note that the critical threshold concentration does not change appreciably by increasing the incubation time. This phenomenon is in accord with the view that a definite ratio of antibiotic per cell must be attained before hemolysis can occur (see below). The above experiment was performed with a sample of perhydrofilipin purified by recrystallization from dioxane (J. DUTCHER, personal communication). The spectrum of this material and filipin in methanol is shown in Fig. 3. There was no evidence for the presence of the conjugated chromophore (peaks at 320, 337, and 355 m#) which is characteristic of filipin. It should be emphasized that these spectra were obtained with solutions containing perhydrofilipin at a concentration approx.

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Fig. 2. C o m p a r a t i v e p o t e n c i e s of filipin a n d perhydrofilipin. T u b e s c o n t a i n e d 0.9 ml of isotonic saline a n d t h e i n d i c a t e d a m o u n t s of antibiotic or d e r i v a t i v e dissolved in o.o 5 ml of d i m e t h y l f o r m a m i d e . T h e r e a c t i o n w a s s t a r t e d b y a d d i t i o n of 0.05 m l of a r a t e r y t h r o c y t e s u s p e n s i o n . O n e series of t u b e s w a s i n c u b a t e d for 2 min, t h e o t h e r for 90 m i n , before r e m o v a l of n o n - l y s e d cells b y c e n t r i f u g a t i o n . I n t h i s e x p e r i m e n t , c o m p l e t e h e m o l y s i s of t h e e r y t h r o c y t e s in I.O m l o f w a t e r g a v e a s u p e r n a t a n t solution w i t h a n a b s o r b a n c e (55 ° mp) of 0.770, w h e r e a s t h e c o r r e s p o n d i n g v a l u e in control tubes, c o n t a i n i n g d i m e t h y l f o r m a m i d e (no antibiotic), w a s o.o3o. Fig. 3. S p e c t r a of filipin a n d perhydrofilipin p r e p a r a t i o n u s e d in e x p e r i m e n t described in Fig. 2.

Biochim. Biophys. Acta, 135 (1967) 835-843

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45 ° times greater than the concentration of filipin. We therefore conclude that the hemolysis observed with the reduced derivative is a property of this compound per se and not due to contamination by traces of unreacted filipin. Saponified filipin Preliminary experiments with the alkali-treated filipin also indicated that this material was hemolytic under the conditions of the experiment shown in Fig. 2. This material was, like perhydrofilipin, far less potent than filipin; the critical threshold concentration was approx. IOO/zg/ml. Saponified filipin has essentially the same spectrum as the parent antibiotic. Attempts to detect and, if present, remove any unreacted filipin from the saponified material by thin-layer chromatography were unsuccessful. Only 0.7% contamination of the saponified preparation by filipin would, however, account for the observed hemolysis. This amount could not have been detected by the techniques which were employed. Therefore, the possibility that the hemolytic activity of the saponified filipin was due to contamination by the parent antibiotic cannot be excluded and further experiments with this material were not attempted. Irradiated filipin ZONDAG, POSTHUMA, AND BERENDSTM have reported that illumination of the polyene antibiotic, pimaricin, with visible light in the presence of flavins results in destruction of the conjugated chromophore. This phenomenon has been used as the basis for an assay of nystatin in the presence of compounds which would otherwise interfere with the spectrophotometric determination of this antibiotic 15. The following experiments demonstrate that filipin exhibits a similar behavior and that, as might be anticipated, treatment of a polyene antibiotic in this manner abolishes biological activity completely. In aqueous media the absorption peaks for filipin are shifted towards slightly longer wavelengths: 357, 339, and 322 m/~ (cf. Fig. 3)- Fig. 4 (Curve A) shows that illumination in the presence of FMN causes a rapid decrease in absorbance at 339 m#; although not indicated a similar absorbance decline was also observed at 357 and 322 m/~. This decrease was appreciably slower when the ravin nucleotide was omitted (Curve B) and did not occur at all in the dark even in the presence of FMN (Curve C). Illumination of filipin in organic solvents had much less effect, in agreement with the original observations of ZONOAG, POSTHUMA AND BERENDS19. Thus, as shown by Curve D, the absorbance of filipin in methanol declined by only 16% of the initial value after 2o-min illumination whereas, under identical conditions, the absorbance of filipin in phosphate buffer had decreased by 80% (Curve A). It should be noted that the absorbance of filipin in organic solvents, such as methanol or dimethylformamide, is significantly greater than the absorbance of the antibiotic in water (cf. Curves A and D, 'zero time'). This observation has some experimental significance as discussed below. LAMPEN et al. 2° first described a similar phenomenon with nystatin and have also reported that this antibiotic is only slowly dialyzable, if at all. On the basis of these observations, they have suggested that the polyenes may be present to some extent as micelles in aqueous media. This conclusion is also supported by the observation of LAMPEN,ARNOW AND SAFFERMAN21 that filipin is sedimentable at low speeds in the centrifuge. This latter finding, which we have Biochim. Biophys. Acla, 135 (1967) 835-843

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Fig. 4. R e q u i r e m e n t s for p h o t o i n a c t i v a t i o n of filipin. C u v e t t e s c o n t a i n e d t h e following: 19.6 # g of filipin a n d o.oi # m o l e o f F M N p e r ml of o.o 5 M p o t a s s i u m p h o s p h a t e (pH 6.9; A, C); I9.6 # g of filipin p e r ml of p h o s p h a t e buffer (B); I 9 . 3 p g of filipin a n d o.oi p m o l e of F M N p e r m l of m e t h a n o l (D). A, B, a n d D were i l l u m i n a t e d as described p r e v i o u s l y ; C was k e p t dark. A t t h e t i m e s indicated, t h e a b s o r b a n c e was m e a s u r e d a t 339 m,u (A, B, C) or 337 m # (D). Fig. 5. Effect of i l l u m i n a t i o n on h e m o l y t i c a c t i v i t y of filipin. T u b e s c o n t a i n e d initially t h e i n d i c a t e d a m o u n t s o f filipin (IO, 5 o, i o o or 5o0 #g) a n d o.i # m o l e of F M N in o.95 ml o f isotonic saline. After i l l u m i n a t i o n for t h e t i m e s s h o w n , o.o 5 m l of a r a t e r y t h r o c y t e s u s p e n s i o n w a s added. Following i n c u b a t i o n for 90 rain at 37 °, i n t a c t cells were r e m o v e d b y c e n t r i f u g a t i o n . I n C u r v e D, filipin was a d d e d a f t e r i l l u m i n a t i o n a n d h e m o l y s i s was d e t e r m i n e d after 2 m i n i n c u b a t i o n w i t h t h e e r y t h r o c y t e s . I n this e x p e r i m e n t , c o m p l e t e h e m o l y s i s of t h e e r y t h r o c y t e s in i.o m l o f w a t e r g a v e a s u p e r n a t a n t solution w i t h a n a b s o r b a n c e (55 ° my) of o.864, w h e r e a s t h e c o r r e s p o n d i n g v a l u e in c o n t r o l tubes, c o n t a i n i n g d i m e t h y l f o r m a m i d e (no antibiotic) w a s o.o66.

been able to confirm, has precluded the use of centrifugation (approx. at IO ooo X g for IO min) as an experimental method by which to measure the amount of filipin which is bound to the erythrocyte membrane. Fig. 5 shows that irradiation of filipin in the presence of FMN results in complete destruction of hemolytic activity. Thus, under the conditions employed, 5 min illumination destroys io/zg filipin (Curve A), 15 min inactivates Ioo/~g (Curve B), whereas 9 ° min is required for the inactivation of 5oo y g of the antibiotic (Curve C). It should be emphasized that these values represent the time necessary to reduce the amount of active antibiotic below the minimum threshold level. With the amount of erythroeytes used in this experiment, filipin induces hemolysis at a concentration of approx, i yg/ml (Fig. 2). Thus the possibility that 499 #g of the antibiotic m a y have been inactivated in less than 9 ° min would not have been detected in the above experiment. Filipin, at concentrations of approx. 5o #g/ml water or greater, is present as a visible suspension. This m a y account for the fact that relatively long periods of irradiation are required for inactivation. I t would probably be better to carry out the reaction under conditions in which the antibiotic is completely soluble (see above) but organic solvents cannot be employed since they inhibit (Fig. 4). Unfortunately, the structure of the irradiated product(s) is not known. POSTHUMA AND BERENDS 2~ have suggested, on the basis of experiments with model compounds, that a t r a n s - c i s rearrangement m a y be involved. We have observed that the antibiotic becomes more water soluble as illumination proceeds. Nevertheless, the fact that irradiation results in complete loss of hemolytic activity provides an important tool. 13iochim. Biopkys. Acta, ~35 (1967) 835"-843

HEMOLYSIS BY FILIPIN

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Fig. 6. Effect of illumination on antifungal activity of filipin. An a q u e o u s suspension, containing 125/~g/ml of filipin and 0.05/zmole/ml of FMN, was illuminated for 90 min. Appropriate aliquots were t h e n added to I25-ml erlenlneyer flasks, containing 25 ml of Fries minima] m e d i u m , to give the final antibiotic concentrations indicated on the abscissa. Flasks were inoculated w i t h approx. 0.2 ml of a dense spore suspension of Neurospora crassa, strain 5297 a. After 44 h g r o w t h a t 3 °0 in the dark, mycelial m a t s were h a r v e s t e d and dried over P~O 5. Results are expressed as percent of control g r o w t h (dry wt.) obtained in flasks containing no antibiotic.

Irradiation of filipin not only destroys hemolytic activity but also abolishes the ability of the antibiotic to inhibit growth of Neurospora. As shown in Fig. 6, I #g/ml of filipin inhibits growth completely; under identical conditions, no effect is obtained with IO #g/ml of the irradiated derivative. In other experiments, even 5o/~g/ml of the irradiated derivative did not inhibit growth. These observations are significant because they illustrate again that antifungal and hemolytic activities are properties of the same molecule.

Effect of filipin and erythrocyte concentration In all earlier investigation, it was demonstrated with amphotericin B, pimaricin and nystatin that both the rate and extent of hemolysis was dependent on antibiotic] cell ratio 13. These antibiotics, however, are relatively weak compared to filipin thus necessitating the use of higher amounts and longer incubation times to achieve the same degree of hemolysis. I t was therefore of some interest to determine whether a similar phenomenon occurs with filipin. Fig. 7 indicates that the rate of hemolysis was markedly influenced by the initial antibiotic concentration. There was a pronounced lag phase before the onset of hemolysis with low levels of filipin. Similar results with amphotericin B suggest that binding and/or penetration of the polyene antibiotics to the erythrocyte membrane m a y be a rate-limiting step in hemolysis 13. In the preceding experiment, a constant number of erythrocytes was employed and the amount of filipin was varied. Results of the converse experiment, using a constant amount of antibiotic and varying amounts of erythrocytes, are shown in Biochim. Biophys. Acta, 135 (1967) 835-843

S. C. KINSKY, R. A. DEMEL, L. L. M. VAN DEENEN

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Fig. 7- Effect of filipin concentration on hemolysis of rabbit erythrocytes. Hemolysis was followed spectrophotometrically. Cuvettes contained initially the following concentrations of filipin in 5 ml of isotonic saline: 6.2 #M (A); 4.6#M (B); 3.1ffM (C); 1.5 ffM (D). The reaction was initiated by addition of 5° ffl of stock erythrocyte suspension (33%, by vol. as determined by hematocrit measurements). The molar ratios of filipin to membrane lipid were calculated as described in the text. Fig. 8. Effect of rabbit erythrocyte concentration on hemolysis by filipin. Hemolysis was folowed spectrophotometrically. Cuvettes contained initially 5 ml of isotonic saline and 3.I ~M filipin. The reaction was initiated by addition of the following amounts of stock erythrocyte suspension (33 %, by vol. as determined by hematocrit measurements) ; 25 ffl (A) ; 50/zl (B) ; IOO #l (C). The molar ratios of filipin to membrane lipid were calculated as described in the text.

Fig. 8. H e m o l y s i s was least extensive, a n d occurred a t the slowest rate, when t h e n u m b e r of e r y t h r o c y t e s was r e l a t i v e l y high. These o b s e r v a t i o n s d e m o n s t r a t e t h a t w i t h filipin, as w i t h t h e o t h e r polyenes, the antib.iotic/cell r a t i o a n d n o t only the a b s o l u t e c o n c e n t r a t i o n of a n t i b i o t i c is a critical factor. I n Figs. 7 a n d 8, we h a v e also i n d i c a t e d t h e m o l a r r a t i o s of filipin to e r y t h r o c y t e lipid. Thus, complete hemolysis was o b t a i n e d when the n u m b e r of filipin molecules a d d e d per molecule of lipid in the e r y t h r o c y t e m e m b r a n e was approx, o.10-o.21, at least u n d e r t h e conditions used. These values are significant because e x p e r i m e n t s to be described in a s u b s e q u e n t p a p e r n h a v e shown t h a t at these molar ratios, filipin p r e f e r e n t i a l l y i n t e r a c t s w i t h lipid m o n o l a y e r s containing cholesterol. NOTE ADDED IN PROOF

(Received September 25th, 1967)

W e have been i n f o r m e d b y Drs. M. E. BERGY a n d T. E. EBLE (The U p j o h n C o m p a n y , K a l a m a z o o , Mich.) t h a t t h e y h a v e r e c e n t l y been able to s e p a r a t e crystalline filipin, which has u p to now been r e g a r d e d as a single chemical e n t i t y , into 3 maj or p e n t a e n e c o m p o n e n t s . These c o m p o n e n t s are closely r e l a t e d chemically as i n d i c a t e d b y e l e m e n t a l analysis, ultraviolet, infrared, a n d N M R spectra. A l t h o u g h these comp o n e n t s show some v a r i a t i o n in biological p o t e n c y , it w o u l d i n d e e d be surprising if t h e y d i d n o t share t h e same basis for selective t o x i c i t y (i.e. i n t e r a c t i o n w i t h m e m b r a n e sterols) since t h e y all induce e r y t h r o c y t e hemolysis, are all g r o w t h i n h i b i t o r y to a v a r i e t y of fungi, and, like t h e original filipin complex, h a v e no a n t i b a c t e r i a l activity.

ACKNOWLEDGEMENTS

Research carried out in the authors' laboratories was supported by U.S. Public Health Service Grant AI-o5114 and a Research Career Development Award Biochim. Biophys. zJcta, 135 (1967) 835-843

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