title: Charge variants of tubulin, tubulin S, membrane-bound and palmitoylated tubulin from brain and pheochromocytoma cells.
abstract: Isoelectric focusing (IEF) of only approximately 1 microg of rat brain tubulin yields 27-30 distinct charge variants in the pH range of 4.5-5.4 with band separations of 0.01-0.02 pH units as detected by silver staining. Variants can be efficiently transferred from the immobilized gradient strip to polyvinylidene difluoride (PVDF) membranes for reaction with monoclonal antibodies. C-terminal-directed antibodies to alpha- and beta-tubulin yield patterns similar to N-terminal-directed antibodies. Removal of the acidic C-termini with subtilisin to form tubulin S increases the pI values by approximately 1 pH unit, leads to a loss in the isoelectric distinction between the alpha- and beta-tubulin variants seen by N-terminal-directed antibodies, and abolishes reactions with all beta-variants and all but three alpha variants by C-terminal-directed antibodies (TU-04 and TU-14). Many, but not all, of the variants are substrates for autopalmitoylation of rat brain tubulin. The distribution of isoelectric variants differs between cytoplasm and membrane fractions from PC12 pheochromocytoma cells. A potential role for different variants is suggested.
authors.affiliation: Laboratory of Biochemistry and Genetics, NIDDK, NIH, Building 8, Room 2A23, 9000 Rockville Pike, Bethesda, MD 20892, USA.
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: Zambito
authors.forename: Leslie
authors.initials: L
authors.lastname: Knipling
authors.forename: J
authors.initials: J
authors.lastname: Wolff
mesh_terms: D000310:Adrenal Gland Neoplasms; D000818:Animals; D001921:Brain / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D019281:Dimerization; D014644:Genetic Variation; D007525:Isoelectric Focusing; D016716:PC12 Cells; D019308:Palmitic Acid / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics* / Q000302:isolation & purification; D010673:Pheochromocytoma / Q000235:genetics*; D011499:Protein Processing, Post-Translational; D051381:Rats; D014404:Tubulin / Q000235:genetics* / Q000302:isolation & purification; D014407:Tumor Cells, Cultured
chemical_list: D010446:Peptide Fragments; D014404:Tubulin; C046279:tubulin S; D019308:Palmitic Acid