Abstract
Zinc ion-induced assembly of tubulin was followed using electron microscopy and turbidimetric measurements. A scheme utilizing repeated cycles of assembly and disassembly was used to prepare tubulin and microtubule-associated proteins (MAPs) (Shelanski, M. L., Gaskin, F., and Cantor, C. R. (1973) Proc. Natl. Acad. Sci. U. S. A. 70, 765-768). Tubulin was further purified by phosphocellulose chromatography to remove the MAPs (Weingarten, M., Lockwood, A. H., Hwo, S-Y, and Kirschner, M. W. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 1858-1862). In tubulin preparations containing MAPs and added GTP, Zn2+-induced sheets of 15 to 60 protofilaments oriented in parallel. In the absence of MAPs and/or added GTP, Zn2+ induced the formation of sheets which wrapped quite specifically and serial sections were often consistent with a tubular structure of approximately 220 nm. The assembly of recycled tubulin + GTP and 0 to 1 mM Zn2+ was analyzed by A350 as a function of time at 30 degrees. The greater the concentration of Zn2+, the shorter the lag time, the faster the rate after the lag, and the greater the plateau value of A350. Although turbidimetric measurements can be used to quantitate microtubules, they are not quantitative for Zn2+-induced sheets.
Highlights
Zinc ion-induced assembly of tubulin was followed using electron microscopy and turbidimetric measurements
Tubulin which has been purified by P-cellulose chromatography is relatively free of microtubule-associated proteins (MAPS)’ and using our assembly conditions will not assemble into microtubules unless a specific microtubule-associated protein called 7 is added back [14, 16]
A, is proportional to the amount of tubulin assembled into microtubules, and the kinetics of assembly are characterized by a lag time, a rate of assembly, and a plateau signifying an equilibrium between tubules and tubulin [2]
Summary
Zinc ion-induced assembly of tubulin was followed using electron microscopy and turbidimetric measurements. In tubulin preparations containing MAPS and added GTP, Zn’+-induced sheets of 15 to 60 protofilaments oriented in parallel. In the absence of MAPS and/or added GTP, Zn2+ induced the formation of sheets which wrapped quite and serial sections were often consistent with a tubular structure of -220 nm.
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