Separation of assembly-competent tubulin from brain microtubule protein preparations using a fast-performance liquid chromatography procedure.
Fast-performance liquid chromatography was used to purify assembly-competent tubulin from porcine brain microtubule protein prepared by two cycles of assembly-disassembly. Microtubule protein (1-100 mg at 1.5-2.5 mg/ml) in buffer consisting of 0.1 M 2-(N-morpholino)ethanesulfonic acid, 0.5 mM MgCl2, 1 mM EGTA, 0.3 M KCl, and 0.02 mM GTP (pH 6.6) was applied to the Mono Q column (anion exchanger). The microtubule-associated proteins, GTP and GDP, eluted in the void volume. The tubulin fraction eluted at 0.45-0.50 M KCl with 65-80% recovery. The tubulin fraction contained trace enzymatic activities when compared with the starting microtubule protein, i.e., less than 1 versus 60 mU/mg/min of nucleoside diphosphate kinase, 0.2 versus 7.0 nmol/mg/min of Mg-ATPase at pH 6.6, and 0.2 versus 88 mU/mg/min of adenylate kinase. Both the Mono Q-purified tubulin and the pelleted microtubules that were assembled in 0.5 mM [3H]GTP contained 0.77 mol of labeled nucleotide/tubulin dimer. The Mono Q-purified tubulin fraction was competent to assemble, i.e., the critical concentration was 0.1 mg/ml in the presence of 0.03 mM taxol and 1 mM GTP at 37 degrees C. The Mono Q-purified tubulin fraction showed trace high-molecular-weight components, which were removed on Mono S (cation exchanger) columns. Alternatively, microtubule protein in buffer was applied to the Mono S column. Tubulin, trace nontubulin proteins, and several enzymatic activities came off in the void volume. A combination of Mono Q-Mono S or Mono S-Mono Q chromatography resulted in highly purified protein.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D001923:Brain Chemistry; D017301:Ca(2+) Mg(2+)-ATPase / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006153:Guanosine Diphosphate / Q000032:analysis; D006160:Guanosine Triphosphate / Q000032:analysis; D046911:Macromolecular Substances; D008868:Microtubule Proteins / Q000302:isolation & purification*; D008970:Molecular Weight; D009701:Nucleoside-Diphosphate Kinase / Q000378:metabolism; D013552:Swine; D014404:Tubulin / Q000302:isolation & purification* / Q000378:metabolism
D046911:Macromolecular Substances; D008868:Microtubule Proteins; D014404:Tubulin; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D009701:Nucleoside-Diphosphate Kinase; D017301:Ca(2+) Mg(2+)-ATPase