THE ANTIMITOTIC PROPERTIES OF THE BENZIMIDAZOLE FUNGICIDE CARBENDAZIM AND AMECHANIS M OF RESISTANCE TO THIS COMPOUND INASPERGILLU S NIDULANS

CENTRALELANDBOUWCATALOGU S

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LEENDERT CORNELISDAVIDSE ,

landbouwkundig ingenieur,gebore n teSin t Laurenso p2 3januar i 1947i sgoed - gekeurd doord epromotor ,dr .ir .J .Dekker ,hoogleraa r ind efytopathologie .

DeRecto rMagnificu sva nd eLandbouwhogeschool ,

J.P.H.va nde rWan t

Wageningen, 18 augustus 1976 r IN Sz0° o i U L.C. Davidse

The antimitotic properties of the benzimidazole fungicide carbendazim and a mechanism of resistance to this compound inAspergillu s nidulans

Proefschrift ter verkrijging vand e graadva n doctor ind elandbouwwetenschappen , op gezagva nd erecto rraagnificus, dr. ir.J.P.H .va nde rWant ,hoogleraa r ind evirologie , inne topenbaa r teverdedige n opvrijda g 12novembe r 1976de snamiddag st evie ruu r ind eaul ava nd eLandbouwhogeschoo lt eWageninge n

2 /$0 ~ /0t/3/S~-<> Aanmij nouder s AanWill y

BIBLIOTHEEK * DER LAWDBO UV HOGE9CH0O> > WAGKNINGEN Stellingen

i Op grondva nhu nwerkingsmechanism e dientd etoepassin gva nbenzimidazoo l verbindingen als fungiciden inland -e ntuinbou w teworde nbeperkt .

II De conclusie vanNachmia se nBaras hda td eresistenti eva nSporobolomyce s roseusmutante n tegenMB Cberus t opee nycrminderd epermeabilitei tva nd e celmembraan,word t onvoldoende doorhu nexperimentel e gegevens ondersteund. Nachmias,A .e nBarash ,I. ,1976 . J.Gen .Microbiol .94 :167-172 .

HI hctdoo r Dassenoy enMeye rwaargenome nmutagen e effectva nbenomy li n Fusarium oxysporum hangtwaarschijnlij k samenme the twerkingsmechanism eva n dcze verbinding. Dassenoy,B .e nMeyer ,J.A. ,1973 . Mutation Res.21 :119-120 .

IV De resultatenva nd edoo rOlso ne nHeat h uitgevoerde experimenten betreffende het voorkomenva ncolchicine-bindend e eiwitten inSaprolegni a feraxe n Allomycesneo-moniliformis ,wettige n nietd edoo rhe ngetrokke nconclusies . Olson,L.W. ,1973 . Arch.Mikrobiol .91 :281-286 . Heath,I.B. , 1975. Protoplasma85 :177-192 . Ditproefschrift .

V De klassificering vanhe therbicid e isopropyl-N-phenylcarbamaatal see nver ­ bindingwelk e zijn effect opd eassemblag e vanmicrotubul iuitoefen t viaee n niet directe interactieme ttubuline ,i sgebaseer d opt eweini g experimentele gegevens.

Coss,R.A. ,Bloodgood ,R.A. ,Brower ,D.L. , Pickett-Heaps,J.D . enMcintosh ,J.R. , 1975. Exptl.Cel lRes .92 :394-398 . NRgt*/ £££

VI De opnameva n systemische fungiciden inplante n isee npassie fproce se n vindt plaats inzowe ld eapoplas t alsd esymplast .

VII Bijd ebestuderin g vand ebiochemisch ebasi sva nd ewaardplant-parasie tre - latiedien t meer gebruik teworde n gemaaktva nd emogelijkhede n diehe t werkenme teel - enprotoplas t culturesva nplante nbiedt .

VIII De studieva nd emoleculair ebasi sva nd egen-om-ge n relatie inwaardplant - parasiet systemenka nleide nto tee ndiepe r inzicht ind eprocesse nwelk eee n rol spelenbi jd egenregulati e ineukaryoten .

IX Hetverdien t aanbeveling eenmutageniteitstest ,welk e isgebaseer d opin - ductieva nmitotisch e recombinatiei ndiploide nva nAspergillu s nidulans, opt eneme ni nhe ttoxiciteitsonderzoe k vanchemisch e verbindingen.

Kafer,E. ,Marshall ,P . en Cohen,G. , 1976. Mutation Res.38 :141-146 .

X Ue juistheid vand eendosymbios e theorieva nLyn nMarguli s tenaanzie nva n het ontstaanva nmicrotubulair e systemen isnie ter gwaarschijnlijk .

Margulis, Lynn, 1976. in "De Microbiologic drie eeuwen na Antoni van Leeuwenhoek", biz. 72-96. Biologische Raad Reeks,Pudoc ,Wageningen .

Proefschrift van L.C. Davidse Wageningen, 12novembe r 1976. Contents

Voorwoord (Dutch)

Introduction 1

Articles: I.Antimitoti c activity ofmethy l benzimidazol-2-yl carbamate 3 (MBC)i nAspergillu s nidulans. Pestic. Biochem. Physiol.3 :317-32 5 (1973).

II.Antimitoti c activity ofmethy l benzimidazol-2-yl carbamate 12 in fungian dit sbindin g tocellula r protein. In"Microtubule s andMicrotubul e Inhibitors",eds .M .Borger s andM .d eBrabander . North-Holland Publ. Comp. (Amsterdam), 483-495 (1975).

III.Th emechanis m ofresistanc e toth eantitubuli n methyl 25 benzimidazol-2-yl carbamate inth efungu sAspergillu s nidulans. Submitted toJ .Cel lBiol .

IV.Metaboli c conversion ofmethy l benzimidazol-2-yl carbamate (MBC) 64 inAspergillu s nidulans. Pestic.Biochem . Physiol.,inpress .

Summary andgenera l discussion 80

Samenvatting (Dutch) 82

Curriculum vitae (Dutch) 84 Voorwoord

Bij het verschijnen vandi t proefschrift wil ik graag alien bedanken die aan het totstan d komenerva nhebbe nbijgedragen . Mijn ouderswi l ik graagnoeme nomda t zijmij n studiemogelij kmaakten . Willy Flachheef t door haar ijver enbekwaamhei dvee lbijgedrage n aanhe tre - sultaat van dit onderzoek. Bij een van de artikelen treedt zijda n ook opal s medeauteur. Adriaan Fuchs heeft met zijn grote belangstelling en kritische kommentaren het onderzoek gestimuleerd.Mij nmanuskripte nwerde n doorhe m vanvel ekant - tekeningen voorzien. Mijn promotor Prof.Dr . Ir.J . Dekker heeft ervoor gezorgd dat ikdi t onder­ zoek konbeginne n en gedurende eenvijfta l jaren konvoortzetten . Zijnvoort - durende belangstelling enopmerkinge n end e grote vrijheid die hij mijver - leende zijnmed ebepalen d geweest voor het verloop vanhe t onderzoek. De diskussies met de ledenva n deWerkgroe p Inwendige Therapie bij Planten, T.N.O. gaven stof totnadenke n en riepen nieuwe ideeenop . Annet Busser en Gerard Voogd hebben tijdens hun ingenieursstudie aanhe ton ­ derzoek meegewerkt.To nRammelo odee d een literatuuronderzoek tijdens zijn ingenieursstudie aand e TH inDelft .Joo p vanDrumpt , Gerrit Nellesteijn en WillemTwijse l hadden hun aandeel inee n vlotverloo p vand ewerkzaamheden . Dehere nJ.W . Brangert,G . Eimers,W.C.T .Middelplaat s en F.J.J.Vo n Planta verzorgden het foto-e n tekenwerk. Jaap Visser heeft een tweetal manuskripten kritisch doorgelezen. Dedame s vand e administratieva n de vakgroep fytopathologie en van deaf - deling tekstverwerking hebben demanuskripte n uitgetypt enbijgedrage n aan de uiteindelijkevormgevin gva nhe t proefschrift. De ledenva nd evakgroe p fytopathologie zorgdenvooree nklimaa twaari nhe t prettig werken was. Het Pudoc droeg zorg voor de vermenigvuldigingva nhe t proefschrift. De Centrale Organisatie T.N.O.,Secti e Landbouwkundig Onderzoek maaktehe t mij mogelijk aandi t onderwerp tewerke n en enkele kongressen tebezoeken . Tevens heeftT.N.O . financieel bijgedragen ind ekoste n van dit proefschrift. Academic Press, Inc.an d Elsevier/North-Holland Biomedical Press B.V. have givenpermissio n to reprint the articles incorporated in this dissertation. Desteu ndi e ik thuiskree g vanWill y end e afleiding dieElle n enPiete rmi j bezorgden,moge noo knie t onvermeldblijven . Hartelijk dankaa n aliendi ehie r genoemd zijn.Hartelij k dank ook aana l degenendi eme tbelangstellin gmij nwer khebbe n gevolgd. Introduction

Sinceth etim ema nbega nt ogro wplant s forfood ,h ewa sconfronte d with theproble mho wt oprotec thi scro p against diseases andpests ,whic h oftencause d aconsiderabl e reduction inyiel d andquality .Th ecause so f diseases andpest swer e initially shrouded inmyster y andmethod s tocontro l themwer e rather imaginative. Inth emiddl e ofth enineteent h century when fungiwer e recognized asth ecaus eo fa numbe ro fplan t diseases thefirs t scientific andsystemati c studies appeared inth efiel d ofplan t disease control. From that timeprogres s incro pprotectio n isclosel y linked with thediscover y anddevelopmen t ofne wchemica l compounds.Th efirs t fungicides used assuc hwer e elementary sulphur,coppe ran dmercur y preparations.In ­ creasing knowledge inchemistr ymad e itpossibl e tosynthesiz ene worgani c compounds andi n193 4th efungitoxi c properties ofsom e organic sulphur com­ poundswer e reported. Inth elas t fortyyear s anenormou s numbero fcompound s have been tested for fungicidal activity andsevera l hundreds have found usei npractice .Mos t of these compounds dono tpenetrat e intoplan t tissues and,consequently ,ca n onlypreven to rprotec t against infection.Absenc eo fpenetratio n isals oth e reasonwh ythes e compounds which mostly appeared tob egenera l cellpoisons , areno tharmfu l toth ehos tplant . Inth elas tte nyear s several compounds have beendiscovere d whichca n penetrate intoth eplan t andar etranslocate d within theplan t tissues.Thes e compounds,whic h areusuall y called systemic fungicides,hav e theadvantag e that theyma yeradicat e established infections,ma yprotec t newly grown parts ofth eplan t andar eles s subject toweathering . Iti seviden t that systemic fungicides have tob eselectiv e inthei r actionwit h respect toth eplan tan d theparasite . Inadditio nt othi s typeo fselectivit y systemic fungicidesar e usually also selectivewithi nth egrou po ffunga l plantpathogens .Som e fungi are very sensitive toa certai n compound,wherea s others areresistant . Soon after theintroductio no fsystemi c fungicides inpractic e negative aspects became apparent.I nsom e instances disease controlwa sn olonge r successful,becaus e fungicide-resistant strains developed inpreviousl y sen­ sitive fungalpopulations .Thi s phenomenon,wel l known ininsec tpes t control, was until recently seldom encountered incontrollin gfunga l diseases andmight , therefore,b einheren t toth eus eo fth ene wsystemi c compounds. Selectivity ofth esystemi c fungicides andresistanc e offung it othes e compounds may be due to adifferentia l uptake of the compound, a differential interaction with target sites or absence of target sites,a differen t con­ version intonon-toxi c or toxic derivatives,o r differences in regulatory systems of the organisms. For adetaile d account of the developments in the field of fungicide research the reader isreferre d to"Systemfungizid e- Systemic fungicides" (eds.H . Lyr andC . Polter,Akademi eVerlag , Berlin), to "Systemic fungicides",2 edition 1976 (eds.R.W . Marsh,Longma n Group Limited, London) and to"Antifunga l compounds",Vol .2 ,197 6 (eds.H.D . Sisler and M.R. Siegel, Marcel Dekker, Inc.Ne w York). Fundamental research on fungitoxic compounds has several aspects. From thepoin t of view of fungal disease control,knowledg e of theirmechanis m of action andpossibl e mechanisms of resistance might elucidate vulnerable sites in fungi. Itmay lead to the development ofne w compounds which specifically interfere with these sites. From the cell biological point ofvie w research on systemic compounds which act specifically may contribute towards ourin ­ sight incel l processes and their regulatory systems.Fro m the toxicological point of view elucidation of target sites makes itpossibl e tocarr y out ad­ ditional specific toxicological tests. Itmigh t be possible that compounds which have passed the general testsma y require further attention on account of the result of aspecifi c test. This thesis dealswit h the mechanism ofactio n ofcarbendazi m or methyl benzimidazol-2-yl carbamate (MBC), one of the systemic benzimidazole fungi­ cides and amechanis m of resistance of fungi to this compound. Biocidal activity of synthetic benzimidazole compounds was first reported in1961 , when the anthelmintic properties ofthiabendazol e were described. Theanti ­ fungal properties of this compound were discovered in 1964.Th e systemic benzimidazole fungicides benomyl and fuberidazole were introduced in1968 . After that time anumbe r ofothe r biologically activebenzimidazol e compounds were found and introduced as anthelmintics likemebendazole ,parbendazol e and fenbendazole, as fungicides like carbendazim, cypendazole and as antitumoral drug like oncodazole. This thesis contains four articles. In the firstpape r the localization of the site of action isdescribed . The second and third paper dealwit h the biochemical aspects of fungitoxicity and themechanis m of resistance in fungi. Sincemetaboli c conversion might play a role inth e mechanism ofre ­ sistance, this aspectwa s also studied. The results of this study are given in the fourthpaper . PESTICIDE BIOCHEMISTRY AND PHYSIOLOGY 3, 317-325

Antimitotic Activity of Methyl Benzimidazol-2-yl Carbamate (MBC) in Aspergillus nidulans

L. C. DAVIDSE

Research Unit for Internal Therapy of Plants, Laboratory of Phytopathology, Agricultural University, Wageningen, The Netherlands

Received March 23, 1973; accepted .May 28, 1973

Mitosis in germ tubes of Aspergillus nidulans was inhibited directly upon addition of methyl benzimidazol-2-yl carbamate to liquid cultures, whereas dry weight increase and DNA and RNA synthesis were progressively inhibited only after a few hours. Micro­ scopic observation of the organism, grown on malt extract agar containing MBC revealed abnormal chromatin configurations. The possible mode of action of MBC through interference with spindle formation is discussed.

INTRODUCTION action of benomyl and the mechanism of re­ sistance of fungi to this systemic fungicide. Methyl benzimidazol-2-yl carbamate (MBC), the conversion product of benomyl, MATERIALS AND METHODS thiophanate-methyl and 2-(3-methoxycar- bonylthioureido)-aniline, is generally ac­ Organism. Aspergillus nidulans bi A-l cepted as the actual fungitoxic principle of Acr A-l, requiring biotin and resistant to these systemic fungicides. Although MBC acriflavine, was used throughout this work. has somewhat inferior systemic fungicidal This strain was kindly provided by Ir. C. J. properties (1), this compound is at least Bos (Department of Genetics, Agricultural as toxic as the parent compound benomyl University, Wageningen) and was originally to Aspergillus nidulans (2) and many other- obtained from the Department of Genetics, fungi (3-5). A difference in toxicity of University of Glasgow. benomyl and MBC has only been found Chemicals. MBC was generously supplied toward Saccharomyces pastorianus (6), but by E. I. Du Pont de Nemours and Co. this appears to be an exception to the rule. (Inc.) (Wilmington, Del.). Calf thymus Hence MBC was the toxicant chosen in DNA and yeast RNA were purchased from this study. Sigma (St. Louis, Mo.) and Fluka AG Information concerning the effect of (Buchs, Switzerland), respectively. MBC on various cellular processes is given Culture methods. The organism was by demons and Sisler (3) and Hammer- grown on malt extract agar in Petri dishes. schlag and Sisler (6). These authors found To prepare conidial suspensions, the coni- an inhibition of DNA, RNA, and protein dia were washed off with sterile water con­ synthesis, which was progressive with time taining one drop of Tween 20 per 20 ml and and which they ascribed to a failure of filtered through two layers of Kleenex normal mitosis or cytokinesis. tissue to remove most of the hyphal frag­ This study was undertaken as part of a ments. The suspension was washed twice research program concerning the mode of with sterile water, and resuspended in sterile water (final conen approx 109 and RNA contents of the cultures. To this conidia per ml). Spores were then added to end, four 20-ml samples of treated and 100 ml of glucose-nitrate medium (7), control suspensions were spun down in supplemented with 500 /ig biotin per liter, centrifuge tubes and washed twice with in a 300-ml Erlenmeyer flask to give a distilled water. To remove low molecular concentration of 107 conidia per ml. weight compounds, samples were incubated Flasks were incubated at 37°C on a in 5 ml ice-cold 0.2 N perchloric, acid Gallenkamp orbital shaker at 150 rpm, (PCA) for 10 min in an ice-bath and centri- for about 10 or 16 hr, depending on the fuged. This process was repeated twice. method used in the inhibition experiments. The pellet was suspended in 4 ml 0.3 N Inhibition experiments. MBC. dissolved KOH and kept overnight at 37°C to in methanol, or methanol alone was added hydrolyze RNA. The suspension was cooled to 300-ml Erlenmeyer flasks containing in an ice-bath and 0.9 ml 2.5 N PCA was either 10 hr old cultures or 100 ml of a added to precipitate DNA. After centrifu- mycelial suspension, which was obtained gation the supernatant was decanted and by filtration of l(i hi' old cultures on a the residue; was washed with 4 ml ice-cold Buchner filter and resuspension of the 0.2 N PCA. The extract and the washing mycelial cake in fresh medium. In (he latter were combined and made up to 10 ml with case also 50-ml Erlenmeyer flasks contain­ 0.2 A' PCA. This fraction was considered ing 10 ml mycelial suspensions were used. the RNA fraction and was assayed for In all instances, the final MBC1 concen­ RNA by the orcinol method (8) with yeast tration in the medium was 4 ,uM ; the RNA as a standard. methanol concentration never exceeded The residue remaining after extracting 0.5% (v v) in treated and control media. RNA, was treated with 1 ml 1.5N PCA for The suspensions wen; incubated as de­ 20 min at 70°C to extract DNA. After scribed above. Samples of treated and centrifugingand decanting; the supernatant, control suspensions, consisting of the whole the residue was again extracted with 1 ml contents, of four 50-ml and two 300-ml 1.5 N PCA in the same way. The super- flasks per treatment were taken at intervals natants were combined and made up to 2.5 and immediately cooled in an ice-bath to ml with 1.5 AT PCA. This fraction was stop growth. considered the DNA fraction and was The contents of the 50-ml flasks were assayed for DNA by the diphenylamine used for dry weight determinations. The method of Burton (9) as modified by Giles contents of the two 300-ml flasks were and Myers (10). Calf thymus DNA was added together and divided in 20-ml por­ used as a standard. tions for dry weight, DNA and KXA de­ Determination of number of nuclei per terminations and, in the experiments with germ tube. Two and a half milliliter samples 10 hr old cultures, also for determining the of treated and control suspensions wen- average number of nuclei per germ tube. pipetted in an equal volume of cold 10% Dry weight determinations. Growth was trichloroacetic acid, kept at 0°C for 30 measured by determining increase in fungal min and centrifuged (7). After washing dry weight of the cultures. Four 10- or with distilled water, the residue was taken 20-ml samples of treated and control up in 0.05 .1/ phosphate buffer, pH 7.2 suspensions were filtered using preweighed to which acridine orange was added, the filter papers. After washing with distilled final concentration being 100 Mg per ml water the filter papers were dried overnight (11). Clumps of germ tubes were broken at 60°C and reweighed. up by agitating the samples in a 50 ml DNA and RNA analysis. DNA and RNA beaker placed in a Metason 1500 ultrasonic synthesis was measured as increase of DNA cleaner (Struers Scientific Instruments, ANTIMITOTIC ACTIVITY OK VIBC

Copenhagen). The material was not dam­ aged by this treatment. The hyphae were viewed with a Zeiss microscope, equipped with an ultraviolet light source (barrier filters 50 and 41, exciter filters I and II). Using this technique the nuclei appear green in red cytoplasm. Nuclei were counted in at least 50 germ tubes (12). Cytological observations. Conidia were allowed to germinate overnight on sheets of dialysis tubing, sterilized by boiling in water, and placed on the surface of malt extract agar (13, 14) with or without 4 fiM MBC. After incubation the sheets were re­ moved from the agar and fixed in modified Helly's solution (15) for 10 min and washed with 70% (v/v) ethanol in water. After hydrolysis for 12 min in 1 N HC1 at 60°C (14) and after washing with distilled water, HOURS the material was stained for at least 1 hr FIG. 1. Effect of 4 (ill MBC on increase of dry with Giemsa (2 ml Giemsa's Losung, weight of mycelium of Aspergillus nidulans. MBC Merck, in 100 ml 0.05 M phosphate buffer, addedat t = 0 to 16hr oldmycelium infresh medium. pH 7.0). The colonies were viewed with a The initial inoculum was 7.9 mg dry wt/10 ml mycelial suspension. Wild photomicroscope.

period. This inhibition proved to be pro­ RESULTS gressive with time so that after 8 hr of MBC (4 nM) did not markedly inhibit incubation with the toxicant synthesis of the increase in dry weight of A. nidulans DNA and RNA was inhibited for almost mycelium during the first 4 hr of the incu­ 100%. At this time, the DNA content of bation period, when an initial inoculum of treated samples was almost doubled, while 7.6 mg dry wt per 10 ml suspension of 16 in the controls the amount of DNA was hr old cultures was used. Strong inhibition more than three times the initial amount. became evident after 4 hr (Fig. 1). Treat­ Inhibition of increase in number of ment of the mycelium with 1 uM MBC nuclei per germ tube was much more during the preexperimental period did not pronounced (Fig. 3). At a concentration of eliminate or reduce the delay in MBC 4 MM, MBC completely inhibited the in­ inhibition of growth. As to increase in crease in number of nuclei per germ tube, DNA and RNA content of the samples a immediately after addition of the toxicant similar pattern was observed (Fig. 2); to 10 hr old cultures. Counting the nuclei however, in this case the inhibition became revealed synchrony of mitosis in individual apparent already after 2 hr. In the latter hyphae as has been reported by Rosen- experiment the inhibition of growth, meas­ berger and Kessel (7). Under these circum­ ured as increase in dry weight during 8 hr stances, inhibition of increase in dry weight of incubation, was about 13% (initial was not noted, while increase in DNA and inoculum 26 mg dry wt/20 ml mycelial RNA content was inhibited for 43 and suspension of 16 hr old cultures). The 13%, respectively, during 4 hr of incubation inhibition of increase in DNA and RNA (initial inoculum 16.4 mg dry wt/20 ml content was about 62% during the same mycelial suspension of 10 hr old cultures). Fm. 2. Effect of 4 /J.\1 MliC on increasi of dry weight (0,9), DXA (•, • and li.\A (A, A) content of mycelial suspensions of Aspergillus nidulans. M KC added at \ = 0 to lfi hr old mycelium in fresh medium. The initial inoculum was 2t> mg dry ui 20 ml mycelial suspension.

With regard to the last experiments, the caused by MBO. Normal phases of mitosis, decreasing RXA content of the germ as were found in control hyphae (Fig. 4B), tubes, viz, from 92 MK UNA mg dry \vt at were not seen in treated hyphae. t = 0 to 69 MR RNA mg dry wt at t = 4, is worth noticing. This phenomenon was not observed in experiments with l(i hr old DISCUSSION germ tubes. The RXA content here was The inhibition of growth of A. nidulans about 55 MgRX A mg dry wt and remained by MHO can be ascribed to an interference the same during the incubation period. with mitosis by the fungicide. Upon ad­ Giemsa-Ht'l staining of MBO treated dition of MBO to liquid cultures the average hyphae revealed a remarkable effect on the DNA content per nucleus increases till structure of the nuclei. While in control nearly twice the initial content (Fig. 5), hyphae the chromatin of the interphase since DNA synthesis is not directly in­ nuclei had a ring-shaped appearance, due hibited. This means that the inhibition of to the size and form of the nucleolus (14, DNA synthesis after 2 hr of incubation in Fig. 4A), in treated hyphae the chromatin the presence! of MBO is the result of was much more contracted and did not arrested mitotic activity. Due to the appear in a ring shape (Fig. 40, D). No asynchronous character of the culture, separate nuclei could be observed; this nuclei will be arrested in increasing number observation and the uneven distribution of in some stage of mitosis, resulting in a irregular masses of chromatin in the hyphae progressive inhibition of DNA synthesis. seems to indicate a disturbance of mitosis The doubling of the DNA content per ANTIMITOTIC ACTIVITY OF MBC

FIG. 3. Effect of 4 MM MBC on increase in dry weight (0,9), DNA (•, •) and RNA (A, A) content of mycelial suspensions and on average number of nuclei per germtube (€),0) of mycelium of A. nidulans. MBC addedat t — 0 to 10 hr old cultures. Initial dry weight 16.0 mg/20 ml mycelial suspension. nucleus after addition of MBC to the treatment with respect to inhibition of culture also means that the Gi period1 DNA synthesis. In the light of our results should be long in comparison with the G2 the doubling of the DNA content of the period of the division cycle. When phase culture of N. crassa conidia after addition Gi is long and phase G2 is short, most of the of MBC is to be expected, since resting nuclei are in phase Gi and upon addition of nuclei in spores have a minimal DNA MBC to the culture, these nuclei can go content and can go through the G1-S-G2 through the S phase, and thus synthesize phases of the division cycle before inhi­ DNA, before the division cycle is blocked bition becomes apparent. With U. maydis in mitosis. On the other hand, when phase sporidia, however, DNA content of cultures Gi is short and G2 is long most of the nuclei only slightly increased upon treatment with are in the latter phase and addition of MBC MBC. As this system is similar to the will result in at most a slight increase of system we used to study the effect of MBC, DNA content per nucleus. The result of this the results of Clemons and Sisler would indirect measurement of the length of suggest that in U. maydis sporidia phase Gi phase Gi and G2 agrees well with the results is short and phase G2 is long. of Bainbridge (12). The effect of MBC on RNA synthesis is Clemons and Sisler (3) reported a dif­ dependent on the age of the germ tubes. ferent response of Neurospora crassa conidia In 10 hr old germ tubes, in which RNA and Ustilago maydis sporidia to MBC content per mg dry weight is decreasing, RNA synthesis is less inhibited than DNA 1Th e (S-) phase of DNA synthesis during inter­ phase is preceded and followed by two phases, synthesis over a 4-hr incubation period. In G(ap)i and G(ap)2, respectively, during which no 16 hr old germ tubes RNA synthesis is DNA synthesis takes place. inhibited to the same extent as DNA syn- DAV I USE

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thesis, indicating a close correlation be­ the antimitotic activity of colchicine in tween DNA and RNA content. In younger human cell cultures (Hi). The mechanism germ tubes this correlation is apparently of action is based on the complex forming- much weaker and inhibition of DNA syn­ ability of colchicine with a protein subunit thesis might not directly result in inhibition of microtubules (17-19). Preliminary ex­ of RNA synthesis. periments showed that A. nidulans was The antimitotic activity of MBC in A. not inhibited by colchicine at concentrations nidulans shows a striking resemblance with of this compound up to 100 Mg/ml in malt ANTIMITOTIC ACTIVITY OF MBC

FIG. 4. Photomicrographs of Giemsa-HCI stained hi/phae of Aspergillus nidulans. A. Untreated; nuclei in

interphase, chromatin ring-shaped. B. Untreated: nuclei in mitosis. C, D. Treated with .'f y.W MBC. Abnormal configurations of dense masses of chromatin.

extract agar. However, as has been shown itself was not inhibitory to the organism. by Haber et al. (20), colcemid, a colchicine This difference in activity could not be derivative, inhibits growth and division of ascribed to a difference in ability of the two Saccharomyces cerevisiae under restricted compounds to permeate the yeast cell, but culture conditions. Also here, colchicine to a difference in affinity of the two agents DAVIDSE

bility of A. nidulans diploids (24), when exposed to sublethal concentrations of bonomyl.

ACKNOWLEDGMENTS The author expresses his gratitude to Dr. A. Fuchs for helpful discussions and for a critical reading of themanuscript . Hefeel s greatly indebted to Prof. Dr. J. Dekker for giving the opportunity to start this work and for his continuous interest.

REFERENCES 1. A. Fuchs, G. A. Van den Berg, and L. C. Davidse, A comparison of benomyl and thio- phanates with respect to some chemical and systemic fungitoxic characteristics, Pestic. Biochem. Physiol. 2, 191(1972) . 2. A. C.Hasti e and S. G.Georgopoulos , Mutational resistance to fungitoxic benzimidazole deriv­ atives in Aspergillus nidulans, J. Gen. Microbiol. 67,37 1 (1971). 3. G. P. Clemons andH . D. Sisler, Localization of FIG. 5. Effect of J, M-M MBC on relative DNA the site of action of a fungitoxic benomyl contentper nucleus. Data arebased on thosein Fig. 3. derivative, Pestic. Riochem. Physiol. 1, 32 (1971). 4. W. A. Maxwell andG.Brody , Antifungal activity for a cellular binding site, which was of selected benzimidazole compounds, Appl. Microbiol. 21, 944(1971) . provisionally identified as a microtubule ">. A. Kaars Sijpesteijn, Biochemical modes of subunit. This difference in affinity to the action of agricultural fungicides, WorldRev. binding site might be due to a minor modi­ Pest Conlr. 9, 85, (1970). fication of the structure of colchicine, for 6. K. S. Hammerschlag and H. D. Sisler, Differ­ ential action of benomyl and methyl-2-ben- colchicine has a NH-CO-CH3 group and zimidazolecarbamate (MBC) in Saccharomyces eolcemid a NH-CH group. 3 pastorianus, Pestic. Biochem. Physiol. 2, 123 The resemblance in the effect of MBC (1972). with that of colchicine and eolcemid on 7. R.F . Rosenberger andM .Kessel , Synchronyo f mitosis and synthesis of macromolecules nuclear replication in individual hyphae of might suggest a similar mode of action, viz, Aspergillus nidulans, J. Bacteriol. 94, 1464 (1967). the formation of a complex between MBC 8. H. N. Munro and A. Fleck, The determination and a subunit of microtubuli, resulting in of nucleic acids in "Methods of Biochemical inhibition of spindle formation. The NH- Analysis" (D. Glick, Ed.),Interscience , Vol. CO-OCH3 side chain of MBC might act 14, p. 145, New York 1966. in the same way as the active group of 9. K. Burton, A study of the conditions and mechanism of thediphenylamin e reaction for colchicine and eolcemid in determining the the colorimetric estimation of deoxyribonu­ binding properties between toxicant and cleic acid, Biochem. J. 62,31 5 (1956). protein. From this point of view the lower 10. K. W. Giles and A. Myers, An improved di­ intrinsic fungicidal activity of ethyl benzi- phenylamine method for the estimation of midazol-2-yl carbamate (EBC) (21-23) deoxyribonucleic acid, Nature {London)206 , 93 (1965). might be due to a lower affinity of this 11 A. J. Clutterbuck and J. A. Roper, A direct toxicant to a microtubule subunit. determination of nuclear distribution of This suggested mode of action of MBC Aspergillus nidulans, Genet. Res.7 , 185 (1966). could also account for the nuclear insta- B. W.Bainbridge , Macromolecular composition

10 ANTIMITOTIC ACTIVITY OK MBC

and nuclear division during spore germination 19. M. L. Shelanski and E. W. Taylor, Isolation of a in Aspergillus nidulans, J. Gen. Microbiol. protein subunit from microtubules. J. Cell 66, 319 (1971). Biol. 34, 549 (1967). 13. A. Fleming and G. Smith, Some methods for 20. J. E. Haber, J. G. Peloquin, H. O. Halvorson, the study of moulds, Trans. Brit. Mycol. Soc. and G. G. Borisy, Colcemid inhibition of cell 27, 13 (1944). growth and the characterization of a col- 14. C. F. Robinow and C. E. Caten, Mitosis in cemid-binding activity in Saccharomyces cere- Aspergillus nidulans, J. Cell Sci. 5, 403 visiae. J. CellBiol. 55, 355 (1972). (1969). 21. J. W. Vonk and A. Kaars Sijpesteijn, Methyl 1.5. C. F. Robinow, Mitosis in the yeast Lipomyces benzimidazol-2-yl carbamate, the fungitoxic lipofer, J. Biophys. Biochem. Cytol. 9, 879 principle of thiophanate-methyl, Pesiic.Sci. 2, (1961). 160 (1971). 16. E. W. Taylor, The mechanism of colchicine 22. T. Noguchi, K. Ohkuma, and S. Kosaka, inhibition of mitosis. J. Cell Biol. 25, 145 Chemistry and metabolism of thiophanate, (1965). Int. Symp. Pestic. Terminal Res. Tel-Aviv, 17. G. G. Borisy and E. W. Taylor, The mechanism Israel, 1971. of action of colchicine. Binding of colchicine 23. T. Noguchi, K. Ohkuma, and S. Kosaka, 3H to cellular protein. /. Cell Biol. 34, .525 Relation of structure and activity in thio- (1967). phanates, 2nd Int. Congr. Pestic. Chem., 18. G. G. Borisy and E. W. Taylor, The mechanism Tel-Aviv, Israel, 1971. of action of colchicine. Colchicine binding to 24. A. C. Hastie, Benlate induced instability of sea urchin eggs and the mitotic apparatus. Aspergillus diploids, Nature (London) 226, J. CellBiol. 34, 535 (1967). 771 (1970).

11 ANTIMITOTIC ACTIVITY OFMETHY L BENZIMIDAZOL-2-YLCARBAHATE INFUNG IAN D ITS BINDINGT OCELLULA R PROTEIN

L.C. Davidse

Research Unit for Internal Therapy of Plants T.N.O. Laboratory of Phytopathology, Agricultural University Wageningen, The Netherlands

1. Summary

Several lines of evidence indicate that methyl benzimidazol-2-ylcarbamate (MBC or carbendazim)and some other benzimidazole derivatives may be considered as antimitotic agents, acting as spindle poisons. A macromolecular MBC receptor with tubulin-like properties appeared to be present in Aspergillus nidulans and other MBC-sensitive fungi. The differences in response of fungi to MBC and the readiness with which changes in response in originally sensitive fungi can be induced may indicate a very specific action. A high specificity of the mechanism of action may also account for the failure of MBC to inhibit both porcine brain tubulin polymerization in vitro and the copolymerization of fungal tubulin into microtubules in a heterologous assembly system with porcine brain tubulin. In view of this, benzimidazole derivatives may prove to be valuable tools to compare biochemical and functional properties of tubulin from different sources. Purification of tubulin in the heterologous assembly system did not involve copurification of the high molecular weight proteins as is normally observed. Incorporation of fungal tubulin into microtubules might prevent their association with structures, composed of these proteins. Th e heterologous assembly system might provide a new approach by which the in vitro i aan d in vivo control of microtubule assembly can be investigated.

2. Introduction

Methyl benzimidazole-2-ylcarbamate (MBC or carbendazim, fig. 1) is one of the benzimidazole derivatives which is highly toxic to some groups of fungi. Partic­ ularly fungi belonging to the Ascomycetes are very sensitive, whereas others be­ longing to the Oomycetes are resistant . Other benzimidazole derivatives like 2-(thiazol-4-yl )benzimidazole (thiabendazole or TBZ, fig. 1) which is also known as an anthelminthic and 2-(2-furyl )benzimidazole (fuberidazole, fig. 1) have a similar spectrum of activity, although these compounds are less active than MBC. In agricultural practice these compounds have become increasingly important for the control of plant diseases. In addition to their high toxicity their systemic behaviour within plants is an important property for effective disease control. Due to efficient translocation these compounds can eradicate established infections and cure the plant. In some cases protection of new growth may be achieved by these systemic compounds.

12 —C— OCH3

%x^ %^ Carbendazim (MBC) Thiabendazole (TBZ)

0 II C_NH-CH2CH2CH2CH3 0 II C-OCH3 V^ ^^" Fuberidazole Benomyl

Fig. 1. Structural formulae of carbendazim (MBC), thiabendazole (TBZ), fuberidazole and benomyl.

Although MBC is used as such in spray formulations, methyl 1-(butylcarbamoyl) benzimidazol-2-ylcarbamate (benomyl, fig. 1) is more widely employed. The reason why benomyl which breaks down to MBC in aqueous solution, is used, is due to its 2 3 slightly superior qualities in a number of applications ' . However, soon after the introduction in practice of the benzimidazole deriva­ tives which were occasionally considered as the final and decisive solution to the control of certain diseases, negative aspects became apparent. In some cases, dis­ ease control was no longer successful, because resistance developed in previously sensitive fungal populations. Also in laboratory experiments resistance could be induced by mutagenic agents. This phenomenon, well known in insect pest control, was seldom encountered in controlling fungal diseases until recently and might, 4 therefore, be inherent to the use of the new systemic compounds . Since the benzimidazole derivatives were assumed to be very specific in their action, scientists became interested in these compounds not only from the point of view of their fungicidal aspects, but also because of their possible use as inves­ tigative tools to study the physiology of fungi. The purpose of this paper is to report recent findings on the mechanism of ac­ tion of MBC and the identification of its site of action. Also the biochemical basis of resistance to this compound will be discussed.

13 3. Mechanism ofactio n

5 The first report concerning themod eo factio n ofMB Cdre w attention toDN A and RNAsynthesi s asth epossibl e targets ofMB Caction . However, inhibitiono f these processes became only evident several hours after addition ofth ecompoun d to liquid cultures ofth efung i studied, viz. Neurospora crassa andUstilag o maydis.Th eobserve d inhibition might therefore beconsidere d asa secondar y effect. Since nuclear and cell division might be affected by MBC, these processes were studied separately from DNA synthesis using synchronous cultures of Saccharomyces cerevisiae and U. maydis or by simply counting nuclei in growing germ tubes of Aspergillus nidulans . These studies revealed that primarily mitosis was inhibited by MBC and that inhibition of DNA and RNA synthesis were secondary effects attributable to mitotic failure. In addition, light microscopic observations of MBC-treated hyphae revealed abnormal chromatin configurations. The antimitotic activity of MBC in fungi resembles mitotic arrest caused by Q colchicine inmammalia n cell cultures .Becaus e ofthi s similarity MBCmigh tb e a membero fa clas so fantimitoti c agents commonly knowna sspindl e poisons,o f 9 which colchicine canb econsidere d asa prototyp e.

4. Induced resistance toMB Can dTB Zi nAspergillu s nidulans

In ordert ostud y more precisely themechanis m ofactio n ofth ebenzimidazol e derivatives andth ebiochemica l basis ofresistance , resistance toMB Can dTB Z was induced inA .nidulan s .I tappeare d thatth eTBZ-resistan t strains couldb e distinguished into twocategories . Strains belonging toth efirs t category which comprised about9 5 %o fth etota l numbero fmutan t strains examined, were also resistant toMBC .A sdistinc t from these strains, strains belonging toth esecon d category which comprised theremainin g 5 %,showe d anincreas e insensitivit yt o MBC. Table 1give s theinhibitor y concentrations ofTB Zo rMB Ccausin g a5 0 % reduction inradia l growth onaga r (ED™ values)fo ron erepresentativ e straino f each category, viz. strain Ran dstrai n 186,a swel l asthos e forth ewil d type strain 003. Genetic analysis proved both resistance andincrease d sensitivity toMB Ct o be caused bymutatio n inth esam e gene, located onlinkag e group VIII .I nadditio n to thisa lowe r resistance toMB Cwa sfoun d tob ecause d bymutatio n oftw oothe r 11,12 genes For biochemical studies, strain R,strai n 186an dstrai n 003 were chosen, becauseo fthei r characteristic behaviour with respect toMBC .

14 Table1

Responseo fAspergillu s nidulans strains R,18 6an d00 3t oMB Can dTBZ . Effects were estimatedb ymeasurin g radial growtho nmal t agar medium containingMB Co r TBZa tdifferen t concentrations. Values.give nar einhibitor y concentrations causinga 5 0X reduction ingrowt h (ED5Q value)

6 ED5Q (ID* M) Strain TBZ MBC R 800 95 •186 195 1.5 003 50 4.5

14 5. Binding studies with C-MBC

The characteristic property of spindle poisons is their interference with the function of microtubules. Colchicine accomplishes this by binding tightly and very specifically to tubulin, the protein dimer of which the microtubule is composed. In order to determine the involvement of a similar binding of MBC to fungal tubulin in the antimitotic action of this compound, cell-free extracts of mycelium 14 of the three A. nidulans strains were incubated with C-MBC. Binding was deter­ mined by the standard gel filtration procedure using Sephadex G-100 columns. Typical elution patterns are shown in fig. 2. From this figure it is evident that radioactivity was present in macromolecular fractions, when extracts of sensitive mycelium were used. The MBC sensitivity of the strains was reflected in the amount of bound radioactivity. Mycelial extracts of the resistant strain did not show binding activity. Thin layer chromatographic analysis of the bound radioactivity showed that this as still present in C-MBC. As distinct from colchicine binding, MBC binding did not require a high tempera­ ture and appeared to be rapidly reversible, as is suggested by the fact that the peak of bound radioactivity showed considerable tailing. In this respect the Q binding mechanism resembles that of the spindle poison podophyllotoxin . The relation between binding of MBC and MBC sensitivity was further studied using normal sensitive strains of Penicillium species and resistant strains which 14 emerged in benomyl-treated crops . Also two naturally resistant fungi were in­ cluded, viz. an Alternaria and a Pythium species. It appeared that only mycelial extracts of sensitive strains showed considerable MBC-binding activity, whereas mycelial extracts from resistant strains did not. These data strongly suggest that MBC binding to fungal cellular material is essential in the antimitotic action of MBC. According to the elution volume of the MBC complex it can be concluded that the molecular weight of the binding species is approximately 110,000 daltons. This value has also been obtained in a more accurate study using a Sephadex G-200 co­ lumn, which was calibrated with proteins of known molecular weight.

FRACTION NUMBER

Fig. 2. Gel fi ltration of 40,000 x g supernatant mycelial extracts of Aspergillus nidulansstrains, containing approximately 40 mg protein per ml, incubated with 2.7 x 10 M 2- C-MBC (spec. act. 11.4 mCi/mmoie)for 120 min. at 4°C. Extracts were prepared in 0.05 M potassiumphosphate buffer, pH 6.8, containing 0.1 M KC1, 5 mM MgCl~, 0.1 mM GTP. Elution profiles of radioactivity of parallel experiments with extracts of strain 003 (x--x), strain 186 (o--o) and strain R (§--•) are ,, shown. The elution profile of Blue Dextran is indicated by A...A (from Davidse ).

The MBC-binding substance was further characterized by chromatography on a DEAE-Sephadex A-50 anion exchanger. This method was employed because of the sup­ posed identity of the MBC-binding substance with fungal tubulin. Elution profiles of radioactivity and protein in experiments with mycelial extracts of strain 186 14 and strain R, incubated with C-MBC are shown in fig 3A and 3B, respectively. With extracts of strain 186 radioactivity was eluted in two peaks, one with the bed volume of the column and the other at 0.6 M KC1, whereas with extracts of strain R the activity was eluted in one single peak with the bed volume of the column. On the basis of the retention properties on DEAE-Sephadex ion exchangers and the estimated molecular weight of 110,000 daltons there is good reason to believe, that the MBC-binding macromolecule is identical with fungal tubulin. Assuming that binding prevents assembly of tubulin into functional microtubules, the mechanism of action of MBC might be similar with that of colchicine and MBC might be considered a spindle poison, especially active in certain fungi. Induced resistance and extra-sensitivity of A. nidulans strains might then be based on changes in affinity of the receptor site to MBC, caused by mutation of the coding gene. Mutations involving resistance and mutations involving increased sensitivity 12 were shown to be about 16 nucleotides apart from each other , corresponding with a distance of about 5 amino acids in the protein. These data are consistent with

16 50 60 70 FRACTION NUMBER FRACTION NUMBER

Fig. 3. DEAE-Sephadex A-50 column chromatography of 40,000 x g supernatant mycelial extracts of strain 186 (A) and strain R (B) of Aspergillus nidulans. A 1.5 x 23 column was loaded with 4 ml of an incubation mixture (for details see text fig. 2) and was eluted first with buffer containing 0.1 li KC1, followed by a linear gradient of 0.1 - 1.0 II KC1. Radioactivity (•--•) and protein (x--x) was -,, determined in 1 ml of each 2 ml fraction ( ) KC1 concentration. (From Davidse ~).

the hypothesis that changes in affinity might be associated with changes in amino acid composition of the MBC-binding site on tubulin, without interfering with its functional properties.

6. Partial purification of the MBC-binding protein

In order to know more about its biochemical properties an attempt was made to 14 purify the MBC-binding protein, using C-MBC as a marker. Since the binding pro­ tein was assumed to be identical with fungal tubulin, a standard tubulin purifi- 15 cation procedure was employed. A 127,000 x g (60 min) supernatant mycelial extract was fractionated with a saturated ammonium sulphate solution in water. The fraction precipitating between 35 and 50 % saturation was taken up in buffer and 14 incubated with C-MBC. The incubation mixture was run onto a DEAE-Sephadex A-50 column and the eluted fractions were assayed for radioactivity. Fractions eluted at c. 0.6 MKC 1 which contained bound MBC and, therefore, the binding protein were

17 subjected to SDS-polyacrylamide electrophoresis (fig. 4). From this figure it is evident that in these fractions a protein is present with similar electrophoretic properties as brain tubulin. According to the specific tubulin purification procedure used, this protein might be considered as fungal tubulin. However, in addition to this protein several other proteins are present, so that from this experiment it can not be definitely concluded that fungal tubulin is responsible for MBC-binding. Further purification of the MBC-binding protein was greatly hindered by the instability of the binding activity. In 40,000 x g supernatant mycelial extracts binding activity rapidly decayed in a first order manner with a half time of 280 min. at 4 C. Guanosine triphosphate (GTP), which is known to stabilize colchicine- -binding activity of tubulin, did not affect the rate of decay. However, in these crude extracts, GTP might be rapidly hydrolyzed, so that no effect can be -5 anticipated. MBC itself, at 10 M, stabilized the binding activity resulting in a half time of decay of 920 min. Possible due to this rapid decay in the absence of MBC hardly any binding activity could be detected in combined and concentrated (vacuum dialysis or ammonium sulphate precipitation) DEAE-Sephadex A-50 fractions, eluted at c. 0.6 H 14 KC1, when these were incubated with C-MBC and analyzed for bound radioactivity by gel filtration on Sephadex G-100 columns. However, the possibility that passage of an extract through this type of column might remove a factor required for the formation of the MBC-protein complex, can not be ruled out. 3 In mycelial extracts showing MBC-binding activity no H-colchicine-binding activity could be demonstrated. Although normally colchicine binding can be seen as an indication of the presence of tubulin, in fungi colchicine probably can not be used as a diagnostic tool. Fungal mitosis appeared to be insensitive to colchicine ' presumably due to low affinity of fungal tubulin to this compound. Although the definite proof has not yet been given the data presented support the idea that MBC is bound to fungal tubulin and that MBC might be used as a diagnostic tool for fungal tubulin.

7. Toxicity of MBC in mammalian cell cultures

20 21 As has been shown ' MBC exhibits antimitotic activity in mammalian cell cultures. Studies of the mechanism of action in mammalian cell extracts might give more information about the character of MBC as a spindle poison. Binding studies 14 with C-MBC and porcine brain extracts revealed that no binding occurred after incubation at 4 C nor at 37 C, although the preparations showed considerable 3 H-colchicine-binding activity. In vitro polymerization of porcine brain tubulin into neurotubules was not affected by MBC. Another benzimidazole derivative, methyl 5-(2-thenoyl)- 22 benzimidazol-2-ylcarbamate, (R 17934), however, proved to be very effective

8. Effects of R 17934 on fungi

In preliminary studies with R 17934 it was shown that this compound could effectively inhibit the growth of fungi. It was found active against A. nidulans even at lower concentrations than MBC. The response of the three above-mentioned strains was similar to that towards MBC, viz. strain R was resistant compared with strain 003, whereas strain 186 appeared to be most sensitive. In binding experiments with C-MBC, R 17934 could effectively reduce the amount of MBC bound to the protein. These facts indicate that at least in fungi R 17934 and MBC have a similar mechanism of action.

9. Heterologous assembly of fungal and porcine brain tubulin

Assuming interference of NBC with microtubule formation during mitosis in fungi, a study of its effect on fungal tubulin polymerization in vitro might add to our understanding of the ability of MBC to act as a spindle poison. Preliminary polymerization experiments with fungal tubulin preparations, purified by DEAE-Sephadex A-50 chromatography were not successful. This could be due to the low amount of protein which could be purified and which was probably not enough to reach the critical concentration essential for tubulin polymeri- 23 zation . On the other hand also cofactors essential for polymerization might 24 25 have been removed during the purification procedure ' Possible interference of MBC with the assembly of fungal tubulin might-also be studied in a heterologous assembly system. Literature data ' report the successful assembly of brain tubulin onto flagellar microtubules from Chlamydo- monas and sea urchin sperm. However, tubulin from the cystoplasmatic pool of 27 Chlamydomonas did not copolymerize with brain tubulin, but prevented assembly . In preliminary experiments using 40,000 x g (60 min) supernatant porcine brain extracts and 127,000 x g (120 min) supernatant mycelial extracts of A. nidulans no polymerization could be obtained, as was shown by analysis of the various fractions by SDS-polyacrylamide gel electrophoresis. Partial purification of the mycelial extract by 35 - 50 %ammoniu m sulphate fractionation and passage of the preparation over a Sephadex G-25 column, equilibrated with reassembly buffer was found necessary to obtain polymerization equal to that in the porcine brain extract, as was revealed by protein determinations in corresponding fractions. That indeed microtubule assembly occurred in the mixture was established by electron microscopy. Microtubules being assembled in the mixture appeared to be similar to those in porcine brain extracts. Fig. 5 shows the results of electro-

19 phoretic analysis of the tubulin preparations after one cycle of assembly and disassembly. It is evident that the assembly product, obtained in the mixture does not contain the two high molecular weight proteins which were found to be always yt po OQ 30 present in tubulin preparations '' ' , purified by the assembly procedure. These proteins were reported to be the components of filaments attached to in 31 vitro assembled brain microtubules (neurotubules) and neurotubules in situ . Besides the tubulin band several other minor bands were visible on the gels loaded with the assembly product of the mixture which did not appear on the gels of the control preparation. These components persisted through a further second assembly and disassembly step. These results prompted us to carry out a heterologous assembly experiment, in which the proteins of the fungal extract were labeled. Proteins were labeled by growing mycelium during the last three hours of the growing period in the presence of 25 uCi 14C-phenylalanine per 1 of medium. About 90 %o f the label was taken up and proteins were labeled at about 44,850 dpm/mg protein as was determined in the 35 - 50 %ammoniu m sulphate preparation after gel filtration on the Sephadex G-25 column. _5 After one cycle of assembly and disassembly HBC (10 M) was added to a sample of the preparation, whereas a similar sample served as control, and a second cycle of assembly was performed. The various fractions were analyzed by SDS-polyacrylamide gel electrophoresis. Gels were sliced and the amount of radioactivity was determined in each segment.

Fig. 4. SDS-polyacrylamide electrophoretic analysis of DEAE-Sephadex A-50 fractions containing MBC-binding protein. Five ml of a 35 - 50 %ammoniu m sulphate preparation in extraction buffer (see text fig. 11 of a 127,000 x g (60 min) supernatant mycelial extract were incubated with C-MBC(final concentration 9 x 10 M; 226,000 dpm/ml) for 120 min. at 4 C. The incubation mixture was loaded onto a 20 ml DEAE-Sephadex column, which was subsequently eluted with 15 ml of a linear gradient of 0.1 - 0.4 M KC1, 15 ml of 0.4 M KC1, 30 ml of a linear gradient of 0.4 - 1.0 M KC1 and 20 ml 1.0 M KC1, respectively, al 1 made up in buffer. Fractions of 2.5 ml were collected and assayed for radioactivity. Fractions from the 0.6 M legion which contained resp. 1670, 2160, 4170, 7230, 5570, 2590 and 1070 dpm bound C-MBC per ml were subjected to electrophoresis (gel A - G). As reference tubulin purified by two cycles of assembly from a porcine brain extract was run on a separate gel (T). Densitometer tracings were made of gel T and gel D (peak fraction of MBC-binding protein). Samples were prepared for electrophoresis as described Gels consisted of 7.5 %acrylamide , 0.13 % bisacrylami de, 0.1 % SDS in 0.1 M sodium phosphate buffer (pH 7.0). Gels were stained for 60 min. in 0.1 %Coomassi e Brilliant Blue in a methanol, water, acetic acid mixture (227:227:46). Destaining was performed by diffusion in 7 %aceti c acid in water.

Fig. 5. SDS-polyacryl amide gel electrophoresis of tubulin, partially purified by one cycle of assembly in porcine brain extract (1) and in a mixture of porcine brain and fungal extract (2). For electrophoretic and staining conditions see fig. 4.

20 zu- T 3 12- m-

3 .8-

.6-

.4- 2- u, J 0 (-) (+)

Fig.4 21 The results of this experiment are given in table 2.

Table 2

Heterologous assembly of fungal and porcine brain tubulin. Two ml of a partially purified mycelial extract (13 mg protein per ml; 44,850 dpm/mg protein) of Aspergillus nidulans strain 003 were mixed with 8 ml of a porcine brain extract (5.5 mg peotein per ml). Two cycles of assembly and disassembly were performed '. In cyle II the effect of MBC (10"b M) was studied. Total radio­ activity (dpm) and its distribution (%) among the various fractions are given. Also the percentages of radioactivity actually present in tubulin subunits as revealed by SDS-polyacrylamide gel electrophoretic analysis are given.

Assembly Radioactivity of the fractions (dpm) mixture H,S C,S cycle l 1,166,000 (100 %) 1,150,000 (98.6 %) 15,750 (1.4 %)

t^S H2S H2P cycle H control 10,500 (100 %) 7,680 (73 %) 2,410 (23 %) MBC 10,500 (100 %) 8,000 (76 %) 2,390 (23 %)

SDS-polyacrylamid e gel electrophoresis Radioactivity of tubulin {% of total radioactivity of the fraction)

CXS H2S H2P cycle H control 49 42 73 MBC 49 47 72

H,S : 45,000 x g (30 min. 25 C) supernatant from first assembly C,S : 45,000 x g (30 min. 4 C) supernatant from resuspended pellet (30 min. 0°C) from first assembly h„S : 45,000 x g (30 min. 25 C) supernatant from second assembly H„P : resuspended pellet (30 min. 0 C) from second assembly

As is evident from this table, copolymerization of radiolabeled fungal protein with porcine brain tubulin under microtubule assembly conditions strongly suggests that heterologous assembly has occurred in a mixture of brain and mycelial extract. Although in the first cycle only 1.4 % of the radioactivity could be recovered in the tubulin preparation, in the second cycle this percentage increased to 23% indicating that actual temperature dependent polymerization of labeled protein, has occurred. Gel electrophoresis proved that tubulin was mainly responsible for this phenomenon, because the amount of radioactivity present in tubulin increased from 49 % in C.S to 73 % in H2P. At the concentration used MBC did not have any effect on polymerization of radioactive protein, although the fungal preparation used did bind MBC, as was determined in similar preparations. An explanation for the inability of MBC to inhibit the assembly of fungal tubulin in a heterologous system can only be tentative, as long as the processes of MbC binding and fungal and procine brain tubulin copolymerization are not

22 completely clear. The inability of MBC to interfere with the heterologous assembly might be explained by supposing that an MBC-fungal tubulin complex is the result 25 32 of interaction of MBC with the suggested Y-state of the tubulin molecule ' and that Y-state fungal tubulin molecules do not take part in the heterologous assembly process. At present experiments along these lines are in progress.

10. Discussion

Although the exact mechanism of action of MBC has not yet been elucidated, several lines of evidence indicate that it acts as a spindle poison in fungi. Its similarity with colchicine in binding to a protein with several biochemical characteristics of tubulin is one of these. The structural relationship of MBC with R 17934, a compound which certainly belongs to this class of antimitotic 22 agents and which probably displays a similar mechanism of action in fungi, also supports this idea. Whether MBC binding to fungal tubulin is comparable with colchicine binding to mammalian tubulin or whether MBC binding requires a specific state of the tubulin molecule is not yet clear. Changes in amino acid composition of the MBC-binding site which might result in affinity changes might explain the phenomena of resistance and increased sensitivity to the benzimidazole derivatives. However, mechanism underlying these phenomena might be more complicated and might also involve changes in amounts of tubulin molecules being able to bind MBC. Although it has been shown that part of the primary structure of the tubulin 33 molecule is strongly conserved during evolution , the colchicine-binding site might have evolved along different lines according to the differential colchicine- -binding properties of fungal and mammalian tubulin. In a comparable way, a binding site for benzimidazole derivatives might have been evolved leading to a differential response of mammalian and fungal cells to MBC. However, modification of the molecule might strongly influence its interaction with tubulin. Benzimidazole derivatives, therefore, might be valuable tools to study differences in biochemical and functional properties of tubulin from different sources. Purification of tubulin in a mixture of porcine brain and mycelial extract did not involve copuri fication of high molecular weight proteins as is normally observed. Supposing polymerization of these proteins being independent of that of microtubules, this phenomenon might be due to an inhibitory action of components 29 31 of the mycelial extract. However, according to literature data ' it is more likely that presence of the high molecular weight proteins in microtubule preparations is caused by a structural linkage of these proteins to microtubules.

23 Their absence in tubulin preparations purified from mixtures of brain and mycelial extracts, might, therefore, indicate that in this case linkage did not occur. This might be due to incorporation of fungal tubulin into microtubules making these less suitable for association with high molecular weight proteins. The incorporation of radioactive fungal protein in microtubule preparations in the heterologous assembly system suggestscopolymerization of fungal and brain tubulin. This means that despite the above mentioned differences between the two tubulin species, binding sites involved in polymerization should be highly conserved during evolution. The heterologous assembly system might provide a new approach by which the in vitro and in vivo control of microtubule assembly can be investigated.

The authorwishes to thank Miss Willy Flach for excellent assistance with the experimental work, and Dr. A. Fuchs for help and advice in the preparation of the manuscript. He feels indebted to Dr.Ir. J. Visser (Department of Genetics, Agricultural University) and Prof.Dr.Ir. J. Dekker for a critical reading of the manuscript.

References

1. Bollen, G.J. and Fuchs, A., 1970, Neth. J. PI. Path. 76, 299. 2. Fuchs, A. et al., 1972, Pestic. Biochem. Physiol. 2, 191. 3. Upham, P.M. and Delp, C.J., 1973, Phytopathology 63, 814. 4. Dekker, J., 1973, 0EPP/EPP0 Bui. 10, 47. 5. Clemons, G.P. and Sisler, H.D., 1971, Pestic. Biochem. Physiol. 1, 32. 6. Hammerschlag, R.S. and Sisler, H.D., 1971, Pestic. Biochem. Physiol. 3, 42. 7. Davidse, L.C., 1973, Pestic. Biochem. Physiol. 3, 317. 8.Taylor, E.W., 1965, J. Cell. Biol. 25, 145. 9. Wilson, L. et al., 1974, Federation Proc. 33, 158. 10. Van Tuyl, J.M. et al., 1974, Neth. J. PI. Path. 80, 165. ll.Hastie, A.C. and Georgopoulos, S.G., 1971, J. Gen. Microbiol. 67, 371. 12.Van Tuyl, J.M., 1975, Meded. Fak. Landb. Wetensch. Gent, 40, in press. 13. Davidse, L.C., 1974, Proc. IV Reinhardsbrunner Symp. 5-11 May Reinhardsbrunn (DDR), in press. 14.Bollen, G.J., 1971, Neth. J. PI. Path. 77, 187. 15. Weisenberg, R.C. et al., 1968, Biochemistry 7, 4466. 16. Weber, K. and Osborn, M., 1969, J. Biol. Chem. 244, 4406. 17.Shelanski, M.L. et al., 1973, Proc. Nat. Acad. Sci. USA 70, 765. 18. Haber, J.E. et al., 1972, J. Cell. Biol. 55, 355. 19. Burns, R.G., 1973, Exptl. Cell Res. 81, 285. 20. Styles, J.A. and Garner, R., 1974, Mutation Res. 26.M77. 21. De Brabander, M. personal communication. 22. De Brabander, M. et al., these proceedings. 23. Borisy, G.G. et al., 1974, Federation Proc. 33, 167. 24.Murphy, D.B. and Borisy, G.G., 1974, J. Cell. Biol. 63, 236a. 25. Weingarten, M.D. et al., 1975, Proc. Nat. Acad. Sci. USA 72, 1858. 26. Binder, L.I. et al., 1975, Proc. Nat. Acad. Sci. USA 72, 1122. 27. Farrell, K.W. and Burns, R.G., 1974, J. Cell Biol. 63, 98a. 28.Dentler, W.L. et al., 1974, Proc. Nat. Acad. Sci. USA 71, 1710. 29.Gaskin, F. et al., 1974, FEBS Letters 40, 281. 30. Kuriyama, R., 1975, J. Biochem. 77, 23. 31.Dentler, W.L. et al., 1975, J. Cell Biol. 65, 237. 32.Kirschner, M.W. et al., 1974, Proc. Nat. Acad. Sci. USA 70, 3594. 33.Luduena, R.F. and Woodward, D.O., 1973, Proc. Nat. Acad. Sci. USA 70, 3594.

24 The mechanism of resistance to the antitubulin methyl benzimidazole-2-yl carbamate in the fungus Aspergillus nidulans

L.C.Davids ee nW .Flac h

ResearchUni tfo rInterna lTherap yo fPlant sT.N.O . Laboratoryo fPhytopathology ,Agricultura lUniversit y Wageningen,Th eNetherland s

ABSTRACT

Theantimitoti ccompoun dmethy lbenzimidazol-2-y lcarbamat e (MBC) formeda comple xi nvitr owit ha protei npresen ti nmycelia lextract so f fungi.Th ebindin gprotei no fAspergillu snidulan sshowe da se to fproper ­ tieswhic hi suniqu efo rtubulin .Bindin goccurre drapidl ya t4 C an dwa s competitively inhibitedb yth eantitubulin soncodazol ean dcolchicine .Othe r antitubulinssuc ha spodophyllotoxin ,vinblastin esulfate ,melatoni nan d griseofulvindi dno tinterfer ewit hbindin go fMBC .Electrophoreti canalysi s ofpartiall ypurifie dpreparation so fth ebindin gprotei nreveale dth e presenceo fprotein swit hsimila rmobilitie sa smammalia ntubuli nmonomers . Hencei ti sconclude dtha tth ebindin gprotei ni sidentica lwit hfunga l tubulin. Theeffec to fMB Co nmycelia lgrowt ho fmutan tstrain so fA .nidulan s waspositivel ycorrelate dwit hth eaffinit yo fth ebindin gsite sfo rthi s compound.Th eapparen tbindin gconstan tfo rMB Can dtubuli nfro ma wild - 5 4 typestrai nwa sestimate da t4. 5x 1 0 ,fro ma resistan tstrai na t3. 7x 1 0, andfro ma strai nwit hincrease dsensitivit yt oMB Ca t1. 6x 1 0 1.mol e Mutantsshowin gresistanc ean dincrease dsensitivit yt oMB Car ecandidate s tohav ealteration si ntubuli nstructure .Lo waffinit yo ftubuli nfo rMB C isprobabl ya commo nmechanis mo fresistanc et othi scompoun di nfungi . Bindingo fcolchicin et oA .nidulan stubuli noccurre da t4 C wit ha n 3 -1 apparantbindin gconstan to f2. 5x 1 0 1.mol e .

25 INTRODUCTION

Inth elas t fifteenyear s severalbenzimidazol e compounds havebee nin ­ troduceda sfungicides ,lik ebenomy l (29),fuberidazol e (58),an dthiabenda ­ zole (66),a santhelmintics , like fenbendazole (5),mebendazol e (68), par- bendazole (1),an dthiabendazol e (12),o ra santitumora ldrug ,lik e oncodazole orR 17934 (3,4). Structural formulaeo fthes e compounds areshow ni nFig .1 . Inrecen tyear s considerable attentionha sbee n givent oth emechanis m of actiono fthes e compounds.Benomy l andit sconversio n product, carbendazim orMBC , interferewit h mitosis infung i (21,39,56),plant s (56)an dmanmalia n cells invivo(59,67 ) andi nvitro(27,59,67) .Mebendazol e induces degenerative changes inintestina l cellso fparasite s after treatment ofthei rhost s(8) . These effects areprobabl y causedb yth einteractio n ofthi s drugwit h cyto­ plasmicmicrotubule s (9,10). Oncodazole interferes with thestructur ean d functiono fmicrotubule s both ininterphas e andmitoti c mammalian cells cul­ tured invitr o (25,26).Microtubule s ofdividin g andnon-dividin g malignant cells inviv oar esimilarl y affected(26) . Although thebiologica l activity ofthes ebenzimidazol e compoundsi s probably based oninterferenc e with theformatio no rfunctionin g ofmicrotu ­ bules,whic har epresen t inal leukaryoti c cells, eukaryotes areno tequall y sensitive toeac hbenzimidazol e compound. Benomyl andMB Cpartl y owethei r success assystemi c fungicides toa relativ e non-toxicity toplant san d animals.Bu tals owithi n fungi,ther ear edifference s insensitivit y tothes e compounds.Fo rinstance ,fung ibelongin g toth eAscomycete sar esensitive , whereas othersbelongin g toth eOomycete sar eresistan t (7).I nadditio nt o natural resistance tobenomy l andMBC ,resistan t strains ofnaturall y sensi­ tive species arefrequentl y found inspraye d crops. Inth elaborator y such strainsca nb ereadil y obtainedb ymutageni c treatment(28) .

The following common names and abbreviations are used: benomyl,methy l 1-(butylcarbamoyl)benzimidazol-2-yl carbamate; fuberidazole, 2-(2'-furyl)benzimidazole; thiabendazole, 2-(4'-thiazolyl) benzimidazole; fenbendazole,methy l 5-phenylthiobenzimidazol-2-yl carbamate;mebendazole , methyl 5-benzoylbenzimidazol-2-yl carbamate;parbendazole ,methy l 5-butyl- benzimidazol-2-yl carbamate; oncodazole or R 17934,methy l 5-(2-thienyl- carbonyl)benzimidazol-2-y l carbamate; carbendazim orMBC ,methy l benzimi- dazol-2-yl carbamate; SDS,sodiu m dodecylsulfate;PKM g buffer, 0.05 M potassium phosphate buffer,p H 6.8, containing 0.1 M KC1 and 0.005M MgCl„; DMSO, dimethylsulfoxide; PNaMg buffer, 0.05 M sodium phosphate buffer,p H 6.8, containing 0.1 M NaCl, and 0.005M MgCl ;MES , 2-(N-morpholino)ethane- sulfonic acid; EGTA, ethyleneglycol-bis-(B-aminoethylether)tetraacetic acid; TEMED,N,N,N',N',-tetramethylenediamine ;VB ,vinblastin e sulfate.

26 C—NH-CHJ-CHJ-CHJ-CHJ

>r-NH-C-OCH3

N carbendazim (MBC) R = H benomyl

R ^ C4H g parbendazole

0 II R = -C- oncodazolefR 17934) ^^^ thiabendazole

R = -C mebendazole

R =..a -S \ fenbendazole ^^ fuberidazole

Fig. 1.Structura l formulae of biologically active benzimidazole compounds.

The curativeactio no fmebendazol e inanima la swel la si nhuma nhelmin ­ thiases implies selectivitywit h respectt ohos tan dparasite. Ultrastructura l studieshav e shown thatupo n treatmento fth ehos tmicrotubule s incell so f theparasit ear ecompletel y destroyed,wherea s cytoplasmican dspindl emicro ­ tubuleso fth ehos t cells remainunaffected ,eve n thoughbot h typeso fcell s havebee n exposed toidentical ,dru g concentrations (9,10). Inviv oexperiment so nth eeffec to foncodazol eo nexperimenta lan d humanneoplasm s have showntha t this compound specifically eliminatesmicro ­ tubules individin gan dnon-dividin g neoplastic cells,wherea s microtubules of interphasenorma l cellsar eapparentl y intact.Microtubule s inmitoti c cells,however ,ar eseriousl y affected(26) . Biochemical studieso nth emechanis mo factio no fbenzimidazol e compounds have showntha tth eantimitoti c actiono fMB Ci nfung ii sprobabl y mediated viabindin gt ofunga l tubulin (22,23). Oncodazole isboun d tomammalia nbrai n tubulina tth ecolchicin ebindin g site (45)an di sa poten t inhibitoro fmicro ­ tubule assembly invitr o (26,27,45). Benomylan dMB Conl y slightly affect this process (27,44).

27 Themolecula rbasi so fselectivit yo fthes ebenzimidazol ecompound si s yetunknown .Differentia luptak eo rmetabolis mma yb eresponsibl efo rth e relativenon-toxicit yo fbenomy lan dMB Ct oanimal san dplant s (36,65).Fo r naturalan dinduce dresistanc ei nfungi ,i tha sbee nfoun dtha tn odifference s inmetabolis mexis tbetwee nMBC-resistan tan dMBC-sensitiv estrain s (24). Here adifferentia lbindin go fMB Ct otubuli ni nstrain sdifferin gi nMB Csensi ­ tivitymigh tunderl yth eselectiv eactio no fthi scompoun d (22,23).Th ese ­ lectiveactio no fmebendazol edoe sno tappea rt ob erelate dt oa differentia l drug-uptakebetwee nhos tan dparasit e (9,10).A differentia linteractio no f thedru gwit hth etarge tinsid eth ecell si sassume dt ob eresponsibl e (9,10). Inthi sstud yth einteractio no fMB Cwit hit srecepto rsit ei nMBC-sensi ­ tivean dMBC-resistan tstrain so fth efungu sAspergillu snidulan sha sbee n investigatedi ndetail .Sinc ei tha sbee nassume dtha tMB Cbind st ofunga l tubulinth eeffec to foncodazol ean do fothe rantitubulin so nMB Cbindin gha s beenstudied .Wit h [ c]MBCa saffinit ylabe lth ebindin gprotei nwa spartial ­ lypurifie dan delectrophoreticall ycharacterized .

MATERIALS AND METHODS

Organisms. Mosto fth eexperiment swer ecarrie dou twit hAspergillu snidulan s biA1AcrA 1 (Strain003 )an dtw omutan tstrain s (Strain18 6an dStrai nR) , whichdiffe ri nMB Csensitivity .Sensitivit yo fthes estrain si nterm so fin ­ hibitoryconcentrations ,whic hcaus ea 50 1reductio ni ngrowt h (EDrn)o nagar , are4. 5 VUMB Cfo rStrai n003 ,1. 5u Mfo rStrai n18 6an d9 5y Mfo rstrai nR . Bothstrain swer eselecte di nth elaborator yafte rU Vtreatmen to fconidi ao f Strain00 3 (69).Geneti canalysi sha sshow ntha tbot hincrease dsensitivit yi n Strain18 6an dresistanc et oMB Ci nStrai nR ha dbee ncause db ya mutatio ni n onesingl egene ,locate do nlinkag egrou pVII I (69,70). Weals ouse dMBC-sensitiv estrain so fPenicilliu mbrevicompactu man d P.corymbiferu man dresistan tstrain so fthes efungi ,whic hemerge di nMBC - treatedcrop s (6).Alternari abrassica ean dPythiu mirregular erepresente dth e naturallyMBC-resistan tfungi .

Culture methods. Conidiao fA .nidulan sstrain swer egrow no na 2%mal textract , 0.1 % bacto-peptone, 2%glucose ,1.5 %aga rmedium .Conidia lsuspension swer epre ­ pareda sdescribe dpreviousl y (21).Conidi ao fPenicilli astrain san dA . brassicaewer eharveste dfro mpotato-dextros eaga ran doatmea lagar ,res ­ pectively.P .irregular ewa smaintaine do npotato-dextros eagar .Myceliu mo f A.nidulan sstrain swa sgrow ni na glucose-nitrat emediu m (57),supplemente d

28 with 1y gbioti npe rml .Myceliu mo fth eothe rfung iwer eculture di nCzapek - Doxliqui dmediu m (Oxoid),supplemente dwit h0.5 % (w/v)yeas textrac tpowde r (Qxoid).Culture swer eincubate do na Gallenkam porbita lshake ra t20 0rp m at37° Cfo rA .nidulan san da t25° Cfo rth eothe rfungi .

Preparation of mycelial extracts. Exponentiallygrowin gmyceliu mwa sharveste d byfilterin go na Biichne rfilter .Th emyceliu mwa swashe dthre etime si ncol d 0.05M potassiu mphosphat ebuffer ,p H6. 8an dfroze na t-2 2C i na previousl y cooledX-Pres sCel lDisintegrato r (LKB-Biotec,Sweden )wit h0. 5m lo fhomo - genizationbuffe rpe rgra mwe tweigh to fmycelium .Th ehomogenizatio nbuffe r consistedo f0.0 5M potassiu mphosphat ebuffer ,p H6.8 ,containin g0. 1M KC 1 and0.00 5M MgCl 2 (PKMgsolution) .Afte r1 hou ra t-22° Cth emyceliu mwa s homogenizedb ypassin gi tfiv etime sthroug hth epress .Th ehomogenat ewa s thawedan dthe nguanosin etriphosphat e (GTP)wa sadde dt oa fina lconcentratio n of0. 1nM .Th esuspensio nwa scentrifuge da t40,00 0x g fo r1 0min .an dth e resultantsupernatan trecentrifuge da t48,00 0x g fo r3 0min .Th e48,00 0x g supernatantwa simmediatel yuse di nbindin gstudies .Al lstep swer edon ea t4 C

Preparation of porcine brain extracts. Freshporcin ebrain swer eobtaine dfro m Stroomberg'sExportslachteri j (Ede,Th eNetherlands )an dimmediatel yprocesse d afterarrival .Bloo dvessel san dmeninge swer eremove dan dth etissu ewa s washedthre ethime swit hic ecol d0.0 5M potassiu mphosphat ebuffer ,p H6. 8 andonc ewit hPKM gsolution .Hundre dgram so ftissu ewer ethe nhomogenize d with10 0m lo fPKM gsolution ,containin g0. 1m MGTP ,usin ga motor-drive n glasshomogenize rwit hTeflo npestl e (Braun,Melsungen ,GDR) .Th ehomogenat e wascentrifuge da t48,00 0x g fo r6 0mi nan dth eresultin gsupernatan twa s fractionatedwit ha neutralize dsaturate dammoniu msulfat esolution .Th e fractionprecipitatin gbetwee n3 5an d50 1saturatio nwa stake nu pi n1 0m lPKMj ; solutioncontainin g 1n MGTP .Thi spreparatio nwa suse di nbindin gassay s eitherdirectl yo rafte rstorag ea t-2 2C .

Binding assays. Varyingamount so fa methanoli csolutio no f[ C]MBCwer e addedt omycelia lo rbrai nextract san dincubate deithe ra t4 or3 7C fo r variousperiod sa sspecifie di nth elegen do fFigure san dTables .Smal lsample s ofa [ H]colchicin esolutio ni nbenzene/ethano l (9/1)wer eplace di nempt y scintillationvial so rcentrifug etube san dth esolven twa sevaporate dt o drynessi na strea mo fnitrogen .Th edrie dcompoun dwa sdissolve ddirectl yi n mycelialo rporcin ebrai nextract .Vial san dtube swer ewrappe di naluminu m foili norde rt opreven tphotodecompositio no fcolchicine .Incubatio nwa sa t

29 4 or3 7C fo rvariou speriod sa sspecified .Potentia linhibitor so fMB C bindingwer eadde da ssolution si ndimethylsulfoxid e (DMSO).Th eDMS Oconcen ­ trationo ftreate dan dcontro lsample sneve rexceede d0.1 %v/v . Bindingo f [ CjMBCo r[ HJcolchicin ewa smeasure db yge lfiltratio no f 1m lo fth eincubatio nmixtur eo na Sephade xG-10 0colum n(2 8x 1. 5cm )wit h PKMgsolutio na selutio nbuffer .Fraction so f1 6drop s (approximately 1ml ) werecollecte dwit ha LK BUltroRa cfractio ncollecto ran dradioactivit yi n eachfractio nwa smeasure di na Nuclea rChicag oMar k ILiqui dScintillatio n Spectrometerwit hBrun oan dChristian' s (13)scintillatio nliquid .Countin g efficiencywa sdetermine db yexterna lstandardizatio nprocedures .Radio ­ activitypresen ti nprotei nfraction swa sconsidere dt orepresen tboun dligand . Bindingo f[ CJMBCwa sals omeasure dwit ha secon dmethod ,whic hha s beenintroduce drecentl yt omeasur ecolchicin ebindin gt otubuli n (62,63). Aliquotso fth eincubatio nmixtur ewer eplace di ncentrifug etube scontainin g anequa lvolum eo fa charcoa lsuspensio n (Merck,Darmstadt ,GDR )a t6 mg/m li n PKMgsolution .Th etube swer eplace di na shake ran dth emixtur ewa sheavil y agitatedfo r1 0mi nan dthe ncentrifuge da t150 0x g fo r5 mi na t4 C .Aliquot s ofth esupernatan twer eassaye dfo rradioactivity .Blanks ,whic hcontaine d 40mg/m lbovin eseru malbumi ni nPKM gsolutio nincubate dwit h [ c]MBC werehandle di nth esam eway .Th edifferenc ei namoun to fradioactivit y foundi nsupernatan taliquot so fsampl ean dblan kwa sassume dt orepresen t bound [ CJMBC BoundMB Cwa sexpresse da sdp mpe runi to fvolum eo fth e originalextract .

Purification of fungal tubulin. Mycelialextract swer eprepare da sdescribe d above,bu twit h0.0 5M sodiu mphosphate ,p H6.8 ,containin g0. 1M NaC lan d 0.005MgCl ~ (PNaMgsolution )a shomogenizatio nbuffer .Th e48,00 0x g super ­ natantmycelia lextrac twa sfurthe rcentrifuge da t127,00 0x g fo r6 0min . Thesolubl eprotein swer efractionate dwit ha neutralize d (pH6. 8afte r2 0: 1 dilution)saturate dammoniu msulphat esolution .Th efractio nprecipitatin g between3 5an d50 %saturatio nwa stake nu pi nPNaM gsolutio ncontainin g 1m MGT P Theresultin gpreparation swer eincubate dwit h [ c]MBCan dru nont oa DEAE-SephadexA-5 0colum n(1 0x 1, 5cm) ,whic hha dbee npreviousl yequilibrate d withPNaM gsolution .Th ecolum nwa ssubsequentl yelute dwit h1 5m lo fa lineargradien to f0.1-0. 4M NaCl ,1 5m lo f0. 4M NaCl ,3 0m lo fa linea r gradiento f0.4-1. 0M NaC lan d2 0m lo f1. 0M NaCl ,respectively ,al lmad eu p inbuffer .Gradient swer eproduce db ya LK BUltrogra dGradien tMixer .Th e eluatewa scontinuousl ymonitore da t25 4n man dfraction so fconstan tvolum e werecollected .Radioactivit ywa smeasure di neac hfraction .

30 Onceth eelutio npropertie so fth eMB Ccomple xwer e established,th ein ­ cubation stepwa somitte dan dth e35-50° sammoniu m sulfatepreparatio nwa s applied directly toth eDEAE-Sephade x column.Fraction swhic hwer e eluted at0.45-0.9 0M NaCl,wer e combined,dialyse d againstbidistille dwater ,an d lyophilized.Dr ysample swer e storeda t-2 2C abov e silicage lunti lanalysis .

Purification of porcine brain tubulin. Twopurificatio nmethod swer eused . Withth efirs tmethod ,a tubuli npreparatio nprepare d asdescribe d above,bu t with PNaMg solutiona sextractio nbuffer ,wa schromatographe d ontoa DEAE - SephadexA-5 0 column inth esam ewa ya sdescribe d above formycelia lextracts . Fractions containing tubulinwer edialyse d againstbidistille dwate ran dlyo ­ philized.Th esecon dmetho d usedwa sa slightl y modified assembly-disassembly procedure according toShelansk ie tal . (61).Fres hporcin ebrain swer ewashe d andhomogenize d in0. 1M ME Sbuffer ,p H6.90 ,containin g 1m M EDTA,0. 1m MGTP , and0. 5m MMgCl „ (MESbuffer) .Th ehomogenat ewa scentrifuge d at40,00 0x g for 10mi nan dth eresultin g supernatant at48,00 0x g fo r6 0mi na t4 C . To achieve assemblyo fmicrotubule s extractswer emixe dwit ha nequa lvolum e ofME Sbuffe r containing 8M glycerol and2 m MGT Pan dincubate d for3 0 min. at 37C .Assemble d microtubuleswer epellete db ycentrifugin g at48,00 0x g at 25C .Afte r resuspensionan ddepolymerizatio no fmicrotubule s inME Sbuffe r at4 C an dcentrifuging ,a secon d assembly cyclewa sperformed .

Molecular weight determination of the MBC-protein complex. Themolecula r weighto fth eMBC-protei n complexwa sdetermine db yge lfiltratio no fa nam ­ monium sulfate fractionated mycelial extract,whic hha dbee n incubatedwit h [ C]MBC ona Sephade xG-20 0colum n (2.5x 3 2cm) ,accordin gt oth emetho d ofAndrew s (2).Th ecolum nwa scalibrate d intw oparalle l runswit h alcohol dehydrogenase (yeast),M W150,00 0an dcytochrom eC (hors e heart),M W12,400 ; andwit h lipoamidedehydrogenas e (pigheart) ,M W100,00 0an da-chymotrypsi n (beefpancreas )M W24,500 , respectively.

Reduction and carboxymethylation of proteins. Proteinswer ereduce db yaddin g '3-mercaptoethanolan dsodiu mdodecylsulfat e (SDS)a tfina l concentrationso f M toth esample san dheatin gfo r2 mi na t10 0C .Th ereduce dprotein swer e dialysed overnighta troo m temperature against 0.01M sodiumphosphat ebuffer , pH 7.0,containin g 0.1s6-mercaptoethano lan d0. HSDS . Protein sampleswer e reducedan dcarboxymethylate d accordingt oth e methodo fCrestfiel d etal .(20 )a smodifie db yRenau de tal .(55) .Protei n solutionswer emad e 1 %i nB-mercaptoethano lan dmixe dwit ha nequa lvolum e ofreducin gsolutio n(0.3 5M Tris-HCl ,p H8.8 ,containin g0.1 2M B-mercapto - ethanol,8 M ure aan d0.1 %EDTA) .Th emixtur ewa sdialyse dovernigh ta troo m temperatureagains tthi ssolution .Lyophilize dprotein swer edirectl ydissolve d inreducin gsolutio nan dincubate dovernigh ta troo mtemperatur eunde rnitrogen . Thereduce dprotein swer ecarboxymethylate db yaddin gon evolum eo fa 1. 1M iodoaceticaci d- 8 M ure asolutio ni n2. 5N NaO Ht onin evolume so fprotei n solution.Th ereactio nwa sallowe dt oprocee dfo ron ehou ri nth edark ,an d thesolutio nwa sthe ndialyse dagains t0.0 1 MTris-HCl ,p H8. 0 fora tleas t 6 hoursa troo mtemperatur eals oi nth edark .

Sodium dodeoylsulfate (SDS) polyaerylamide gel electrophoresis. TwoSD Ssys ­ temswer eemployed .Th efirs ton ewa sbase do ntha to fWebe ran dOsbor n(71) . Reducedo rcarboxymethylate dprotei nsample swer eru no n6 c mgel s (7.5%(w/v ) acrylamide,0.13 % (w/v)bisacrylamide ,0.075 % (w/v)ammoniu mpersulfate , 0.075% (v/v)TEME Di n0. 1M sodiu mphosphat ebuffer ,p H7.0 )a t4 0Vol tfo r about6 hours .Th esecon dsyste mwa ssimila rt oth ediscontinuou sSD Ssyste m asha sbee ndescribe db yLuduen aan dWoodwar d (51).Carboxymethylate dprotei n sampleswer eprepare dfo relectrophoresi sa sdescribe dan dru no n9 c mgel s at4 m Ape rge lfo rapproximatel y 2.5hours . Gelswer estaine dfo r1 5hour si n0.015 %Coomassi eBrillian tBlu ei n methanol/aceticacid/wate r (45/9/46).Destainin gwa sperforme db ydiffusio n inmethanol/aceti cacid/wate r (2/3/35).

Protein determination. Proteinwa sdetermine daccordin gt oth emetho do fLowr y etal . (50)a smodifie db yHartre e (40)wit hbovin eseru malbumi na sa stan ­ dard.

Chemicals. [2- CJMethylbenzimidazol-2-y lcarbamat e (spec.act .11. 4mCi/mmole ) waspurchase dfro mInternationa lChemica l& Nuclea rCorporatio n (Irvine,Cal. , USA).Stoc ksolution swer eprepare di nmethano la tapproximatel y 1200 vM [ C]MBC. Radiochemicalpurit yo fth epreparatio nwa schecke da tinterval s bythin-laye rsilic age lchromatograph yo nDC-Alufoli e6 0F 25 4(Merck ,Darm ­ stadt,GDR )wit hethylacetat esaturate dwit h0.0 5M potassiu mphosphat ebuffer , pH6.8 ,a ssolvent .Chromatogram swer escanne dwit ha Nuclea rChicag oActi - graphII Iradiochromatograph y systeman dthe ncu ttransversel yint o1 c msec ­ tions,whic hwer eplace di nscintillatio nvial scontainin gBrun oan dChris ­ tian'sscintillatio nliqui dan dcounted .Purit ywa salway sfoun dhighe rtha n 97%. [Ring C-methoxyl- HJcolchicin e (spec.act .3. 8Ci/mmole )a sa solutio n

32 inbenzene/ethano l (9/1)wa sobtaine dfro mNe wEnglan dNuclea r (Boston,Mass. , USA).Th epreparatio ndilute dt oa specifi cactivit yo f20-20 0mCi/mmol ewa s eitheruse da ssuc ho rafte rbein gpurifie daccordin gt oth eprocedur edes ­ cribedbelow . Methyl5-(2-thieny l carbonyl)-benzimidazol-2-yl carbamate (oncodazoleo r R17934 )wa sa gif to fDr .M .D eBrabande r (JanssenPharmaceutica ,Beerse , Belgium).Colchicin ewa spurchase dfro mMerc k (Darmstadt,GDR) .Griseofulvi n andvinblastin esulfat ewer eobtaine dfro mSigm a (St.Louis ,Mo. ,USA) . Mela­ toninan dpodophyllotoxi nwer epurchase d fromFluk a (Buchs,Switzerland )an d AldrichChemica lCo .(Milwaukee ,Wise ,USA) ,respectively .

Purification of [ HJcolchicine. Analiquo to fth e [H ]colchicin esolutio n inbenzene/ethano lwa sdilute dwit hunlabelle dcolchicin ei nth esam eso ­ lution,givin ga concentratio no f1 0 Mcolchicin ea tapproximatel y 10 dpm per10 0p isolution .Tw ohundre dp io fthi ssolutio nwer eapplie da sa smal l bandt oa silic age lplat e (DC-Alufolie6 0F 254 ,Merck ,Darmstadt ,GDR )an d chromatographedi nchloroform/aceton/diethylamin e (5/4/1)ove ra distanc eo f 17c mwit hunlabelle dcolchicin ea sa reference .Th ecentr epar to fth e[ H j colchicineban d (visibleunde rU Va t25 4nm )wit hRf-valu e0.5 7an da 1-cm - broadban dcorrespondin g toth eRf-valu eo fa nunknow nradiolabele dcompoun d X, whichwa sfoun dt ob epresen ti nou rcolchicin epreparation ,wit hRf - value0.4 6wer ecu tou tan dtransferre d toa centrifug etube .Th esilic age l wasscrape dof fan dsubsequentl yelute d4 time swit h 1m lmethanol .T oavoi d photodecompositiono fth ecompounds ,exposur eo fsample st oligh twa skep tt o aminimu mdurin gth eprocedure .Tube san dchromatograph y tankwer ewrappe di n aluminumfoil .Approximatel y 1.3x 1 0 dpmwer erecovere d in[ H]colchicin e and 1.4x 1 0 dpmi ncompoun dX .T oanalyz eth epurifie dpreparation saliquot s werespotte dont oa chromatogra man ddevelope dwit hth esam esolven ta suse d before.Chromatogram swer ecu ttransversel y into1 c msection swhic hwer e placedi nscintillatio nvial scontainin gBrun oan dChristian' sscintillatio n liquidan dcounted .Radioactivit ypresen ti nsection scorrespondin gt oth e Rf-valueso fcolchicin ean dcompoun dX wa s8 6an d84°6 ,respectivel yo fth e totalradioactivit yrecovered .I nth ecas eo f [H]colchicin e 11%o fth e radioactivitywa spresen ti na thir dcompoun dwit hRf-valu e0.83 .Thi scom ­ pound isprobabl y identicalt otha tfoun db yBoris yan dTaylo r (11),whic hwa s shownt ob eforme ddurin gstorag eo fL H Jcolchicin epreparation spurifie db y silicage lchromatography .

33 RESULTS

Evaluation of charcoal assay. Thege lfiltratio nmetho dfo rmeasurin gMBC - bindingactivit yi nmycelia lextracts ,a suse di npreviou swor k (22,23]give s reproducibleresults .Howeve rth eassa yi slaboriou san dno tsuitabl efo r multipledeterminations .Therefore ,i nth epresen tinvestigatio na secon d methodwa suse di nadditio nt oth eSephade xG-10 0assay .Th eusefulnes so f thismethod ,whic hi sbase do nseparatio no ffre eMB Cfro mboun dMB Cb yad ­ sorptiont ocharcoal ,wil lb ediscusse dfirst . Theeffectivenes so fcharcoa li nadsorbin gfre eMB Cwa sdetermine di n solutionso fBS Aa t4 0mg/m li nPKM gbuffer .Mor etha n99.5° 6o fth etota l radioactivitypresen ti nth eincubatio nmixtur ecoul db eremove da t [ clMBC concentrations,rangin gfro m1-4 0yM ,whic hcorrespon dwit h2.5-10 0x 1 0 dpm/ml.Remova lo ffre eMBC ,however ,ma yno tinvolv eremova lo fboun dMB C whichi spresen ti nmycelia lextract sincubate dwit hMBC .Therefor eth echar ­ coalassa ywa scompare dwit hth ege lfiltratio nmethod .A si seviden tfro m TableI eve nhighe rvalue sfo rboun dMB Cwer efoun dwit hth echarcoa lassa y thanwit hth ege lfiltratio nmethod .Sinc eincubatio no fmycelia lextract s with [ C]MBC inth epresenc eo fa specifi cinhibito ro fMB Cbindin g (see below)resulte di ncomplet eadsorptio no fradioactivit yt ocharcoal ,th e higherdegre eo fbindin gfoun dwit hth echarcoa lassay ,i sno tdu et oin ­ completeadsorptio no ffre eMB Ct ocharcoa li nmycelia lextracts .Hence , lessdissociatio no fMB Cfro mth ecomple xdurin gth echarcoa lassa ytha n duringth ege lfiltratio nprocedur ei sprobabl yresponsibl efo rth ehighe r degreeo fbindin gfound . Duet odissociatio nth evalu eo fboun dMB Cdecrease swit hth elengt ho f theincubatio nperio dwit hcharcoa l (Fig.2) .I nth estandar dprocedur emix ­ tureswer eshake nfo r1 0mi n withcharcoal ,s otha tdisturbanc eo f

TableI Comparisono fcharcoa lan dSephade xG-10 0assay sfo rMB Cbindin g

Assay bounddpm/m lextrac t pinoleboun dMBC/m gprotei n charcoal 31.4xl03 30 SephadexG-10 0 22.3x103 22

Freshlyprepare dmycelia lextrac to fA .nidulan sStrai n 186a t4 1m gprotei n perm lwa sincubate dwit h [ c]MBCa t5. 7y M (14.2x10 dpm/ml)fo r2 h at4 C .Bindin gassay swer ecarrie dou ta sdescribe dunde rMaterial san d Methods.

34 2.0

\ 1.5-

°1. 0

0.5-

0.0 I -1 10 15 20 Time (mm)

Fig.2 .Effec to fincubatio ntim edurin g thecharcoa lassa yo nth equantit y ofboun dMBC . Amycelia lextrac to fA .nidulans ,Strai n00 3a t3 8m gprotei npe rm lwa s incubatedwit h [ c]MBCa t21. 2u M (53.0x10 dpm/ml)fo r2 h a t4°C .Bindin g wasdetermine dwit hth echarcoa lassay ,i nwhic hth elengt ho fth eincubatio n periodwa svaried .

equilibriumwa sminimu man dremova lo ffre eMB Cwa smaximum .

Selectivity of MBC binding. MBCbindin gwa sfoun di n48,00 0x g supernatan t extractso fmycelia lhomogenate si nPKM gsolutio nafte rth eextract swer ein ­ cubatedwit h [ c]MBCa t4 C (22,23).Bindin gactivit yappeare dt ob ecorrelat e withth ei nviv oMB Csensitivit yo fth especie so rstrai nexamined .Tabl eI I givesdetaile dbindin gdat aobtaine dwit hth eSephade xG-10 0assa yfo rvariou s MBC-sensitivean dMBC-resistan tfunga lspecie so rstrains .Wherea sextract so f sensitivestrain sdi dbin dMB Ci nquantitie sbetwee n3 an d 18%o fth equantit y added,resistan tstrain sdi dno tbin dmor etha n0.6 %o fthi samoun twhic hi s hardlyabov ebackground . Sincei tha sbee nsuggeste d (22,23)tha tth eMBC-bindin gsubstanc ei s identicalwit hfunga ltubulin ,th eMBC-bindin gpropertie so fmammalia nbrai n tubulinwer einvestigated .A si seviden tfro mTabl eII Iporcin ebrai ntubuli n didno tbin dMB Ci nsignifican tamount sa t4 C no ra t3 7C .A sexpecte dth e preparationshowe d[ H jcolchicin ebindin gactivit ywhic himplie sth epresenc e ofnativ etubuli ni nth epreparatio nused .Therefore ,i tca nb econclude dtha t

35 Table II.MB C binding in48,00 0x g supernatant mycelial extracts of fungal species and strains,whic h differ inMB C sensitivity

MBC cone. boundMB C Species Strain ED 50 protein cone. -3 -2 uM mg/ml (dpm/ml)xl0 yM (dpm/ml)xl0 pmole/mg protein

Aspergillus 003 4.5 38 64.0 2.6 56.4 6.0 nidulans 186 1.5 42 68.2 2.7 122.0 11. 5

R 55 40 68.2 2.7 3.2 0.3 + Penicillium S < 22 67.0 2.7 41.4 7.6 brevicompactum R > 2C;00+ 20 56.0 2.2 2.2 0.4

Penicillium S < 2+ , 24 66.0 2.6 20.8 3.4 corymbiferum R > 2000+ 38 56.8 2.3 1. 4 0.1 Alternaria > 1000§ 37 64.0 2.6 3.7 0.4 brassicae Pythium > 1000§ - 65.0 2.6 2.5 irregulare

Mycelial extracts were incubated for2 hour s at4 C wit h L CJMBC. Bindingwa sdetermine d byge lfiltratio n of 1m lo fth eincubatio n mixture through a SephadexG-10 0column . Inhibitory concentration causing a50 %reductio n ingrowt h onaga r medium. Data from Bollen (28)fo rbenomyl . Data from Bollen andFuch s (21)fo rbenomyl . Not determined.

Table III.MBC-bindin g and colchicine-binding activity ofpartiall y purified porcine brain tubulin

Exp. Binding agent Temp. Concentration Binding activity dpm/ml uM dpm/ml pmole/mg protein

1 [14C]MBC 4°C 15.3x10 6.1 2.1x10 0.3 2 [1AC]MBC 37°C 19.5x10* 7.8 4.1x10 0.6 3 L Hjcolchicine 37°C 1.2xl06 3.0 48.6x10 45.0

A porcine brain extract purified byammoniunijsulfat e fractionation (35-50%) at 27m g proteinpe rm lwa sincubate d with [ c]MB C and [H ]colchicine , respectively, for2 h a tth etemperature s indicated. Thetubuli n preparation had been stored for0 (exp.1) ,1 1 (exp.2 )an d 13day s (exp.3 )a t-21°C , respectively.

36 mammaliantubuli nha sn oo ra tleas ta lo waffinit yfo rMBC .Thes efinding s arei nagreemen twit hth eobservatio ntha tMB Ca ta concentratio nwhic hi s lethalfo rMBC-sensitiv efung idoe sno tinterac twit hi nvitr omicrotubul e assemblyi nbrai nextract s (23,27,44). Forfurthe rwor kth ethre eAspergillu snidulan sStrain s003 ,18 6an dR wereselected ,merel ybecaus eo fth efac ttha tthei rgeneti cbackgroun di s known (69,70).

Molecular weight of the MBC complex. Todetermin eth emolecula rweigh to f theMB Ccomple xa partiall ypurifie dmycelia lextract ,whic hha dbee nincu ­ batedwit h [ c]MBC,wa sge lfiltrate dthroug ha calibrate dSpehade xG-20 0 column.Boun dradioactivit ywa selute di na singl epea kan da sca nb esee n inFig .3 th eelutio nvolum ecorrespond swit ha molecula rweigh to fth eMB C complexo fapproximatel y 110,000.Sinc ethi svalu ei sver ysimila rt otha t foundfo rth ecolchicine-tubuli ncomple x (17), theelutio nbehaviou ro fbot h complexeswa scompare do na Sephade xG-20 0colum n (1.5x 2 7cm) .Th eelutio n volumeo fth eMB Ccomple xappeare dt ob esimila rt otha to fth ecolchicine - tubulincomplex .

20- 15- V alcoholdehydrogenase

'CL.MBC-COMPLEX 10- lipoamide dehydrogenase f 8- o x 6. i 4-

N» ot-chymotrypsin 2 2

cytochromec \

75 100 125 150 Elution volume (ml)

Fig. 3.Molecula r weight determination of theMBC-protei n complex. 5m l of an ammoniumsulfate fractionated (35-50%)mycelia l extract (40,000x g) at 38m g protein perm l was incubated with [ CJMB C at 8u M for 2h at 4 C and gel filtrated through a calibrated Sephadex G-200column . The labelled complex was eluted as a single peak at an elution volume corresponding to amolecula r weight of approximately 110,000.

37 6-i

5-

* 3-

IT) 2 • £ Q- S. 1-

30 60 90 120 Incubation time(min. )

Fig. 4.Tim e course ofMB C binding. Amycelia l extract ofA . nidulans,Strai n 003, at 38m g protein perm l was incubated with [ c]MB C at 1.1 yM (2.7x10 dpm/ml)fo rvaryin g periods of time at 4C . The quantity of bound MBC was determined with the charcoal assay.

Factors which influence MBC binding. Bindingo fMB Ct oit srecepto rwa snearl y completewithi n1 5minute so fincubatio no fth emycelia lextrac twit h [ CJMBC (Fig.4) . Onlya sligh tincreas ei nboun dradioactivit ywa sfoun dthereafter . Thereforea nincubatio nperio do f1 hou ro rmor ewit h [ CJMBC,a sroutinel y usedi nou rexperiments ,ca nb econsidere dt ob elon genoug hfo rmaximu m binding. MBC-bindingactivit ywa sinfluence db yp Ha si sillustrate d inFig .5 . Maximumbindin gappeare dbetwee np H6.5-6.8 .Whethe rp Haffecte dbindin go f MBCindirectl ythroug ha neffec to nstabilit yo fth erecepto ro rdirectl yha s notbee ninvestigated . MBC-bindingactivit yo fmycelia lextract swa sno tstable .A t4 C th e abilityt obin dMB Cdecaye daccordin gt ofirs torde rkinetic s (Fig.6) . The half-timefo rinactivatio nwa s6. 5hours .Sucrose ,MB Can dglycero lstabilize d thebindin gactivity .Know nstabilizer so fcolchicine-bindin gactivit yo f tubulinlik evinblastin esulfat ean dGT Pdi dno tsignificantl yincreas eth e stability. Theinfluenc eo ftemperatur eo nMBC-bindin gactivit ywa sals oexamined . When48,00 0x g supernatan tmycelia lextract swer eincubate dfo r1 h a t3 7C aconsiderabl edenaturatio no fprotein stoo kplace .Therefore ,thes eextract s werecoole di nic efo rabou ton ehou ran dcentrifuge dbefor ebindin gactivit y wasmeasured .Sinc efo runknow nreason sth echarcoa lassa yapplie dt oheate d

38 10

\

—I— —I— —|— —I 5.5 6.0 6.5 7,0 7.5 pH Hours

Fig. 5.(left )Effec t of pH onMB Cbinding . Twom l samples of a 127,000x g (1h ) supernatantmycelia l extract ofA . nidulans, Strain 186,a t 24m g protein perml ,wer e adjusted todifferen t pH-valueswit h 50y l of aH„P0 , orKO H solution,respectively ,o f different strenght. Onem l of the resulting preparationwa s incubatedwit h L cjMBC at 11u M (27.5x10 dpm/ml)fo r 1h at 4°C.Th e quantity of bound MBCwa s determined with the charcoal assay.Th e pH (22C )wa s determined in the other ml of thepreparation .

Fig. 6. (right). Stability of theMBC-bindin g activity under variouscondi ­ tions at 4C .Thre em l of amycelia l extract of A. nidulans,Strai n 186,a t 24m g protein perm lwer emixe d with an equalvolum e of a solutiono f respectively, 1m M vinblastine sulfate (VB),2 m M GTP, 8M glycerol,22. 8y M [ c]MB C (56.8x10 dpm/ml), and 2M sucrose inPKM g solution.A s control preparation 3m l extract was dilutedwit h 3m l PKMg solution.A t the times indicated [ cjMBCwa s added (28.4x10 dpm/ml) to aliquots of themixture s except to thatwhic h already contained [ CjMBC. After incubation for 15mi n at 4C the quantity ofboun dMB Cwa s determined with thecharcoa l assay using appropriate blanks.Bindin g activitywa s expressed aspercen t of initial binding directly aftermixin g the solutions (510dpm/5 0 yl of 1 : 1dilute d extract). The half-times of decay (tj)o f theMBC-bindin g activity have been calculated from the slopes of thelines .

extracts gave anomalous binding data,bindin g activitywa s determinedwit h the gel filtration method. Binding activity of the supernatant was 11% of that of control sampleswhic hwer e kept at 4 Cdurin g incubation and centrifuging of the experimental sample.Bindin g activity inth e control samplewa s determined with the charcoal assay.Thi s result indicates that loss ofbindin g activity isenhance d by high temperature. Incubating mycelial extractswit h 0.1 %trypsi n for onehou r at 4 Cbefor e incubationwith [ CjMBCresulte d ina 55°slos s ofbindin g capacity.

39 Centrifuginga t127,00 0x g fo r1 hou ra t4 C o fth e48,00 0x g super ­ natantlowere dth ebindin gactivit yo fth eresultin gsupernatan tt o70-80 %o f thato fcontro lsample swhic hwer eno tcentrifuge dan dwer estore da t4 C fo r theduratio no fth erun .Thi sresul tsuggest stha tth eMBC-bindin gactivit y mightb epartiall yassociate dwit hsom eparticulat efraction .Th enatur eo f thisbindin gactivit yha sno tbee nstudie dfurther .

Affinity for MBC in extracts of Aspergillus nidulans Strain 003, Strain 186 and Strain R. Sincei tha sbee nsuggeste d (22,23)tha tdifference si naffinit y ofMB Cbindin gsite sfo rMB Cmigh tb eth ebiochemica lbasi so fth eselectivit y ofMBC ,w edetermine dapparen tbindin gconstant s (K)an dnumbe ro fbindin g sites (S) i nmycelia lextract so fth ethre eA .nidulan sstrains .Tota lMB C bindingwa smeasure da sa functio no ffre eMB Cconcentratio nan dth edat awer e plottedi ndoubl ereciproca lform ,accordin gt oth eequation :

1 + (11) V [boundMBC ] /s V K.S •V [freeMBC ]

Theconcentratio no fboun dMB Cha sbee nexpresse da sboun ddp mpe rvolum e ofmycelia lextrac tan dth econcentratio no ffre eMB Ci nuM .Result so fa numbero fexperiment sar esummarize di nFig .7 .Fro mth eslope so fth eline s

o •7* 12

Kl I mole-'] 1 003 4.51*11 ]x10 ! in I6(*005]x106 Q. Q 37(;05 1x10 ^i o Maximum bound MBC _- pmoles/mg protein -^~ 003 TJ 330 1*10,1] c L -J 186 26,41* 1.1) o _Q R 18.91* 2.11

2 3 1/[free MBC] (/JM-1] Fig. 7.Bindin g of [ c]MB C tomycelia l extracts of A.nidulans ,Strai n003 , Strain 186an d StrainR . Mycelial extracts of Strain 003,Strai n 186,an d Strain R at 38,3 9 and 40m g protein per ml,respectively ,wer e incubated with [ CjMB C at increasing concentrations for 1h at 4°C.Th e quantity of bound MBCwa s determined with the charcoal assay.Th e concentration of free MBC was calculated from thedif ­ ferences between total and bound MBC.Value s of binding constants K (l.mole ) and maximum binding capacity (pmoles/mg protein)wit h their respective standard deviations are given as determined in at least four experiments.

40 andth eintercepts ,value so fS , expresse da smaximu mbindin g capacityi n pmolespe rm gprotei nan dth eapparen tbindin g constantswer e calculated. Evidently thenumbe ro fbindin g sites inextract so fth ethre e strainsar e about equal,bu tth erespectiv ebindin g constantsdiffe r considerably.Th e reciprocals ofth ebindin g constants,whic h areidentica lwit h theMB C concentrationwhic hwil lhalf-maximall ysaturat eth eMBC-bindin g site,ar e 2.2,0. 6an d2 7y Mfo rextract so fStrai n003 ,Strai n 186an dStrai nR , respectively.Thes e values correspondwit h theinhibitor y concentrationo f MBC causing a 50% reduction ingrowt h (seeTabl e 2).Thi s strongly suggests thatth erespons eo fth ethre eA .nidulan s strains toMB Ci sgoverne db yth e affinityo fth erecepto r sitefo rMBC .

Inhibition of MBC binding by antimitotic agents .I norde rt ocharacteriz e theMBC-recepto rsit ei nfungi ,severa l compoundswhic h interact insom e waywit h tubulin,wer e testedfo rinhibitor y effectso nMB Cbinding .A firstscreenin gwa scarrie dou tb yaddin g these compounds simultaneously r14- , with L CJMBC,a tvaryin g concentrations tomycelia l extractso fA .nidulan s Strain 186.Podophyllotoxi nan dvinblastin e sulfate,whic h compoundsar e knownt obin dt otubuli n (74,75)di dno treduc eMB Cbindin ga ta concentratio n of50 0yM .Melatoni nwhic hha sbee nshow nt ointerfer ewit h colchicine binding tomammalia nbrai n tubulin (76)an dwhic hwa sactiv ei nth estento r oralmigratio n assay forantitubuli ndrug s (52), wasals o ineffectivea t 500yM .Griseofulvin ,whic h affects spindle functioning inA .nidulan s(19 ) and selectively interfereswit h invitr omicrotubul e assembly (73),di dno t r14 i affectMB Cbindin ga t100 0yM ,eithe rwhe n added simultaneously with [ CJMBC orafte rextract sha dbee n incubated for1 hou rbefor e additiono f [ CJMBC. r14T Qncodazolewa sinclude d inth einhibitio n experimentsbecaus e infung i theactio no fthi scompoun dha sbee nsuggeste d tob esimila rt otha to f MBC (23).Thi s assumptionwa sbase do na simila r responseo fth ethre e strains toth egrowth-inhibitor yactio no foncodazole . ED™ valuesfo r mycelial growtho naga rwer e0.5 5y Mfo rStrai n003 ,0.2 3y Mfo rStrai n18 6 and 20y Moncodazol e forStrai nR . Oncodazole appearedt ob ea ver y effective inhibitoro fbindin g inth e primary test.A t5 0yM ,thi s compound inhibitedMB Cbindin g for90% ,i f assayeda t6. 0y Mwit h thege lfiltratio nmethod . Although thestandar d antitubulin colchicinedi dno tinhibi t mycelial growtho naga ro fan yo fth ethre eA .nidulan s strainsa tconcentration su p to 10mM ,a sligh t inhibitiono fMB Cbindin g inmycelia l extractswa snotice d at50 0yM .

41 The ability ofth elatte rtw ocompound s toinhibi tMB Cbindin gwa sin ­ vestigated infurthe r experiments,i nwhich mycelial extracts were preincubated foron ehou r eitherwit h oncodazole orwit h colchicine atincreasin g concen­ trations.Afte r thisperio d [ C]MBCwa sadde d andth emixture s werein ­ cubated foranothe rhour . Figs. 8an d9 sho wth eresul to fthi s experiment. Iti seviden t that oncodazole isa muc h morepoten t inhibitor ofMB Cbindin g than colchicine.Wherea s oncodazole gavea 50° &inhibitio no fMB Cbindin ga t a molar ratioo foncodazol e toMB Co f0.1 8 :1 ,colchicin e couldno teve n achieve thisa ta mola r ratioo f83 0: 1 . Inhibitiono fMB Cbindin g became apparent inextract s ofStrai n 186a tlowe r colchicine concentrations thani n extracts ofStrai n003 . The effecto foncodazol e onth epreforme d MBCcomple xwa sals o studied. r14 l [ CJMBCwa sadde d toa mycelia l extractan dafte r 1hou ro fincubation , oncodazolewa sadde dan dth emixture swer e incubated foranothe rhour . Inthi s case inhibitionb yoncodazol ewa smuc h lower than that foundwhe n extracts were preincubated with theinhibito r (Table IVexp . 1). However,afte r2 0 hourso fincubatio nwit hbot h compounds,inhibitio n valueswer e similar (Table IV,exp .2) .Evidentl y after thisperio d equilibriumha dbee nesta ­ blished,whic h confirms thereversibl e character ofMB Cbinding . These results suggest that oncodazolema ycompetitivel y inhibitth e

Table IV.Effec t of oncodazole onMB C binding

% inhibition ofMB C binding Time atwhic h com- exp. 1 exp.2 pounds were added: 2h after initiation 20h after initiation t=0h t=lh of experiment of experiment

rl4I [ CJMBC 0 0 oncodazole ['c ]MB C 94 52 [ c]MB C oncodazole 41 54 [14C]MB C + 62 54 oncodazole Mycelial extracts of A. nidulans Strain 186wer e incubated with oncodazole at 10yM 4 and [ c]MB C at 2.7 uM (6.8x10 dpm/ml) in exp. 1an d 5.4 yM (13.6x10 dpm/ml) in exp. 2,respectively , in the order as indicated. Bound MBC was assayed 2h (exp. 1)an d 20h (exp. 2)afte r initiation of the expe­ riment by the charcoal method. The quantity of bound MBC in the absence of oncodazole (0%inhibition )wa s 1010dpm/5 0 yl extract (exp. 1)an d 540 dpm/50 ul extract (exp. 2),respectively .

42 1.0 2.0 40 10.0 Oncodazole (juM )

Fig. 8. Inhibition ofMB C binding by oncodazole. Aliquots of amycelia l extract of A. nidulans,Strai n 186 at 30m g protein perm l were incubated with increasing concentrations of oncodazole for 1h and then for an additional hour with [ C]MB C at 5.5 uM (13.8x10 dpm/ml) at 4C . BoundMB C was assayed by the charcoal method. The quantity of bound MBC in the absence of oncodazole (0%inhibition )wa s 1000dpm/5 0 ul extract.

1.0 2.0 5.0 10.0 Colchicine (mM)

Fig. 9. Inhibition ofMB C binding by colchicine. Aliquots ofmycelia l extracts ofA . nidulans,Strai n 003 and Strain 186a t 30m g and 29m g protein per ml,respectively ,wer e incubated with increasing concentrations of colchicine for 1h at 4 C and then for an additional hour with [ CJMBC at 11.6u M (29.0x10 dpm/ml) at 4°C. Bound MBCwa s assayed by the charcoal method. The quantity of bound MBC in the absence of colchicine (0% inhibition)wa s 980dp m (Strain 003)an d 950 dpm (Strain 186)pe r 50y l extract.

43 bindingo fMBC .Sinc ethi sca nonl yb eestablishe da tequilibrium ,mycelia l extractsdilute d 1: 1 wit ha solutio no f2 M sucros ei nPKM gbuffe rt osta ­ bilizeMBC-bindin gactivit ywer eincubate dfo r2 0hour swit h [ c]MBCa tin ­ creasingconcentration swit ho rwithou tinhibitor .Dat afro ma nexperimen t withoncodazol ea sinhibito rar eplotte di ndoubl ereciproca lfor mi nFig .1 0 andthos ewit hcolchicin ei nFig .11 .I ti seviden tfro mthes efigure stha t bothcompound sinhibi tMB Cbindin gi na competitiv emanner ,sinc eonl yth e slopeo fth elin eha sbee nchange dbu tno tth eintercept .Inhibitio nconstant s (K.)ca nb ecalculate dusin gth eequation :

1 + 1 AboundMBC ]= /so /K.So- V +[l ] /K±) •V [free MBC] whereI i sth econcentratio no ffre einhibitor .Since ,however ,th econcen ­ trationo ffre einhibito rcanno tb ereadil ydetermined ,th evalu eo fth e totalinhibito rconcentratio nha sbee nuse dt oestimat eth einhibito ncon ­ stant.Fo roncodazol ethi sestimatio nprobabl yresult si na valu ewhic hi s highertha nth erea lvalue ,becaus eth econcentratio no fbindin gsite si so f thesam eorde ra stha to fth eoncodazol eadded ,resultin gi na hig hproportio n ofboun doncodazole .Fo rcolchicine ,whic hha sbee nuse da thighe rconcen ­ trations,th evalu ecalculate dca nb econsidere da rea lestimatio no fth e inhibitionconstant . Thelowe rpotenc yo fcolchicin et oinhibi tMB Cbindin gi nextract so f Strain003 ,compare dwit htha tobserve di nextract so fStrai n186 ,suggest s adifferentia laffinit yo fth ebindin gsit efo rcolchicine . Sincei tha sbee nshow ntha toncodazol ean dcolchicin ebin dt omammalia n tubulina tth esam esit e (45),competitiv einhibitio no fMB Cbindin gi nmy ­ celialextract sb ythes etw ocompound sindicate stha tth eMBC-bindin gsit ei s locatedo nfunga ltubulin .

Colahicine-binding activity of mycelial extracts. Competitiveinhibitio no f MBCbindin gt ofunga ltubuli nimplie sbindin go fcolchicin et ofunga ltubulin . Sincereport so ncolchicin ebindin gi nfung iar erathe rscarc ean dsomewha t controversial (18,38,43,46,54),th ecolchicine-bindin gactivit yo fmycelia l extractso fA .nidulan swa sinvestigated . Preliminaryexperiment si nwhic hmycelia lextract swer eincubate dwit h [H]colchicin ea t1 0yM ,showe dtha tu pt o b%o ftota lradioactivit ywa sboun d tomacromolecule s (TableV ,exp .1) .Sinc ethi samoun twa sunexpectedl yhigh , thenatur eo fthi sboun dradioactivit ywa sinvestigated .Thinlaye rchromato ­ graphic (TLC)analysis ,wit hchloroform/aceton/diethylamin e (5/4/1)a ssolvent ,

44 Oncodazole ,-0.8x10-7M

1/ [FreeMBC] (/JM-

Fig. 10. Inhibition ofMB C binding by oncodazole. Amycelia l extract of A. nidulans, Strain 186a t 33m g protein per ml was diluted 1 :1 wit h a solution of 2M sucrose in PKMg buffer,,Aliquots of the solution were incubated with increasing concentrations of [ CjMBC with (•-•) and without (o-o) 1u M oncodazole for 20h at 4 C. Bound MBC was assayed by the charcoal method. The concentration of freeMB C was calculated from the difference between total and bound MBC.

o

d-20 in CM E16 Colchicine 2Q. Ki-0.4x10_3M

CD Noinhibito r o **•« -O

0.5 1.0 1.5 20 1/[FreeMBC ](yuM- 1)

Fig. II. Inhibition ofMB C binding by colchicine. Amycelia l extract ofA . nidulans,Strai n I86a t 31 mg protein perm l was diluted 1 : 1wit h a solution of 2M sucrose inPKM gbuffer,.Aliquot s of the solutionwer e incubated with increasing concentrations of [ C]MB C with (•-•) and without (o-o) 1m M colchicine for 20h at A C. Bound MBC was assayed by the charcoal method. The concentration of freeMB C was calculated from thedifferenc e between total and bound MBC.

45 TableV . Binding of[H jcolchicin e inmycelia l extracts

+ + Exp. Binding agent Concentration Bound radioactivity %boun d uM dpm/ml dpm/ml

1 [H]colchicin e 10 3.7x10 2.1x10" 5.7 from stock [ H]colchicin e 1.2x1 0 1.3x10 1. 1 purified 1.3x10' 6.7x10 52.0 [3R!X. * purified

[ H]colchicin e and [Hj Xwer e purified according to themetho d described underMaterial s and Methods. Bound radioactivity was determined with the Sephadex G-100 assay after mycelial extracts ofA . nidulans Strain 003 at 41 mg/ml (exp. 1)o r StrainR at 42m g protein perm l (exp. 2,3 )ha d been incubated with the radio­ chemicals for 2h at 4 C. of Sephadex G-100 column fractionswhic h contained bound radioactivityre ­ vealed thatth eradiolabe lwa spresen t ina Compoun d X,whic hwa sno t identical with colchicine. Furthermore,i ncontras twit h known colchicine-tubulincom ­ plexes,th ecomple xwa sperchlori c acid (PCA)stable ,sinc e after addingPC A ata fina l concentration of0. 5N t oth efraction smentione d abovean dcen - trifuging themixtur e about 70% ofth eradioactivit y remained associated with thepellet .Washin g thepelle twit h ethanol/ether (3/1), removed mosto fth e radiochemical,whic hwit hTL Canalysi swa sidentifie d asCompoun d X.Sinc ei t was presumed that this radiochemicalwa spresen t asa nimpurit y inou r [II] - colchicine preparation, thelatte rwa spurifie d (fordetail s seeMaterial san d Methods) andbindin g experimentswer e performedwit h thepurifie d compounds. Results ofthes eexperiment s aregive n inTabe lV ,exp .2 an d3 .A si seviden t about 50?oo fth eradioactivit y present inCompoun d Xwa sboun d tocomponent s of themycelia l extract.Wit h purified [ll]colchicin e less radioactivitywa s bound thanwit h thenonpurifie d preparation.Thes e results indicate that binding activity ofmycelia l extracts upon incubationwit h unpurified [Hj - colchicine ismainl ydu et oassociatio n ofth eradiochemica l impurity Xwit h macromolecularcomponents . Sincepurificatio n of[H]colchicin epreparation s is laborious and100 1 purity canneve rb eachieved , infurthe r experiments amor e specific binding

46 assaywit hDEAE-Sephade xA-5 0io nexchange rha sbee nused .Mycelia lextract s wereincubate dwit h [H ]colchicin ea t50 0u M (approximately 19.6x 1 0 dpm/ml) for1 hou ra t4° Can dru nont oDEAE-Sephade xcolumns .Th eelutio npatter no f radioactivity,whic hi sshow ni nFig .12 ashow stw ohig hpeak san don ever y smallpeak .TL Canalysi sreveale dtha tradioactivit y infractio n4 ,whic hcon ­ tainedth emajorit yo fth eprotei nwhic hwa sno tadsorbe dt oth eio nexchanger , waspresen ti nth efor mo fCompoun dX ,wherea sradioactivit yelute da tth etw o otherpeak srepresente dcolchicine .Th ehig hpea ki sdu et ofre ecolchicin e sincei ti selute da tth ebe dvolum eo fth ecolumn .Th esecon d [H]colchicin e peakwa selute da tapproximatel y0.5 2M NaCl .Sinc ecolchicine-tubuli ncom ­ plexesar echaracteristicall yelute da tthi sioni cstrengt h (seeals onex t sections)thi sresul tsuggest sth eformatio no fa colchicine-funga ltubuli n complexi nth eincubatio nmixture .Tha tindee dcolchicin ewa selute da s boundt oprotei nan dno ti na fre efor mwa sdetermine db yge lfiltratio n ofa naliquo to ffractio n2 7o na Sephade xG-2 5colum n (1.5x 2 0cm) ,im ­ mediatelyupo nelutio no fthi sfraction .Abou t 37%o fth eradioactivit yre ­ coveredi nth eeluate ,elute dassociate dwit hprotei ni nth evoi dvolum eo f ® 3.0n 3.0

r20 - 2.0= 3 e 1.0 .9 .8 o 1.0 o .7 •1.0 H 1 , Si 50 o iz •3 11/ \ •2 0.0 V41 0.0 10 20 30 10 20 30 40 Fractionnumbe r Fraction number Fig. 12.DEAE-Sephade xA-5 0colum nchromatograph yo fmycelia lextract so f A. nidulans,Strai n 186incubate dwit h [H]colchicin e at4 C (a)an d3 7C (b ) for 1hour . 48,000x g supernatan tmycelial.extract s at3 2 (a)an d2 9 (b)m g proteinper,m lwer e incubatedwith -50 0p M [HJcolchicin e (19x10 dpm/ml(a ) and 12.2x10 dpm/ml(b)) .4 m l of the incubationmixtur e (a)o rsupernatan to f thecentrifuge dincubatio nmixtur e(b )wer eloade dont oDEAE-Sephade xcolumn s and eluteda sdescribe dunde rMaterial san dMethods . Thegraph ssho wth e elutionpatter no fradioactivity . NAC1concentration .

47 thecolumn . Whenmycelia lextract swer eincubate da t37° Cfo r1 h wit h [H]col ­ chicinean dafte rcentrifugin gboun dcolchicin ewa sassaye dwit hth eDEAE - Sephadexmethod ,th equantit yo fboun dcolchicin eseeme dt ob elowe rtha ntha t afterincubatio na t4 C (Fig.12b) .Sinc eth ebindin gsit efo rcolchicin ei s evidentlyidentica lwit htha to fMBC ,whic happeare dt ob edenature dunde r theseconditions ,thi sresul twoul db eexpected .I tshoul db efurthe rnotice d thatn osignifican tamoun to fradioactivit ywa selute dassociate dwit hth e bulko fth eprotein .Thi ssuggest stha tCompoun dX eithe rdi dno tbin da tthi s temperatureo rwa sassociate dwit hcomponents ,whic hwer eremove dfro mth e extractb ycentrifuging .Thes epossibilitie swer eno tstudie dfurther . Sinceth eDEAE-Sephade xbindin gassa yi sno tsuitabl efo rquantitativ e measurementsdu et odissociatio no fth ecolchicine-tubuli ncomple xdurin gth e procedure,n oattempt swer emad et odetermin ei nthi swa yth ebindin gconstan t forcolchicin ean dfunga ltubulin .

Purification of fungal tubulin. Asha sbee nshow ni nth epreceedin gsections , theMBC-bindin gprotei nca nb eassume dt ob eidentica lwit hfunga ltubulin . Thismake sMB Ca suitabl etoo lfo rth epurificatio no ffunga ltubulin ,ana - loguoust oth eus eo fcolchicin ei npurificatio nmethod sfo rmammalia ntubulin . Witha standar dpurificatio nprocedur efo rtubuli nth efollowin gresult swer e obtained.MBC-bindin gactivit yi n48,00 0x g supernatan tmycelia lextract s couldb eselectivel yprecipitate dwit hammoniu msulfat ebetwee n3 5an d50 % saturation (TableVI) .Hig hspee dcentrifugin gan dammoniu msulfat efrac ­ tionationtogethe rresulte di na preparatio nwhic hcontaine dapproximatel y 50°*o fth einitia lbindin gactivity .Th especifi cactivit yo fthi spreparatio n wasincrease dabou ttwo-fold . Inpreviou swor k (22,23)i tha sbee nshow ntha tth eMB Ccomple xwa sre ­ tainedo na DEAE-Sephade xA-5 0anio nexchange ran dcoul db eselectivel yelute d ata ioni cstrengt ho f0. 6M KC1 .Therefore ,DEAE-Sephade xcolum nchromato ­ graphywhic hha sprove nt ob eusefu li npurificatio no ftubuli nfro mothe r organisms (cf.74) ,wa sinclude da sa fina lpurificatio nste pfo rfunga ltubu ­ lin.Thi sste pwa sfirs tstandardize dwit hboun d [ C]MBC.A partiall ypuri - r14 •, fiedpreparatio nwa sincubate dwit h [ CJMBCan dapplie dt oth ecolumn .Par t ofth eelutio npattern so fradioactivit yobtaine dwit hpreparation so fStrai n 003,Strai n186 ,an dStrai nR ar eshow ni nFig .13 .Fo rStrai n186 ,par to f theradioactivit ywa selute da sa distinc tpea ka tapproximatel y0.5 2M NaCl , whereaswit hStrai n00 3onl ya smal lshoulde rwa sobserve di nth eelutio n

48 TableVI .Purificatio n ofMBC-bindin g protein frommycelia l extract

Ammonium sulfate Total protein % of total binding Specific binding fractionation range (mg) activity recovered activity (pmole/ mg protein)

0-20 5 0.4 3.7 20- 35 72 12.0 7.7 35- 50 92 74.1 37.5 > 50 173 13.6 3.6

Mycelial extract (11.5ml ) of A. nidulans, Strain 003 at 29m g protein per ml was fractionated with a saturated ammonium sulfate solution inwate r between limits indicated. The preparations were incubated with [ c]MBC at 10.9 uM and bound MBC was assayed by the charcoal method.

TableVII .Ge l filtration of DEAE purified MBC-protein complex on a Sephadex G-100 column

Strain 003 186

Radioactivity of DEAE-purified [ C]MBC-protei n complex (dpm/ml) 3,140 15,220 rI4 i Recovery of bound l CJMBC after gel filtration on Sephadex G-100 (dpm/ml) 701 8,410 % recovery of bound [14C]MBC 22 55

48,000x g supernatant mycelial extracts of A. nidulans Strain 003 at 38m g protein perm l and of Strain 186a t 37m g protein perm l in PKMg buffer,were incubated with [ c]MB C at 7.8 uM (19.4x10 dpm/ml) and 8.1 uM (20.2x10 dpm/ml), respectively, for 2h at 4C 10m l of the incubationmixtur e was run onto aDEAE-Sephade x A-50 column equilibrated with PKMg buffer, as des­ cribed under Materials and Methods,bu t using potassium buffers instead of sodium buffers.Radioactivit y of the fractionswa s determined immediately after they had been eluted. 1m l of the fraction containing the peak of bound MBC was filtered immediately through a Sephadex G-100 column.

49 20 25 30 Fraction number

Fig. 13.DEAE-Sephade x A-50 column chromatography of partially purified mycelial extracts ofA . nidulans,Strai n 003,Strai n 186 and Strain R. 127,000x g supernatant mycelial extracts were fractionated with a saturated ammonium sulfate solution (seeMaterial s and Methods). 5m l of theprepara ­ tions containing 15m g (Strain 003) 12m g (Strain 186)an d 19,mg (Strain R) protein perm l were incubated with [ CJMBC at 5.1 uM (12.8x10 dpm/ml)an d run onto DEAE-Sephadex columns as described under Materials andMethods . Graphs show part of the elution pattern of radioactivity. •-•, Strain003 ;

o-o, Strain 186;• — •t Strai nR . NaCl concentration.

patterna tthi s salt concentration.A sexpecte dwit h StrainR n osignifican t amountso fradioactivit ywer e eluteda tNaC l concentrationso f0. 4M an d higher. Whether eiutiono fradioactivit ya t0.5 2M NaC lwa scause db yelutio n ofth eMBC-protei n complex itselfo rb ydissociatio no fMB Cfro mth eDEAE - bound complex,du et oincreasin g salt concentration,wa sdetermine db yrechro - matographyo fth efraction s concernedo nSephade x G-100columns .I tappeare d (TableVII )tha tdependin go nth estrai nuse d considerable amountso fradio ­ activitywer e boundt oprotein ,whic h indicatesth eelutio no fpurifie dMBC - protein complex fromth ecolumn . Insubsequen texperiment sn o [ C]MBCwa spresent ,an dfraction s eluted at0.45-0.9 0M NaC lwer e consideredt ocontai nth epurifie d bindingprotein . .Th eMBC-bindin g activityo fDEAE-purifie dprotei nha sno tye tbee ninvesti ­ gated indetail .I na preliminar y experiment,i nwhic ha crud emycelia l extract fractionatedwit hammoniu m sulfatewa spasse d throughth eDEAE-Sephade x column andth eresultin g purified protein solutionwa sconcentrate d witha Sartoriu s

50 ultrafiltration device,approximatel y 5°so fth e initialbindin g capacity of thecrud eextrac twa s recovered.Th e specific activity ofthi s preparation was raised toabou t 13-fold of that ofth e crude extract.Thi s rather low yield indicates aconsiderabl e losso fbindin g activitydurin g theprocedure , whichwa sprobabl y due to the instability ofth ebindin gprotein . Proteins isolated from thethre estrain s ofA .nidulan sb y DEAE-Sephadex columnchromatograph ywer e analysedwit h thecontinuou s SDSsystem . Gels loadedwit h reducedprotei n samples offractio n 24-30 (Fig. 13)fro m the experimentwit h Strain 186an d StrainR are showni nFig . 14an d 15,respec ­ tively.A s reference,reduce d porcinebrai n tubulinpurifie db y onepoly ­ merization cyclewa s runo na separat e gel.O ngel s loadedwit hprotein s from Strain 186 three closely-spacedbands ,amon g others,wer epresen twhic h have a comparablemobilit y asth eporcin ebrai nmonomer swhic hmov e together in thissystem .Th ebandin gpattern s ofgel s loadedwit h reducedprotein s from corresponding fractions derived from Strain00 3wer e very similar (not shown). Ingel s loadedwit hprotein s from strainR ,however ,onl y oneban dwa s present at amigratio ndistanc ewhic h iscomparabl ewit h that ofbrai ntubulin . Thedifferenc ewa smor epronounce dwhe nprotein swer e reduced and car- boxymethylated beforeanalysi swit h the same system.Gel s loadedwit hDEAE - purifiedproteins ,elute d at 0.45-0.90M NaCl, from Strain003 ,Strai n 186an d StrainR are shown inFig . 16a.Carboxymethylate d DEAE-purified porcine brain tubulinwa s run asreferenc e protein. It iseviden t that thepreparatio n of StrainR differe d from those ofStrai n 003 and Strain 186. Ingel s loaded withprotein s fromth e latter twostrains ,tw odistinc tband s arepresen twit h a comparablemobilit y as theban d representingporcin ebrai n tubulin and one bandwhic hha dmove d slightly faster.Wit h StrainR twowea kband s arepresen t atthi smigratio n distancewherea s otherband s showed aboutequa l staining as correspondingband s present inth e other two gels. When identicalprotei npreparation s ofth e three strainswer e runo n discontinuous SDS gels, twomajo rband s ofunequa l staining densitywer e foundwit h similarmobilit y as theband s on thereferenc e gel (Fig. 16b).Th e faster-moving band on thereferenc e gel isdu e to 3-tubulinan d theslower - moving one toa-tubuli n(51) . Whengel s frombot h SDSsystem s arecompare d it iseviden t thattw obands , which are found tob e separated inth e continuous system,mov e together inth e discontinuous system.Wit h respect toth e staining densities of theband s it seems thaton e of the twoslower-movin g bands of the continuous systemmove s togetherwit h the fastermovin g band in thediscontinuou s system.

51 1 1 1

• ... Iw 1 6

I [ 24 25 26 27 28 29 30 Fig. 14.Electrophoreti c analysis of proteins isolated from A.nidulans , Strain 186b y DEAE-Sephadex column chromatography. Samples of fractions 24-30 (seeFig . 13, Strain 186)wer e reduced and ana­ lysed on the continuous SDS system. Reduced porcine brain tubulin (T)purifie d by one polymerization cyclewa s run as areference .

24 25 26 27 28 29 30 T Fig. 15.Electrophoreti c analysis of proteins isolated from A.nidulans , Strain Rb y DEAE-Sephadex column chromatography. Samples of fractions 24-30 (see Fig. 13,Strai n R)wer e reduced and analysed on the continuous SDS system. Reduced porcine brain tubulin (T)purifie d by one polymerization cycle was run as areference .

52 (B)t:tJ\ p I

^4

mm I •SB

003186 R 003186R T

Fig. 16.Electrophoreti c analysis of proteins isolated from A.nidulans , Strain 003,Strai n 186 or Strain R, by DEAE-Sephadex column chromatography. Lyophilized proteins from pooled fractions eluted at 0.45-0.90 M NaCl were carboxymethylated and run in the continuous SDS system (a)an d thediscon ­ tinuous system (b).Carboxymethylate d DEAE-purified porcine brain tubulin was run as areference .

Bothge lsystem s couldb ecorrelate d inth efollowin gmanner . Itwa s found thatheatin ga DEAE-purifie dpreparatio nfo r1 h a t37°C ,coolin gi n icean dcentrifuging ,resulte d ina supernatan t inwhic hupo nelectrophore ­ sis inth econtinuou s SDSsyste mth etw oslowe rmovin gband swer eno tpresen t (Fig. 17a,b). Theseband s appeared tob epresen t ingel s loadedwit hth e pelletedmaterial .Heat-dependen t precipitationo fprotein swa sno treversibl e by cold,no rwa sth eproces s inhibitedb yC a ,colchicin eo rMBC ,whic h eli­ minatesth epossibilit y thatmicrotubul e assembly isresponsible .Apparentl y denaturationb yhea to fth eprotein s concerned causesprecipitatio nwhic hi s inagreemen twit h theobservatio n thatmycelia l extractswhic har eheate d at3 7C an dcentrifuged , showrathe r lowMBC-bindin gactivity . Uponelectrophoresi s inth ediscontinuou s systemth eprecipitate d material runsmainl ya stw obands ,wit h similarmobilit ya sa -an dB-tubuli n frompor ­ cinebrai n (Fig.17c) .Becaus eo fthi sbehaviou r itca nb econclude d that theseband s representbot h tubulinmonomer s ofA .nidulans .Th ethir d faster- movingband incontinuou s SDSgel swhic h showeda simila rmobilit ya sB-tubuli n inth ediscontinuou s system,apparentl y represents aprotei nwhic h copurified with fungal tubulini nthi spurificatio nprocedure . Iti seviden t thatth epurificatio nprocedur e applied toextract so f

53 StrainR resulte d insignificantl y lessa - and 3-tubulin,tha nwa s obtained with asimila rprocedur e applied toextract s of Strains00 3an d 186.Althoug h a somewhat lower initial tubulinconcentratio nmigh tb epartl y responsible (seeFig . 7),i t iscertainl y not theonl y factor involved.A reason for the failure topurif y tubulino fStrai n Rmigh tb e a lowaffinit y of this tubulin forDEAE-Sephadex .A low affinity ofbrai n tubulin forDEAE-Sephade xha sbee n reported (47,53)an d seems tob e relatedwit hphosphorylatio n of theprotein . At any rate,th edifferen tbehaviou r of tubulino f Strain R in comparison with thato fbot h other strains,suggest s chemical differences of theprotei n itselfo ro f factors associated with tubulin,a n ideawhic h is compatible with theobservatio n thatdifference s exist inaffinit y ofth e tubulins of the three strains toMBC . The results of the electrophoretic analysis indicate thatMB Cmigh tb e used asa naffinit y label topurif y fungaltubuli n fromMBC-sensitiv e strains ofA . nidulans. Itsupport s the idea that thisprotei n is theprimar y target ofMB Caction .

DISCUSSION

TheMBC-bindin gprotei n inA .nidulan s ischaracterize d by ase to fpro ­ pertieswhic h isuniqu e for tubulin (cf. 14).Bindin g activity couldb ese ­ lectively fractionated with ammonium sulfatebetwee n 35an d 50% saturation. TheMBC-protei n complexwa s retained onDEAE-Sephade x columns and itsmole ­ cularweight wa s estimated at 110,000.Bindin g activitywa s labilean dcoul d be stabilized by sucrose,glycero l andMB C itself.Bindin gwa s competitively inhibitedb y knownantitubulin s sucha soncodazol e and colchicine.Electro ­ phoreticanalysi s ofpartiall ypurifie dpreparation s ofth eMBC-protei ncom ­ plex, showed thepresenc e ofprotein swit h similarmobilitie s asmammalia n tubulinmonomers .O n thebasi s of these results itca nb e concluded that the MBC-bindingprotei n is identicalwit h fungaltubulin . MICbindin g to fungal tubulinwa s rapid,reversibl e and ditno t require ahig h temperature.A s hasbee n reported recently (45),bindin g of oncodazole tora tbrai n tubulinshow s similar features. Inthi srespec tMB C andoncoda ­ zoleresembl epodophyllotoxin ,a compoun dwhic h competeswit h colchicine for the colchicine binding siteo n tubulin fromdifferen t sources (75).However , podophyllotoxin didno t inhibitMB Cbindin gwhic h suggest thatfunga l tubulin hasn oo ra t leasta lowaffinit y for this compound. From thedat agive n inFig . 7th enumbe ro fMB Cbindin g sitesca nb e determined.Assumin g thaton emolecul e ofMB C isboun dpe rmolecul e oftubulin ,

54 43 4-> 43 T3 O C 43

sua) r-H 4-> o -o TO co CTO3 <- <> , 3 x; « « u 4-1 i-i £ o ra S S-l H x D. >i o 43 43 ^.u u CO CD TO

4-J X c o TO 43 TO TO

a) u -a D- o a) •~- 3 TO T3

co 43 >> 4-1 CO -H T3 a) co 4J O -a TO CO . cu a) u 43 to • TO 3 43 o 3 3 • C 3 1 Si i 3

a) a) •!- I 4J !- C 4 O C

0) 4=

O ' 3 1 I x cd V4 TO M> 6 M CH X ~ TO T3 O 4-1 >^ CO e O~ •C U CO TO O O 00 U •H 43 O CU o la) CK

55 one cancalculat e that tubulin is0.2-0.4 %b yweigh t of totalprotei npresen t inmycelia l extracts.Thi svalu e resembles thevalu e of0.6 % found forth e colcemid-bindingprotei n inSaccharomyce scerevisia e (38)whic hprove d tob e ingoo d correspondencewit h the figure of0.36° scalculate d from theestimat e of thenumbe r ofmicrotubule s pernucleus . Although colchicinewa s evidentlyboun d totubuli no fA .nidulan s the binding reactionshowe d someunusua l features.Th e rateo fcomple x formation seemed tob e rapid andbindin gwa s not temperature dependent,whic h properties are incontras twit h those of colchicinebindin g tomammalia n tubulin (cf.74) . Moreover,affinit y ofA . nidulans tubulin tocolchicin ewa s rather lowcom ­ paredwit h thato fmammalia n tubulin.A lowaffinit y of,presumably , tubulin from S.cerevisia e for colchicine hasbee nreporte db y Haber et al.(38) . Burns (18)wa s notabl e todemonstrat e colchicinebindin g inSchizosaccharomyce s pombe andJockusc h (46)di dno t find colchicine-binding proteins in Physarum polycephalum.Heat h (43)reporte d thepresenc e oftw obindin g components in Saprolegnia feraxwit h a lowaffinit y for colchicine,on e ofwhic hwa s trichloroacetic acid (TCA)stable .A TCA-stablebindin g proteinwa s also foundb yOlso n (54)i nAllomyce smoniliformis .However ,th e last two authors didno t investigate the identity of theboun d radiolabel. Since inou r colchicine-binding experiments the formation ofa PCA-stable labelled complex couldb e ascribed toth epresenc e of an impurity inou r [H Jcolchicin e preparation,th e results ofthes e authors should be interpretedwit hcaution .

Lowaffinit y ofA .nidulan s tubulin forcolchicin e isprobabl ypartl y responsible for thefailur e of this compound to inhibitmycelia lgrowth . Cellmembran e permeability might alsopla y arol e sincen o effect ongrowt h wasnotice d at concentrations 25-timeshighe r thanthos ewhic h areneede d tohalf-maximall y saturate thebindin g sites invitro . Incompariso nwit h MBCan d oncodazole thisrati o israthe rhigh ,sinc e these compounds gavea 50°ireductio n ingrowt h already atconcentration s 2-3 timeshighe r thanth e estimatedvalu e of theirrespectiv e dissociationconstants . To ourknowledg edat a concerning affinity of fungal tubulin forcolchi ­ cine are restricted tothos e already mentioned.W e think that lowaffinit y isa characteristic property of fungal tubulin.Thi s assumption isbase d on the fact that fungi are commonly resistant toth e antimitotic actiono f colchicine (cf.42) ,whic h seems onlypartiall y causedb y a lowpermeabilit y of the cellmembran e (38,64)an d on the fact thatmicrotubule s are supposed topla y anessentia l role infunga lnuclea rdivisio n (34,35). Untilno wn o suitable agentha sbee n foundwhic h specifically disruptsmicrotubule s in

56 fungi (42).Becaus e of theirhig h fungitoxicity,MB C andoncodazol e are potential candidates,sinc e frombot h compounds it isknown ,tha t they inducedisappearanc e ofmicrotubule s inmammalia n cells (25-27). Thedifferentia l growth response ofth e twomutan t Strains Ran d 186, incompariso nwit h that ofth ewil d type Strain003 ,i sprobabl ybase d ona difference inaffinit y of their tubulins forMBC .Thi s idea isals o supported by the fact thatn odifference s are found inuptak e ordetoxicatio n ofMB C between thevariou s strains(24) . Genetic analysisha s shown thatmutation s toresistanc e andt o increased sensitivity tookplac e inth e same gene (69,70).Althoug h itca nb e assumed thatthi sgen e codes fortubulin ,thi s canonl yb edefinitel y concludedwhe n differences are found between theprimar y structures of tubulino f the three strains.A difference inaffinit y forMB Cmigh t alsob e causedb y differences inpost-translationa lmodification s of tubulin,suc h asphosphorylatio n (30,31), glycosylation (32)o rassociatio nwit h tau-like factors (15,16,72).A dif ­ ferentmodification ,rathe r thana single aminoaci d substitutionmigh t also explain the failure topurif y tubulin from Strain R. Mutation toresistanc e or increased sensitivity didno t affect growth rateno r sporulation of the strains (ref.22 ,69 ,Fig . 1),whic h indicates normal assembly and functioning ofmicrotubules .However ,diploid s carrying bothmutation swer e notstabl e aswa s evident from increased sectoring of diploid colonies .Sinc enon-disjunctio n ofchromosome s isthough t tob e mainly responsible for sectoring,increase d sectoring indicates improper functioning ofmicrotubules .Thi smigh tb e causedb y thepresenc e oftw o incompatible types of tubulins inthes e diploids. Increased sectoringha s alsobee n found inordinar yA .nidulan s diploidswhe n they are exposed to MBC at sublethal concentrations (37,41,49). Griseofulvin induces a similar effect (37,48). These observations are compatiblewit h the idea thatbindin g ofMB C totubuli n interfereswit h the assembly of tubulin intomicrotubules . The action ofMB C and themechanis m ofresistanc ewa s only studied in detail inA . nidulans,bu t thedat a inTabl e Isugges t that themechanis m of resistance found heremigh tb e agenera l type ofresistanc e infungi .Sinc e infung ivariou s types ofmitosi s are found (35)i twoul db e interesting to knowwhethe r resistance and sensitivity toMB C isrelate d toa certai n type ofmitosis .

1 VanTuyl ,J.M. , personal communicationan d ownobservation .

57 Untilno wn oevidenc eha sbee npresente d thatresistanc e toothe r anti- tubulinsmigh tb ecause db ya simila rmechanism . Resistancet oantitubulin s has recentlybee ndiscusse db yFree dan dOhlsson-Wilhel m (33).A dru gexclu ­ sionmechanis mwa sfoun dt ooperat ei nsevera l instances.Th ebiochemica l basiso fresistanc ewhic h appearedt ob especifi ct oth eselectin gagent , hasno tye tbee nstudied . Severalpossibilitie s existt oexplai nth eselectivit yo fthes ebenzi - midazolecompound s ingeneral .I nadditio nt oa differentia l uptakeo rmeta ­ bolism,a differen t affinityo ftubuli n fromvariou s sourcesfo ra certai n benzimidazole compoundmigh tpla ya role .Thi s isillustrate db yth efac t thatn obindin gwa sfoun dbetwee nporcin ebrai n tubulinan dMBC ,althoug ha t identical concentrationso fMB Cconsiderabl e binding tofunga l tubulinwa s found.A differenc e inaffinit ybetwee n tubulins fromdifferen t sourcesfo r a certainbenzimidazol e compound mightb ecause db ysimila r factors already discussedabove . Iti snot eworth yher e thatdespit e differences inaffinit ybetwee n porcinean dA .nidulan s tubulint oMBC ,th etw otype sar eabl et ocopoly - merize invitr o (23,60).Apparently ,bindin g sites involved inpolymerizatio n havebee nhighl y conserveddurin g evolutioni ncontras twit hth ecolchicin e bindingsite . Undoubtedly,thes ebenzimidazol e compoundswil l becomeusefu la sexperi ­ mental toolsi nth estud yo fmicrotubul e structurean dfunctio ni ncells . Theiruse ,however ,i nagricultur e asfungicide sand ,quantitativel y ona minor scale,i nveterinar ymedicine ,shoul db ereconsidere d fromth epoin t ofvie wo fthei rmechanis mo faction . Interferenceo fMB Cwit h nuclear divisioni nmammalia ncell sha sbee n foundt ooccu r invitr o (27,59,67)an d inviv o (59,67). This impliesa potentia l genetic riskfo rman .Th etoxicolog y and genetic effectso fbenzimidazol e compoundshav e recentlybee n reviewed by Seller (59).W eagre ewit hhi mtha tth eus eo fpesticide swit h this type ofactio n shouldb erestricted .

ACKNOWLEDGEMENTS

Wear egratefu l toProf.Dr.Ir .J .Dekker ,Dr .A .Fuch san dDr.Ir .J .Visse r (Dept.o fGenetics ,Agricultura l University)fo rcomment so nth emanuscrip t andt oMrs .Brouns-Murra y (Pudoc,Wageningen ,Th eNetherlands )fo rcorrect ­ ingth eEnglis htext .

58 REFERENCES

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63 Metabolic conversion of methyl benzimidazol-2-yl carbamate (MBC) in Aspergillus nidulans

L.C. Davidse

Research Unitfo rInterna l Therapyo fPlant s T.N.O. Laboratory ofPhytopathology ,Agricultura l University Wageningen,Th eNetherland s

ABSTRACT

Methyl benzimidazol-2-yl carbamatewa smetabolize db yAspergillu s nidulans mycelium totw ometabolites ,on eo fwhic hwa sidentifie d asmethyl - S-hydroxybenzimidazol-2-yl carbamate.Thi s compoundwa sfurthe r convertedt o a second metabolitewhic hwa sno tidentified . Conversion ratewa shighes t whenth ecultur emediu mwa sdeplete d ofnutrients .Especiall y inage dcon ­ centrated mycelial suspensions conversionwa srapi dan dcomplete . Since strains differing inMB Csensitivit y metabolizedMB Ca tequa l rates,conver ­ siono fMB Ct oth enon-fungitoxi c metabolitesha sn obearin go nth emechanis m ofresistanc e tothi s compound.

INTRODUCTION

Since theintroductio no fth esystemi c fungicides methyl 1-(butyl- carbamoyl)benzimidazol-2-y l carbamate (benomyl)an d1,2-bis-(3-methoxy - carbonyl-2-thioureido)benzene (thiophanate-methyl)severa l reportshav e been published about theirnon-biologica l andbiologica l conversion inplants , animalsan dmicroorganisms . Inaqueou s andnon-aqueou s mediabot h compoundsar ereadil y converted tomethy lbenzimidazol-2-y l carbamate (MBC) (1-3),whic h isgenerall y

The following abbreviations are used:MBC ,methy l benzimidazol-2-yl carba­ mate; 2-AB, 2-aminobenzimidazole; 5-OH-MBC,methy l 5-hydroxybenzimidazol-2-yl carbamate.

64 regarded asth eactua l fungitoxic compound. Conversiono fthiophanate-methy l ispH-sensitiv e (4)an dth econversio n rate isincrease d byplan t extracts (5,6]. Inplants ,MB Ci sth eonl y fungitoxic compound recovered after root treatmentwit hbenomy lo rthiophanate-methy l (7-10).Metabolis m studiesi n plantswit h radiolabeled compounds showed thatMB Cwa sfurthe r decomposed via 2-aminobenzimidazole (2-AB) (11-13)int obenzimidazole ,o-aminobenzonitril e and aniline (14).B-Glycoside so fMB Can d2-A Bar eals o found. Inanimal s benomyl andthiophanate-methy l aremetabolize d viaMB Ct o methyl S-hydroxybenzimidazol-2-yl carbamate (S-OH-MBC)an dit s 4-hydroxy- analog which compounds areeliminate d fromth eorganis m asglucuronid e and/or sulphate conjugates (9,15-17). Microorganisms candegrad e benomyl (18,19). Thisproces s involvesth e formationo f2-A Ba sa nintermediat e andth eliberatio n of CO? from [2- C] MBC,whic h indicates that ring cleavage occurs (20,21). Infungi , thiophanate- methyl isconverte d toMB Ca ta rat ewhic h suggests theparticipatio no f fungal metabolic activity inthi sproces s (4,22).A smino rmetabolite s 5-OH- MBCan da nunknow n substance areals o found. Since 5-OH-MBCi sles s fungitoxic thanMB C (22,23),MB Cmetabolis mi n fungi mightb eregarde d asa detoxicatio n mechanism. Detoxication might play a rolei nth eresistanc e offung it othi s compound. Thepurpos e ofthi s studywa st odetermin e themetaboli c fateo fMB Ci n Aspergillus nidulans andit spossibl e relation tomutationall y induced resistance inthi s fungus.

MATERIALS AND METHODS

Organism. Aspergillus nidulansbiA 1AcrA1 , requiring biotin,resistan tt o acriflavine andwit hwild-typ e sensitivity toMB C (Strain 003) (24)wa s used inmos t experiments. StrainR ,resistan t toMB Can dStrai n 186,highl y sensitive toMB C (25),wer e used todetermin ewhethe r mutant strains with a differential response toMB Cals o differed inrat eo fmetabolism .

Chemicals. Methyl benzimidazol-2-yl carbamate (MBC)an dmethy l 5-hydroxy- benzimidazol-2-yl carbamate (5-OH-MBC)wer e giftso fE.I .D uPon td eNemour s and Co. (Inc.) (Wilmington,Del .USA) .Benomy lwa spurchase d from AAgrunol (Groningen,Th eNetherlands )a sa 50° swettabl e powder (Benlate). 2-Amino- benzimidazole (2-AB)an dbenzimidazol e werepurchase d fromK & K Laboratorie s (Inc.) (Plainview,N.Y. , USA)an dKoch-Ligh t Laboratories (Ltd.) (Colnbrook,

65 England), respectively.[2- c]MBC (spec.act . 11.4mCi/mmole )wa s purchased from International Chemical andNuclea r Corporation (Irvine,Cal. ,USA) .

Culture methods. Conidia ofAspergillu s nidulans strainswer e grown ona 2 % maltextract ,0.1 1Bacto-Peptone , 2%glucose ,1.5 1aga rmedium . Conidial suspensionswer e prepared asdescribe d previously(24) . Inmetabolis m experimentsmyceliu mwa s either grown in Czapek-Dox liquidmediu m (Oxoid)o rglucose-nitrat e medium (26).Bot hmedi awer esupple ­ mentedwit h 1u gbioti npe rml . Incubationwa s at 37C o na Gallenkam p orbital shaker at 200rpm .Wit h Czapek-Doxmedium ,benomy l (50°sWP )o rMB C was added asa sterile aqueous suspension orsolutio n inmethanol ,respec ­ tively,eithe r simultaneously with spores orafte r an initial growth period. Sterility of theaqueou sbenomy l suspensionwa s achievedb y first dissolving themateria l inethanol ,afte rwhic h sterilewate rwa s added.Methano lcon ­ centration inth emedi aneve r exceeded 1 %(v/v) . Mycelium grown for 15hour s inglucose-nitrat emediu mwa sharveste d by filtration on aBiichne rfilte r andwashe d three timesb y resuspension in fresh medium and filtration.Th emycelia l cakewa s resuspended inglucose - nitratemediu m or in0.0 5 Mpotassiu m phosphatebuffer ,p H 6.8 ata concen ­ tration of 1g we tweigh t ofmyceliu mpe r 25m l medium.MB C or [ CJ MBC was added to themycelia l suspensions eitherdirectl y or after an initial incubation period.

Dry weight determinations. Dryweigh t ofmyceliu mwa s determined as described previously(24) .

Determination of MBC. MBC concentration incultur e filtrateswa smeasure d by a thin-layer chromatographic bioassay (TLC-bioassay) (27).A 50o r 100y l aliquot of thecultur e filtratewa s spotted on asilic a gel plate (DC Alufolie Kieselgel F254 ,Merck ,Darmstadt ,W . Germany). Chromatogramswer e developed eitherwit h chloroform/methanol (97/3) (Solvent A), ethyl acetate (Solvent B)o r ethyl acetate,saturate dwit h 0.05 M potassium phosphate buffer,p H 6.8 (Solvent C). After drying,chromatogram swer e sprayedwit ha spore suspension ofPenicilliu mbrevicompactu m innutrien t solution (27) and incubated ina humi d chamber at 25C .Afte r 3day so f incubation, inhibition zoneswer e replicated ontracing-pape r (SchoellershammerHoch - transparent,N r 205glatt ;Diire nW . Germany),cu t out andweighe d intripli ­ cate.Th evalue s obtained area naccurat e estimationo fth e sizeo f the inhibition zones.Amount s ofMB Cpresen t inth e aliquotswer e calculated

66 withth eregressio n equationy = a + b Inx ,wher e aan db ar econstants , y isth erelativ e sizeo fth einhibitio n zone (mg)an dx i sth eamoun to f MBC fng).Th eregressio n constanta an dth eregressio n coefficientb o fthi s equationwer e calculated bynon-linea r regression analysiso fth erelatio n between sizeo finhibitio n zonesan dknow n amounto fMBC ,wit ha Dieh l Algotronic calculator.Th enon-linea rcorrelatio n coefficient andt valu e (n= 14 )wer emostl y higher than0.9 9an d30 ,respectively . With SolventC , whichprove d togiv eth ebes t correlation,quantitie s ofMB Ci nth erang eo f 3- 6 0n gcoul d accuratelyb edetermined . This corresponds toa lowest detection levelo f0.1 5y MMBC ,whe n 100y l aliquots arespotted . TheMB Cconten t ofth emyceliu mwa sals o estimatedb yth eTLC-bioassay . Mycelium, thatha dbee n exposed toMB Cwa sharveste d anddried . Usually 300m go fth edr ymateria lwa ssuspende d in3 m l 50%methano l inwate ran d allowedt ostan d for2 4hour s atroo m temperature.Th emycelia l residuewa s thenspu n downan daliquot s ofth esupernatan twer e analysedb yth e TLC- bioassay.

Analysis and identification of radiolabelled metabolites. Radioactivityi n r141 culture filtrateso fmycelia l suspensions exposed to [ CJMBCwa smeasure d by liquid scintillation counting ofaliquot s ina Nuclea r ChicagoMar kI Liquid Scintillation Spectrometer,wit h Brunoan dChristian' s (28)scintilla ­ tion fluid. Counting efficiencywa sdetermine d byth eexterna l standardization procedure. •I A C-Metabolites incultur e filtrates ofmycelia l suspensions exposedt o [ C]MBCwer e tentatively identified with thin-layer cochromatography using MBC,2-AB ,benzimidazol ean d5-OH-MB Ca sreferences .A ssolvents ,Solven tC and ethylacetate/acetic acid/chloroform (55/ 5 / 1 )(Solven tD )wer e used. Rpvalue swer e determined from radioscans ofth echromatogram swit ha Nuclea r ChicagoActigrap h III radiochromatography system. Reference compounds were localized under UV-light. To determine theproportio no fradioactivit y presenta sMBC ,chromato ­ gramswer ecu tint o segments,an dplace d inscintillatio n vials containing thesam e scintillation liquida smentione d above.Radioactivit y presenti n segments corresponding with theR fvalu eo fMB Cha sbee n expressed asper ­ centage oftota l radioactivity recovered.

RESULTS

Analysis ofth efat eo fMB Ci ngrowin g cultures ofMBC-sensitiv e fungi,

67 sucha sA .nidulan s implies thenee d ofa ver y sensitive method ofMB C estimation.A ssuch ,th eTLC-bioassa y ishighl y suitable,becaus e itca n detectMB Ci nnanogra m quantities (29),eve ni ncomple xmedia . Culture filtrates containingMB Ca tconcentration s whichwer e sublethal toA .nidulans , Strain 003,coul d readilyb eanalyse d asi sshow n inFig .1 .I nfact ,i nth e experiment concerned, cultureswer e incubatedwit hbenomy la t2 u Mbu ti t canb eassume d (1)tha t under theexperimenta l conditions used, this com­ poundwa scompletel y converted toMBC .Althoug h growthwa sno taffected , cultures treated withbenomy l showed ayellow-brow n discoloration after1 6 hourso fincubation . Upon further incubation theculture s became more darkly coloured andafte r4 0hour s themyceliu mwa snearl y black,wherea sth e culture filtratewa sligh tbrown .Non-treate d culturesdi dno tsho wan y discoloration. Analysis ofth ecultur e filtrate revealed thatth eMB Cconcentratio nwa s initially constant butdecrease d below thedetectio n level after 32hour so f incubation.Whe nMB Cwa sadde d tonon-inoculate dmediu m adjusted todifferen t pHvalue sn odecreas e inMB Cconcentratio nwa sobserved . Therefore itca nb e concluded that fungal metabolic activitywa sresponsibl e forth eeliminatio n ofMB Ci ninoculate d cultures.

hours

Fig. 1.Growt h ofA . nidulans Strain 003 in liquid Czapek-Dox medium, contain­ ingbenomy l at 2uM , •- •; control,o - o. 10m l medium in 25m l Erlenmeyer flasks were inoculated with spores at a final concentration of 5.10 spores perml . Inset shows the inhibition zones caused by MBC on thin-layer chroma- tograms (Solvent A)o f aliquots of the culture filtrate after different times of incubation.

68 To investigatewhethe r theMB Celiminatio nproces sha dbee n inducedb y MBCa nexperimen twa scarrie dou ti nwhic h cultureswer e incubatedwit hMB C at2 an d4 uM ,respectively ,fo rdifferen t times.MB Cwa sadde dt oth ecul ­ tures simultaneously with inoculationo rafte r3 0hour swhe n growthwa s exponential. Fig. 2show sth edecreas e inMB Cconcentratio nunde r thevariou s con­ ditions.Compariso no fcurv eA an dC wit h curve Ban dD ,respectively , indi­ cates thatMB Celiminatio n fromth ecultur e filtrate started immediately uponadditio no fMB Ct o30-hour-ol d culturesa ta rat e comparablewit h that ofMB Celiminatio ni nculture swhic hha dbee n continuously exposed toMBC . Whenculture swer e initially incubatedwit h 2y MMB Can dafte r 30hour sa

c 3-

70 80 hours

Fig. 2.Eliminatio n ofMB C from culture filtrates ofA .nidulan s Strain003 , growing in liquid Czapek-Dox medium. 100m l medium in 300m l Erlenmeyer flasks were inoculated with spores at a final concentration of 7.10-5 spores per ml.MB C was added either simultaneously with spores (2 and 4yM , res­ pectively) or after 30hour s of incubation. Part of the cultures towhic h MBC (2yM )wa s added at t= 0 h received an additional amount ofMB C (2yM ) at t= 30h . MBC concentrations in culture filtrates were determined with theTLC-bioassa y (Solvent B).o - oA , 2y M at t= 0h ; •- • B, 2y M at t = 30 h;• -• C, 4y M at t= 0h ;• -• D, 4y M at t= 30h ; A -A E, 2 yM at t= 0h and 2y M at t= 30 h. ,minima l detectable concentration.

69 second portiono fMB Cwa sadded ,th erat eo feliminatio n (curve E)wa sfoun d similar totha twhe nMB Cwa sadde d only oncet oth eculture s growing exponen­ tially 30hour s after inoculation (curveD) . Also inthi s experiment brownish discoloration ofmyceliu m andcultur e filtratewa snoticed .Th ecolou rwa sdarke r inculture s athighe rMB Ccon ­ centrations andafte r incubation forlonge rperiods . The results ofthi s experiments suggest thatMB Celiminatio nwa sno t inducedb yMBC ,bu twa sdetermine db ycertai npropertie s ofth eculture . To determine whether differences exist betweenA .nidulan s strainsi n eliminatingMB Cfro mth ecultur e medium, Strain00 3wit hwild-typ e sensitivity toMBC ,an dStrai nR whic h isresistant ,wer e compared. Resultso fthi s experiment aregive ni nTabl e I.Fro mth eamount s ofMB Crecovere d after different timeso fincubation ,i ti seviden t that elimination ofMB Cfollowe d a similarpatter n inculture s ofbot h strains.Thes e results indicate that herea tleast ,eliminatio n ofMB Ci sno trelate d toth emechanis m ofresis ­ tance tothi s compound. MBC couldno tb edetecte d inth emycelia l fractions ofth ecultures , although themyceliu mwa sprocesse d insuc ha wa ytha teve nth epresenc eo f H ofth eamoun t initially added toth eculture s should havebee n readily

ble I.Eliminatio n of MBC from culture filtrates of A. nidulans Strain 003 nd Strain R.

Recovery of MBC in culture filtrates (% of the amount initially added) Incubation time (h) Strai n 003 Strain R

i m 2 uM 1 uM 2y M

21 90 103 104 84 29 48 49 54 72 45 a - - 69b - - -

100m l liquid Czapek-Doxmediu m in 300m l Erlenmeyer flasks were inoculated with spores giving a final concentration of 10 spores per ml.MB C was added simultaneously with spores. Solvent Bwa s used in the TLC-bioassay. amount ofMB C below lower limit of detection. at this timeMB C could not be detected in themyceliu m (lower limit of detection 1%o f the amount initially added).

70 demonstrated. Itshoul d be noted that inoculum densities used in this experiment were higher than those used in the experiment described in Fig. 2.Tha t elimination ofMB C at this higher inoculum density became apparent after a shorter incubationperio d suggested thatMB Cwa s eliminated atmaximu m rates at the end of the exponential growth phase.Therefore ,th eproces s was further studied with concentrated mycelial suspensions (1 go fwe t weight mycelium per 25m l medium) inglucose-nitrat emedium . Since thismediu m promotes rapid growth,nutrient s arc rapidly consumed and the stationary growth phase is readily attained. Table IIgive s the results of this experiment. As is evident from the dryweigh t determinations,growt h of the cultureswa s rapid and sometime between 5an d 10hour s after initiation of the experiment the stationary phasewa s attained. After 15hour s dryweigh t decreased, indi­ cating that lysis occurred. Rates ofMB C elimination from the culture fil­ trates was highest during the stationary phase.Withi n 5hour s cultures in thisphas e eliminated over 90t>o f the amount of MBC initially added.

This system seemed tob e most suitable for studying the elimination

Table II.Effec t of age of culture onMB C elimination from culture filtrates of concentrated mycelial suspensions of A. nidulans Strain003 .

Time after Dry weight Recovery of MBC after initiation of (mg/25 ml) 5h of incubation(% ) experiment (h)

0 146 30 5 224 < 10a 10 234 < 10 15 210 < 10 20 169 25 25 143 57 30 123 70

Mycelium grown for 15hour s in glucose-nitrate medium was harvested, washed and resuspended in fresh medium at a concentration of 1g we t weight of mycelium per 25m l medium. At different times during incubation MBC was added (4pM)an d the amount ofMB C recovered in culture filtrates after 5 hours of incubation was determined by the TLC-bioassay (Solvent B).Dr y weight determinations weremad e every 5 hours, lower limit of detection.

71 process inmor e detailwit h [ C]MBC.Whe n [ c]MB Cwa sadde dt oage d concentrated mycelial suspensions inth estationar y phase,MB Cwa srapidl y eliminated fromth emedium ,a sshow nb yth eTLC-bioassay ,bu tth eamoun to f radioactivity inth emediu m remained constant (Fig.3) .Thu smetabolis mo f MBCi sresponsibl e forth eeliminatio n phenomenon.Th etim e requiredt o metabolize 50°6o fth eMB Cwa sabou t 2.5hour s forbot h strains. Similar 14 results were also obtained with Strain 186.Sinc e C-metabolites appeared tob epresen t inth ecultur e filtrates,aliquot so fthes ewer e analysedb y TLC. Radioscans (Fig.4 )showe d that during incubationa Metabolit e (I) was formed,whic hwa sconverte d intoa secon d Metabolite (II)upo n prolonged incubation.Tentativ e identification ofth emetabolite swa sperforme db y cochromatographyo faliquot so fcultur e filtrateswit h authentic samples ofMBC ,2-AB ,benzimidazol e and5-OH-MBC . From Table IIIi ti sclea r that

hours rl4 1 Fig. 3.Metaboli c conversion of L CjMBC by aged concentrated mycelial sus­ pensions ofA . nidulans Strain 003 and Strain R. Mycelium grown for 15hour s in glucose-nitrate medium was harvested,washe d and resuspended in fresh medium. After 15hour s of incubation [ cjMBC (spec. act. 1.6 mCi/mmole)wa s added giving a final concentration of approximately 4 pM. After different times of incubation theMB C concentration of filtrates (o- o, Strain003 ; • - •, Strain R)wa s determined with the TLC-bioassay (Solvent C). Radio ­ activity (A - A, Strain 003;A - A, Strain R)wa s measured in 1m l of the culture filtrates.

72 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 Rf value Rf value

I MBC n I MBC n h

Fig. 4. Radioscans of chromatograms of culture filtrates of concentrated mycelial suspensions ofA . nidulans Strain R exposed to [ CjMBC. For details of incubation seeFie . 3.

73 Metabolite Icochromatographe dwit h 5-OH-MBCi nth etw osolven t systems used (Can dD) . Metabolite IIremaine d onth eorigi n ineac ho fth esolven t systems A andD ,an di nsevera l other systems tested (ethanol/ammonia (70/30); hexcine/ethyl acetate/meth.-.mol (10/10/1);ethylacetate/dioxane/methanol / ammonia (160/20/5/0.5); ethyl acetate/chloroform/acetic acid (15/85/2); butanol/ethanol/water (4/1/1);butanol/ammoni a (4/1); ethylacetate/isopro- panol/ammonia (35/45/20);an dchloroform/methano l (90/10),N ofurthe r attemptwas ,therefore ,mad et oidentif yMetabolit eII . MBCwa sno tmetabolize d incultur e filtrates,obtaine db ycentrifugin g or filtratingmycelia l suspensions after different timeso fincubation . Crude supernatantmycelia l extracts preparedb yhomogenizin g frozenmyceliu m with anX-Pres s (LKB-Biotec,Sweden )i n0.0 5M potassiu mphosphat e buffer, didno tmetaboliz eMB Cno rdi dth emycelia l debris. Sincemetabolis mwa sa tit shighes t rate inth estationar y growthphase , MBC conversionwa sstudie d inconcentrate dmycelia l suspensions eithermad e up inglucose-nitrat emediu mo ri n0.0 5M potassiu m phosphatebuffer ,p H r1 4 i 6.8. I CJMBCwa sadde d immediately after resuspensiono fth emycelium . Aliquots ofth emediu mwer e analysedb yTL Can dth eproportio no fradio ­ activity present inMB Cwa sdetermined . Fig.5 show s theresults .I nth e buffer inwhic h nogrowt h occurred,th einitia l rateo fmetabolis mwa srapi d but slowed downupo n further incubation.Afte r 3.5hour s 50%o fMB Cwa s

Table III.Chromatographi c characteristics of benzimidazole derivatives and of C-metabolites found in culture filtrates of concentrated mycelial suspensions of A. nidulans Strain R exposed to L cjMBC.

va lue Rf Compound Solvent C S Dlvent D

MBC 0.56 0.73 2-AB 0.00 0.45 Benzimidazole 0.23 0.17 5-OH-MBC 0.29 0.63

Metabolite I 0.29 0.64 Metabolite II 0.00 0.00

For details of incubation seeFig . 3.

74 Fig. 5.Effec t of incubation medium onmetabolis m of [ CJMBC by concentrated mycelial suspensions of A. nidulans Strain 003.Myceliu m grown for 15hour s in glucose-nitrate medium was harvested, washed and resuspended in fresh glucose-nitrate medium or 0.05 M potassium phosphate buffer,p H 6.8 [ c]MB C (spec. act. 11.4mCi/mmole )wa s added giving a final concentration of appro­ ximately 4yM . After different times of incubation dry weight (A -A , glucose-nitrate medium; A.- A, buffer) and percentages of radioactivity present inMB C (o- o,glucose-nitratemedium ; •- •,buffer )wer e determined.

metabolized.Afte r 24hour s (notshow ni nFig .5 )ther ewa sstil l some radioactivity inMBC ,wherea smos to fth eradioactivit ywa spresen ti n Metabolite Ian dMetabolit e II.I nglucose-nitrat emedium ,i nwhic h mycelial growthoccurred ,th erat eo fmetabolis mwa srathe r low,bu tincrease dwit h time.Afte r 7.5hour s 50°so fMB Cwa smetabolized .Afte r 24hour s allo fth e radioactivity present inth emediu mwa spresen t inMetabolit e II.Durin gth e first8 hour so fincubatio nth etota l amounto fradioactivit y inth emedi a wasunaffected ,indicatin g thatn ofixatio no fradiolabele d compoundsi n mycelium occurredo rn ovolatil e degradationproduct swer e formed.Afte r2 4

75 hours of incubation,however ,i nsuspension s inglucose-nitrat emedium ,th e amounto f radioactivity inth emediu mha ddecrease d toapproximatel y 70% of the amount initially added.Wit hmycelia l suspensions inbuffe rn o decrease inamoun to fradioactivit y inth e filtrateswa s observed.

DISCUSSION

Inmyceliu m ofA .nidulan sMB Cwa smetabolize d toS-OH-MB Cwhic h init s turnwa s converted toa compoun d (Metabolite II)whic h could notb eidenti ­ fied.Bot h compounds are less fungitoxic thanMBC ,sinc e otherwise they would havebee ndetecte d inth eTLC-bioassay .Th emetaboli c conversiono f MBC inA .nidulan s issimila r totha t reportedb y Yasuda et al. (22)i n Pelliculariasasaki i andAlternari amali .Thes e authors identified 5-OH-MBC as amino rmetabolit e alongwit hMB C as themajo rmetabolit ewhe n cultures of these fungiwer e exposed toradiolabele d thiophanate-methyl. Inaddition , they found twoothe r labelled compounds,whic hwer e not identified.On eo f thesewhic h remained at theorigin ,upo nTLC-chromatography ,migh tb eiden ­ ticalwit hMetabolit e II.Th eothe rone ,Metabolit e X,wa s notdetecte d in culture filtrates ofA .nidulans .Sinc e radiolabeled thiophanate-methyl was used,thi s compoundmigh tb e aconversio nproduc t of thiophanate-methyl other thanMBC .Formatio no fa n intermediate fungitoxic conversionproduc t in the invitr o conversiono fthiophanate-methy l hasbee n reported(30) . The rate ofmetabolis mwa smaximu munde r conditions,a twhic h the culturemediu mbecome s depleted ofnutrients .Normally ,thes e conditions are attained aftera certai nincubatio nperiod ,dependin g on the initial amounto f inoculum. Inconcentrate d mycelial suspensions in glucose-nitrate medium inwhic h arapi d depletion ofth emediu m occurs,th e timerequire d to metabolize 50 Io fth eMB Cwa s 7.5 hours,whe nMB Cwa s initially present ata concentration of 4 \M. Aged concentratedmycelia l suspensions,incubate d for 15hour sprio r toaddin gMB Cmetabolize d 50°so fthi s amount in2. 5hours . Mycelium inbuffe r andhenc eunde r starvation conditions,initiall ymeta ­ bolizedMB C at ahig h ratebu t this slowed downupo nprolonge d incubation. After 24hour s of incubation theamoun tmetabolize dwa s less thantha tb y a similar curve inglucose-nitrat e medium. The observation that cultures inth epresenc e ofMB C atsubletha lcon ­ centrations rapidly turned dark-brown suggests thatmelani nwa s synthesized. Synthesis ofmelani n isfavoure dunde r conditions ofautolysi s (31)an d is mediated by theactio no f apheno l oxidase (32).Sinc e similar cultural conditionspromot ehydroxylatio n ofMB C to 5-OH-MBCan d conversion ofthi s

76 product toMetabolit e II,th esyste m responsible forMB Cmetabolis m might parallel thesyste m involved inmelanogenesis . Hence,th efina l Metabolite IImigh tb ea noxidatio nan dcondensatio n producto f5-OH-MBC . Strainswit h altered sensitivity toMB Cdi dno tsho wan ydifferenc ei n rateo fMB Cmetabolism . Therefore,difference s inmetaboli c activity cannot account forth edifferen t behaviouro fthes e strains towards MBC. Since MBC isevidentl y metabolized within thecells ,an dbecaus en odifference s exist inth erat eo fmetabolis m insensitiv e andresistan t strains,resis ­ tancet oMB Cdoe sno tsee mt ob erelate d todifference s inuptak e ofth e compound. Recently itha sbee n shown that differences insensitivit y arei nfac t related todifference s ininteractio nbetwee nMB Can dit srecepto r site, ofwhic h thepresenc e andsignificanc e inth emechanis m ofactio no fMB Cha s been demonstrated (33-34).

ACKNOWLEDGEMENTS

Iwis h tothan kMis s Willy Flach forhe rexcellen t technical assistancean d Dr.A .Fuch s andProf.Dr .J .Dekke r fora critica l readingo fth emanuscript . Ia mgratefu l toMrs .E .Brouns-Murra y (Pudoc,Wageningen ,Th eNetherlands ) for correcting theEnglis h text.

REFERENCES

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77 10. Y. Soeda, S. Kosaka, and T. Noguchi, Identification of alkyl 2- benzimidazole-carbamate as amajo r metabolite of thiophanates fungicide in/on the bean plant,Agr . Biol. Chem. 36.81 7 (1972). 11. M.R. Siegel,an d A.J. Zabbia,Jr .Distributio n and metabolic fate of the fungicide benomyl indwar f pea,Phytopatholog y 62,63 0 (1972). 12. M.R. Siegel.Distributio n and metabolism of methyl-2-benzimidazole carbamate, the fungitoxic derivative of benomyl, in strawberry plants,Phytopatholog y 63,89 0 (1973). 13. A. Ben-Aziz,an d N. Aharonson,Dynamic s of uptake, translocation, and disappearance of thiabendazole and methyl-2-benzimidazole carbamate in pepper and tomato plants,Pestic . Biochem. Physiol. 4, 120 (1974). 14. J.P. Rouchaud, J.R. Decallonne, and J.A.Meyer ,Metaboli c fate of methyl-2-benzimidazole carbamate inmelo n plants, Phytopathology 64, 1513 (1974). 15. J.A.Gardiner , R.K. Brantley and H. Sherman, Isolation and identification of ametabolit e of methyl l-(butylcarbamate)-2-benzimidazole carbamate in rat urine,J . Agr. Food Chem. 16, 1050 (1968). 16. J.J. Kirkland,Metho d for high-speed liquid chromatographic analysis of benomyl and/or metabolites residues in cowmilk , urine, feces and tissues,J . Agr. Food Chem. 21, 171 (1973). 17. J.A.Gardiner ,J.J . Kirkland, H.L. Klopping and H. Sherman,Fat e of benomyl in animals,J . Agr.Foo d Chem. 22,41 9 (1974). 18. A. Helweg,Microbia l breakdown of the fungicide benomyl, Soil Biol. Biochem. 4, 377 (1972). 19. M.R. Siegel,Benomyl-soil-microbia l interactions, Phytopathology 65, 219 (1975). 20. A. Fuchs,an d G.J. Bollen, Benomyl,afte r seven years, in "System- fungicide - Systemic fungicides",p . 121, (H.Ly r and C. Polter, Eds.)Akademie-Verlag , Berlin, 1975. 21. A. Fuchs,an d F.W. deVries , in preparation. 22. Y. Yasuda,S .Hashimoto ,Y . Soeda, and T. Noguchi,Metabolis m of thio- phanate-methyl by pathogenic fungi and antifungal activity of its metabolites,Ann . Phytopath. Soc. Japan 39,4 9 (1973). 23. J.W.Eckert , and M.L. Rahm,Fungistati c activity of esters of benzimidazol-2-yl carbamic acid and related compounds,Pape r given at 3r Intern.Congr . Pestic. Chem. (IUPAC), Helsinki, 3-9 July, 1974. 24. L.C. Davidse,Antimitoti c activity ofmethy l benzimidazol-2-yl-carbamate (MBC) inAspergillu s nidulans. Pestic. Biochem. Physiol. 3, 317 (1973). 25. J.M. van Tuyl,L.C . Davidse, and J. Dekker,Lac k of cross resistance to benomyl and thiabendazole in some strains of Aspergillus nidulans. Neth. J. PI. Path. 80, 165 (1974). 26. R.F. Rosenberger and M. Kessel,Synchron y of nuclear replication in individual hyphae of Aspergillus nidulans,J . Bacteriol. 94, 1464 (1967). 27. A.L. Homans, and A. Fuchs,Direc t bioautography on thin-layer chromato- grams as ametho d for detecting fungitoxic substances,J . Chromatogr. 51,32 7 (1970). 28. G.A. Bruno,an d J.E. Christian,Determinatio n of Carbon-14 in aqueous bicarbonate solutions by liquid scintillation counting techniques, Anal. Chem. 33, 1216 (1961). 29. A. Fuchs,D.L . Fernandes,an d F.W. deVries ,Th e function of anMBC - releasing deposit of benomyl and thiophanates inplan t roots and soil,Neth . J. PI. Path. 80,7 (1974). 30. A. Fuchs,G.A . van den Berg, and L.C. Davidse,A comparison of benomyl and thiophanates with respect to some chemical and systemic fungi­ toxic characteristics,Pestic . Biochem. Physiol. 2, 191 (1972).

78 31. A.T. Bull,Chemica l composition of wild-type andmutan t Aspergillus nidulans cellwalls .Th e nature of polysaccharide and melanin constituents,J . Gen.Microbiol . 63,7 5 (1970). 32. A.T. Bull, and B.L.A. Carter,Th e isolation of tyrosinase from Aspergillus nidulans,It skineti c andmolecula r properties and some consideration of its activity invivo ,J . Gen.Microbiol . 75, 61 (1973). 33. L.C. Davidse,Mod e of action ofmethy l benzimidazol-2-yl-carbamate (MBC)an d some biochemical aspects of acquired resistance against this fungicide inAspergillu s nidulans, in "Systemfungizide- Systemic fungicides",p . 137 (H.Ly r and C. Polter, Eds.), Akademie-Verlag,Berlin , 1975. 34. L.C, Davidse,Antimitoti c activity ofmethy l benzimidazol-2-yl carbamate in fungi and itsbindin g to cellular protein, in "Microtubules and Microtubule Inhibitors",p . 483 (M.Borgers ,an d M. de Brabander, Eds.), North-Holland Publishing Company, Amsterdam, 1975.

79 Summary and general discussion

Systemic benzimidazole fungicides arewell-know n forthei r pronounced ability tocontro l alarg enumbe ro ffunga lplan t diseases.O nth eothe rhan d development ofresistanc e infung it othes e compounds isa swel lwidely,known . Biochemical aspects ofbot h fungitoxic actionan dresistanc e infung i areth esubjec t ofthi s thesis.I tcontain s four articles describing a)th e mechanismo factio no fcarbendazi mo rmethy l benzimidazol-2-yl carbamate, b)a mechanis m ofresistanc e tothi s compound andc )it smetaboli c conversion. Mosto fth ewor kwa sdon ewit hAspergillu s nidulans,becaus e this fungusi s genetically well defined andne wmutant s canb ereadil y characterized. Three strainswer e used,on estrai nwit hwil d type sensitivity,on ewit ha nin ­ creased sensitivity andon ewit ha decrease d sensitivity toMBC .Th ebehavi ­ ouro fbot hmutan t strainswa sdu et oa mutatio n inth eben A locus. Inth efirs t paper itha sbee n shown thatMB Ci sa neffectiv e inhibitor ofmitosi s inAspergillu s nidulans.Synthesi s ofDN Aan dRN Aappeare d alsot o be affectedbu tthi s couldb eascribe d toth edisturbanc e ofth ecel lcycle , duet oinhibitio n ofmitosis .Hence ,mitosi s canb econsidere d theproces s first affectedb ythi s compound. Inmammalia n cells potent inhibitors ofmitosi s like colchicine,podo - phyllotoxin andvinblastin e sulphate exert their actionvi ainterferenc e with functioning andassembl y ofmicrotubules ,th eelement s ofth espindle .Thes e compounds bindt otubulin ,th edimeri c subunit ofmicrotubules ,preventin gi n thiswa ymicrotubul e formation. Onth eassumptio n thatth eantimitoti c activity ofMB Cmigh tb ebase do n a similar binding,theMBC-bindin g properties ofmycelia l extracts werein ­ vestigated.Th eresult s ofthes e studies arepresente d inth esecon dan d thirdpaper .A nMBC-bindin g proteinwa sfoun dt ob epresen t inmycelia lex ­ tractswhich snowed characteristic properties oftubulin .MB Cbindin gwa s competitively inhibitedb ycolchicin e andoncodazole ,a benzimidazol e com­ poundwhich binds tomammalia n tubulina tth ecolchicin e binding site. Partial purification ofth ebindin g activity resulted ina protei n pre­ paration inwhich thetw otubuli nmonomer s predominated. These resultsin ­ dicate thatMB Ci sboun d tofunga l tubulin.Bindin gprobabl y preventsmicro ­ tubule formation orfunctionin g which leadst odisturbanc e ofmitosis . Studyo fth eactio no fMB Ca tth emolecula r level also ledt oth eelu ­ cidationo fa mechanis m ofresistanc e offung it othi s compound. Resultso f

80 binding experiments with anumbe r ofMBC-sensitiv e andMBC-resistan t fungal species and strains suggested that the affinity of thebindin gprotei nfo rMB C determined the fungal response to the action ofMBC .Evidenc e in favour of this hypothesis was obtained fromdetaile d binding experiments with three A. nidulans strains.Result s clearly indicated the relation between degree of sensitivity forMB C and magnitude of thebindin g constant forMB C and tubulin. Inth e fourth paper the conversion ofMB C to anon-toxi c metabolite has been described. Conversionwa s found tooccu r in three strains ofA . nidulans examined and inno n of these cases itha d anybearin g on themechanis m of resistance to this compound. IVheatherconversio n ofMB Cplay s any role in the mechanism of resistance in other fungi remains tob e elucidated. The identification ofMB C asa n antitubulin, acompoun d which binds to tubulin,indicate s that antitubulins can be successful fungicides.However , awid e spread and frequent use of such compounds does not seem advisable for a number of reasons.Thei r action based on the specific interference with one target site ispossibl y subjected toan y change in this site.A mutation leading to adecrease d affinity can finally result in the development ofa resistant fungal population which cannot be controlled effectively. The use of these compounds which are selectivewit h respect toplan t pathogenic fungima y alsohav e ecological implications.A shift in thedyna ­ mic equilibrium between the various components of themicroflor a in the soil and on the surface of theplan t isconceivable .Naturall y resistant,a swel l as resistant strains ofnormall y sensitive plant pathogens might be favoured in theirdevelopment ,givin g rise to increased disease incidence. Thepotentia l hazard of large scale use ofcompound s with this type of action should also be kept inmind .Mitosi s inal l eukaryotes follows abasi c pattern,an d innearl y all eukaryotes microtubules play an essential role in the separation of the chromosomes.Th e structure ofmicrotubule s seems tob e highly conserved during evolution and, therefore,agent s interfering with microtubules might be active in awid e range of organisms.Althoug h NBC is relatively non-toxic tomammals ,whic h isprobabl y due to arapi d metabolic conversion and excretion,a swel l as to low affinity ofmammalia n tubulin for this compound, its ability to induce abnormalities innuclea r division in some cells ofthes e organisms has beendemonstrated . Such aneffec t inrepro ­ ductive cells may have severe consequences.Th e use ofcompound s which act on mitosis through interference withmicrotubules ,therefore ,implie s apotentia l genetic risk formen . Whatever may be the future ofpractica l use ofbenzimidazol e compounds inagricultur e ormedicine ,the ywil l undoubtedly continue tob e valuable tools inth e study of the structure and functioning ofmicrotubules .

81 Samenvatting

Systemische benzimidazool fungiciden genietenee nalgemen ebekendhei di n land-e ntuinbou wvanweg ehu nuitgesproke n kwaliteitente naanzie nva nd ebe - strijdingva nee ngroo t aantal schimmelziekten.Anderzijd s isevenwe lhe t vermogenva nschimmel s om resistent teworde n tegen dezemiddele noo kzee r bekend.D ebiochemisch e aspektenva nfungitoxicitei tva ndez emiddele ne n resistentieva nschimmel s ertegen zijnhe tonderwer pva ndi tproefschrift .I n eenvierta l artikelenword t aandacht besteed aana )he twerkingsmechanism e van carbendazim ofmethy l benzimidazol-2-yl carbamaat (MBC),b )ee nresisten - tiemechanisme tegendi tmidde le nc )d eomzettin gva ndez e verbinding.D e meesteexperimente nwerde nuitgevoer dme tAspergillu s nidulans omdatdez e schimmel genetisch goed gedefinieerd ise nnieuw e mutanten gemakkelijk kunnen worden gekarakteriseerd. Drie stammenwerde n gebruikt,waarva nee nsta md e normale gevoeligheid hadvoo rMB Ce nd eander e tweeee ntoegenomen , respektie- velijkee nafgenome n gevoeligheid vertoonden,tengevolg eva nmutati ei nd e benA locus. Het eerste artikel isgewij d aand elokaliserin gva nd eplaat sva n werkingva nMB Ci nd egebruikt e toetsschimmelAspergillu s nidulans.MB Cblee k een effektieve remmerva nd ekerndelin g tezijn ,terwij l daarnaast effekten werden gevondeno pDN Ae nRN Asynthese . Deze laatste effekten konden worden toegeschrevenaa nd eontregelin gva nd ecelcyclu s tengevolgeva nd egeremd e kerndeling.D ekerndelin g konderhalv e beschouwd worden alshe teerst e proces dat doorMB Cwer d beinvloed. Remmersva nd ekerndelin gva ndierlijk e cellen zoals colchicine,podo - phyllotoxine envinblastin e sulfaat oefenenhu nwerkin gui tdoo r interaktie methe tfunktionere no f de assemblage vanmicrotubuli ,d eelemente nva nd e kernspoel. Deze stoffenworde n gebondenaa ntubuline ,ee ndimee r eiwitda t beschouwdma gworde nal sd ebouwstee nva nd emicrotubuli .Dez e binding belem- mertd eassemblag eva ntubul ito tmicrotubuli . Metdi tvoorbeel dvoo r ogenhebbe nw ehe tMB Cbindend e vermogenva n mycelium extrakten onderzocht.D eresultate nva ndi tonderzoe k zijnbe - schreven ind etweed ee nd ederd epublikatie .He tblee kda ti nmyceliu mex ­ traktenee nMBC-binden d eiwit aanwezigwa sme tee naanta l eigenschappen karak- teristiek voor tubuline.MB Cbindin gko nkompetitie fworde n geremd door col­ chicinee nd ebenzimidazoo l verbinding oncodazole.Va noncodazol ei sbeken d dathe tword t gebondenaa ntubulin eo pd ecolchicin e bindingsplaats.N age -

82 deeltelijke zuivering van debindingsaktivitei t uitmyceliu m extrakt konme t behulpva nelektrofores e worden vastgesteld dat ind epreparate n de beide tubulinemonomere n aanwezigwaren . Uit deze resultaten konworde n gekonklu- deerd datMB C bindt aan schimmeltubuline en dat binding de funktionering of vorming vanmicrotubul i belemmert. Dit leidtwaarschijnlij k tot eenver - storing vand e kerndeling. Debestuderin g van hetwerkingsmechanism e vanMB C verschafte tegelijker- tijd inzicht inhe tmechanism e vand e resistentie van schimmels tegen deze verbinding. Uit bindingsexperimentenme t een aantalMBC-gevoelig e enre - sistente schimmelsoortene n stammenwer d de aanwijzing verkregen dat deaf - finiteitva n debindingsplaat s voorMB C het gedrag van de schimmel tenop - zichte vanMB C zoukunne n bepalen. Bindingsexperimenten met drie stammen vanA .nidulan s toonden inderdaad aan dat demat e van gevoeligheid voor MBC end e grootte van de bindingskonstante voor MBC en tubuline eennauw e samen- hang vertoonden. Ind evierd e publikatie isd e omzetting vanMB C inee n niet-fungitoxische verbinding beschreven.Omzettin g konworde n aangetoond inkulture s van een drietalA . nidulans stammen.E r kon geen relatieworde n vastgesteld tussen mate van gevoeligheid voor MBC enmat e van omzetting. Inhoeverr e dit ook voor andere schimmels geldt isno g een open vraag. De aan de engelse samenvatting toegevoegde algemenediskussi e is gewijd aand e implikaties vanhe tpraktisch e gebruik van deze middelen in land- en tuinbouw.

83 Curriculum vitae

Leendert Cornells Davidsewer d op2 3januar i 1947t eSin t Laurensge - boren.Hi jbehaaj.d ei n196 4he teinddiplom aHBS- B aand eChristelijk eHB S voorWalchere n teMiddelburg . Hetzelfde jaar liethi jzic h als studentin - schrijven aand eLandbouwhogeschoo l teWageningen ,waa rhi ji njun i196 5 hetpropaedeutisc h examen,i nmaar t 1969he tkandidaatsexame n richting Plantenziektenkunde eni njanuar i 1972he tdoctoraalexame n richting Planten- ziektenkundeme tal shoofdva k Fytopathologie enal skeuzevakke n deBiochemi e end eFysiologi ede rPlanten ,me tlo faflegde . Van 1januar i 1972to t1 me i 1972wa shi jwerkzaa mal swetenschappelij k assistent,e nva n1 me i197 2to thede n alsgastmedewerker, i ndiens tva nd e Centrale Organisatie TNO,Secti e Landbouwkundig Onderzoek,o phe tLaboratoriu m voor Fytopathologie,Binnenhave n 9 teWageningen .

84