Isolation of mammalian brain tubulin by amino-activated gel chromatography.
In the present study, we report the isolation of the acidic structural protein tubulin using a number of amino-activated gels. Crude 100,000 g supernatant derived from sheep brain was applied to gels activated with either aminohexyl, aminoethyl, argininyl, diethylaminoethyl, lysinyl and polylysinyl residues and eluted with three distinct sequential buffer changes (pH 6.5): (i) 0.025-0.4 M morpholinoethanesulphonic acid; (ii) 0.076 and 0.379 M ammonium sulphate in 0.025 M morpholinoethanesulphonic acid; and (iii) 0.8 M sodium chloride in 0.025 M morpholinoethanesulphonic acid. Tubulin was recovered from all columns in an enriched form. However, the elution profile and purity, as judged by [3H]colchicine binding and electrophoresis, varied with the ligand. Hydrophobic gels, such as diethylaminoethyl and aminohexyl, required elution with high-ionic-strength buffers (0.8 M sodium chloride) and significant inhibition of [3H]colchicine activity resulted. This problem was avoided with the hydrophilic ligands such as arginine, polylysine and aminoethyl. Manipulation of elution conditions enabled complete elution of tubulin from arginine-activated gels in 2.5% ammonium sulphate without detectable losses of [3H]colchicine binding activity and with purity comparable to that achieved using diethylaminoethyl Sephacel.
Lacey
E
E
McMaster Laboratory, CSIRO Division of Animal Health, N.S.W. Australia.
Snowdon
K L
KL
D000588:Amines; D000818:Animals; D001923:Brain Chemistry; D002850:Chromatography, Gel / Q000379:methods*; D003078:Colchicine / Q000378:metabolism; D005782:Gels; D008024:Ligands; D012756:Sheep; D014404:Tubulin / Q000302:isolation & purification* / Q000378:metabolism
D000588:Amines; D005782:Gels; D008024:Ligands; D014404:Tubulin; D003078:Colchicine