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: Anna Maria Zambito, Leslie Knipling, J Wolff
• Journal: Biochimica et biophysica acta (Biochim Biophys Acta) Vol. 1601 Issue 2 Pg. 200-7 (Dec 16 2002) ISSN: 0006-3002 [Print] Netherlands
• PMID: 12445483 (Publication Type: Journal Article)

Chemical References

• Peptide Fragments
• Tubulin
• tubulin S
• Palmitic Acid

Topics

• Adrenal Gland Neoplasms
• Animals
• Brain (metabolism)
• Cell Membrane (metabolism)
• Dimerization
• Genetic Variation
• Isoelectric Focusing
• PC12 Cells
• Palmitic Acid (metabolism)
• Peptide Fragments (genetics, isolation & purification)
• Pheochromocytoma (genetics)
• Protein Processing, Post-Translational
• Rats
• Tubulin (genetics, isolation & purification)
• Tumor Cells, Cultured