Depletion of acetylated alpha-tubulin during microtubule purification from bovine brain gray and white matter regions.
We have followed the fate of acetylated alpha-tubulin during microtubule (MT) purification from both gray matter- and white matter-enriched bovine brain regions, using quantitative immunoblot assays employing well characterized monoclonal antibodies specific for acetylated alpha-tubulin and all beta-tubulins. Our results show that crude homogenates from both gray matter and white matter brain regions contain the same proportion of acetylated alpha- to total tubulin. We have found that the acetylated isoform cycles more efficiently with MTs from gray matter than with MTs from white matter. However, the resultant purified MT preparations from both gray and white matter regions are greatly depleted in the acetylated isoform compared to the tubulin in the initial homogenates because most of the acetylated alpha-tubulin from both tissue sources partitions with the cold-insoluble fraction of the initial brain homogenate. A low percentage of the acetylated alpha-tubulin in brain homogenates does become incorporated into MTs initially, but this subpopulation of acetylated alpha-tubulin then becomes associated with a cold- and calcium-insoluble fraction of the MT preparation. These results demonstrate that the standard brain MT and tubulin preparations used by most investigators for in vitro studies are greatly depleted in acetylated alpha-tubulin, and thus provide poor model systems for the analysis of the function of this tubulin isoform.
Kim
H
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Department of Cell Biology, University of Alabama, Birmingham 35294-0005.
D000107:Acetylation; D000818:Animals; D000906:Antibodies; D001921:Brain / Q000648:ultrastructure*; D002118:Calcium; D002417:Cattle; D002458:Cell Fractionation / Q000379:methods; D002499:Centrifugation, Density Gradient / Q000379:methods; D002852:Chromatography, Ion Exchange; D003080:Cold Temperature; D015151:Immunoblotting; D008870:Microtubules / Q000648:ultrastructure*; D010487:Periaqueductal Gray / Q000648:ultrastructure*; D012995:Solubility; D014404:Tubulin / Q000302:isolation & purification* / Q000378:metabolism
D000906:Antibodies; D014404:Tubulin; D002118:Calcium