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 stable to colchicine than bovine microtubules. In order to further characterize this difference, the effect of the colchicine analogue 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cyclo heptatrien-1-one (MTC) was studied on assembly, as measured by turbidity and sedimentation analysis, and on polymer morphology. MTC has the advantage to bind fast and reversible to the colchicine binding site of tubulin even at low temperatures. It was found to bind to one site in cod brain tubulin, with affinity (6.5 +/- 1.5) x 10(5)M-1 at both low or high temperature, similarly to bovine brain tubulin. However, the effect of the binding differed. At substoichiometric concentrations of MTC bovine brain microtubule assembly was almost completely inhibited, while less effect was seen on the mass of polymerized cod microtubule proteins. A preformed bovine tubulin-colchicine complex inhibited the assembly of both cod and bovine microtubules at substoichiometric concentrations, but the effect on the assembly of cod microtubules was less. At higher concentrations (5 x 10(-5) to 1 x 10(-3) M), MTC induced a large amount of cold-stable spirals of cod proteins, whereas abnormal polymers without any defined structure were formed from bovine proteins. Spirals of cod microtubule proteins were only formed in the presence of microtubule associated proteins (MAPs), indicating that the morphological effect of MTC can be modulated by MAPs. The effects of colchicine and MTC differed. At 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 spirals previously found when cod microtubule proteins polymerize in the presence of high Ca2+ concentrations. The present data show that even if the colchicine binding site is conserved between many different species, the bindings have different effects which seem to depend on intrinsic properties of the different tubulins.
de Pereda
J M
JM
Centro de Investigaciones Biologicas, Madrid, Spain.
Wallin
M
M
Billger
M
M
Andreu
J M
JM
D000818:Animals; D001665:Binding Sites; D002417:Cattle / Q000378:metabolism*; D003078:Colchicine / Q000378:metabolism*; D005399:Fishes / Q000378:metabolism*; D008868:Microtubule Proteins / Q000648:ultrastructure*; D013045:Species Specificity; D014334:Tropolone / Q000031:analogs & derivatives* / Q000378:metabolism
D008868:Microtubule Proteins; C030370:2-methoxy-5-(2',3',4'-trimethoxyphenyl)tropone; D014334:Tropolone; D003078:Colchicine