Journal
BIOCHEMISTRY
Volume 42, Issue 18, Pages 5349-5357Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi027293o
Keywords
-
Categories
Funding
- NCI NIH HHS [CA 39821, CA77263] Funding Source: Medline
- NIAID NIH HHS [AI49749] Funding Source: Medline
- NIGMS NIH HHS [5T32 GM07260] Funding Source: Medline
Ask authors/readers for more resources
Six human aalpha-tubulin and seven human beta-tubulin isotypes, each of which can undergo posttranslational modifications, have been detected by the reverse transcriptase-polymerase chain reaction. This repertoire of tubulin isotypes plays a role in development and in the building of specialized microtubule-based structures. In cell lines, the relationship between resistance to microtubule-interacting drugs and altered tubulin isotype expression profiles is often established by quantitation of cDNA and/or Western blot analysis. Tubulin mutations in major isotypes are detected by sequencing cDNA, but more analysis of expression of tubulin mutations at the protein level, to assess their role in drug resistance, is needed. We utilized a Taxol-based purification and high-resolution isoelectrofocusing combined with a mass spectrometry-based analysis of tubulin. This approach has allowed the separation and relative quantitation of tubulin isotypes having a difference in isoelectric point values of 0.01, without the need for two-dimensional gel electrophoresis. The specificity of tubulin isotype antibodies also has been established. In cell lines resistant to microtubule-stabilizing drugs that express heterozygous tubulin mutations, the relative amount of mutant tubulin expression has been determined. In these cell lines, the absence of betaII- and betaIVa-tubulin has been demonstrated, and an increased level of expression of betaIII-tubulin in resistant cells has been confirmed, indicating that this tubulin isotype is a unique marker of resistance.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available