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Differential binding of methyl benzimidazol-2-yl carbamate to fungal tubulin as a mechanism of resistance to this antimitotic agent in mutant strains of Aspergillus nidulans.

The antimitotic compound methyl benzimidazol-2-yl carbamate (MBC) formed a complex in vitro with a protein present in mycelial extracts of fungi. The binding protein of Aspergillus nidulans showed a set of properties which is unique for tubulin. Binding occurred rapidly at 4 degrees C and was competitively inhibited by oncodazole and colchicine. Other inhibitors of microtubule function such as podophyllotoxin, vinblastine sulfate, melatonin, and griseofulvin did not interfere with binding of MBC. Electrophoretic analysis of partially purified preparations of the binding protein revealed the presence of proteins with similar mobilities as mammalian tubulin monomers. Hence it is concluded that the binding protein is identical with fungal tubulin. The effect of MBC on mycelial growth of mutant strains of A. nidulans was positively correlated with the affinity of the binding sites for this compound. The apparent binding constant for MBC and tubulin from a wild type was estimated at 4.5 X 10(5), from a resistant strain at 3.7 X 10(4), and from a strain with increased sensitivity to MBC at 1.6 X 10(6) liters/mol. Mutants showing resistance and increased sensitivity to MBC are candidates to have alterations in tubulin structure. Affinity of tubulin for MBC is probably a common mechanism of resistance to this compound in fungi. Low affinity of tubulin for MBC is probably a common mechanism of resistance binding constant of 2.5 X 10(3) liters/mol.

Although the biological activity of these benzimidazole compounds is probably based on interference with the formation or functioning of microtubules, which are present in all eukaryotic cells, eukaryotes are not equally sensitive to each benzimidazole compound. Benomyl and MBC partly owe their success as systemic fungicides to a relative nontoxicity to plants and animals. But also within fungi, there are differences in sensitivity to these compounds. For instance, fungi belonging to the Ascomycetes are sensitive, whereas others belonging to the Oomycetes are resistant (7). In addition to natural resistance to benomyl and MBC, resistant strains of naturally sensitive species are frequently found in sprayed crops. In the laboratory such strains can be readily obtained by mutagenic treatment (28).
The curative action of mebendazole in animal as well as in human helminthiases implies selectivity with respect to host and parasite. Ultrastructural studies have shown that upon treatment of the host microtubules in cells of the parasite are completely destroyed, whereas cytoplasmic and spindle microtubules of the host cells remain unaffected, even though both types of cells have been exposed to identical drug concentrations (9,10).
In vivo experiments on the effect of oncodazole on experimental and human neoplasms have shown that this compound specifically eliminates microtubules in dividing and nondividing neoplastic cells, whereas microtubules of interphase normal cells are apparently intact. Microtubules in mitotic cells, however, are seriously affected (26).
Biochemical studies on the mechanism of action of benzimidazole compounds have shown that the antimitotic action of MBC in fungi is probably mediated via binding to fungal tubulin (22,23). Oncodazole is bound to mammalian brain tubulin at the colchicine-binding site (45) and is a potent inhibitor of microtubule assembly in vitro (26,27,45). Benomyl and MBC only slightly affect this process (27,44).
The molecular basis of selectivity of these benzimidazole compounds is yet unknown. Differential uptake or metabolism may be responsible for the relative nontoxicity of benomyl and MBC to animals and plants (36,65). For natural and induced resistance in fungi, it has been found that no differences in metabolism exist between MBCresistant and MBC-sensitive strains (24). Here a differential binding of MBC to tubulin in strains differing in MBC sensitivity might underly the selective action of this compound (22,23). The selective action of mebendazole does not appear to be related to a differential drug uptake between host and parasite (9, 10). A differential interaction of the drug with the target inside the cells is assumed to be responsible (9,10).
In this study the interaction of MBC with its receptor site in MBC-sensitive and MBC-resistant strains of the fungus Aspergillus nidulans has been investigated in detail. Since it has been assumed that MBC binds to fungal tubulin the effect of oncodazole and of other inhibitors of microtubule function on MBC binding has been studied. With [14C]MBC as affinity label, the binding protein was partially purified and electrophoretically characterized.

Organisms
Most of the experiments were carded out with A. nidulans biA1 AcrA1 (strain 003) and two mutant strains (strains 186 and R), which differ in MBC sensitivity. The sensitivity of these strains, in terms of inhibitory concentrations which cause a 50% reduction in growth (mean inhibitory dose, IDs0) on agar, is 4.5 t~M MBC for strain 003, 1.5 /xM for strain 186, and 95 /~M for strain R. Both strains were selected in the laboratory after UV treatment of conidia of strain 003 (69). Genetic analysis has shown that both increased sensitivity in strain 186 and resistance to MBC in strain R had been caused by a mutation in one single gene, located on linkage group VIII (69,70).
We also used MBC-sensitive strains of Penicillium brevicompaetum and P. corymbiferum as well as resistant strains of these fungi, which emerged in MBC-treated crops (6). Alternaria brassicae and Pythium irregulare represented the naturally MBC-resistant fungi.
nidulans strains was grown in a glucose-nitrate medium (57)

Preparation of Mycelial Extracts
Exponentially growing mycelium was harvested by filtering on a B6chner filter. The mycelium was washed three times in cold 0.05 M potassium phosphate buffer, pH 6.8, and frozen at -22~ in a previously cooled X-Press Cell Disintegrator (LKB Produkter, Stockholm, Sweden) with 0.5 ml of homogenization buffer per g wet weight of mycelium. The homogenization buffer consisted of 0.05 M potassium phosphate buffer, pH 6.8, containing 0.1 M KCI and 0.005 M MgClz (PKMg solution). After 1 h at -22"C the mycelium was homogenized by passing it five times through the press. The homogenate was thawed and then guanosine triphosphate (GTP) was added to a final concentration of 0.1 raM. The suspension was centrifuged at 40,000 g for 10 min and the resultant supemate recentrifuged at 48,000 g for 30 re.in. The 48,000-g supernate was immediately used in binding studies. All steps were done at 4~ mediately processed after arrival. Blood vessels and meninges were removed and the tissue was washed three times with ice cold 0.05 M potassium phosphate buffer, pH 6.8, and once with PKMg solution. 100 g of tissue was then homogenized with 100 ml of PKMg solution, containing 0.1 mM GTP, using a motor-driven glass homogenizer with Teflon pestle (B. Braun Apparatebau, Melsungen, Germany). The homogenate was centrifuged at 48,000 g for 60 min and the resulting supernate was fractionated with a neutralized saturated ammonium sulfate solution. The fraction precipitating between 35 and 50% saturation was taken up in 10 ml PKMg solution containing 1 mM GTP. This preparation was used in binding assays either directly or after storage at -22~

Binding Assays
Varying amounts of a methanolic solution of [14C]MBC were added to mycelial or brain extracts and incubated either at 4 ~ or 37"C for various periods as specified in the legend of figures and tables. Small samples of a [aH]colchicine solution in benzene/ethanol (9/1) were placed in empty scintillation vials or centrifuge tubes and the solvent was evaporated to dryness in a stream of nitrogen. The dried compound was dissolved directly in mycelial or porcine brain extract. Vials and tubes were wrapped in aluminum foil to prevent photodecomposition of colchicine. Incubation was at 4 ~ or 37~ for various periods as specified. Potential inhibitors of MBC binding were added as solutions in dimethylsulfoxide (DMSO). The DMSO concentration of treated and control samples never exceeded 0.1% vol/vol.
Binding of [~4C]MBC or [aH]colchicine was measured by gel filtration of 1 ml of the incubation mixture on a Sephadex G-100 column (28 x 1.5 cm) with PKMg solution as elution buffer. Fractions of 16 drops (approximately 1 ml) were collected with a LKB UItroRac fraction collector, and radioactivity in each fraction was measured in a Nuclear-Chicago Mark I Liquid Scintillation Spectrometer (Nuclear-Chicago Corp., Des Haines, Ill.) with Bruno and Christian's (13) scintillation liquid. Counting efficiency was determined by external standardization procedures. Radioactivity present in protein fractions was considered to represent bound ligand.
Binding of [14C]MBC was also measured with a second method, which has been introduced recently to measure colchicine binding to tubulin (62,63). Aliquots of the incubation mixture were placed in centrifuge tubes containing an equal volume of a charcoal suspension (Merck A. G. Inc., Darmstadt, West Germany) at 6 rag/ ml in PKMg solution. The tubes were placed in a shaker and the mixture was heavily agitated for 10 rain and then centrifuged at 1,500 g for 5 rain at 40C. Aliquots of the supernate were assayed for radioactivity. Blanks, which contained 40 mg/ml bovine serum albumin in PKMg solution incubated with [~4C]MBC were handled in the same way. The difference in amount of radioactivity found in supernatant aliquots of sample and blank was assumed to represent bound [14C]MBC. Bound MBC was expressed as disintegrations per minute per unit of volume of the original extract.

Purification of Fungal Tubulin
Mycelial extracts were prepared as described above, but with 0.05 M sodium phosphate, pH 6.8, containing 0.1 M NaC1 and 0.005 MgCI~ (PNaMg solution) as homogenization buffer. The 48,000-g supernatant mycelial extract was further centrifuged at 127,000 g for 60 min. The soluble proteins were fractionated with a neutralized (pH 6.8 after 20:1 dilution) saturated ammonium sulfate solution. The fraction precipitating between 35 and 50% saturation was taken up in PNaMg solution containing 1 mM GTP.
The resulting preparations were incubated with The eluate was continuously monitored at 254 nm and fractions of constant volume were collected. Radioactivity was measured in each fraction.
Once the elution properties of the MBC complex were established, the incubation step was omitted and the 35-50% ammonium sulfate preparation was applied directly to the DEAE-Sephadex column. Fractions which were eluted at 0.45-0.90 M NaCI, were combined, dialyzed against bidistilled water, and lyophilized. Dry samples were stored at -22~ above silica gel until analysis.

Purification of Porcine Brain Tubulin
Two purification methods were used. With the first method, a tubulin preparation prepared as described above, but with PNaMg solution as extraction buffer, was chromatographed onto a DEAE-Sephadex A-50 column in the same way as described above for mycelial extracts. Fractions containing tubulin were dialyzed against bidistilled water and lyophilized. The second method used was a slightly modified assembly-disassembly procedure according to Shelanski et al. (61). Fresh porcine brains were washed and homogenized in 0.1 M 2-(N-morpholino)ethane sulfonic acid (MES) buffer, pH 6.90, containing 1 mM EDTA, 0.1 mM GTP, and 0.5 mM MgCI2 (MES buffer). The homogenate was centrifuged at 40,000 g for 10 min and the resulting supemate at 48,000 g for 60 min at 4~ To achieve assembly of microtubules extracts were mixed with an equal volume of MES buffer containing 8 M glycerol and 2 mM GTP and incubated for 30 min at 37~ Assembled microtubules were pelleted by centrifuging at 48,000 g at 25~ After resuspension and depolymerization of microtubules in MES buffer at 4~ and centrifuging, a second assembly cycle was performed.

Molecular Weight Determination of the MBC-Protein Complex
The molecular weight of the MBC-protein complex was determined by gel filtration of an ammonium sulfate fractionated mycelial extract, which had been incubated with [~4C]MBC on a Sephadex G-200 column (2.5 x 32 cm), according to the method of Andrews (2). The column was calibrated in two parallel runs with alcohol dehydrogenase (yeast), mol wt 150,000 and cytochrome c (horse heart), mol wt 12,400; and with lipoamide dehydrogenase (pig heart), mol wt 100,000 and a-chymotrypsin (beef pancreas), mol wt 24,500, respectively.

Reduction and Carboxymethylation of Proteins
Proteins were reduced by adding/3-mercaptoethanol and sodium dodecyl sulfate (SDS) at final concentrations of 1% to the samples and heating for 2 rain at 100~ The reduced proteins were dialyzed overnight at room temperature against 0.01 M sodium phosphate buffer, pH 7.0, containing 0.1% /3-mercaptoethanol and 0.1% SDS.
Protein samples were reduced and carboxymethylated according to the method of Crestfield et al. (20) as modified by Renaud et al. (55). Protein solutions were made 1% in/3-mercaptoethanol and mixed with an equal volume of reducing solution (0.35 M Tris-HCl, pH 8.8, containing 0.12 M fl-mercaptoethanol, 8 M urea, and 0.1% EDTA). The mixture was dialyzed overnight at room temperature against this solution. Lyophilized proteins were directly dissolved in reducing solution and incubated overnight at room temperature under nitrogen. The reduced proteins were carboxymethylated by adding one volume of a 1.1-M iodoacetic acid-8 M urea solution in 2.5 N NaOH to nine volumes of protein solution. The reaction was allowed to proceed for 1 h in the dark, and the solution was then dialyzed against 0.01 M Tris-HCl, pH 8.0, for at least 6 h at room temperature also in the dark.

SDS-Polyacrylamide Gel Electrophoresis
Two SDS systems were employed. The first one was based on that of Weber and Osborn (71). Reduced or carboxymethylated protein samples were run on 6-cm gels (7.5% [wt/vol] acrylamide, 0.13% [wt/vol] bisacrylamide, 0.075% [wt/vol] ammonium persulfate, 0.075% in 0.1 M sodium phosphate buffer, pH 7.0) at 40 V for about 6 h. The second system was similar to the discontinuous SDS system as has been described by Luduena and Woodward (51). Carboxymethylated protein samples were prepared for electrophoresis as described and run on 9-cm gels at 4 mA per gel for approximately 2.5 h.

Protein Determination
Protein was determined according to the method of Lowry et al. (50) as modified by Hartree (40)  Chromatograms were scanned with a Nuclear-Chicago Actigraph III radiochromatography system (Nuclear-Chicago Corp.) and then cut transversely into 1-cm sections, which were placed in scintillation vials containing Bruno and Christian's scintillation liquid and counted. Purity was always found higher than 97%.
[Ring C-methoxyl-3H]colchicine (sp act 3.8 Ci/mmol) as a solution in benzene/ethanol (9/1) was obtained from New England Nuclear (Boston, Mass.). The preparation, diluted to a sp act of 20-200 mCi/mmol, was used either as such, or after being purified according to the procedure described below.

Purification o f [3H] Colchicine
An aliquot of the [aH]colchicine solution in benzene/ ethanol was diluted with unlabeled colchicine in the same solution, giving a concentration of 10 -4 M colchicine at approximately 10 a dpm per 100 p.1 solution. 200 txl of this solution was applied as a small band to a silica gel plate (DC-Alufolie 60 F 254, Merck A. G., Inc.) and chromatographed in chloroform/acetone/diethylamine (5/4/1) over a distance of 17 cm with unlabeled colchicine as a reference. The center part of the [aH]colchicine band (visible under UV at 254 nm) with an Rr value of 0.57 and a 1-cm broad band corresponding to the Ry value of an unknown radiolabeled compound X, which was found to be present in our colchicine preparation, with an Rf value of 0.46 was cut out and transferred to a centrifuge tube. The silica gel was scraped off and subsequently eluted four times with 1 ml methanol. To avoid photodecomposition of the compounds, exposure of samples to light was kept to a minimum during the procedure. Tubes and the chromatography tank were wrapped in aluminum foil. Approximately 1.3 x 10 ~ dpm were recovered in [aH]colchicine and 1.4 x 10 ~ dpm in compound X. To analyze the purified preparations, aliquots were spotted onto a chromatogram and developed with the same solvent as used before. Chromatograms were cut transversely into 1-cm sections which were placed in scintillation vials containing Bruno and Christian's scintillation liquid and counted. Radioactivity present in sections corresponding to the Re values of colchicine and compound X was 86 and 84%, respectively of the total radioactivity recovered. In the case of [aH]colchicine 11% of the radioactivity was present in a third compound with an R e value of 0.83. This compound is probably identical to that found by Borisy and Taylor (11), which was shown to be formed during storage of [3H]colchicine preparations purified by silica gel chromatography.

Evaluation of the Charcoal Assay
In addition to the gel filtration method for measuring MBC binding, in this work a second method was used, which is based on separation of free MBC from bound MBC by adsorption to charcoal. This method was developed because it is rapid and suitable for multiple determinations.
Activated charcoal appeared to be very effective in adsorbing free MBC in solutions of bovine serum albumin at 40 mg/ml in PKMg buffer. More than 99.5% of the amount of MBC could be removed from the solution, when the compound was initially present in concentrations up to 40 /xM. In mycelial extracts incubated with MBC, however, removal of free MBC may not involve removal of bound MBC. Therefore the charcoal assay was compared with the gel filtration method. The amount of bound MBC as determined with the charcoal assay was about one and one-half times as high as that found with the gel filtration method. Since incubation of mycelial extracts with [14C]MBC in the presence of a specific inhibitor of MBC binding (see below) resulted in complete adsorption of radioactivity to charcoal, the higher degree of binding found with the charcoal assay, is not due to incomplete adsorption of free MBC to charcoal in mycelial extracts. Hence, less dissociation of MBC from the complex during the charcoal assay than during the gel filtration procedure is probably responsible for this phenomenon.
Due to dissociation the value of bound MBC decreases with the length of the incubation period with charcoal (Fig. 2). In the standard procedure mixtures were shaken for 10 min with charcoal, so that disturbance of equilibrium was minimum and removal of free MBC was maximum.

Selectivity of MBC Binding
MBC binding was found in 48,000-g supernarant extracts of mycelial homogenates in PKMg solution after the extracts were incubated with [~4C]MBC at 4~ (22,23). Binding activity appeared to be correlated with the in vivo MBC sensitivity of the species or strain examined. Table  I gives detailed binding data obtained with the Sephadex G-100 assay for various MBC-sensitive and MBC-resistant fungal species or strains. Whereas extracts of sensitive strains did bind MBC in quantities between 3 and 18% of the quantity added, resistant strains did not bind more than 0.6% of this amount which is hardly above background.
Since it has been suggested (22,23) that the MBC-binding substance is identical with fungal tubulin, the MBC-binding properties of mammalian brain tubulin were investigated. Native porcine brain tubulin, partially purified by ammonium sulfate fractionation, did not bind MBC in significant amounts at 4 or 37~ indicating that mammalian tubulin has no or at least a low affinity for MBC. This is in agreement with the observation that MBC at a concentration which is lethal   (7) for benomyl. ND, not determined.
for MBC-sensitive fungi does not interact with in vitro microtubule assembly in brain extracts (23,27,44). For further work the three A. nidulans strains 003,186, and R were selected, merely because of the fact that their genetic background is known (69,70).

Molecular Weight of the MBC Complex
To determine the molecular weight of the MBC complex, a partially purified mycelial extract, which had been incubated with [14C]MBC, was gel filtrated through a calibrated Sephadex G-200 column. The bulk of the bound radioactivity was eluted in a single peak and as can be seen in Fig. 3 the elution volume corresponds with a molecular weight of the MBC complex of approximately 110,000. Since this value is very similar to that found for the colchicine-tubulin complex (17), the elution behavior of both complexes was compared on a 1.5 x 27-cm Sephadex G-200 column. The elution volume of the MBC complex appeared to be similar to that of the colchicine-tubulin complex. Etution votume (mD

Factors which Influence MBC Binding
The binding of MBC to its receptor was rapid and increased to a plateau within 15 min of incubation at 4~ Therefore an incubation period of 1 h or more with [14C]MBC, as routinely used in our experiments, can be considered to be long enough for maximum binding.
MBC-binding activity was influenced by pH as is illustrated in Fig. 4. Maximum binding appeared between pH 6.5 and 6.8. Whether pH affected binding of MBC indirectly through an effect on stability of the receptor or directly has not been investigated.
MBC-binding activity was not stable. At 40C the ability of mycelial extracts to bind MBC decayed according to first order kinetics (Fig. 5). The half time for inactivation was 6.5 h. Sucrose, MBC, and glycerol stabilized the binding activity. Known stabilizers of colchicine-binding activity of tubulin like vinblastine sulfate and GTP did not significantly increase the stability.
The decay of the binding activity was enhanced by high temperature. Binding activity of extracts, which had been heated for 1 h at 37~ cooled in 10-   ice for another hour, and centrifuged to remove heat-denatured proteins, was 11% of that of control samples which were kept at 4~ during heating and centrifuging of the experimental sample. Since for unknown reasons the charcoal assay applied to heated extracts gave anomalous binding data, binding activity was measured with the gel filtration method. Binding activity in the control sample was determined with the charcoal assay. Incubating mycelial extracts with 0.1% trypsin for 1 h at 4~ before incubation with [14C]MBC resulted in a 55% loss of binding capacity.
Centrifuging at 127,000 g for 1 h at 4~ of the 48,000-g supernate lowered the binding activity of the resulting supernate to 70-80% of that of control samples which were not centrifuged and were stored at 4~ for the duration of the run. This result suggests that the MBC-binding activity might be partially associated with some particulate fraction. The nature of this binding activity has not been studied further.

Affinity for MBC in Extracts of A. nidulans Strains 003, 186, and R
Since it has been suggested (22,23) that differences in affinity of MBC-binding sites for MBC might be the biochemical basis of the selectivity of MBC, we determined apparent binding constants (K) and the number of binding sites (So) in mycelial extracts of the three A. nidulans strains.
Total MBC binding was measured as a function of free MBC concentration and the data were plotted in double reciprocal form (11).
Results of a number of experiments are summarized in Fig. 6. From the slopes of the lines and the intercepts, values of So, expressed as maximum binding capacity in picomoles MBC per milligram protein and the apparent binding constants were calculated. Evidently the number of binding sites in extracts of the three strains are about equal, but the respective binding constants differ considerably. The reciprocals of the binding constants, which are identical with the MBC concentration which will half-maximally saturate the MBC-binding site, are 2.2, 0.6, and 27 /zM for extracts of strains 003, 186, and R, respectively. These values correspond with the inhibitory concentration of MBC causing a 50% reduction in growth (see Table I). This strongly suggests that the response of the three A. nidulans strains to MBC is governed by the affinity of the receptor site for MBC.

Inhibition of MBC Binding by Antimitotic Agents
To characterize the MBC-receptor site in fungi, several compounds which interact in some way with tubulin, were tested for inhibitory effects on MBC binding. A first screening was carried out by adding these compounds simultaneously with [14C]MBC at varying concentrations to mycelial extracts of A. nidulans strain 186. Podophyllotoxin and vinblastine sulfate, which compounds are known to bind to tubulin (74, 75) did not reduce MBC binding at a concentration of 500 /zM. Melatonin which has been shown to interfere with colchicine binding to mammalian brain tubulin (76) and which was active in the Stentor oral migration assay for inhibitors of microtubule function (52), was also ineffective at 500 ttM.      Since it has been shown that oncodazole and colchicine bind to mammalian tubulin at the same site (45), competitive inhibition of MBC binding in mycelial extracts by these two compounds indicates that the MBC-binding site is located on fungal tubulin.

Colchicine-Binding Activity of Mycelial Extracts
Competitive inhibition of MBC binding to fungal tubulin implies binding of colchicine to fungal tubulin. Since reports on colchicine binding in fungi are rather scarce and somewhat controversial (18,38,43,46,54), the colchicine-binding activity of mycelial extracts of A. nidulans was investigated.
Preliminary experiments in which mycelial extracts were incubated with [aH]coichicine at 10 /,tM, showed that up to 6% of total radioactivity L. C. DAVIDSE AND W. FLACH Differential Binding of MBC to Fungal Tubulin was bound to macromolecules (Table II, exp. 1). Since this amount was unexpectedly high, the nature of this bound radioactivity was investigated. Thin-layer chromatographic (TLC) analysis, with chloroform/acetone/diethylamine (5/4/ 1) as solvent, of Sephadex G-100 column fractions which contained bound radioactivity revealed that the radiolabel was present in a compound X, which was not identical with colchicine. Furthermore, in contrast with known coichicinetubulin complexes, the complex was perchloric acid (PCA) stable, because after adding PCA at a final concentration of 0.5 N to the fractions mentioned above, and centrifuging the mixture, about 70% of the radioactivity remained associated with the pellet. Washing the pellet with ethanol/ ether (3/1) removed most of the radiochemical, which was identified as compound X with TLC analysis. Since it was presumed that this radiochemical was present as an impurity in our [aH]colchicine preparation, the latter was purified (for details see Materials and Methods) and binding experiments were performed with the purified compounds. Results of these experiments are given in Table II, exps. 2 and 3. As is evident about 50% of the radioactivity present in compound X was bound to components of the mycelial extract. With purified [3H]colchicine, less radioactivity was bound than with the nonpurifled preparation. These results indicate that binding activity of mycelial extracts upon incubation with unpurified [aH]colchicine is mainly due to association of the radiochemical impurity with macromolecular components.
Since purification of [3H]colchicine preparations is laborious and 100% purity can never be achieved, in further experiments a more specific binding assay with DEAE-Sephadex A-50 anion exchanger has been used. Mycelial extracts were incubated with [3H]colchicine at 500 ~M (approximately 19.6 x 108 dpm/ml) for 1 h at 4~ and run onto DEAE-Sephadex columns. The elution pattern of radioactivity, which is shown in Fig. 9 a shows two high peaks and one very small peak. TLC analysis revealed that radioactivity in fraction 4, which contained the bulk of the protein which was not adsorbed to the ion exchanger, was present in the form of compound X, whereas radioactivity eluted at the two other peaks represented colchicine. The high peak is due to free colchicine since it is eluted at the bed volume of the column. The second [aH]colchicine peak was eluted at approximately 0.52 M NaCl. Since colchicine-tubulin complexes are characteristically eluted at this ionic strength (see also next sections) this result suggests the formation of a colchicine-fungal tubulin complex in the incubation mixture. That indeed colchicine was eluted as bound to protein and not in a free form was determined by gel filtration of an aliquot of fraction 27 on a Sephadex G-25 column (1.5 x 20 cm), immediately upon elution of this fraction. About 37% of the radioactivity recovered in the eluate, eluted associated with protein in the void volume of the column.
When mycelial extracts were incubated at 37~ for 1 h with [aH]colchicine and after centrifuging bound colchicine was assayed with the DEAE-Sephadex method, the quantity of bound colchicine seemed to be lower than that after incubation at 4~ (Fig. 9b). Since the binding site for colchicine is evidently identical with that of MBC, which appeared to be denatured under these conditions, this result would be expected. It should be further noticed that no significant amount of radioactivity was eluted associated with the bulk of the protein. This suggests that compound X either did not bind at this tempera-  ture or was associated with components which were removed from the extract by centrifuging. These possibilities were not studied further.
Since the DEAE-Sephadex-binding assay is not suitable for quantitative measurements due to dissociation of the colchicine-tubulin complex during the procedure, no attempts were made to determine in this way the binding constant for colchicine and fungal tubulin.

Purification of Fungal Tubulin
As has been shown in the preceding sections, the MBC-binding protein can be assumed to be identical with fungal tubulin. This makes MBC a suitable tool for the purification of fungal tubulin, analogous to the use of colchicine in purification methods for mammalian tubulin. With a standard purification procedure for tubulin the following results were obtained. MBC-binding activity in 48,000-g supernatant mycelial extracts could be selectively precipitated with ammonium sulfate between 35 and 50% saturation. Highspeed centrifuging and ammonium sulfate fractionation together resulted in a preparation which contained approximately 50% of the initial binding activity. The specific activity of this preparation was increased about twofold.
In previous work (22,23) it has been shown that the MBC complex was retained on a DEAE-Sephadex A-50 anion exchanger and could be selectively eluted at an ionic strength of 0.6 M KC1. Therefore, DEAE-Sephadex column chromatography which has proven to be useful in purification of tubulin from other organisms (cf. 74), was included as a final purification step for fungal tubulin. This step was first standardized with bound [14C]MBC. A partially purified preparation was incubated with [14C]MBC and applied to the column. Part of the elution patterns of radioactivity obtained with preparations of strains 003,186, and R are shown in Fig. 10. For strain 186, part of the radioactivity was eluted as a distinct peak at approximately 0.52 M NaC1, whereas with strain 003 only a small shoulder was observed in the elution pattern at this salt concentration. As expected with strain R no significant amounts of radioactivity were eluted at NaCl concentrations of 0.4 M and higher.
Whether elution of radioactivity at 0.52 M NaC1 was caused by elution of the MBC-protein complex itself or by dissociation of MBC from the DEAE-bound complex, due to increasing salt concentration, was determined by rechromatography of the fractions concerned on Sephadex G-100 columns. It appeared (Table III)   x 104 dpm/ml), respectively, for 2 h at 4"C. 10 ml of the incubation mixture was run onto a DEAE-Scphadex A-50 column equilibrated with PKMg buffer, as described under Materials and Methods, but using potassium buffers instead of sodium buffers. Radioactivity of the fractions was determined immediately after they had been eluted. 1 ml of the fraction containing the peak of bound MBC was filtered immediately through a Sephadex G-100 column.
of radioactivity were bound to protein, which indicates the elution of purified MBC-protein complex from the column.
In subsequent experiments no [t4C]MBC was present, and fractions eluted at 0.45-0.90 M NaCI were considered to contain the purified binding protein. The MBC-binding activity of DEAE-purified protein has not yet been investigated in detail. In a preliminary experiment, in which a crude mycelial extract fractionated with ammonium sulfate was passed through the DEAE-Sephadex column and the resulting purified protein solution was concentrated with a Sartorius ultrafiltration device (Sartorius Mem-branFilter GmbH, G6ttingen, West Germany), approximately 5 % of the initial binding capacity of the crude extract was recovered. The specific activity of this preparation was raised to about 13-fold of that of the crude extract. This rather low yield indicates a considerable loss of binding activity during the procedure, which was probably due to the instability of the binding protein.
Proteins isolated from the three strains of A. nidulans by DEAE-Sephadex column chromatography were analyzed with the continuous SDS system. Gels loaded with reduced protein samples of fractions 24-30 ( Fig. 10) from the experiment with strains 186 and R are shown in Figs. 11 and 12, respectively. As reference, reduced porcine brain tubulin purified by one polymerization cycle was run on a separate gel. On gels loaded with proteins from strain 186 three closely spaced bands, among others, were pres-FIGURE 11 Electrophoretic analysis of proteins isolated from A. nidulans strain 186 by DEAE-Sephadex column chromatography. Samples of fractions 24-30 (see Fig. 10, strain 186) were reduced and analyzed on the continuous SDS system. Reduced porcine brain tubulin (T) purified by one polymerization cycle was run as a reference. ent which have a comparable mobility as the porcine brain monomers which move together in this system. The banding patterns of gels loaded with reduced proteins from corresponding fractions derived from strain 003 were very similar (not shown). In gels loaded with proteins from strain R, however, only one band was present at a migration distance which is comparable with that of brain tubulin.
The difference was more pronounced when proteins were reduced and carboxymethylated before analysis with the same system. Gels loaded with DEAE-purified proteins, eluted at 0.45-0.90 M NaCI, from strains 003,186, and R are shown in Fig. 13a. Carboxymethylated DEAE-purified porcine brain tubulin was run as reference protein. It is evident that the preparation of strain R differed from those of strains 003 and 186. In gels loaded with proteins from the latter two strains, two distinct bands are present with a comparable mobility as the band representing porcine brain tubulin and one band which had moved slightly faster. With strain R, two weak bands are present at this migration distance whereas other bands showed about equal staining as corresponding bands present in the other two gels.
When identical protein preparations of the three strains were run on discontinuous SDS gels, two major bands of unequal staining density were found with similar mobility as the bands on the reference gel (Fig. 13 b ). The faster moving band on the reference gel is due to/3-tubulin and the slower moving one to a-tubulin (51).
When gels from both SDS systems are compared it is evident that two bands, which are found to be separated in the continuous system, move together in the discontinuous system. With respect to the staining densities of the bands it seems that one of the two slower moving bands of the continuous system moves together with the faster moving band in the discontinuous system.
Both gel systems could be correlated in the following manner. It was found that heating a DEAE-purified preparation for 1 h at 37~ cooling in ice and centrifuging, resulted in a supernate in which, upon electrophoresis in the continuous SDS system, the two slower moving bands were not present (Fig. 14a, b). These bands appeared to be present in gels loaded with the pelleted material. Heat-dependent precipitation of proteins was not reversible by cold, nor was the process inhibited by Ca 2 § colchicine, or MBC, which eliminates the possibility that microtubule assembly is responsible. Apparently denaturation by heat of the proteins concerned causes precipitation which is in agreement with h6UP.E 13 Electrophoretic analysis of proteins isolated from A. nidulans strains 003, 186, or R, by DEAE-Sephadex column chromatography. Lyophilized proteins from pooled fractions eluted at 0.45-0.90 M NaC1 were carboxymethylated and run in the continuous SDS system (a) and the discontinuous system (b). Carboxymethylated DEAE-purified porcine brain tubulin was run as a reference.
Fmu~ 14 Electrophoretic analysis of proteins isolated from A. nidulans strain 186 by DEAE-Sephadex column chromatography. Pooled fractions eluted at 0.45-0.60 M NaC1 were heated for 1 h at 370C, cooled in ice, and centrifuged at 40,000 g for 10 rain. Aliquots of the nonheated (N), heated (H) and supernatant (S) protein solution, and the pellet (P) were either reduced and run in the continuous SDS system (a) or carboxymethylated and run in both SDS systems (b, c). DEAE-purified porcine brain tubulin (T), either reduced (a) or carboxymethylated (b, c) was run as a reference.
the observation that mycelial extracts which are heated at 37~ and centrifuged, show rather low MBC-binding activity.
Upon electrophoresis in the discontinuous system the precipitated material runs mainly as two bands, with similar mobility as c~-and/3-tubulin from porcine brain (Fig. 14c). Because of this behavior it can be concluded that these bands represent both tubulin monomers of A. nidulans. The third faster moving band in continuous SDS gels which showed a similar mobility as fl-tubulin in the discontinuous system, apparently represents a protein which copurified with fungal tubulin in this purification procedure.
It is evident that the purification procedure applied to extracts of strain R resulted in significantly less a-and fl-tubulin, than was obtained with a similar procedure applied to extracts of strains 003 and 186. Although a somewhat lower initial tubulin concentration might be partly responsible (Fig. 6), it is certainly not the only factor involved. A reason for the failure to purify tubulin of strain R might be a low affinity of this tubulin for DEAE-Sephadex. A low affinity of brain tubulin for DEAE-Sephadex has been reported (47,53) and seems to be related with phosphorylation of the protein. At any rate, the different behavior of tubulin of strain R in corn-parison with that of both other strains, suggests chemical differences of the protein itself or of factors associated with tubulin, an idea which is compatible with the observation that differences exist in affinity of the tubulins of the three strains to MBC.
The results of the electrophoretic analysis indicate that MBC might be used as an affinity label to purify fungal tubulin from MBC-sensitive strains of A. nidulans. It supports the idea that this protein is the primary target of MBC action.

DISCUSSION
The MBC-binding protein in A. nidulans is characterized by a set of properties which is unique for tubulin (cf. 14). Binding activity could be selectively fractionated with ammonium sulfate between 35 and 50% saturation. The MBC-protein complex was retained on DEAE-Sephadex columns and its molecular weight was estimated at 110,000. Binding activity was labile and could be stabilized by sucrose, glycerol, and MBC itself. Binding was competitively inhibited by known inhibitors of microtubule function such as oncodazole and colchicine. Electrophoretic analysis of partially purified preparations of the MBC-protein complex, showed the presence of proteins with similar mobilities as mammalian tubulin mono-mers. On the basis of these results it can be concluded that the MBC-binding protein is identical with fungal tubulin.
MBC binding to fungal tubulin was rapid, reversible, and did not require a high temperature. As has been reported recently (45), binding of oncodazole to rat brain tubulin shows similar features. In this respect MBC and oncodazole resemble podophyllotoxin, a compound which competes with colchicine for the colchicine-binding site on tubulin from different sources (75). However, podophyllotoxin did not inhibit MBC binding which suggests that fungal tubulin has no or at least a low affinity for this compound.
From the data given in Fig. 6 the number of MBC-binding sites can be determined. Assuming that one molecule of MBC is bound per molecule of tubulin, one can calculate that tubulin is 0.2-0.4% by weight of total protein present in mycelial extracts. This value resembles the value of 0.6% found for the colcemid-binding protein in Saccharomyces cerevisiae (38) which proved to be in good correspondence with the figure of 0.36% calculated from the estimate of the number of microtubules per nucleus.
Although colchicine was evidently bound to tubulin of A. nidulans the binding reaction showed some unusual features. The rate of complex formation seemed to be rapid and binding was not temperature dependent, which properties are in contrast with those of colchicine binding to mammalian tubulin (cf. 74). Moreover, affinity of A.
nidulans tubulin to colchicine was rather low compared with that of mammalian tubulin. A low affinity of, presumably, tubulin from S. cerevisiae for colchicine has been reported by Haber et al. (38). Burns (18) was not able to demonstrate colchicine binding in Schizosaccharomyces pombe and Jockusch (46) did not find colchicine-binding proteins in Physarum polycephalum. Heath (43) reported the presence of two binding components in Saprolegnia ferax with a low affinity for colchicine, one of which was trichloroacetic acid (TCA) stable. A TCA-stable-binding protein was also found by Olson (54) in Allomyces moniliformis.
However, the last two authors did not investigate the identity of the bound radiolabel. Since in our colchicine-binding experiments the formation of a PCA-stable-labeled complex could be ascribed to the presence of an impurity in our [aH]colchicine preparation, the results of these authors should be interpreted with caution.
Low affinity of A. nidulans tubulin for colchi-cine is probably partly responsible for the failure of this compound to inhibit mycelial growth. Cell membrane permeability might also play a role since no effect on growth was noticed at concentrations 25 times higher than those which are needed to half-maximally saturate the binding sites in vitro. In comparison with MBC and oncodazole this ratio is rather high, since these compounds gave a 50% reduction in growth already at concentrations two to three times higher than the estimated value of their respective dissociation constants.
To our knowledge data concerning affinity of fungal tubulin for colchicine are restricted to those already mentioned. We think that low affinity is a characteristic property of fungal tubulin. This assumption is based on the fact that fungi are commonly resistant to the antimitotic action of colchicine (cf. 42), which seems only partially caused by a low permeability of the cell membrane (38,64) and on the fact that microtubules are supposed to play an essential role in fungal nuclear division (34,35). Until now no suitable agent has been found which specifically disrupts microtubules in fungi (42). Because of their high fungitoxicity, MBC and oncodazole are potential candidates, since from both compounds it is known, that they induce disappearance of microtubules in mammalian cells (25)(26)(27).
The differential growth response of the two mutant strains R and 186, in comparison with that of the wild-type strain 003, is probably based on a difference in affinity of their tubulins for MBC. This idea is also supported by the fact that no differences are found in uptake or detoxication of MBC between the various strains (24).
Genetic analysis has shown that mutations to resistance and to increased sensitivity took place in the same gene (69,70). Although it can be assumed that this gene codes for tubulin, this can only be definitely concluded when differences are found between the primary structures of tubulin of the three strains. A difference in affinity for MBC might also be caused by differences in post-translational modifications of tubulin, such as phospho~ rylation (30,31), glycosylation (32), or association with tau-like factors (15,16,72). A different modification, rather than a single amino acid substitution might also explain the failure to purify tubulin from strain R.
Mutation to resistance or increased sensitivity did not affect growth rate nor sporulation of the strains (references 22 [ Fig. 1] and 69 [Fig. 1]), which indicates normal assembly and functioning of microtubules. However, diploids carrying both mutations were not stable as was evident from increased sectoring of diploid colonies. 2 Since nondisjunction of chromosomes is thought to be mainly responsible for sectoring, increased sectoring indicates improper functioning of microtubules. This might be caused by the presence of two incompatible types of tubulins in these diploids. Increased sectoring has also been found in ordinary A. nidulans diploids when they are exposed to MBC at sublethal concentrations (37,41,49). Griseofulvin induces a similar effect (37,48). These observations are compatible with the idea that binding of MBC to tubulin interferes with the assembly of tubulin into microtubules.
The action of MBC and the mechanism of resistance was only studied in detail in A. nidulans, but the data in Table I suggest that the mechanism of resistance found here might be a general type of resistance in fungi. Since in fungi various types of mitosis are found (35) it would be interesting to know whether resistance and sensitivity to MBC is related to a certain type of mitosis.
Until now no evidence has been presented that resistance to other agents which bind to tubulin might be caused by a similar mechanism. Resistance to compounds of this type has recently been discussed by Freed and Ohlsson-Wilhelm (33). A drug exclusion mechanism was found to operate in several instances. The biochemical basis of resistance which appeared to be specific to the selecting agent, has not yet been studied.
Several possibilities exist to explain the selectivity of these benzimidazole compounds in general. In addition to a differential uptake or metabolism, a different affinity of tubulin from various sources for a certain benzimidazole compound might play a role. This is illustrated by the fact that no binding was found between porcine brain tubulin and MBC, although at identical concentrations of MBC considerable binding to fungal tubulin was found. A difference in affinity between tubulins from different sources for a certain benzimidazole compound might be caused by similar factors already discussed above.
It is noteworthy here that despite differences in affinity between porcine and A. nidulans tubulin to MBC, the two types are able to copolymerize in vitro (23,60). Apparently, binding sites involved 2 Van Tuyl, J. M., personal communication and own observation.
in polymerization have been highly conserved during evolution in contrast with the colchicine-binding site.
Undoubtedly, these benzimidazole compounds will become useful as experimental tools in the study of microtubule structure and function in cells. Their use, however, in agriculture as fungicides and, quantitatively on a minor scale, in veterinary medicine, should be reconsidered from the point of view of their mechanism of action. Interference of MBC with nuclear division in mammalian cells has been found to occur in vitro (27,59,67) and in vivo (59,67). This implies a potential genetic risk for man. The toxicology and genetic effects of benzimidazole compounds have recently been reviewed by Seiler (59). We agree with him that the use of pesticides with this type of action should be restricted.
We are grateful to Prof. Dr. Ir. J. Dekker, Dr. A. Fuchs, and Dr. It. J. Visser (Department of Genetics, Agricultural University, Wageningen, The Netherlands) for comments on the manuscript and to Mrs. Brouns-Murray (Pudoc, Wageningen, The Netherlands) for correcting the English text.