Purification of tubulin by fast-performance liquid chromatography.
Purification of tubulin by fast-performance liquid chromatography.
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通过快速高效液相色谱纯化微管蛋白。
DOI：
10.1111/j.1749-6632.1986.tb38437.x
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发表时间：
1986
影响因子：
5.2
通讯作者：
Roychowdhury,S
中科院分区：
文献类型：
--
作者：
Gaskin,F;Roychowdhury,S
Fast performance liquid chromatography (FPLC) was used to purify tubulin from porcine microtubule protein (MTP) prepared as previously described using two cycles of assembly-disassembly.'Pharmacia Mono Q (anion exchanger) column chromatography is a fast and reproducible way to purify as little as 1 mg or as much as 100 mg tubulin. The procedure is done at 4-8" C, and the purified tubulin is competent to assemble with 0.02 m M taxol or in 4 M glycerol plus 10% dimethyl sulfoxide (DMSO). The purification scheme is described at the end of this paper. FIGURE 1 demonstrates the chromatography profile of a typical experiment that took 85 rnin to purify 38 mg tubulin. This tubulin fraction contains trace enzyme activities, that is,< Ol mu/mg/min nucleoside diphosphate kinase (NDPK), 0.1 nmoles/mg/min Mg-ATPase, and~ 0. 2 mU/mg/min myokinase activities. Using the same assay conditions, MTP contains 60 mU/mg/min NDPK, 7Q nmoles/mg/min Mg-ATPase, and 87.5 mu/mg/min myokinase. The NDPK and myokinase activities were done as described in references 2 and 3. ATPase activity was determined using ATPy3'P and published procedures in 0.1 M 2-N (morpholino) ethanesulfonic acid (MES), 1 m M MgCI,, and 1 mM ethylene glycol bis (P-aminoethyl ether)-N, N, N, N'-tetracetic acid (EGTA), pH 6.6, 37OCS4 FIGURE 2 demonstrates that a high molecular weight (HMW) component copurifies with tubulin on Mono Q columns and that this component can be removed by Mono S (cation exchanger) chromatography. Tubulin comes off in the void on this latter column. Alternatively, MTP can be put on the Mono S column, followed by the Mono Q column. Tubulin and some microtubule-associated proteins (MAPs) come off in the void from the Mono S column, and this fraction is treated as in steps 4-10 in the purification scheme for the Mono Q column. In our purification scheme, guanosine triphosphate (GTP) and the major (MAPs) come off in the void on the Mono Q column. Tubulin-nucleotide is isolated in the tubulin fraction using MTP containing 0.5 mM GTP that was incubated 20 min at 4" C, 30 min at 37OC, and 10 min at 4 C. The nucleotide/tubulin ratio was determined by precipitating the protein with 50% ethanol at pH 5.6 and measuring the nucleotide concentration at AZs2. The protein contained a nucleotide/tubulin ratio of 2.07. This number is similar to the nucleotide/tubulin ratio of 1.87 that we find for pelleted microtubules that were assembled in 0.5 m M GTP. Both the Mono Q purified tubulin and pelleted microtubules contained 0.77 moles labeled nucleotide/tubulin dimer. Thus most of the exchangeable nucleotide appears to stay bound to tubulin during Mono Q chromatography. In one step it is possible to separate active tubulin nucleotide from the main enzyme contaminants, MAPs, and unbound nucleotide. The purification of tubulin from MTP using Mono Q, Mono S columns is as
