Effects of Various Anions, Glutamate and GTP on Microtubule Assembly In Vitro

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Abstract
Microtubule (MT) assembly from one-cycle porcine brain tubulin was analyzed by viscometry, turbidimetry, sedimentation and electron microscopy in the presence of various kinds of anions and different amounts of GTP. Using K or Na salts, the effectiveness for increasing the viscosity of tubulin solution was in the order glutamate, fluoride, acetate >chloride>bromide>iodide. The magnitude of tubulin polymerization was measured as a function of the K glutamate concentration by the sedimentation method. The amount of assembled MT's was saturated even at a glutamate concentration of 0.05 M and the tubulin-MT equilibrium remained stationary until the glutamate concentration was increased to 0.9 M. On the other hand, the specific viscosity increased with increasing glutamate concentration from 0.05 to 0.35 m. Furthermore, at GTP concentrations ranging from 0.4 to 1.4 mM, tubulin solution containing 0.1 M glutamate exhibited a stable and constant plateau value of turbidity. However, simultaneous viscosity measurements indicated that the viscosity increase was dependent on the GTP concentration. The maximum viscosity attained at 1.4 mM GTP was twice that attained at 0.4 mM GTP. Viscometry again did not accurately reflect the mass concentration of the assembled MT's. After the viscosity of a tubulin solution with low GTP content had reached a plateau, additional GTP caused the viscosity to increase to twice the original level without any change in turbidity. The apparent GTPase activity of the tubulin solution was 0.002 μmol P1 released/mg/min, which caused a significant amount of GTP to be hydrolyzed during a long incubation. These results suggested that the changes in the viscosity of MT suspension without any change in the mass concentration reflect changes in the flexibility of MT's, probably caused by reversible loosening of a part of the MT surface lattice depending on the concentrations of glutamate and GTP.

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