Preparation and characterization of posttranslationally modified tubulins from Artemia franciscana.

Abstract

Tubulin heterogeneity within eukaryotic cells is generated by differential gene expression and posttranslational modification of alpha- and beta-tubulin gene products, either as heterodimers or when polymerized into microtubules. The characterization of posttranslationally modified tubulins from the crustacean Artemia franciscana is presented, although tubulins from other sources can be studied with these procedures. Tubulin is prepared from cell free extracts by taxol-induced assembly and centrifugation of microtubules through sucrose cushions, which also yields microtubule-associated proteins, or it is purified to apparent homogeneity by relatively simple chromatographic procedures and assembly/disassembly steps. To detect posttranslationally modified tubulins protein samples are electrophoresed in sodium dodecyl sulfate (SDS) polyacrylamide gels, blotted to nitrocellulose membranes and probed with isoform-specific antibodies. Isotubulins, for which gene-encoded amino acid differences and posttranslational modifications generate charge variations, are resolved in two-dimensional gels using isoelectric focusing followed by SDS polyacrylamide gel electrophoresis, a procedure useful for resolution of microtubule-associated proteins. Isoforms patterns are visualized by Coomassie blue and/or silver staining and individual isoforms are identified by antibody reactivity on Western blots. Tubulin isoforms are localized in Artemia by immunofluorescent staining of larvae. The focus of this chapter is the purification of tubulin from a nonneural source and characterization of tyrosinated, detyrosinated, and nontyrosinatable alpha-tubulins using polyclonal antibodies made to carboxy-terminal peptides of each isoform.