The Role of GTP Binding and Microtubule-Associated Proteins in the Inhibition of Microtubule Assembly by Carbendazim

B. S. Winder, C. S. Strandgaard and M. G. Miller

Department of Environmental Toxicology, One Shields Avenue, University of California, Davis, California 95616

Received August 9, 2000; accepted September 29, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The fungicide carbendazim (CBZ) is known to disrupt microtubular structures in the testis and to cause testicular toxicity in rats. To investigate the mechanism underlying the toxicity of CBZ, tubulin and microtubule-associated proteins (MAPs) were isolated from rat testis and brain using two techniques. The effects of CBZ on MT assembly were compared with the known microtubule (MT) disruptors, colchicine and nocodazole. CBZ (100 µM) had no effect on the assembly of MTs from MAP-containing tubulin isolated with one cycle of glycerol-dependent assembly and disassembly while colchicine (40 µM) and nocodazole (12.5 µM) strongly inhibited the assembly reaction. Similarly, formation of MTs from tubulin prepared with two cycles of glycerol-dependent assembly was strongly inhibited by colchicine and nocodazole but only weakly by CBZ. All three compounds inhibited the assembly of MTs from MAP-free tubulin isolated with glutamate. However, the inhibition by CBZ was reversed by the inclusion of high-molecular-weight MAPs and not by unrelated protein (bovine serum albumin, BSA). Addition of nocodazole to assembled MTs caused immediate depolymerization, whereas CBZ did not directly cause depolymerization. However CBZ was an effective inhibitor of the polymerization of depolymerized tubulin. In competitive binding assays, CBZ was found to inhibit the binding of guanosine triphosphate (GTP) to tubulin. The data suggest that CBZ interferes with initial events of MT polymerization, specifically GTP binding, and that MAPs moderate this effect.

Key Words: carbendazim; GTP binding; tubulin; microtubule assembly; microtubule-associated proteins; nocodazole; colchicine.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Microtubules (MTs) play a central role in the growth and division of cells, intracellular trafficking, and maintenance of cell morphology. Carbendazim (CBZ) and its parent, benomyl (BNL), are benzimidazole carbamates whose fungicidal properties derive from their ability to interfere with the assembly of fungal MTs (Davidse, 1986Go). Chronic, subchronic, and acute exposure of rats and mice to BNL causes male reproductive damage, manifested as decreased testicular weights, early sloughing of germ cells, and atrophy of seminiferous tubules (Carter and Laskey, 1982Go; Hess et al., 1991Go). While the acute systemic toxicity of BNL in the rat is low (LD50 approx. 10 g/kg), a single dose of 100 mg/kg results in a testicular lesion (Hess et al., 1991Go). It has been speculated that the testicular toxicity of these compounds derives from the same MT-dependent mechanism as the fungicidal activity (Davidse and Flach, 1977Go).

Until recently it was unclear whether BNL itself or its metabolite, CBZ, was responsible for the observed testicular toxicity. Lim and Miller (1997) measured testicular levels of BNL and CBZ and found that the extent of testicular damage could be directly correlated with the concentration of CBZ and not BNL. CBZ was also 2-fold more potent than BNL as a testicular toxicant. The testicular lesion following CBZ treatment is characterized by premature sloughing of spermatids, loss of Sertoli cell apical cytoplasm (Lim and Miller, 1997Go; Nakai and Hess, 1997Go), and collapse of intermediate filaments (Hess and Nakai, 2000Go).

The formation of MTs is a multi-step, temperature-dependent process. In the absence of a microtubule-organizing center (MTOC) such as the centrosome, the process starts with a nucleation event to generate the structures from which the MTs grow. Next is an elongation phase during which heterodimers of {alpha} tubulin assemble into polymers. Whether the polymers form MTs, sheets, ribbons, or other structures is influenced by the assembly conditions including buffer composition and the presence of microtubule-associated proteins (MAPs). Elevated temperatures (30–37°C) stimulate assembly while lower temperatures cause MTs to disassemble. Magnesium ions and guanosine triphosphate (GTP) are generally required for MT assembly, with the latter bound at the exchangeable (E) and non-exchangeable (N) sites of the ß and {alpha} subunits, respectively. GTP is hydrolyzed during the formation of MTs but GTP hydrolysis per se appears not to be required for elongation of the polymer, which can be supported by non-hydrolyzable analogs of GTP (Dye and Williams, 1996Go; O'Brien and Erickson, 1989Go) as well as guanosine diphosphate (GDP; Carlier and Pantaloni, 1978). There appears to be a requirement for GTP in the initial formation of the nuclei from which the polymer grows, as this nucleation step is supported by tubulin with GTP-bound (GTP-tub) but not by GDP-tub (Karr et al., 1979Go; O'Brien and Erickson, 1989Go). Thus, an early step in the formation of MTs is the binding of GTP by tubulin, although the role of GTP hydrolysis in MT polymerization is complex and not fully understood (Dye and Williams, 1996Go).

The present studies have examined the ability of CBZ to alter the assembly of MTs from tubulin prepared from rat brain and testis. This work evaluated the effects of MAPs on MT assembly in the presence and absence of CBZ, as well as the effects of CBZ on GTP binding. The effects of CBZ were compared with the response to the known MT disrupters, colchicine (CLC) and nocodazole (NOC).


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and animals.
Except where otherwise specified, all reagents were from Sigma-Aldrich Chemical Co. (St. Louis, MO). [3H]-GTP was from DuPont NEN (Wilmington, DE). Male Sprague Dawley rats (100–130 days old) were obtained from Charles River (Hollister, CA) and maintained at 22°C on a 12/12 h light/dark cycle, with food and water provided ad libitum.

Tubulin preparation.
MAP-containing and MAP-free tubulin were prepared from rat brain and testis by 2 different methods, using either glycerol or sodium glutamate. To prepare tubulin-containing MAPs, the procedure of Shelanski et al. (1973) was used with modification. Rats were killed by CO2 inhalation and the brains and testes homogenized in MES buffer (0.1 M MES, pH 6.4, 1 mM EGTA, 1 mM MgCl2, 1 mM dithioerythritol, 1% aprotinin and 200 µM PMSF [phenylmethyl sulonylfluoride]). To enhance MT depolymerization, the homogenates sat on ice for 15 min prior to centrifugation at 100,000 x g for 60 min at 4°C. Tubulin was prepared by either one or two cycles of heat-induced assembly (37°C, 60 min) in the presence of glycerol (4 M) and GTP (1 mM). Each cycle was followed by centrifugation at 37°C at 100,000 x g for 30 min to pellet assembled MTs. The pellets were resuspended by sonication and incubated on ice to promote disassembly. Centrifugation (100,000 x g, 30 min at 4°C) removed protein aggregates. The tubulin-containing supernatant from the cold centrifugation step of the second cycle was filtered through glass wool, snap frozen in methanol/dry ice, and stored at –80°C.

For the preparation of tubulin substantially free of MAPs, 1 M glutamate was used in a batch preparation technique modified from Hamel and Lin (1981). The tissues were homogenized in 0.1 M MES (pH 6.75) with 1 mM EGTA, 0.5 mM MgCl2, 1% aprotinin and 200 µM PMSF. Samples sat on ice for 15 min prior to centrifugation at 100,000 x g as described above. GTP (0.1 M) was added to the supernatants, which were gently mixed for 60 min in the cold with DEAE-Sephacel (1 ml/10 g tissue) previously equilibrated with 1 M Na glutamate, pH 6.6. After the Sephacel had settled for 15 min on ice, the supernatant was removed and the resin was rinsed with washing buffer (1 M Na glutamate, 0.1 M NaCl, 0.1 mM GTP, pH 6.6) and pelleted at 100 x g. This wash step was repeated 2 times. Tubulin was eluted from the resin with elevated salt by incubation with 1 ml elution buffer (1 M Na glutamate, 1 M NaCl, 0.1 mM GTP, pH 6.6) on ice for 15 min with occasional gentle mixing. The resin was pelleted as before and the supernatant saved. This step was repeated for a total of 5 times. Glycerol (4 M) and GTP (1 mM) were added to the pooled supernatants and the tubulin allowed to polymerize at 37°C for 30 min. Assembled MTs were pelleted at 100,000 x g, 30 min 37°C. The pellets were resuspended in 1 M Na glutamate, sonicated, and allowed to depolymerize on ice for 30 min. Following a final centrifugation at 100,000 x g, depolymerized tubulin was collected in the supernatant and snap frozen. Residual nucleotides in the supernatant were removed by passage over Sephadex G25 minicolumns prior to use.

Preparation of MAPs.
MAPs were isolated according to Neely and Boekelheide (1988) as modified from Vallee (1982), snap frozen, and stored at –80°C. Briefly, MTs and associated MAPs were polymerized in the presence of taxol, glycerol and GTP, then pelleted by centrifugation through sucrose. MAPs were dissociated from the MTs with 750-mM NaCl and collected in the supernatant after pelleting the MTs. Prior to use, MAPs solutions were desalted by passage through Sephadex G25.

Assembly assay.
The assembly of tubulin into MTs was monitored turbidometrically at 350 nm in a Shimadzu recording spectrophotometer with a thermostatically regulated cuvette holder. Tubulin assembly mixtures included 2 mM Mg2+, and inhibitors or vehicles. No difference in the assembly was observed irrespective of whether MgCl2 or MgSO4 was used. Protein concentrations were determined with a bicinchoninic acid protein assay kit (Sigma, St Louis, MO) with BSA as standard. The reactions were initiated by the addition of 0.5–1.0 mM GTP and elevation of the cuvette temperature to 37°C. Optical densities were recorded at 2-min intervals through one or more cycles of heating to 37°C and cooling to less than 10°C. Tubulin samples were either preincubated or co-incubated with CBZ dissolved in dimethylformamide (DMF), NOC in dimethyl sulfoxide (DMSO), or CLC in water. The volume of added vehicle was 1% or less of the final reaction volume.

For experiments in which MAPs were added to MAP-free tubulin, MAPs:tubulin ratios from 1:4 to 1:15 (µg/µg) were tested with qualitatively similar results. The higher MAPs:tubulin ratios were used routinely as they gave more pronounced results.

[3H]-GTP binding to tubulin.
The effects of CBZ on the binding of GTP by tubulin were analyzed by the DEAE cellulose filter method of Borisy (1972). Tubulin was incubated with CBZ for 20 min on ice, prior to the addition of 5 µM [3H]-GTP. The reaction mixtures were diluted with PEM (80 mM PIPES, pH 6.8, 1 mM MgCl2, 1 mM ethylene glycol bis(aminoethyl ether) [EGTA]) and poured on to stacks of 3 DEAE cellulose filter disks (Whatman DE81, Fisher Scientific) on a vacuum manifold. The filters were rinsed twice with 4 ml 8 mM PEM containing 10 mM KCl to reduce nonspecific nucleotide binding to the filters. Protein-bound nucleotide on the filters was quantified by liquid scintillation spectroscopy after elution overnight in scintillation fluid (Emulsifier-Safe, Packard Inst., Meriden, CT).

Analysis of GTP- and GDP-tubulin.
The effects of CBZ on GTP hydrolysis were assayed by HPLC separation and quantitation of GDP and GTP according to Melki et al. (1996). MAP-free tubulin was incubated with 1 mM GTP for 30 min on ice. Unbound GTP was removed by passage over Sephadex G25 to yield a 1:1 GTP:tubulin complex. CBZ or vehicle (DMF) was added to the GTP:tubulin. After incubation for 2–40 min, 100 µl samples were transferred to 12 µl of ice cold 40% perchloric acid to precipitate protein, which was removed by centrifugation The ratio of GDP to GTP in solution was determined by HPLC on a Waters HPLC system equipped with an Alltech Adsorbosphere Nucleotide column. The samples were resolved by isocratic elution of the column with a mobile phase of methanol/60 mM NH4NHPO4 (25:75) containing 5 mM tetrabutylammonium sulfate as an ion-pair reagent.

Unless otherwise indicated, experiments were performed 3 times. Statistical significance of treatment effects was determined with a Student's 2-tailed t-test, p < 0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Tubulin Purified with Glycerol
Tubulin prepared with glycerol from either brain or testis contained large numbers of MAPs after one cycle of polymerization/depolymerization. The protein patterns on denaturing polyacrylamide gels were different from the 2 tissues, indicating differences in the numbers and types of MAPs isolated from the respective sources (Fig. 1AGo). A second cycle of glycerol-promoted assembly/disassembly and resulted in preparations enriched in tubulin but still containing MAPs, although reduced in numbers and amount (Fig. 1BGo). In contrast to the MAPs-containing glycerol tubulin preparations, tubulin prepared with glutamate was depleted of MAPs (Fig 1CGo).



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FIG. 1. Tubulin isolation with glycerol or glutamate. Tubulin prepared from testis (T) and brain (B) with one cycle of glycerol-promoted polymerization/depolymerization, by the method of Shelanski et al. (1973), was resolved on 10% SDS (sodium dodecyl sulfate)–polyacrylamide gels, according to Laemmli (1970) (A). After 2 cycles of glycerol polymerization, tubulin was substantially enriched, especially in testis (B). Tubulin prepared with glutamate, according to Hamel and Lin (1981), was largely MAP-free (C). Molecular weights (in kDa) are on the left.

 
The assembly of MTs from brain and testis tubulin prepared by the one-cycle glycerol method is shown in Figure 2Go. This figure is a composite of 4 assembly reactions and, as shown in Figure 1Go, the brain and testis preparations contained differing amounts of tubulin and MAPs. To facilitate comparison among runs and between tissues, the data are presented as the change in optical density per milligram total protein. The assembly of MTs is sensitive to the relative abundance of tubulin in the preparation and it was noted that the maximum assembly for testis tubulin was lower than for brain, reflecting the lower proportion of tubulin to total protein in the testis preparations. In both tissues, the assembly of MTs was markedly inhibited by CLC and NOC as observed by others (Deery and Weisenberg, 1981Go; Friedman and Platzer, 1978Go; Hoebeke et al., 1976Go). In contrast, carbendazim had no significant effect on these MAP-containing testis or brain tubulin preparations (Fig. 2Go).



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FIG. 2. Inhibition of assembly of tubulin prepared with one-cycle glycerol. Microtubule assembly was initiated by elevation of the temperature to 37°C and monitored as the change in optical density at 350 nm in the presence of 1 mM MgCl2, 1 mM GTP and CBZ (100 µM), NOC (12.5 µM), and CLC (40 µM). The temperature was later lowered, as shown in the bottom graph (C). The graphs are composite runs from at least 3 separate experiments for testis (A) and brain (B) tubulin. Tubulin assembly was followed as the change in optical density standardized to protein concentration to facilitate comparison. Solid lines and filled symbols: inhibitors. Dashed lines and open symbols: vehicle controls.

 
After two cycles of polymerization with glycerol, fewer MAPs were present and slight CBZ sensitivity was seen in brain but not testis tubulin (Fig. 3Go). Interestingly the maximum assembly of MTs in these preparations was higher for testis than for brain. This corresponds to a greater increase in the apparent tubulin-to-MAPs ratio in testis versus brain during the second cycle of purification (Fig 1BGo), as well as to different patterns of MAPs in the 2 tissues. The shape of the curves for DMSO suggests a slight vehicle effect or experimental differences in these runs, which was manifested as a delay in the start of assembly. Nevertheless, NOC and CLC caused significant inhibition compared with their respective vehicle controls in both brain and testis tubulin.



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FIG. 3. Inhibition of assembly of tubulin prepared with 2-cycle glycerol. The assembly of microtubules from testis (A) and brain (B) tubulins isolated with 2 cycles of glycerol-promoted polymerization was assayed as in Figure 2Go. The temperature was later lowered, as shown in the bottom graph (C). The graphs are composite runs from at least 3 separate experiments. Tubulin assembly was followed as the change in optical density standardized to protein concentration to facilitate comparison. Solid lines and filled symbols: inhibitors. Dashed lines and open symbols: vehicle controls.

 
MAP-free Tubulin and the Addition of MAPs
The MAP-depleted tubulin prepared with glutamate was extremely sensitive to the inhibitory effects of CBZ (Fig. 4Go). A possible role for MAPs in the modulation of CBZ sensitivity was further tested with MAP-depleted tubulin, which was allowed to assemble with and without added MAPs and CBZ. Comparison of the plateau level of assembly of CBZ-treated samples with their respective controls at 24 min in Figure 4Go shows that the assembly of MTs from testis tubulin was much more inhibited by 100 µM CBZ in the absence of MAPs (92%) than was the assembly in the presence of MAPs (31%). Similarly CBZ inhibited assembly of brain tubulin by 84% in the absence, but only 34% in the presence of MAPs (Fig. 5Go).



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FIG. 4. Effects of MAPs on CBZ inhibition of MAP-free testis tubulin assembly. MAP-free testis tubulin was isolated by the method of Hamel and Lin (1981), using sodium glutamate to stabilize tubulin and DEAE Sephacel to separate tubulin from MAPs. These preparations were substantially free of MAPs as assessed by SDS–PAGE. To test the effects of MAPs on the inhibition of tubulin polymerization by CBZ, MAP-free tubulin from testis (1.13 mg/ml) was assayed for assembly, as in Figure 2Go, in the presence and absence of CBZ (100 µM), without (A) and with (B) testis MAPs (94 µg/ml). The temperature cycle is shown in C. These are representative curves from 3 separate experiments.

 


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FIG. 5. Effects of MAPs on CBZ inhibition of MAP-free brain tubulin assembly. MAP-free brain tubulin was isolated and assayed as for Figure 4Go. To test the effects of MAPs on the inhibition of tubulin polymerization by CBZ, MAP-free tubulin from brain (1.95 mg/ml) was assayed for assembly, as in Figure 2Go, in the presence and absence of CBZ (100 µM) and without (A) and with (B) brain MAPs (130 µg/ml). The temperature cycle is shown in C. These curves are representative of 3 separate experiments.

 
To test whether the CBZ inhibition was MAPs-specific or just the result of nonspecific MT stabilization or binding of CBZ by protein, BSA was added to MAP-free tubulin in place of MAPs. At BSA concentrations comparable to those of MAPs (0.2 mg/ml), there was no effect of BSA on CBZ inhibition of tubulin assembly, suggesting that the decrease in CBZ inhibition is MAPs-specific (data not shown).

CBZ Acts on Tubulin
To determine whether CBZ's effects were primarily on assembling tubulin or whether it could disrupt already-formed MTs, CBZ (100 µM) was added to MAP-free tubulin in which MT assembly had reached a steady state at 37°C as shown by no further increase in the optical density. The cuvettes were cooled to <10°C, to allow disassembly, then another thermal cycle was initiated. Figure 6Go depicts the changes in the optical density of tubulin preparations treated in this fashion. CBZ inhibited assembly when present at the start of the assembly reaction (Fig. 5AGo), but had no apparent effect on assembled MTs (Fig. 6BGo). However, once the MTs had disassembled, CBZ inhibited assembly during the second thermal cycle, suggesting that CBZ affected the initiation of assembly rather than directly disrupting MTs. This contrasts with the effects of NOC on MTs. When NOC (12.5 µM) was added to MTs at steady state there was an immediate loss of turbidity, indicating depolymerization of the MTs (Fig. 6AGo). No MT assembly was seen during the second thermal cycle in the presence of NOC. Similar to CBZ, CLC (40 µM) did not cause depolymerization of the MTs and did inhibit MT assembly during the second cycle. The cold-induced depolymerization observed after CLC was not as extensive as that observed after either CBZ or NOC.



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FIG. 6. Differential sensitivity of microtubules vs. tubulin to CBZ and NOC. MAP-free testis tubulin was assayed for assembly through 2 thermal cycles. (A) NOC (12.5 µM) or DMSO vehicle were included prior to initiation of assembly, as in Figure 2Go (NOCini, DSOini), or were added 15 min after microtubule assembly plateaued (NOCdel, DSOdel). (B) CBZ (100 µM) or DMF vehicle were added at 15 min also (CBZdel, DMFdel). At 30 min, the temperature was lowered to promote depolymerization. A second cycle of assembly was initiated by raising the temperature to 37°C for 20 min, followed by cooling to 5°C. Brain tubulin assayed in identical fashion gave comparable results. Open symbols: vehicle controls. Closed symbols: inhibitors.

 
CBZ and the Interaction of GTP with Tubulin
It was postulated that an early step in MT assembly, with which CBZ could interfere, is GTP binding. This was tested both in assembly assays and in competitive binding assays. In competitive binding assays on DEAE cellulose filter disks, CBZ prevented the binding of [3H]-GTP to tubulin from brain (Fig. 7Go) and testis in a dose-dependent fashion. The data for CBZ inhibition of GTP binding were linearized in a logit plot according to Bylund and Yamamura (1990). The logit transformation is the natural logarithm of the ratio of the percent bound to 100, minus the percent bound. The IC50 is 50% binding and the logit of 50% [ln (1)] is 0. From the graph, CBZ was seen to inhibit GTP binding in brain with an IC50 of 74.1 (SD = 1.2) µM from duplicate experiments. A similar experiment with testis gave an IC50 of 60 µM. If CBZ and GTP compete for binding at a single or overlapping site, the addition of excess GTP should prevent or reverse the effects of CBZ on assembling MTs. To test this, CBZ was added to tubulin at the assembly plateau as in Figure 6Go, but with and without excess GTP. For both testis and brain, the addition of GTP significantly decreased the inhibition of assembly by CBZ during the second cycle, compared to CBZ alone (Fig. 8CGo). Thus GTP partially rescued MT assembly from CBZ inhibition. That this effect was due to inhibition of GTP binding versus inhibition of GTP hydrolysis was shown by adding CBZ to tubulin previously loaded with GTP and following the rate of GDP formation by HPLC (Fig. 9Go). There was no statistically significant difference in the amount of GDP formed in the presence of CBZ versus vehicle.



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FIG. 7. CBZ inhibits the binding of [3H]-GTP by tubulin. Brain tubulin was incubated with CBZ for 20 min on ice, then with [3H]-GTP. Tubulin with bound [3H]-GTP was isolated on DEAE-cellulose filters and quantitated by liquid scintillation spectroscopy. The data were linearized by plotting ln [percent bound/(100-percent bound)] vs. CBZ concentration (Bylund and Yamamura, 1990Go). The IC50 is at 50% bound, the natural log of which is 0 on the ordinate axis. Similar results were obtained from 2 different tubulin preparations assayed in triplicate.

 


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FIG. 8. GTP rescue of tubulin assembly from CBZ inhibition. (A) Plot of brain tubulin through 2 cycles of assembly. CBZ (100 µM) was added to 2 samples (circles) and the assembly was followed through cold disassembly and another cycle of assembly/disassembly. Similar results were obtained with testis tubulin. DMF is the vehicle control. (B) Temperature of reaction cuvettes through 2 thermal cycles. When the maximum assembly during the second cycle, in the presence of CBZ +/- GTP, is shown as a percentage of the first cycle prior to inhibitor addition (C), GTP is seen to rescue tubulin assembly in both brain and testis. GTP added to the vehicle did not affect the level of assembly in the second cycle. Statistics: Student's t-test; p < 0.05.

 


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FIG. 9. Effects of CBZ on GTP hydrolysis by tubulin. Tubulin was preloaded with GTP, then incubated with CBZ or vehicle on ice for 20 min. The protein was precipitated and the supernatant analyzed by HPLC for GTP and GDP. Br CBZ: brain + CBZ; Ts CBZ: testis + CBZ; DMF: dimethylformamide vehicle. The GDP:GTP ratio is presented ± SD.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The ability of CBZ to inhibit tubulin assembly in vitro was compared with that of NOC and CLC. The 3 compounds used in this study inhibited MT polymerization. However, unlike NOC or CLC, CBZ's ability to inhibit tubulin assembly was largely abolished in the presence of MAPs for both brain and testis tubulin preparations. This was observed both with tubulin isolated with MAPs in the presence of glycerol and with MAP-free tubulin isolated with glutamate, to which MAPs were added back. The MAPs effect was thus independent of the method of isolation and apparently specific to CBZ. This may represent overlapping binding sites for MAPs and CBZ; however, since most MAPs bind at or near tubulin's C-terminus, an allosteric modification of the CBZ binding site following MAP binding is more likely. The relative lack of CBZ sensitivity in testis versus brain after 2 cycles of glycerol purification, which removed substantial numbers of MAPs and resulted in different protein-banding patterns between testis and brain (Fig. 1Go), suggested that specific MAPs modulate CBZ's effect (Fig. 3Go). Whether the MAPs remaining after 2 cycles of glycerol assembly are tissue specific is unknown. Nevertheless, the final removal of these MAPs with glutamate gave the dramatic CBZ sensitivity seen in Figure 4Go.

The selection of CLC and NOC as positive controls in the present study was based in part on published competitive binding studies with CLC. From these studies it had been suggested that the benzimidazoles bind in or near the CLC binding site and perhaps operate through a similar mechanism (Davidse and Flach, 1977Go; Russell et al., 1992Go). However, several observations from the present work suggest dissimilar mechanisms of action between the two compounds, CBZ and NOC. When added to preformed MTs, only NOC stimulated their depolymerization whereas CBZ and CLC had no apparent effect, suggesting a differential ability of MTs to bind NOC versus CBZ and CLC. Indeed, the fact that CLC binds free tubulin rather than MTs has been used to explain the observed stability of MTs exposed in vitro to high concentrations of CLC (Farrell and Wilson, 1984Go). CBZ also appears to bind only free tubulin, since it was not until the MTs disassembled that CBZ and CLC were able to prevent further re-polymerization under assembly-promoting conditions. NOC and CBZ are also dissimilar in that NOC is reportedly much more tightly bound by tubulin (Friedman and Platzer, 1978Go). Furthermore, whereas NOC has been associated with an increase in GTPase activity (Mejillano et al., 1996Go), there was no evidence that CBZ stimulated GTP hydrolysis in the present studies.

In contrast to NOC, CBZ and CLC were more alike, since neither caused depolymerization of MTs and both inhibited subsequent repolymerization. In addition, the in vivo effects of CBZ and CLC are similar in that CLC can induce pathological changes in the testis (Allard et al., 1993Go) that resemble the premature sloughing of germ cells seen with CBZ (Hess et al., 1991Go; Nakai et al., 1993Go). There are, however, differences between CBZ and CLC associated with stage-specific sensitivity. Allard et al. (1993) reported that stages IX–XIV seminiferous tubules were the most sensitive to CLC. With CBZ, sloughing was seen starting in stage VII (Parvinen and Kormano, 1974Go), one h after exposure, and from stage VI onward, 2 h post-exposure (Nakai et al., 2000Go). At 3-h post-CBZ exposure, sloughing was seen in all stages except III–V (Nakai and Hess, 1994Go). In the testis, the stage specific sensitivity of Sertoli cells to CBZ vs. CLC may be related to the stage-dependent presence of various MAPs and/or chaperone proteins. Several of the MAPs are known to stabilize MTs or promote assembly upon binding near the carboxyl terminus of tubulin (Maccioni and Cambiazo, 1995Go). The modulation of CBZ's assembly inhibition by these MAPs would therefore likely be indirect and involve allosteric effects. On the other hand, certain of the chaperonins, recently recognized for their involvement in the assembly of MTs as well as the folding and heterodimerization of the tubulins, have subunits that bind to and co-purify with tubulin (Roobol et al., 1999Go). It remains to be determined which of these MAPs or MAP-like proteins modulate CBZ binding and the mechanisms involved.

It was noted that, when added to assembling MAP-free tubulin shortly (2 min) after the addition of GTP, CBZ was a much less potent inhibitor than when it was present prior to GTP addition. The dependence of inhibition on the sequence of the addition of CBZ and GTP indicates that CBZ does not displace bound GTP. Rather it appears that CBZ prevents GTP binding, thereby interfering with the initiation of assembly. This scenario is further supported by the competitive binding experiments in which CBZ inhibited GTP binding to tubulin in vitro in a dose-dependent fashion. The IC50 of 74 µM that we obtained for CBZ inhibition of GTP binding in the brain is in good agreement with the IC50 of 90 µM reported by Friedman and Platzer (1978) for the inhibition of brain tubulin assembly by CBZ. Similarly, if CBZ inhibits GTP binding by direct competition for a common or overlapping binding site, then excess GTP should competitively prevent CBZ inhibition. Indeed, in assembly assays in which excess GTP was added to MTs after the addition of CBZ, the inhibition of assembly during the second cycle was significantly reduced.

A mechanism for binding competition between CBZ and GTP is provided by the observation that the amino acids involved in CBZ binding in fungal tubulin overlap the GTP binding site. Based on studies of fungal sensitivity and resistance to CBZ following site-directed mutagenesis, amino acids 6, 165–167, and 198–200 are thought to be involved in the binding of CBZ (Fujimura et al., 1992Go; Jung et al., 1992Go). Comparison of published tubulin sequence data indicates that these amino acids are conserved between fungal and mammalian tubulins. According to the model presented by Nogales et al. (1998), amino acids 3–8 are thought to contact phosphates on GTP/GDP while amino acids 166–171 are very near the ribose. This is in concordance with the photolabeling of tubulin within amino acids 3–35 by 8-azido-GTP (Jayaram and Haley, 1994Go) and amino acids 155–174 by GTP (Hesse et al., 1987Go). Thus GTP and CBZ binding sites overlap at amino acid 6 and in the region of amino acids 165–167.

The levels of CBZ in these experiments that inhibit assembly and GTP binding were approximately 5-fold lower than those measured in testis following in vivo ip exposure to CBZ, which caused testicular lesions in rats (Lim and Miller, 1997Go). This further enhances the significance of GTP-binding inhibition in the etiology of CBZ toxicity. In these experiments, the responses of brain and testis to CBZ inhibition of tubulin assembly and GTP binding in vitro were similar. However, this does not rule out a differential tissue-specific sensitivity to CBZ in vivo. For example, it is well known that in the various stages of spermatogenesis there is differential expression of proteins. This would also offer an explanation of the stage-specific toxicity reported for CBZ treatment. In neuronal tissue, there is no corollary to the cyclical expression of MAPs in the testis.

In summary, we have found that CBZ is an effective inhibitor of the in vitro assembly of brain and testis tubulin, an effect that is strongly moderated by MAPs. Our data support a mechanism for CBZ action in which CBZ inhibits the binding of GTP by tubulin, thereby interfering with the initiation of tubulin polymerization and the formation of MTs.


    NOTES
 
1 To whom correspondence should be addressed. Fax: 530–752–3394. E-mail: mgmillersears{at}ucdavis.edu.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Allard, E. K., Johnson, K. J., and Boekelheide, K. (1993). Colchicine disrupts the cytoskeleton of rat testis seminiferous epithelium in a stage-dependent manner. Biol. Reprod. 48, 143–153.[Abstract]

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