Vinblastine-induced formation of tubulin polymers is electrostatically regulated and nucleated.
Vinblastine promotes tubulin polymerization as measured by turbidity at 400 nm. Unlike microtubule assembly, this mode of polymerization does not require GTP and, in fact, GTP inhibits polymerization, as do other G nucleotides in the potency order: GtetraP > or = GTP > GDP > GMP > no nucleotide. Inhibition is not nucleoside-specific as ATP, ADP, and CTP also inhibit, and inorganic oligophosphates are as inhibitory as nucleotides in the order tetraphosphate approximately triphosphate > pyrophosphate >> phosphate. Inhibition of polymerization is a rough function of the number of anionic charges and can be mimicked by suramin or tartrate. It is not due to sequestration of magnesium or to debinding of vinblastine. The anion-induced decrease in turbidity generation is reflected in the amount of tubulin that is pelletable, but even in the absence of turbidity significant pelletable tubulin persists which can be assessed by 90 degree light scattering. Formation of this polymer is less sensitive to anions. Shearing of GTP-inhibited and vinblastine-induced samples promotes turbidity and addition of seeds made from vinblastine polymers leads to rapid increases in turbidity in a concentration-dependent manner. Adjustment of the vinblastine concentration permits the demonstration of a latent period for polymerization that can be shortened by polymer seeds. Vinblastine-induced polymerization shows a critical concentration, and, in the presence of GTP, two distinct critical concentrations can be identified. We conclude that charge-charge interactions play a significant role in the formation of vinblastine-induced polymers, and that their formation is a two-step process resembling a nucleation/elongation mechanism.
Rai
S S
SS
Laboratory of Biochemical Pharmacology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Wolff
J
J
D006160:Guanosine Triphosphate / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D011108:Polymers / Q000737:chemistry; D055672:Static Electricity; D014404:Tubulin / Q000737:chemistry*; D014747:Vinblastine / Q000494:pharmacology*
D011108:Polymers; D014404:Tubulin; D014747:Vinblastine; D006160:Guanosine Triphosphate; D008274:Magnesium