Complete amino acid sequence of alpha-tubulin from porcine brain.

Abstract

The amino acid sequence of a-tubulin from porcine brain was determined by automated and manual Edman degradation of eight sets of overlapping peptides. It comprises 450 residues plus a COOH-terminal tyrosine that is present only in 15% of the material. A region of 40 residues at the COOH-terminus is highly acidic, mainly due to 16 glutamyl residues. This high concentration ofnegative charge suggests a region for binding cations. At least six positions, most of them around position 270, are occupied by two amino acid residues each. Several of these exchange sites were assigned to specific peptides by analysis of the purified corresponding fragments. These data indicate four a-tubulins in porcine brain. Although a-tubulin on the whole is unrelated to other proteins, there are regions that can be correlated to sequences of the myosin head, to actin, to tropomyosin, and to troponins C and T.Tubulins occur in all eukaryotic cells as the constituents of microtubules, which participate in cell division, intracellular transport and secretion processes, ciliary and flagellar movement, morphogenesis, and cell orientation. Tubulins from widely differing species and cell types appear to be remarkably similar regarding composition, molecular weight, binding of cytostatic and psychopharmacological drugs, immunological crossreactivity, and capacity to copolymerize. Yet even within one cell, there are several types of microtubules that have differing stabilities and assemble into distinct organelles at various times. Knowledge of the primary structure should clarify whether there is just one tubulin for all functions or whether there exists a family of similar proteins. It will also facilitate mapping of binding sites for various ligands, production of antibodies to well-defined antigenic sites, matching of protein structure with that of messengers and genes, and investigation of functionally defective tubulin mutants. Comparison of the structure with those of known proteins may give hints for experiments regarding tubulin function.Tubulin in solution is assumed to exist as a heterodimer of two chains, a and f, each with a molecular weight4of 50,000, and very similar amino acid compositions. Yet functional differences have been reported. For example, only a-tubulin (from blood platelets) binds cyclic AMP (1) and only /3-tubulin binds exchangeable GTP (2). Here we present the sequence of the a-chain from porcine brain and report on the general strategy used. MATERIALS AND METHODSWe have purified tubulin from porcine brain by a modification of the methods used by Eipper (3) and by Luduena et al. (4). The 100,000 X g brain supernatant in 0.05 M sodium pyrophosphate buffer (pH 7.0) was incubated with 0.1 mM colchicine for 15 min at 37°C before chromatography on DEAE-cellulose with a linear gradient of 0.1-0.3 M sodium chloride. Tubulin was identified by the fluorescence of its complex with colchicine (5). The preparation was reduced, alkylated with iodoacetic acid, and assayed for protein impurities by disc gel electrophore...

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Interactions of actin and tubulin with human deoxyhemoglobin

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Direct Photoaffinity Labeling by Dolastatin 10 of the Amino-terminal Peptide of β-Tubulin Containing Cysteine 12

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