Fraction Of Tubulin Research Articles

Ionic requirements and subcellular localization of tubulin tyrosinolation in human polymorphonuclear leukocytes.

We previously reported a specific stimulation of polymorphonuclear leukocyte (PMN) tubulin tyrosinolation as induced by the peptide chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fmet-leu-phe) and the Ca2+ ionophore A23187 that is coupled to the NADPH oxidase-mediated stimulation of the PMN respiratory burst. The present study demonstrates that the presence of extracellular Ca2+ is necessary for fmet-leu-phe- and A23187-induced stimulation of PMN tubulin tyrosinolation, as indicated by the complete inhibition of the response by the addition of 1 mM EGTA to the extracellular medium. Methoxyverapamil (10(-5) M), a putative calcium channel blocker, completely inhibited the fmet-leu-phe-induced stimulation of tubulin tyrosinolation in PMN, but did not inhibit the A23187-induced response. Moreover, the calmodulin-binding drugs, trifluoperazine, fluphenazine, or chlorpromazine, at concentrations of 1 to 10 microM, caused significant inhibition of fmet-leu-phe- or A23187-induced stimulation of tubulin tyrosinolation. In related studies, enzymatic [14C]-tyrosinolation in isolated subcellular fractions of PMN revealed the presence of native tubulin in PMN fractions that were enriched in plasma membranes, the specific granules, or the azurophil granules. Most interestingly, tubulin tyrosine ligase (ligase), primarily a cytoplasmic enzyme, was detected in association with the PMN azurophil granule-rich fraction. Immunoautoradiography with the alpha-tubulin antibody YL 1/2 of isolated PMN subcellular fractions demonstrated a preferential stimulation of tyrosinolation of tubulin associated with the plasma membrane-rich fraction of fmet-leu-phe-stimulated cells. A significant stimulation was also observed in the cytoplasmic tubulin fraction. Consistent with the findings of in vitro tyrosinolation studies with PMN subcellular fractions, tyrosinolated tubulin was detected in the azurophil granule-enriched fractions isolated from both resting and fmet-leu-phe-stimulated cells. The antibody YL 1/2, which reacts with tyrosinolated alpha-tubulin and not with the detyrosinolated form, showed significant cross-reaction with several nontubulin PMN proteins.

Inhibition of plant cell proteolytic activities that degrade tubulin

The requirement for proteinase inhibitors during the chromatographic isolation of tubulin from cultured cells of rose ( Rosa sp. cv. Paul's scarlet) was examined by NadodecylSO 4-polyacrylamide gel electrophoresis, electron microscopy and immunoblotting. Tubulin fractions isolated in the absence of proteinase inhibitors showed substoichiometric ratios of α-subunit to β-subunit, and low molecular weight polypeptides, one (~32 Kd) of which coassembled with polymers. Electron microscopy revealed polymorphic structures, including C- and S-shaped ribbons and free protofilaments. Immunoblotting experiments with IgGs to the individual α- and β-subunits showed that some of the low molecular weight polypeptides were fragments of proteolytically degraded subunits. The use of low micromolar concentrations of the synthetic proteinase inhibitors leupeptin hemisulfate and pepstatin A protected tubulin from endogenous proteolytic activities during the isolation procedure and resulted in increased tubulin purity.

A modified colchicine-binding assay for the measurement of total and microtubule-derived tubulin in rat liver

The usual measurement of liver tubulin by the colchicine-binding assay does not take into account the accelerated decay of the colchicine-binding capacity of tubulin when liver supernatants, especially those containing microtubule-derived tubulin, are incubated at 37°C. This results in marked underestimations. Our findings indicate that this alteration is due to an inhibitor of colchicine-tubulin binding in liver supernatants that is probably extracted from particulate fractions. The inhibitory activity is decreased by dilution of the supernatants and by increasing the concentration of colchicine. However, the former modification decreases the sensitivity of the assay and the latter increases the nonspecific binding of colchicine to liver proteins other than tubulin. Assessment of the decay and correction for it by calculating the initial binding capacity results in complete recovery of brain tubulin from liver supernatants and values for microtubule-derived tubulin that closely correspond to those expected from simultaneous morphometric assessment of liver microtubules by electron microscopy. The modified method also indicates that the fraction of liver tubulin assembled in microtubules is greater than previously reported.

Organization and expression of a-tubulin genes in Drosophila melanogaster: One member of the a-tubulin multigene family is transcribed in both oogenesis and later embryonic development

Genomic clones containing α-tubulin sequences were isolated from a library of Drosophila melanogaster DNA and identified by a hybridization-selection and in vitro-translation procedure. The in vitro translation products were identical to the two electrophoretic variants of α-tubulin present in Drosophila embryos. They co-assembled with an embryonic tubulin fraction to form microtubules in vitro and generated the same partial proteolytic fragments as Drosophila α-tubulins. Hybridization in situ to polytene chromosomes revealed that the α-tubulin sequences constitute a multigene family localized on the right arm of chromosome 3 at sites 84 B3–6, 84 D4–8 and 85 E6–10. Clones hybridizing to these sites corresponded to the three major α-tubulin sequences in genomic DNA. The α-tubulin sequences at 84 B3–6 were present twice per haploid genome, embedded in a large duplicated DNA segment. The sequences of the three genomic α-tubulin genes showed considerable divergence, making it possible to conclude that both of the α-tubulin variants present in embryos are encoded by the genes at 84 B3–6. Furthermore, the abundance of this α-tubulin messenger RNA changes with the requirements for microtubule synthesis during embryo development. The genes at 84 B3–6 encoded both the stored maternal mRNA of the oocyte and the major mRNA transcribed during embryonic development.

Chapter 3 Assembly-Disassembly Purification and Characterization of Microtubule Protein without Glycerol

Publisher Summary This chapter presents the methods for isolating microtubule protein from hog and beef brain, along with the procedures for fractionating microtubule protein into separate tubulin and microtubule-associated protein (MAP) fractions. Microtubules are prominent components of the cytoplasmic matrix and perform important functions as cytoskeletal elements for the determination of cell shape and as key elements in intracellular motility such as mitosis and the translocation of cell organelles. The purification methods described in the chapter are based on the temperature dependence of microtubule polymerization. Microtubule assembly is an equilibrium process involving free tubulin subunits and microtubule polymers. Warm temperature (37°C) shifts the equilibrium to favor polymer, whereas cold temperature (0°C) shifts the equilibrium to favor free dissociated subunits. An assembly–disassembly procedure that does not employ glycerol was originally used to purify microtubules from extracts of hog brain. MAPs bind to tubulin in large quantity and co-purify in constant stoichiometry to tubulin through several successive cycles of in vitro assembly–disassembly.

Carboxyl terminal tyrosine metabolism of alpha tubulin and changes in cell shape: Chinese hamster ovary cells

Chinese hamster ovary cells maintained in their epithelial-like form do not incorporate tyrosine post-translationally into α tubulin even though a significant fraction of the soluble tubulin is tyrosinated and tubulin: tyrosine ligase is present. Incubation with dibutyryl cyclic AMP + testosterone, which leads to a change in cell shape, immediately activates this metabolic pathway. Podophyllotoxin, which prevents the conversion to a fibroblast-like morphology, significantly inhibits this activation.

Tubulin pools in differentiating neuroblastoma cells.

The distribution of tubulin in soluble, reversibly stabilized (assembled) and insoluble forms has been determined in neuroblastoma cells undergoing microtubule-dependent neurite elongation. Procedures were developed to obtain reproducible tubulin fractions and to assay total tubulin. Radioimmunoassays showed that both differentiated and nondifferentiated cell contained approximately 4 pg of tubulin per cell, of which 3-10% was in an insoluble, particulate form. The amount of tubulin assembled in differentiated cells was four to five times greater than in nondifferentiated cells, constituting 48-63% and 11-16% of the total tubulin pool in the respective cell types. Calculation of the concentration of soluble tubulin indifferentiated cells (approximately 0.8 mg/ml) and nondifferentiated cells (approximately 1.6 mg/ml) indicates that a critical concentration of subunits probably does not limit the induction of microtubule formation during neurite elongation.

Reassembly of flagellar B (alpha beta) tubulin into singlet microtubules: consequences for cytoplasmic microtubule structure and assembly.

B(alpha beta) tubulin was obtained from a homogeneous class of microtubules, the incomplete B subfiber of sea urchin sperm flagellar doublet microtubules, by thermal fractionation. The thermally derived soluble B tubulin fraction (100, 000 g-h) repolymerizes in vitro, yielding microtubule-like structures. The microtubule-associated protein (MAP) composition and certain assembly parameters of thermally derived B tubulin are different from those reported for sonication-derived flageller tubulin and purified vertebrate tubulin. The "microtubules" reassembled from thermally prepared B tubulin are composed of 12-15 protofilaments (73% possess 14 protofilaments). A certain number possess a single "adlumenal component" applied to their inside walls, regardless of the number of protofilaments. Following the first cycle of polymerization, 81% of the B tubulin and essentially 100% of the MAPs remain cold insoluble. Evidence suggests that B tubulin assembles faithfully into a B lattice, creating a j seam between two protofilaments that are laterally bonded in a A-lattice configuration. The significance of these seams is discussed in relation to the mechanism of microtubule assembly, the stability of observed ribbons of protofilaments, and the three-dimensional organization of microtubule-associated components.

Microtubule assembly and disassembly at alkaline pH.

Although it is now apparent that the intracellular pH may rise considerably above neutrality under physiological conditions, information on the effect of alkaline pH on microtubule assembly and disassembly is still quite fragmentay. We have studied the assembly/disassembly of bovine brain microtubule protein at alkaline pH in vitro. When microtubules are assembled to a new steady state at pH less than 7 and pH is then made more alkaline, they undergo a rapid disassembly to a new steady state. This disassembly is reversed by acidification. The degree of disassembly is determined largely by the pH- dependence of the critical concentration, which increases five to eight times, from pH 7 to 8. A fraction of assembly-incompetent tubulin is identified that increases with pH, but its incompetency is largely reversed with acidification. Measurements of microtubule lengths are used to indicate that disassembly occurs by uniform shortening of microtubules. A comparison of shortening by alkalinization with dilution suggests that the intrinsic rate of disassembly is accelerated by increasing pH. The capacity for initiating assembly is progressively lost with incubation at alkaline pH (although some protection is afforded by sulfhydryl-reducing agents). However, direct assembly from depolymerized mixtures is possible at least up to pH 8.3, and the steady state achieved at these alkaline pH values is stable. Such preparations are readily disassembled by cold and podophyllotoxin (PLN). Disassembly induced by PLN is also markedly enhanced at alkaline pH, suggesting a corresponding enhancement of "treadmilling." The implications of physiological events leading to alkaline shifts of pH for microtubule assembly/disassembly are discussed, particularly in the light of recent hypotheses regarding treadmilling and its role in controlling the distribution of microtubules in vivo.

Tubulin synthesis in sea urchin embryos II. Ciliary a tubulin derives from the unfertilized egg

We have determined the fraction of tubulin of the A microtubule of ciliary doublets that is synthesized in sea urchin embryos during cleavage. Cleavage-stage Stronglyocentrotus purpuratus embryos were radiolabeled with leucine; tubulin from isolated ciliary A microtubules was purified by selective extraction and two-dimensional gel electrophoresis. The specific activity of the tubulin chains was 0.1–0.2 times that of whole-embryo protein. We calculate from this that only 2–4% of ciliary A tubulin is synthesized during cleavage. It remains unresolved whether distinct metabolic compartments exist in the cleavage-stage tubulin pool.

Ca2+- and calmodulin-stimulated endogenous phosphorylation of neurotubulin.

Ca2+ plays a major role in the functional use of tubulin in brain and other tissues. It activates an endogenous tubulin kinase system in brain cytosol, tubulin, and presynaptic nerve terminal fractions prepared from rat brain. Activation of the Ca2+ tubulin kinase system was modulated by the Ca2+ receptor protein calmodulin. The concentrations of Ca2+ and calmodulin required to produce a half-maximal stimulation of the tubulin kinase were 0.8 microM and 0.4 micrograms, respectively. Ca2+ -calmodulin tubulin kinase activity was very unstable after death, and procedures were developed to stabilize the activity of this enzyme system. Evidence is presented demonstrating that the Ca2+ -calmodulin tubulin kinase system is distinct from the previously described cyclic AMP-Mg2+ tubulin kinase. The results suggest that Ca2+- and calmodulin-stimulated phosphorylation of tubulin may be a major biochemical mechanism modulating some of calcium's effects on tubulin and may play a significant role in mediating some of calcium's actions on cell functions.

Effects of the principal hydroxy-metabolites of benzene on microtubule polymerization

The principal hydroxy-metabolites of benzene - phenol, catechol and hydroquinone - possess characteristics and produce toxicity similar to those reported for certain inhibitors of microtubule polymerization. In this study we examined the effects of phenol, catechol and hydroquinone on purified microtubule polymerization and the decay of tubulin-colchicine binding activity. Hydroquinone, but not catechol or phenol, inhibited microtubule polymerization and accelerated the decay of tubulin-colchicine binding activity. The latter effect was shown to be dependent on the concentration of GTP. Hydroquinone did not directly complex with GTP or ATP but bound to the high molecular weight fraction of tubulin. concentration ratios of hydroquinone to tubulin resulting in altered activity were low, suggesting a specific interaction, presumably at the tubulin-GTP binding site. The acceleration of tubulin-colchicine binding activity decay was completely prevented under anaerobic conditions, indicative of an oxidative mechanism. These studies suggest that hydroquinone, which auto-oxidizes, may interfere with microtubule function, nucleotide binding or both and that this mechanism may be involved in eliciting the wide range of cytoskeletal-related abnormalities observed in cells exposed to benzene in vivo or its metabolites in vitro.

An apparent paradox in the occurrence, and the in vivo turnover, of C-terminal tyrosine in membrane-bound tubulin of brain.

Tubulin tyrosine ligase catalyzes the reversible addition of tyrosine to the C-terminus of tubulin alpha chains. By using ligase and carboxypeptidase A in conjunction, we have previously shown that brain cytoplasmic tubulin exists in three forms: 15-40% already has C-terminal tyrosine, another 10-30% can accept additional tyrosine, and about one-half is an uncharacterized species which is not a ligase substrate. A membrane-bound fraction of brain tubulin, purified by vinblastine precipitation from a detergent extract, has been found to differ by the complete absence of preexisting tyrosine. The membrane fraction from which tubulin was extracted also contained masked forms of both ligase and a distinct detyrosylating enzyme, which can be released by detergent extraction. The turnover of alpha-chain C-terminal tyrosine in vivo was studied by incubating brain mince with labeled tyrosine, or injecting it intracerebrally, under conditions where protein synthesis was inhibited. Tyrosine appeared to turn over to about the same extent in membrane-bound, as in soluble, tubulin. This apparently paradoxical result was not due to ATPase in the membrane fraction, which might have allowed ligase-catalyzed exchange between free and fixed tyrosine. Authentic [14C]tyrosylated tubulin added to the brain membrane fraction was not detyrosylated or subject to endoprotease digestion during subsequent procedures to isolate tubulin. The unexpected finding that tubulin tyrosylated at the C-terminal in vivo appears to be in the "non-substrate" fraction points toward a possible resolution of the paradox.

Total tubulin and its aminoacylated and non-aminoacylated forms during the development of rat brain.

The amount of total tubulin in the soluble fraction of rat brain was measured by a method based on the purification of tubulin previously labeled by incorporation of [14C]tyrosine in the C terminus of its alpha-chain. The tubulin content decreased from 2.01 to 1.30 nmol/mg protein when the animals passed from 4 to 30 days of age and then remained practically constant. The amounts of aminoacylated and non-aminoacylated tubulin present in the soluble brain extracts were determined from the incorporation of [14C]tyrosine into the free acceptor sites of tubulin preparations, that were preincubated without carboxypeptidase A or with this enzyme to eliminate tyrosine and phenylalanine from the C terminus of the alpha-chain of tubulin. The values obtained were corrected for the inactivation of tubulin to accept [14C]tyrosine that occurred during the isolation and incubation of the soluble fractions. The ratio non-aminoacylated/aminoacylated tubulin increased from 1.62 +/- 0.03 in the 4-day-old rats to 2.11 +/- 0.17 in the 120-day-old rats. The aminoacylatable tubulin, that is the sum of aminoacylated plus non-aminoacylated tubulin, decreased from 1.71 to 0.75 nmol/mg protein from 4-day-old to 30-day-old rats respectively and then remained practically constant. The amount of aminoacylatable tubulin is lower than that of total soluble tubulin. Therefore there is a fraction of tubulin that is unable to accept tyrosine. This non-aminoacylatable tubulin fraction increases with the age of the animal so that in the 120-day-old rats this tubulin species accounts for 48% of the total soluble tubulin.

An ATPase activity associated with brain microtubules

A persistent ATPase/GTPase activity has been found to be associated with highly recycled bovine brain microtubules. A GTP regeneration system was introduced to minimize the inhibitory effects of this hydrolase on microtubule polymerization. The characteristics of the ATPase indicate that it is not involved in GTP-induced mictrotubule polymerization, but is directly involved in ATP-induced polymerization. ATP-induced polymerization was also shown to require stoichiometric amounts of GDP, but higher levels of GDP inhibited both microtubule formation and the ATPase activity. An ammonium sulfate fractionation procedure was devised to separate microtubule protein into an ATPase-rich fraction and a pure tubulin fraction. The pure tubulin fraction polymerized in the presence of GTP, but not in the presence of ATP and GDP. In contrast, the ATPase-rich fraction polymerized with either ATP or GTP. It is still not known whether the microtubule associated ATPase plays a significant role in cellular microtubule function.

Assembly of nonneural microtubules in the absence of glycerol and microtubule-associated proteins

Microtubule protein from Ehrlich ascites tumor cells purified by an in vitro polymerization process in the absence of glycerol and calcium chelators contains several accessory proteins but lacks the high molecular weight proteins which are present in neurotubulin. DEAE-Sephadex chromatography of two-times cycled tubulin removes these nontubulin proteins, resulting in pure tubulin, as critically examined by sodium dodecyl sulfate gel electrophoresis. This tubulin can readily assemble into microtubules in assembly buffer, at low magnesium concentrations, without glycerol and at tubulin concentrations above 0.8 mg/mL. Electron microscopy shows that the tubules are identical with normal microtubules. When the purified tubulin fraction was reduced and carboxymethylated, a significant minor protein component could be observed electrophoretically, migrating between alpha- and beta-tubulin. At present, the identity and function of this protein are not known. The results demonstrate that the in vitro assembly of tubulin from Ehrlich ascites tumor cells does not require high molecular weight proteins or gamma-like factor(s) as has been proposed for the neurotubulin system.

Capability of tubulin and microtubules to incorporate and to release tyrosine and phenylalanine and the effect of the incorporation of these amino acids on tubulin assembly.

Abstract— Incorporation of [14C]tyrosine into the C‐terminal position of α‐tubulin of rat brain cytosol was 10‐fold higher for non‐assembled than for assembled tubulin. The incorporation into tubulin from disassembled microtubules was higher than into non‐assembled tubulin; therefore, the low incorporation into microtubules was not due to a lower acceptor capacity of their tubulin constituent.[14C]Tyrosine was released from assembled and non‐assembled [14C]tyrosinated tubulin by the action of an endogenous carboxypeptidase. Release from non‐assembled tubulin was shown by incubating a tubulinyl‐[14C]tyrosine preparation in the presence of CaCl2 at a concentration that abolished microtubule formation. Release from microtubules was inferred from the observation that the percentages of [14C]tyrosine released and the decrease of the specific radioactivity of the recovered microtubules were practically identical and did not change after a 10‐fold dilution of the incubated microtubules.[3H]Phenylalanine was released from a preparation of tubulinyl‐[3H]phenylalanine also by an enzymatic activity.The capacity of a tubulin preparation to incorporate tyrosine was increased 43% by pre‐treatment with endogenous carboxypeptidase.Tubulin tyrosinated in vitro was assembled to the same extent as native tubulin. After a mixture of tubulinyl‐[14C]tyrosine and tubulinyl‐[3H]phenylalanine was partially assembled, the ratio of 14C/3H found in the microtubules was the same as in the non‐assembled tubulin fraction.

Characterization and in vitro polymerization of Tetrahymena tubulin.

Tetrahymena tubulin was purified from the cell extract using DEAE-Sephadex A-50 ion-exchanger and ammonium sulfate precipitation. About 2.2% of the total protein in the 20,000 X g supernatant was recovered as DEAE-Sephadex-purified tubulin fraction. Applying the temperature-dependent polymerization-depolymerization method to this fraction in the presence of Tetrahymena outer fibers as a seed, almost pure tubulin was obtained. Tetrahymena tubulin dimer showed different behavior on SDS-polyacrylamide gels from porcine brain tubulin, and showed very low affinity for colchicine, amounting to about one-twentieth of the binding to porcine brain tubulin. The tubulin fraction failed to polymerize into microtubules by itself. Addition of a small amount of the ciliary outer fiber fragment induced polymerization as demonstrated by viscometric measurements, but the reconstituted microtubules were very unstable in the absence of glycerol. Microtubule-depolymerizing agents such as Ca2+ ions, low temperature, or colchicine all inhibited in vitro polymerization. Although Tetrahymena tubulin purified by the polymerization-depolymerization method could copolymerize with porcine brain microtubules, the DEAE-Sephadex-purified tubulin fraction suppressed the initial rate of porcine brain microtubule assembly in vitro. There seemed to be no differences between cytoplasmic tubulin and outer fiber tubulin in colchicine binding activity or SDS-gel electrophoretic behavior, or between the fine structure of both reconstituted microtubules observed by electron microscopy.

Tubulin synthesis in sea urchin embryos: Almost all tubulin of the first cleavage mitotic apparatus derives from the unfertilized egg

Developing embryos of Stronglyocentrotus purpuratus were exposed to [ 14C]leucine between fertilization and metaphase of the first cleavage division. At metaphase, mitotic apparatus was isolated from the embryos and tubulin was extracted from mitotic apparatus. The specific activity of the tubulin fraction was only 0.2 to 0.4 times the specific activity of whole embryo protein. We calculate from this result that no more than 0.4% of the tubulin of the first cleavage mitotic apparatus could be synthesized following fertilization.

In Vitro Polymerization of Flagellar and Ciliary Outer Fiber Tubulin into Microtubules

About 10--20% of the total protein in the outer fiber fraction was solubilized by sonication in a solution containing 5 mM MES, 0.5 mM MgSO4, 1.0 mM EGTA, 1.0 mM GTP, and 0 or 50 mM KC1 at pH 6.7. The sonicated extract was shown by analytical centrifugation to consist largely of a 6 S component (tubulin dimer), having a molecular weight of 103,000, as determined by gel filtration, and possessing a colchicine-binding activity of 0.8 mole per tubulin dimer. The tubulin fraction failed to polymerize into microtubules by itself. Addition of a small amount of the ciliary outer fiber fragments or reconstituted short brain microtubules, however, induced polymerization, as demonstrated by viscosity of flow birefringence changes as well as light or electron microscopic observations. The growth of heterogeneous microtubules upon mixing outer fiber tubulin with DEAE-dextran-decorated brain microtubules was observed by electron microscopy. Microtubules were reconstituted from outer fiber tubulin without addition of any nuclei fraction when a concentrated tubulin fraction was warmed at 35degree. A few doublet-like microtubules or pairs of parallel singlet microtubules that were closely aligned longitudinally could be observed among many singlet microtubules. Unlike other fiber microtubules, the reconstituted polymers were depolymerized by exposure to Ca2+ ions, high or low ionic strength, colchicine, low temperature or SH reagents. No microtubules were assembled under these conditions.