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Scheme of the procedure for the purification of lung tubulin The method indicated in the Experimental section was followed, and the protein present in each step of purification was characterized by electrophoresis. 1984
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Tubulin from pig lung was quantitatively determined, isolated and characterized. It accounted for about 0.3-0.4% of the total soluble protein of pig lung, as measured by colchicine binding or radioimmunoassay. Purified tubulin was obtained by several cycles of polymerization and depolymerization in the presence of dimethyl sulphoxide and 2H2O as st...
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... to achieve microtubule polymerization. Lung extracts were accordingly used at protein concentrations of 40mg/ml for the poly- Pig lung tubulin merization of tubulin at 35°C. The extracts also contained 10% (v/v) dimethyl sulphoxide, 4M-glycerol and 70% (v/v) 2H20. Microtubule protein was further purified by an additional polymerization cycle. Fig. 3 shows the protocol for the isolation of lung tubulin by successive cycles of polymerization-depolymerization and the protein composition at the different steps of ...
Citations
... Protein IEF was performed as described by Diez et al. (4). ...
Purified brain tubulin subjected to an exhaustive phosphatase treatment can be rephosphorylated by casein kinase II. This phosphorylation takes place mainly on a serine residue, which has been located at the carboxy-terminal domain of the beta-subunit. Interestingly, tubulin phosphorylated by casein kinase II retains its ability to polymerize in accordance with descriptions by other authors of in vivo phosphorylated tubulin. Moreover, the V8 phosphopeptide patterns of both tubulin phosphorylated in vitro by casein kinase II and tubulin phosphorylated in vivo in N2A cells are quite similar, and different from that of tubulin phosphorylated in vitro by Ca/calmodulin-dependent kinase II. On the other hand, we have found an endogenous casein kinase II-like activity in purified brain microtubule protein that uses GTP and ATP as phosphate donors, is inhibited by heparin, and phosphorylates phosphatase-treated tubulin. Thus it appears that a casein kinase II-like activity should be considered a candidate for the observed phosphorylation of beta-tubulin in vivo in brain or neuroblastoma cells.
... These gels were silver-stained[37]. Isoelectric focusing was done in a LKB multiphor apparatus following the method of O'Farrell[38]and the modifications described by Diez et al.[39]. A mixture of ampholines (LKB) pH 3.5-5/pH 4-6.5 in a volume ratio of 4: 1 was employed. ...
MAP2 purified without heat treatment was compared to MAP2 prepared with the usual boiling step. No significant differences were found by isoelectric focusing, peptide mapping, fluorescence spectroscopy and circular dichroism. Hydrodynamically, MAP2 is a monomer with s degree 20,w = 3.5 +/- 0.1 S and Mr = 220 000. The molecular mass was measured by sedimentation equilibrium and verified by gel chromatography in denaturing solvent and dodecyl sulfate gel electrophoresis at different acrylamide concentrations. The high frictional ratio, f/fmin = 3.7, indicated that MAP2 was clearly not globular but had either a very elongated shape or an unordered expanded structure. Circular dichroic results were consistent with a predominantly unordered structure independently of the preparation procedure. This supports the notion that MAP2, in solution, is a very flexible and non-compact protein. Examination of the circular dichroism of MAP2 as a function of temperature indicated that the thermal stability of this protein was probably due to its mostly unordered structure and the fact that the little ordered structure present was resistant to thermal denaturation.
Tubulin was purified from the brain of the catfishHeteropneustes fossilis by cycles of temperature-dependent assembly and disassembly. Fish tubulin assembles into microtubules in the absence of high
molecular weight microtubule associated proteins. Its subunits comigrate with goat brain α andβ tubulin subunits and is composed of 4 major α andβ tubulins each as analyzed by isoelectric focusing and two dimensional gel electrophoresis. Peptide mapping showed it to be
very similar to goat brain tubulin. Polymerization of catfish brain tubulin occurs optimally between 18–37°C and the critical
protein concentrations of assembly at 18°C and 37°C are the same, as opposed to mammalian brain tubulins.
The proportion of tubulin and its isoform pattern have been analyzed at different stages in the development of Drosophila melanogaster. Tubulin proportion varied during development, the highest proportion being found at embryogenesis where two α- and β- (one of them transitory) tubulin subunits were found. During the larval stage, the proportion of total tubulin decreased but new α-isotubulins were identified. These α-isotubulins were also present at the adult stage and all of them could be incorporated into microtubules assembled in vitro.
Tubulin was estimated to account for 0.3% of the total soluble protein in Trichinella spiralis cytosolic fractions. Tubulin from T. spiralis was partially purified by precipitation with either taxol or vinblastine sulphate. Immunoblotting with alpha- and beta-tubulin monoclonal antibodies revealed the presence of tubulin in T. spiralis partially purified preparations. Electrophoretic mobility of T. spiralis tubulin in sodium dodecyl sulphate-polyacrylamide gels was very similar to that shown by pig brain tubulin. Further studies with colchicine binding assays indicated that T. spiralis tubulin has binding features similar to that of tubulin from other nematodes: colchicine association constant = 8.1 x 10(-4) M and competitive inhibition of colchicine binding by podophyllotoxin, with an inhibition constant of 1.3 x 10(-6) M. Finally, inhibition of colchicine binding by several benzimidazoles (mebendazole, fenbendazole, oxibendazole and albendazole) was investigated. All the benzimidazoles inhibited colchicine binding in a competitive manner, with inhibition constant values ranging from 1.4 x 10(-7) M (mebendazole) to 3.9 x 10(-6) M (fenbendazole).
A cold-labile fraction of microtubules with unusual properties was isolated from the brain of the Atlantic cod (Gadus morhua). The yield was low, approximately six times lower than that for bovine brain microtubules. This was mainly caused by the presence of a large amount of cold-stable microtubules, which were not broken down during the disassembly step in the temperature-dependent assembly-disassembly isolation procedure and were therefore lost. The isolated cold-labile cod microtubules contained usually only a low amount of microtubule-associated proteins (MAPs). Three high molecular mass proteins were found, of which one was recognized as MAP2. Cod MAP2 differed from mammalian brain MAP2; it was not heat stable and had a slightly higher molecular mass. In contrast to mammalian MAPs, MAP1 was not found in the cold-labile fraction of microtubules. A new heat-labile MAP of higher molecular mass (400 kilodaltons) was however present, as well as a heat-stable protein of slightly lower molecular mass than MAP2. These MAPs showed similar tubulin-binding characteristics as bovine brain MAPs, since they coassembled with taxol-assembled bovine brain microtubules consisting of pure bovine tubulin. In spite of the fact that Ca2+ bound equally to cod and porcine tubulins, it did not inhibit cod microtubule assembly even at high concentrations (greater than 1 mM). In contrast, rings, spirals, and macrotubules were formed. The results show that there are major differences between this fraction of cod microtubules and microtubules from mammalian brain.
The amount of microtubule protein present in the total soluble protein from brains of Alzheimer's disease patients and from brains of non-Alzheimer age-matched controls, were determined by radioimmunoassay. No differences were found in the amount of tubulin or microtubule-associated protein MAP2 present in either group. However, the amount of tau protein or MAP1 from the brains of Alzheimer's disease patients was about half of that present in their control counterparts.
Posttranslational modifications of tubulin were analyzed in mouse brain neurons and glia developing in culture. Purified tubulin was resolved by isoelectric focusing. After 3 weeks of culture neurons were shown to express a high degree of tubulin heterogeneity (8 α and 10 β isoforms), similar to that found in the brain at the same developmental stage. Astroglial tubulin exhibits a less complex pattern consisting of 4 α and 4 β isoforms.
After incubation of neuronal and glial cells with 3H-acetate in the presence of cycloheximide, a major posttranslational label was found associated with α-tubulin and a minor one with β-tubulin. The acetate-labeled isotubulins of neurons were resolved by isoelectric focusing into as many as 6 α and 7 β isoforms, while those of astroglia were resolved into only 2 α and 2 β isoforms. The same α isoforms were also shown to react with a monoclonal antibody recognizing selectively the acetylated forms(s) of α-tubulin [38]. Whether acetate-labeling of α-tubulin in these cells corresponds to the acetylation of Lys40, as reported for Chlamydomonas reinhardtii [30, 33, 34], is discussed according to very recent data obtained by protein sequence analysis.
Tubulin phosphorylation was analyzed by incubation of cell cultures with 32PO4. No phophorylation of α-tubulin isoforms was detected. A single β-tubulin isoform (β′2), expressed only in neurons, was found to be phosphorylated. This isoform is similar to that previously identified in differentiated mouse neuroblastoma cells [16, 18, 21].
1. The presence of microtubule-associated protein MAP-1B in all mammalian tissues tested, as well as in brain, has been demonstrated by immunoblotting using a monospecific polyclonal antibody. 2. The expression of brain MAP-1B is developmentally controlled, as it is less abundant in adult than in newborn rat brain, where it is a major microtubule assembly promoting factor. 3. The level of MAP-1B in tissues other than brain is lower than it is in brain; but the relative ratios of MAP-1B to tubulin are very similar in all tissues, thus differing from the observed for MAP-2 or tau. 4. The amount of MAP-1B in non-nervous tissues seems not to be under developmental control. 5. These results are consistent with a role for MAP-1B in the assembly of microtubules in most cells.
