COMPARATIVE-STUDY OF THE COLCHICINE BINDING-SITE AND THE ASSEMBLY OF FISH AND MAMMALIAN MICROTUBULE PROTEINS

Isolated microtubules from cod (Gadus morhua) are apparently more stab le to colchicine than bovine microtubules. In order to further charact erize this difference, the effect of the colchicine analogue 2-methoxy -5-(2,3,4-trimethoxyphenyl)-2,4,6-cyclo heptatrien-1-one (MTC) was stu died on assembly, as measured by turbidity and sedimentation analysis, and on polymer morphology. MTC has the advantage to bind fast and rev ersible to the colchicine binding site of tubulin even at low temperat ures. It was found to bind to one site in cod brain tubulin, with affi nity (6.5 +/- 1.5) x 10(5)M(-1) at both low or high temperature, simil arly to bovine brain tubulin. However, the effect of the binding diffe red. At substoichiometric concentrations of MTC bovine brain microtubu le assembly was almost completely inhibited, while less effect was see n on the mass of polymerized cod microtubule proteins. A preformed bov ine tubulin-colchicine complex inhibited the assembly of both cod and bovine microtubules at substoichiometric concentrations, but the effec t on the assembly of cod microtubules was less. At higher concentratio ns (5 x 10(-5) to 1 x 10(-3)M), MTC induced a large amount of cold-sta ble spirals of cod proteins, whereas abnormal polymers without any def ined structure were formed from bovine proteins. Spirals of cod microt ubule proteins were only formed in the presence of microtubule associa ted proteins (MAPs), indicating that the morphological effect of MTC c an be modulated by MAPs. The effects of colchicine and MTC differed. A t 10(-5)M colchicine no spirals were formed, while at 10(-4)M and 10(- 3)M, a mixture of spirals and aggregates was found. The morphology of the spirals differed both from vinblastine spirals and from the spiral s previously found when cod microtubule proteins polymerize in the pre sence of high Ca2+ concentrations. The present data show that even if the colchicine binding site is conserved between many different specie s, the bindings have different effects which seem to depend on intrins ic properties of the different tubulins. (C) 1995 Wiley-Liss, Inc.