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

Abstract

The primary structure ofporcine brain (3-tubulin was determined by automated and manual Edman degradation of six sets of overlapping peptides. The protein consists of 445 amino acid residues and has a minimum of six positions that are heterogeneous, indicating at least two (3-tubulins in porcine brain. Com-parison of the optimally aligned sequences of a-tubulin and 13-tubulin indicates that 41% of their primary structures are identical. A region rich in glycyl residues is similar both in sequence and predicted secondary structure to the phosphate binding loop of several nucleotide binding enzymes. fi-Tubulin contains a highly acidic COOH-terminal region that resembles the NH2-terminus of troponin T.Microtubules are helical arrays of alternating globular units of a-tubulin and 83-tubulin, each having a molecular weight of 50,000, a similar amino acid composition, and a similar overall shape (for review, see ref. 1). Yet these subunits seem to differ in function, as indicated by the binding of exchangeable GTP to f3-tubulin (2) and by the participation of a tissue-specific /3-tubulin in the meiotic spindle and other stages of spermatogenesis in Drosophila (3, 4). P-Tubulin mutants have been shown to cause reduced binding of cytostatic drugs (5,6) and were used to demonstrate the involvement of microtubules in nuclear movement in germinating spores of Aspergillus (7). A knowledge of the primary structure of ,3-tubulin, along with that of a-tubulin, could be helpful in understanding how different tubulins function, how the assembly of microtubules into various organelles is regulated, and how certain drugs and nucleotides are bound with high affinity. The sequence of a-tubulin has already shown several variants of this protein in brain (8). Regional similarities were found to muscle proteins, indicating the possibility ofcommon biochemical features in muscular and microtubular movement. Here we present the sequence of l3-tubulin from porcine brain and compare it with that of the a-chain. MATERIALS AND METHODSTubulin was purified from porcine brain as described (8). The 100,000 X g brain supernatant in 0.05 M sodium pyrophosphate buffer, pH 7.0, was chromatographed on DEAE-cellulose with a linear gradient of0. 1-0. 3 M sodium chloride. The protein was reduced and alkylated with iodoacetic acid and assayed for contaminating proteins by disc gel electrophoresis in the system of Yang and Criddle (9) using 8% polyacrylamide gels. a-and ,/3chains were separated on hydroxylapatite in 0.1% NaDodSO4 with a linear gradient of 0.2-0.4 M sodium phosphate (10). Fractions were assayed for purity by gel electrophoresis as above. Only }3-chain preparations containing <5% impurities were used for sequence determination.To remove NaDodSO4, the protein was dialyzed against 1 mM ammonium bicarbonate, then, the solution was concentrated by vacuum evaporation and brought to pH 5.5 with acetic acid, and the protein was precipitated with 9 vol of ice-cold acetone. The supernatant was discarded after 2 hr at -200C, and the precipit...

A novel tau‐tubulin kinase from bovine brain

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Characterization of the Taxol Binding Site on the Microtubule

“…The amino acid sequence obtained was identical with those amino acids in the CNBr fragment 268 -293 of pig brain ␤-tubulin (21). No identifiable amino acid could be detected at the position corresponding to amino acid 282 in our sequencing studies.…”

“…4C) was digested with clostripain and the resulting tritium-labeled peptide, after purification by C8 reverse phase chromatography, was subjected to N-terminal Edman sequence analysis. The amino acid residues depicted on the bar diagram come from the published pig sequence (21). The radioactivity (cpm) released during each cycle was determined.…”

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

“…There are only three such sites in tubulin. One is located in the ␣-subunit (positions 306 -307) (45) and two in the ␤-subunit (positions 31-32 and positions 304 -305) (46). In our previous studies with decyl-agarose-purified ␤-tubulin cross-linked to colchicine analogs (3, 4), we had noted a number of secondary cleavage sites derived from the initially formed larger peptides.…”