Characterization of brain microtobule proteins prepared by selective removal of mitochondrial and synaptosomal components

T. L. Karr, H. D. White, D. L. Purich

Research output: Contribution to journalArticlepeer-review

78 Scopus citations

Abstract

Tubulin purified by the method of Shelanski, M.L., Gaskin, F., and Cantor, R.C. ((1973) Proc. Natl. Acad. Sci. U.S.A. 70, 765-768) contains significant levels of glutamate dehydrogenase resulting from hypotonic shock of mitochondria. Presumably, the anionic character of tubulin leads to ionic associations with compartmentalized proteins not normally associated with tubulin in vivo. A new sucrose extraction method for tubulin purification, which maintains cellular organelle integrity during extraction while producing high yields, is described. Briefly, this involves extraction of cerebral cortical tissue in a sucrose medium and minor modification of the Shelanski protocol. Assays of two mitochondrial enzymes, cytochrome c oxidase and glutamate dehydrogenase, indicated that the sucrose extraction method contains 10- to 20-fold less enzyme activity than the former hypotonic method. Sodium dodecyl sulfate-polyacryalmide gel electrophoresis indicated distinctly less protein contamination of the microtubules obtained by the sucrose extraction procedure at all stages of purification. High molecular weight microtubule-associated proteins had altered electrophoretic behavior, and the slowest migrating band was consistently larger in the new protocol. The microtubule protein from the sucrose extraction method demonstrated normal assembly kinetics as well as Ca(2+)-, drug-, and cold-induced depolymerization. The significance of the lower critical concentration for assembly (0.09 mg/ml versus 0.16 mg/ml) and the altered microtubule-associated proteins composition are currently under investigation.

Original languageEnglish (US)
Pages (from-to)6107-6111
Number of pages5
JournalJournal of Biological Chemistry
Volume254
Issue number13
StatePublished - Jan 1 1979
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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