Carbamate formation on tubulin: CO2/bicarbonate buffers protect tubulin from inactivation by reductive methylation and carbamoylation and promote microtubule assembly at alkaline pH.
Carbamate formation on tubulin: CO2/bicarbonate buffers protect tubulin from inactivation by reductive methylation and carbamoylation and promote microtubule assembly at alkaline pH.
复制标题
微管蛋白上的氨基甲酸酯形成：CO2/碳酸氢盐缓冲液可保护微管蛋白免于因还原甲基化和氨基甲酰化而失活，并促进碱性 pH 下的微管组装。
DOI：
10.1021/bi00403a027
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发表时间：
1988
期刊：
影响因子：
2.9
通讯作者：
Berlin,RD
中科院分区：
文献类型：
--
作者：
Clark,RW;Volpi,M;Berlin,RD
Materials and Methods Materials. GTP, MES, PIPES, NaHC03, Na2C03-H20, glycylglycine, and NaCNBH3 were obtained from Sigma Chemical Co. NaCNBH3 used in all experiments was periodically recrystallized as described by Jentoft and Dearborn (1979). HCHO was purchased from Aldrich Chemical Co. and KNCO from Fisher Scientific. Radiolabeled HI4CHO (53 mCi/mmol) was obtained from New England Nuclear and KN14CO (51 mCi/mol) from Amersham. For preliminary experiments,[14C] formaldehyde was purified by anionic ex-change (Jentoft & Dearborn, 1979). However, since 90% of the total radioactivity was recovered after chromatographyand 98% precipitable by 5, 5-dimethyl-2, 3-cyclohexanedione (Lewis et al., 1961), this purification step was later omitted. The fluorescent probe pyranine was purchased from Kodak. Preparation of Microtubule Protein and Tubulin. Bovine brain microtubule protein was isolated by the procedure of Asnes and Wilson (1979), including three cycles of polymerization-depolymerization in 20 mM sodium phosphate, 100 mM sodium glutamate, 1 mM EGTA, and 0.5 mM MgCl2, pH 6.75. Theprotein preparation was stored at-80 C. Except where indicated, on the day of the experiment, aliquots of protein were passed through a final polymerization-depolymerization cycleto yield a preparation of microtubule protein consisting of approximately 80% tubulin and 20% high mo-lecular weight microtubule-associated proteins (MAPs)(4X microtubule protein). When required, purified tubulin (PC-tubulin) was obtained from 3xtubulin by phosphocellulose chromatography. Chromatography was performed in 50 mM PIPES, pH 6.7, using 2.5-3 mg ofprotein/mL of column bed volume. Both PC-tubulin and 4Xmicrotubule protein were exchanged into the appropriate buffer by serial chromatog-raphy using two PD-10 molecular sieve columns (Pharmacia). Removal of free amino groups, ie, glutamate, was essential since it is a substrate for both reductive methylation and carbamate formation. The final proteinconcentration was assayed by the biuret method (Gornall, 1949) using bovine serum albumin as standard. Steady-State Microtubule Assembly. To study the effects of HC03 “/C02 and reductive methylation or carbamoylation on the assembly of microtubule proteins, two types of exper-iments were performed. In most experiments, microtubule protein was first incubated with formaldehyde/NaCNB3 or cyanate in the presence or absence of HC03~/C02. Free reactants were removed by rapid gel filtration, and the two modified samples and a control were then assembled in identical MES buffer (withoutHC03 “/C02) and compared. In some experiments where direct effects ofHCO~/C02 on assembly were studied, an aliquot of microtubule protein was chemically modified (MES buffer) in the absence of HC03~/C02 and the free reactants were removed as described above. This sample was then divided and was assembled either in MES plus HC03"/C02 or in MES buffer alone. For experiments in which HC03~/C02 was employed only during the chemicalmodification step, subsequent assembly was monitored by using standard 1.0-cm quartz cuvettes. In experiments in which assembly was performed with or without HC03"/C02, samples were placed in 2-mL glass vials, adjusted with NaOH or NaHC03, and the vials sealed with rubber sleeve stoppers. For samples to be exposed toC02, the septum was provided with input and exit needles to allow for the continuous delivery of moistened C02. After 6-7 min, the samples were removed by syringe and transferred into pre-gassed cuvettes. These closed cuvettes (Suprasil quartz, micro …
