Regulation of cytoplasmic tubulin carboxypeptidase activity during neural and muscle differentiation: characterization using a microtubule-based assay.
Regulation of cytoplasmic tubulin carboxypeptidase activity during neural and muscle differentiation: characterization using a microtubule-based assay.
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神经和肌肉分化过程中细胞质微管蛋白羧肽酶活性的调节：使用基于微管的测定进行表征。
DOI：
10.1021/bi00140a021
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发表时间：
1992
期刊：
影响因子：
2.9
通讯作者：
Bulinski,JC
中科院分区：
文献类型：
--
作者：
Webster,DR;Modesti,NM;Bulinski,JC
材料与方法[14C]酪氨酸（necc - 289e,~ 500 mCi/mmol）来源于New England Nuclear Research Products， Boston， MA。组织培养化学品、粉末状培养基和血清均来自gibco， Inc.(Grand Island， NY)；所有其他化学品均从Sigma Chemical Co.（St.）获得。路易斯，密苏里州)或费舍尔科学（匹兹堡，宾夕法尼亚州）。高分子TCP底物的制备。10毫克/毫升解决猪或牛脑微管蛋白(包括微管蛋白和地图)是由两个周期随温度而变的装配/拆卸净化(法兰,1986)和放射性标记的有0.1毫米(14 c)酪氨酸在每上孵化PEM (EGTA 0.1 Mpipes, pH值6.7,0.1毫米,0.1毫米MgCl2)补充KC1 0.1米,12.5毫米MgCl2, 3毫米ATP, 1毫米三磷酸鸟苷和0.22毫克的部分纯化的微管蛋白酪氨酸连接酶(~ 100 pmol /(模仿);murufushi, 1980]，最终体积为2ml。然后用DEAE或磷酸纤维素纯化微管蛋白
Materials and Methods Materials.[14C] Tyrosine (NEC-289E,~ 500 mCi/mmol) was obtained from New England Nuclear Research Products, Boston, MA. Tissue culture chemicals, powdered media, and sera were obtained fromGIBCO, Inc.(Grand Island, NY); all other chemicals were obtained from Sigma Chemical Co.(St. Louis, MO) or Fisher Scientific (Pittsburgh, PA). Preparation of Polymeric TCP Substrate. A 10 mg/mL solution of porcine or bovine brain microtubule protein (including both tubulin and MAPs) was prepared by two cycles of temperature-dependent assembly/disassembly purification (Vallee, 1986) and was radiolabeled with 0.1 mM [14C] tyrosine during a 45-min incubation in PEM (0.1 Mpipes, pH 6.7, 0.1 mM EGTA, 0.1 mM MgCl2) supplemented with 0.1 M KC1, 12.5 mM MgCl2, 3 mM ATP, 1 mM GTP, and 0.22 mg of partially purified tubulin tyrosine ligase [~ 100 pmol/(miming); Murofushi, 1980], in a final volume of 2 mL. Tubulin was then purified by either DEAE or phosphocellulose