Interaction with phospholipids as a possible mode of action of 3-phenylindole on Aspergillus niger

PESTICIDE

BIOCHEMISTRY

AND

Interaction

PHYSIOLOGY

6, 422429

(1976,

with Phospholipids as a Possible Mode of Action of 3-Phenylindole on Aspergillus niger

H. H. HOPPE,’ Institute

jar

A. KERKENAAR, Organic

ASD A. KAAHS SIJPESTEI~?;*

Chemistry

T.lrO,

I’irecht,

The Ntztherlands

Received September 16, 1975; accepted January 8, I Q76 Low concentrations of 3-PI(3-phenylindole) inhibit several uptake and biosynthetic processes in liquid cultures of Aspergillus nigw. The incorporation of 32P, into phospholipids, [Wluridine into nucleic acids, and [Wlphenylalanine into proteins was inhibited and t,he uptake of all three precursors was reduced. Studies on an in vitro interaction between 3-phenylindole and phospholipids showed, that at about. equimolar conc:entrat.ions 3-phenylindole prevents phospholipids from swelling in water. At the same ratio a decrease of the absorption intensity of the NH-band of 3-phenylindole was observed in ir spectra. Moreover, the maxima of the uv spectrum of 3-phenylindole shifted after addition of a sonicated phospholipid suspension. It is suggested that binding to phospholipids is the first effect of 3-phenylindole. Apparently this affects, consequently, several membrane-bound reactions, i.e., t,ransport and biosynthetic processes. INTRODUCTION

3-Phenylindole (3-PI)3 is an antimicrobial compound active towards many fungi and gram-positive bacteria (1). The lipid-soluble toxicant is rapidly taken up or adsorbed by the mycelium of Aspergillus niger. It inhibits mycelial growth and at higher concentrations also germination of spores but does not kill t.he fungus. 3-PI markedly affect,s the composit.ion of the lipid fraction inducing a decrease in phospholipid concentration with a coincident increase in free fatty acids. Under growth conditions the compound exerts a slight stimulation of 32P leakage from the cells. 1 Present address : Institut fiir Pflanzenpathologie und Pflanzenschutz, Georg August UniversitBt, 34 GBttingen, Federal Republic of Germany. * To whom requests for reprints should be sent. 3 The following abbreviations are used: 3-P& 3phenylindole; ‘*Pi, [32P]orthophosphate; TLC, thinlayer chromatography; MIC, minimum growth-inhibiting concentration; NMR, nuclear magnetic resonance; EPR, electron paramagnetic resonance.

The fungistatic activity is reversible by addition of phospholipids to the medium. The present paper reveals t#he effect, of 3-PI on several metabolic processes, all of which seem explicable in view of the observation that’ 3-PI binds t.o phoq)hc Ilipids. MATERIAL

Cultwe procedures. Most experiments were performed wit,h SO-hr-old small pellets of A. n.igel grown in shake culture in glucose mineral salts medium pH 6.9 as described earlier (1). 3-PI was added to the autoclaved medium from an awtone solution.

Final

acetone

oonec>ntrrttion

was

0.67&. “Pi imorporation ithto p/mph/lipids. I’cllets of A. ,rz.igerwere grown in IO-ml portions of nut#rirnt solution. The flasks contained about E-20 mg of mycclium dry weight. Then 2 or 5 pg of 3-1’1 per ml were added. After suitable intervals “Pi was added by replawment of th(L original 422

Copyright 0 1976 by Academic Press. Inc. All rights of reproduction in any form reserved.

AND METHOIjS

INTERACTION

OF

3-PHENYLINDOLE

medium by a new medium with a reduced phosphate content (0.004% KZHPO,, O.OOl’% KH,POJ containing 2.5 &i of [32P]orthophosphate/ml and the same amount of fungicide as used previously. A control flask without the fungicide was treated in the same way. The replacement of the medium was necessary to avoid extensive dilution of 32Pi and reduction of its uptake by the unlabelled phosphate in the original medium. After incubation for 2 hr with 32Pi the mycelium was harvested by filtration, rinsed with 10 ml of ice-cold distilled water and freeze dried. The first samples were already taken after 1 hr of incubation with 32Pi. Largely following the method of Bligh and Dyer the freeze-dried mycelium of each flask was extracted for at least 12 hr in a centrifuge tube with 4.75 ml of CH30H:CHC13:HzO (=2:1: 0.8) (2). Thereafter 1.25 ml of CHC13 and 1.25 ml of Hz0 were added. After centrifugation radioactivity was measured in 0.5 ml of the upper CHaOH-water layer and in 0.5 ml of the lower CHCl3 layer. Incorporation of 32Pi and [14C]acetate into individual lipids fractions. A niger pellets were grown in 50-ml portions of nutrient solution before addition of 5 lg of 3-PI/ml. The isotopes were applied as described before. Final radioactivity was 2.5 MC!i of [32P]orthophosphate/ml of nutrient solution and 0.2 PCi of sodium [1J4C]acetate/ml (specific activity : 59.3 mCi/mmol). Samples were incubated, with 3’Pi for 2 hr and with [14C]acetate for 0.5 hr. One hundred milligrams of the freeze-dried mycelium were extracted following the method of Bligh and Dyer (2). Lipids were dissolved in 0.5 ml of CHCl3. Labelled lipids in 0.04 ml of the total lipid extract were separated by one-dimensional TLC using the solvent system hexane: diethyl ether : acetic acid (70 : 30 : 1) for neutral lipids and the system CHC13: CHBOH : acetic acid: Hz0 (170 : 25 : 25 : 6) for phospholipids (3). TLC plates (Silica gel 60 F25a precoated, 0.25 mm thick, 20 X 20 cm) were purchased from Merck.

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[‘“C] Uridine incorporation into nucleic acids. [UJ4C]Uridine was added to A. niger pellets grown in lo-ml portions of nutrient solution as described for 32Pi in the phospholipid synthesis experiments. Final activity of [14C]uridine was 0.15 pCi/ml of nutrient solution (specific activity : 58 mCi/ mmol). After incubation for 30 min, the mycelium was harvested, freeze dried, treated overnight with 3 ml of 96% ethanol largely following the method of Clemons and Sisler (4) and, after addition of 3 ml of water, extracted for at least 4 hr. Thereafter the mixture was centrifuged and the pellet was again extracted twice with 3 ml of 50% ethanol. The residue was freeze dried. Radioactivity was assayed after oxidizing ca. 10 mg of dry material and also in 0.5-ml samples of the 50’% ethanol extracts. [14C]Phen&aZanine incorporation into proteins. [UJ4C]L-phenylalanine incorporation was measured in the same way as described for [14C]uridine. Final radioactivity of C’4C]phenylalanine was 0.1 pCi/ml of nutrient solution (specific activity : 522 mCi/mmol). Uptake experiments. In the 32Pi-uptake experiments 32Pi and 3-PI were added together to 50-hr-old cultures of A. niger by replacement of the nutrient solution, by a medium wit’h reduced phosphate content as described before. The final 32Pi activity was 1.2 pCi/ml of nutrient solution. After various intervals, the mycelium was filtered and rinsed with 10 ml of ice-cold distilled water. After freeze drying, 10 mg of dry material was treated with 1 ml of 0.1 M Na3P04 for 24 hr to solubilize the labelled compounds (5). Then dioxane containing scintillation solution was added for radioactivity measurements. Uptake of [14C]phenylalanine and [‘“Cluridine was determined in the same way measuring at intervals the radioactivity of the nutrient solution in O.l-ml samples. The same isotopes were used as in the incorporation experiments. At the st,art of

424

HOPPE,

KERKEiYAAR

dNO

the experiment.s the nukicnt solution contained 0.1 PCi of each labelled precursor/ml. Radioactivity measurement. Radioactivity measurement, was performed with a Packard scintillation counter. Whrn radioactivity of lipids was to be dekrmined the CHCl, was removed under a stream of X2 and the residue dissolved in 15 ml of counting solution consisting of 5 g of PI’0 and 0.3 g of dimethyl-POPOP per lit.cr of t,oluenc. Substances dissolved in water n-cw counted in dioxanr containing 120 g of nnphthalcne, 4 g of PPO, and 50 mg of POPOP/litw. For determination of the incorporation of precursors into grotcins and nucleic acids the freeze-dried cell residue was combusted with a TriCarb-Packard sample oxidizer. 14C02 was trapped with ethanolamine and counted with a scintillator on the basis of I’PO and 1,4-bis(3-methyl-st,yryl)benzcne. Radioactivity of thin-layer chromatograms KBS measured with a Berthold scanner. Spectroscopic an.alysis. I,ipid dispersions used for uv speet,roscopy were prepared by pipctting 80 mg of phospholipids (from egg yolk, Merck, Darmstadt) dissolved in CHCla into a X0-ml round-bottomed flask. After removing the solvent in a rotary (vapor&or the phospholipids dried :IS :t

KA.4RS

STJPESTETJN

thin lnyrr onto the surfwtt of the flask ww suspended in 1 ml of 50 mIII KC1 solut,ion (6). The suspension (20 mg of phonpholipids/ml) n-as sonic:ltt~d for 3 min in an iw Mb. 120r uv qwtrcwopy the lipid suspc~nsion was dilutcad to 100 pg of phospholipidsjml \vith 50 m.l/ I<(‘1 w)ntaining 10 pg/ml of 3-1’1. Samplrs for jr spec*troscopy wre propared by dissolving t,hc phospholipids (5%) tog&her with the fnngicid(b ;it different, ratios in CHCI:,; t’hcreaftw :L &in film of the solution n-as brought onto KRr pelkts and the solvent \Y:LS cwlporatwl rrndw :t stream of nitrogen.

Eject of S-PI IJ~(, ~~l~(~s~~l~oli~)itb,wcleic acid, arad proteirl sydhesis. A study of the cffwt of 3-1’1 on phospholipid synthesis rcveakd that treatment, of A. rrifl~ with 2 and 5 pg/ml had a pronounwd inhibitory effect on the incorporation of “‘1’; into phospholipids which KM slrcad?~ widcnt, 1 hr afkr fungicide application. At, 4 hr the rate of “Pi inctorporat,ion intcv phospholipids of t,hca t!rcated ti:tmplw W:LS on12 shout 15-300/, of the wntrol (I’ip. I).

/-100

IO0

BC

60 40

20

20

FIQ. 1. Effect of J-PI on the incorporafion of a2Pi info phospholipida (A), C’T]tmdinc, into nucleic acids (B), und [14C]phunyZalanine into proteins (C) oJ A. nigvr. (0), Treated with 2 pg/ml; ( l ), treated with 5 pg/rnl. Incubation with z21’i wu.s curried out Johr 2 or 1 hr in the fLrst sccrnplv. lncorporrition oJ the ‘“C-labelled precursors was measu.rc,d ozxr a 30-min pf,riod. Incubation lcifh ihe prcvursors ended at the time the point uppears in the jigurc. S-PI wa.s prtsmf continuously Jrom zero timr

INTERACTION

OF

%PHENYLINDOLE

[r4C]phenylalanine into proteins (Fig. 1). Nucleic acid synthesis was rather strongly affected. After 2 hr, 5 pg of 3-PI/ml inhibited uridine incorporation into nucleic acids 50%; shorter incubation periods with the fungicide had no clear effect on the radiolabel of nucleic acids. Incorporation of Q4C]phenylalanine into the protein fraction was inhibited less than 20% by 2 pg 3-PI/ml after 6 hr. Five pg 3-PI reduced protein synthesis 4 and 6 hr after application to about 5Oa/o of the controls. Inhibition of protein synthesis was less pronounced than that of nucleic acid and phospholipid synthesis. At 6 hr after fungicide application we observed in some experiments in the 2 pg/ ml treated samples a weakening of the fungicidal effect. In another experiment the rate of inhibition of phospholipid synthesis during the period 16-18 hr after application of 2 and 5 pg of 3-PI/ml was only 10 and SO%, respectively, indicating a degradation of the compound by the fungus at lower fungicide concentrations. Measuring radioactivity in the water phases of the extracts it was observed that the amount of labelled water-soluble compounds was also reduced in the treated mycelium but to a lesser extent t’han the inhibition of synthesis; this indicates that 3-PI may have interfered also with the uptake of 32Pi, [14C]uridine, and [14C]phenylalanine. Among the three aspects of cell metabolism investigated in this first part, phospholipid synthesis was t’he process most rapidly and severely inhibited by 3-PI. Effect of S-PI on 32Pi and [14C]acetate incorporation into individual lipids. In view of the early inhibitory effect on the incorporation of 32Pi into phospholipids it was determined whether this effect was specific for an individual phospholipid fraction or whether all phospholipids were affected in the same way. 32Pi and [*“Clacetate were used because these two precursors are taken up by the cell by completely different transport systems, enter

WITH

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425

independent pools, and label different parts of the lipid molecules. The labelled phospholipids extracted 2 and 4 hr after fungicide application were separated by TIC into four fractions (phosphat’idyl inositol, phosphatidyl choline + phosphatidyl serine, phosphatidyl ethanolamine, and diphosphatidyl glycerol + phosphatidic acid) and radioscanned. The 32P activity in the lipid extract could be attributed nearly exclusively to lipid-bound phosphate. Inorganic 32Pj was present only in trace amounts ( < 1%). 3-PI reduced the incorporation of ““Pi into all phospholipid fractions 2 and 4 hr after fungicide application. No specificity in the inhibition of phospholipid synthesis was detected. With [14C]acetate as precursor very similar results were obtained, the incorporation of [14C]acetate and 32Pi into phospholipids being inhibited by 3-PI to about the same extent. After 4 hr, but not after 2 hr the incorporation of [14C]acetate into the neutral lipids (sterols, sterol esters, free fatty acids, and triglycerides) was reduced by 5 /lg of 3-PI/ml. In these same experiments 3-PI appeared to reduce the 32Pi uptake from the medium but not that of [14C]acetate. In order to determine more clearly whether the inhibition of synthesis was the consequence of reduced precursor uptake, we measured the uptake of 32Pi, [14C]uridine, and [14C]phenylalanine by A. niger after 3-PI treatment. E$ect of S-PI on 32Pi, [14C]uridine, and [14C]phenyZalanine uptake. Control uptake of 32Pi occurred rather slowly under conditions applied, still being in the linear phase even after 6 hr of uptake. Phenylalanine, however, was taken up very quickly, after 2 hr most of the radioactivity being present in the fungus. 3-PI reduced the uptake of all three precursors (Fig. 2). The 32Pi uptake was quantitatively more affected than the uptake of uridine or phenylalanine. In the case of phenylalanine, differences between the con-

426

HOPPE,

KERKEXAAR

.4X1)

trols and the treated samples were observed only in the first 2 hr after isotope application. In the later phase, when apparently the rate of uptake by the control became limited by low concentrations of phenylalanine in the medium, differences in uptake between control and treated samples gradually decreased and, aft’er 6 hr, uptake of thr Yj Hg/ml treat,ed samples rcwhed 90-95y0 of the control. Since phosphate uptake is known to depend st’rongly on the pH of the medium (5) it was checked whether the differences in phosphate uptake between treated and cwnt,rol samples might be due to different pH vnluw of the medium. After the relat,ivcly short incubation period with 32Pi, howewr, there was no difference in pH between t,he samples. The above results showed that Z-PI interferes with several biosynthct’ic and uptake processes. Inhibition of phospholipid synthesis belonged to t*he early efferts of the fungicide, suggesting probably that this inhibition might br the cause for th(l decrraw in phospholipid concentration at later stages aftw fungicide application (1). Howevw, comparing the biosynthesis cxpwimmts with the quantitative phospholipid dct,wminations it, has to be taken into caonsidcration that in the cast of t)hc

K.4ARS

biosynthesis esperimrnts t,h(l nut rifbnt sulution had to be rrplarcd and th:lt th(b new medium containocl th(a :~mount of 3-PI :tpplicLd b>- wplawmt~nt plus t tics amount rctaincd in hyphuc. It is tliffic~ult to (*alwl:Lttk t hc> (x:wt 3-1’1 c7)nc~c~ntr:~tic111in tIi(a medium ; ho\~cvc~r, th(> rwults strl~v\-, ttlat at least t tic> 1II<’ inhibits phcwpholipid synthesis. Although inhibition (tf phospholipid synthesis is prc~h:~bl~. uot th(t primary ~aiiw of growth inlrihition, it might help to (axplain thaw lo\vcst. phospholipid (~ontmt of 41s t rw~c~d \\ith 5 and 10 pg;‘ml for 1-2 dn>x ‘1’11~~f:wt, that it

is

2

4

6

hr

I0teractiou

Z-PI innw form

FIG.

2. EJect (A),

A. niger. compounds radioactivity radioactivity

of S-PI

on the uptake (0), treated were added together at t = present in the mycelium; from the nutrient solution Untreated;

2

funglade

t0

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tklc’

fuilgi(‘id(’

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LL-.

--ii I

after

pOSSibl(h

addition of phospholipids to tht\ medium did alwady l(xd to th(s suggwtion that’ phospholipids might rrwt \vitll 3-1’1 in the medium (1). ~2 simil:~r intcwc~tion with phospholipids present in t hcs t)ic,nlc,mbriln~?s might explain the different t4fcsc*ts of &PI on m&bolism? bcwuw it is I;llo\vn that phospholipids aw involwd in m:\ny biosynthetic* and transport ~)r’cww;c~s (7). Somr cspc~rinicnts ww( c.:lrriclcl out. to study irr ~*itw intwnctiori twt \\ w&n 3-1’1 and phospholipids.

I-3

SIJPESTEIJN

6

hr

‘5

.-Am-J 30

60

“IIP

oppl’cot’on

of ““P;(A), [W]uridins (H), and [14C]phenylalaninr: (C) by with 2 rg/ml; ( l ), trralcd with 5 pg/ml. 3-PI and 1abtGd 0 to the medium. 32P, rrptak(, was determined measuring the in the case of [K’jurklinc and [‘4C]phmylalanirt.p ,uptake of (NS) was analysed.

INTERACTION

OF 3-PHENYLINDOLE

easily dispersable in salt solutions. Mixtures of phospholipids with 3-PI at the ratios 1:0.25 and 1:0.5 (v/v) formed a transparent film which did not mix with a 50 mM KC1 solution. Thus, at these ratios swelling and liposome formation of phospholipids in salt solutions was suppressed by 3-PI. A mixture at a ratio of 1:0.05 exhibited the behavior of pure phospholipids. At ratios of 1 :O.l and 1:0.25 liposome formation was reduced. The uv spectra of a 10 Hg/ml solution of 3-PI in 50 mM KC1 and in a 100 pg/ml phospholipid dispersion in 50 mM KC1 are given in Fig. 3. The fungicide in 50 mik? KC1 has absorption peaks with maxima at 221 and 265 nm, respectively. In the phospholipid suspension both maxima show bathochromic shifts of about 5 and 10 nm, respectively. With lower phospholipid concentrations this effect gradually decreased and concentrations lower than 20 fig/ml were ineffective. 3-PI dissolved in hexane showed the same spectrum as in 50 mM KC1 solution. Addition of phospholipids to hexane had no effect on the position of the absorption maxima of 3-PI. The main difference in the infrared spectra of the mixture phospholipids-3-PI (1:0.5 and 1:0.25, v/v), compared with the spectra of the single components in a solid film on KBr pellets, was a strong decrease in the intensity of the NH-band at 3400 cm-l compared with other absorption peaks of 3-PI. DISCUSSION

An earlier survey of effects on Aspergillus niger revealed a decrease of phospholipid content in the mycelium 2 and 3 days aft’er application of low concentrations of the fungicide (1). Evidence has now been presented that 3-PI even at concentrations below the MIC affects several biosynthetic and uptake processes. This makes it difficult to localize the primary site of action of the compound. Taking into consideration that it was possible to

WITH

427

PHOSPHOLIPIDS

210

250

300

2 350 h (nm)

Fro. 3. Ultraviolet spectra oj S-PI in 50 mM KC1 solution (A) and in a sonicated suspension of 100 ppm phospholipids (of egg yolk) in 50 mM KC1 (B).

reverse the antimicrobial action by addition of phospholipids to the medium it was studied whether possibly the lipophilic 3-PI interacts with phospholipids. Studies on liposome formation, uv and ir spectra strongly indicated in vitro interaction between phospholipids and 3-PI. Interaction could already be observed at ratios of phospholipids to 3-PI of 1:0.1-l :0.2 (v/v). This corresponds to a molar ratio of 1: 0.51: 1, calculated on the basis of an average molecular weight for the phospholipids of 1000. Suppression of liposome formation, shifts in uv, ir, and NMR spectra have been observed at similar ratios following the interaction between phospholipids and the antibiot.ics Chlorothricin, polymyxin B, and gramicidin S. For these compounds it is postulated that phospholipids are their receptor molecules in bacterial membranes (8-10). In contrast to 3-PI, however, these antibiotics cause a rapid destruction of the membranes, killing the bacteria. In the case of 3-PI, membranes are not destroyed but membrane function is affected. It is known that phospholipids play an important role in many transport and biosynthetic reactions. This subject was recently reviewed by Fourcans and Jain (7). Bilayer fluidity seems to be important for

42s

HOPPE,

KEHKENAAR

AND

the activity of membrane bound enzymes as well as for transport processes. Reduction of membrane fluidity by increase in degree of saturation of phospholipid fatty acids results in an inhibition of phospholipid, RNA and DNA synthesis in Exherichia coli. As in our experiments, protein synthesis was lntclr aff’&ed (ll,, 12). %Rlcthylindole, one of the main end products of tryptophan fermentation in rumen (IS), is known to induce acutch pulmonary edema and emphysema in cahtle (14) ; it also has slight antimicrobial activity (15). Tissue damage by this compound is related to its lipophilic properties. It is explained by an interaction with biological membranes. On the basis of NJIR and EPR studies it is suggested that] 3-methylindole interacts with lecithin, with t,he ordered regions of the alkyl chains (16, 17) also perturbing the structure of spin-labelled membrane proteins (16). A similar interaction with membrane phospholipids is proposed for 3-PI. Although it is difficult to discuss the nature of the interaction with phospholipids on the basis of the tentative spectrosropic studies, the results give a strong indication that such an interaction exists. Infrared sp&ra show t)hat’ the NH group of the indole ring may bc involved in this intcraction. This group plays also an important role in the antimicrobial activity, because substitution at position 1 or 2 in the indole nucleus suppressrs antimicrobial activity (15). The &ion of 3-PI can best be summarized as a rapid interaction of this lipid-soluble compound with phospholipids in the membranes of the fungus. The observed drcrcaasein several uptake and biosynthetic processes during the first l&6 hr after fungicide application may be the immediate result of the binding of 3-1’1 to the membranes. As found earlier (1) 5 pg/ml of 3-PI causes only a slight decline in total phospholipid content after

KSARS

SIJI’ES’I’ETJN

24 hr but about 50$‘, decrease after 48 hr, whereas in the untreated cells this content showed an increase in the s:~me period. One might expect that once :i decrease in phospholipid conknt has stwted, this would (~ausc :I shift in the! rat io phospholipid : S-PI rclsulting in :tn gr:ulual inwww in thcl primary c+fwt of :(-1’1. At, thcb same timtt n-h(Jn phospholipid cc~nc~:ntrntion d(Acr(xas(q growth stops :tnd dry wight starts to dwreaw indiwt’ing probably that’ at thcsr I:ltcbr st:lgw after fungicide application dec:rcaxc* (rf phospholipid (*ontent might inducae Icakagc. Thest> lntclr effects caouldb(sthcb immcdiatc (~auscof growt,h inhibition. The relatively long lag-phastb betwtrtn th(b early inhibition of uptake :md biosynthrsis prococ’ssc’sand thud inhibition of growth may indicate that, thth tirst c~ff~~c~t,s, which arc’ w\-c&rncd partially at’tcr thp first hours, havtl no s(‘v(ar(’ (:onsqucn(*(~s for the ~11. (;ron-th inhibition i’ollows immrdiatf>ly, when about 24 hr I:ltc?r phospholipid caonttlnt dcc*rc>ascls dutt iI 1inhibition of synthesis, to stimulnt~r~ddtLgr:ld:&n, OI to both, induwd by th(> intrir:rcl1ion of 3-1’1 with phospholipids in th(b mcnl/)ran(~s. No concalusiveexplanation VU t)(a0ffcrc.d for the mechanism of rc&t:tnc~c~ of thfx 3-PI-rcsistwnt strains of :I . )/I’!qc~..

We are thankful to Dr. J. CT. Overeem for his helpful discussious on the sperf loFic.opic experimeuts and his geueral iuterest in the subject. H. H. Hoppe thanks the J)e\ctsc+rr Forsc.hungsgemeirlsrhaft for financial sltpport

ftEFEHES(‘ES

1. H. H. Hoppe, A. Kerkenaar. and A. Kaars Sijpesteijrl, On the mode of action of 3phenylindole taowards A sprrgilkcs Niger, P~slic~. Hiochrm. Physiol. 6, 413 (1976). 9. E. G. Hligh and W. J. I)yer, A rapid method of total lipid extract.iorl and purification, Canad. J. Biochem. Physiol. 37, 911 (1959). 3. M. Kates, ‘Techniques of Lipidology,” p. 43.5, North-Holland American Elsevier, Amsterdam/London/New York, I !)72,

INTERACTION

OF 3-PHENYLINDOLE

4. G. P. Clemons and H. D. Sisler, Localization of the site of action of a fungitoxic benomyl derivative, Pestic. Biochem. Physiol. 1, 32 (1971). 5. H. S. Lowendorf, C. L. Slayman, and C. W. Slayman, Phosphate transport in Neurospora. Kinetic characterization of a constitutive, low-affinity transport system, Biochim. Biophys. Acta 373, 369 (1974). 6. A. D. Bangham, J. de Gier, and G. D. Greville, Osmotic properties and water permeability of phospholipid liquid crystals, Chem. Phys. Lipids 1, 225 (1967). 7. B. Fourcans and M. K. Jain, Role of phoapholipids in transport and enzymic reactions, Advan. Lipid Res. 12, 147 (1974). 8. W. Pache and D. Chapman, Interaction of antibiotics with membranes: Chlorothricin, Biochim. Biophys. Acta 255, 348 (1972). 9. W. Pache, D. Chapman, and R. Hillaby, Interaction of antibiotics with membranes: Polymyxin B and gramicidin S, Biochim. Biophys. Acta 255, 358 (1972). 10. M. Teuber, Action of polymyxin B on bacterial membranes. III. Differential inhibition of cellular function in Salmon&a typhimwium, Arch. Microbial. 100, 131 (1974). 11. M. Glaser, W. H. Bayer, R. M. Bell, and

WITH

12.

13.

14.

15.

16.

17.

PHOSPHOLIPIDS

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P. It. Vagelos, Regulation of macromolecular biosynthesis in a mutant of E. coli defective in membrane phospholipid biosynthesis, Proc. Nat. Acad. Sci. USA 70, 385 (1973). L. R. Kass, The antibacterial activity of 3-decynoyl-N-acetylcysteamine, J. Biol. Chem. 243, 3223 (1968). M. T. Yokoyama and J. R. Carlson, Dissimilation of tryptophan and related indolic compounds by ruminal microorganisms in vitro, Appl. Microbial. 27, 540 (1974). J. It. Carlson, M. T. Yokoyama, and E. 0. Dickinson, Induction of pulmonary edema and emphysema in cattle and goats with 3-methylindole, Science 1’76, 298 (1972). W. H. Dekker, H. A. Selling, and J. C. Overeem, Structure activity relationships of some antifungal indoles, J. Agric. Food Chem. 23, 785 (1975). T. M. Bray, H. E. Sandberg, and J. R. Carlson, An EPR study of structural perturbations induced by 3-methylindole in the proteins and lipid regions of erytrocyte membranes, Biochim. Biophys. Acta 382, 534 (1975). T. M. Bray, J. A. Magunson, and J. R. Carlson, Nuclear magnetic resonance studies of lecithin-skatole interaction, J. Biol. Chem. 249, 914

(1974).