The role of GTP binding and microtubule-associated proteins in the inhibition of microtubule assembly by carbendazim

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-associat ed proteins (MAPs) were isolated from rat testis and brain using two techni ques. The effects of CBZ on MT assembly were compared with the known microt ubule (MT) disrupters, colchicine and nocodazole. CBZ (100 muM) had no effe ct on the assembly of MTs from MAP-containing tubulin isolated with one cyc le of glycerol-dependent assembly and disassembly while colchicine (40 muM) and nocodazole (12.5 muM) strongly inhibited the assembly reaction. Simila rly, formation of MTs from tubulin prepared with two cycles of glycerol-dep endent assembly was strongly inhibited by colchicine and nocodazole but onl y weakly by CBZ. All three compounds inhibited the assembly of MTs from MAP -free tubulin isolated with glutamate. However, the inhibition by CBZ was r eversed by the inclusion of high-molecular-weight MAPs and not by unrelated protein (bovine serum albumin, BSA). Addition of nocodazole to assembled M Ts caused immediate depolymerization, whereas CBZ did not directly cause de polymerization. However CBZ was an effective inhibitor of the polymerizatio n of depolymerized tubulin. In competitive binding assays, CBZ was found to inhibit the binding of guanosine triphosphate (GTP) to tubulin. The data s uggest that CBZ interferes with initial events of MT polymerization, specif ically GTP binding, and that MAPs moderate this effect.