Isolation of bovine brain microtubule protein without glycerol: Polymerization kinetics change during purification cycles

Abstract

Microtubule protein was isolated from bovine brain without glycerol by three cycles of polymerization, utilizing conditions for maximum assembly. Maximum polymerization occurred over a narrow GTP concentration range in crude extracts, with peak polymerization at 2.5 m m GTP. Peak polymerization occurred at 1.5 m m GTP and 0.5 m m MgCl 2 after three polymerization cycles. In a typical purification, 72 mg of microtubule protein was obtained per 100 g (wet weight) of cerebral cortex. Of this, a minimum of 52% was tubulin and 24% was high molecular weight-microtubule associated proteins (HMW-MAPs). Microtubule polymerization kinetics changed during purification cycles. In initial cycles, the total quantity of microtubules decreased significantly after peak levels of assembly were attained. In the third cycle, the decrease was minimal and only apparent at high protein concentrations. Peak viscosity correlated with tubulin and HMW-MAPs, but the magnitude of the decrease was independent, suggesting that some other component, eliminated during purification, was responsible for the decrease.