Charge variants of tubulin, tubulin S, membrane-bound and palmitoylated tubulin from brain and pheochromocytoma cells.
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.
Zambito
Anna Maria
AM
Laboratory of Biochemistry and Genetics, NIDDK, NIH, Building 8, Room 2A23, 9000 Rockville Pike, Bethesda, MD 20892, USA.
Knipling
Leslie
L
Wolff
J
J
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
D010446:Peptide Fragments; D014404:Tubulin; C046279:tubulin S; D019308:Palmitic Acid