Tubulin from cultured tobacco cells: isolation and identification based on similarities to brain tubulin

Autor: Philip Filner, Narendra S. Yadav
Rok vydání: 1983
Předmět:
Zdroj: Planta. 157:46-52
ISSN: 1432-2048
0032-0935
Popis: The microtubule protein, tubulin, was isolated from most other proteins of cell suspension cultures of Nicotiana tabacum L. by its copolymerization with cow-brain tubulin. Cow-brain tubulin was added to the soluble protein fraction of extract from (35)S-labeled tobacco cells and subjected to two cycles of temperature-dependent assembly-disassembly (copolymerization). When analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) about 70% of the radioactivity in the twice copolymerized protein was found in a prominent doublet migrating close to the doublet of brain tubulin. When analyzed by two-dimensional isoelectric-focusing-SDS-PAGE the radioactive doublet behaved like the doublet of brain tubulin. Limited proteolysis of the individual polypeptides of the coublets showed that, while the peptide maps of the leading radioactive band and of the β-subunit of brain tubulin were virtually indistinguishable, the maps of the trailing radioactive band and of the α-subunit of brain tubulin, though similar, were not identical. Most of the copolymerized (35)S-labeled protein also behaved like brain tubulin during gel filtration and ion-exchange chromatography. It is concluded that the doublet of radioactive polypeptides isolated by copolymerization with brain tubulin are tobacco tubulin polypeptides that have, in their native as well as denatured forms, properties very similar to, but not identical with, cow brain tubulin. Apparently, tubulin has been highly conserved during evolution.
Databáze: OpenAIRE
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