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1.
Richard J. Schimmel 《Biochimica et Biophysica Acta (BBA)/General Subjects》1975,399(1):181-190
Colchicine was found to be taken up by adipose tissue and therein to bind to a soluble macromolecule not sedimented by centrifugation for 2 h at 100 000 × g. A similar binding occurred when soluble extracts of adipose tissue were incubated with colchicine. The binding reaction is temperature dependent and shows a pH optimum between 6.8 and 7.0. Double reciprocal plots of colchicine concentration versus amounts of colchicine bound to protein in the steady state disclosed an apparent Km of 0.250 to 1.5 ωM. The colchicine binding activity of soluble tissue extracts decreased when the extracts were incubated at 37°C. Addition of guanosine triphosphate and Mg2+ retarded the loss of colchicine binding activity. The molecular weight of the colchicine complex was estimated to be 115 000 and its sedimentation coefficient 5.8 S. All of these characteristics are remarkably similar to those of the protein tubulin which has been isolated from other tissues. Since it is now well known that tubulin is a protein subunit of cytoplasmic microtubules, it is suggested that the previously reported metabolic effects of colchicine on adipose tissue result from the dissolution of microtubules by colchicine. 相似文献
2.
THE MECHANISM OF ACTION OF COLCHICINE : Colchicine Binding Properties of Sea Urchin Sperm Tail Outer Doublet Tubulin 总被引:14,自引:5,他引:9
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The thermal depolymerization procedure of Stephens (1970. J. Mol. Biol. 47:353) has been employed for solubilization of Strongylocentrotus purpuratus sperm tail outer doublet microtubules with the use of a buffer during solubilization which is of optimal pH and ionic strength for the preservation of colchicine binding activity of chick embryo brain tubulin. Colchicine binding values were corrected for first-order decay during heat solubilization at 50°C (t½ = 5.4 min) and incubation with colchicine at 37°C in the presence of vinblastine sulfate (t½ = 485 min). The colchicine binding properties of heat-solubilized outer doublet tubulin were qualitatively identical with those of other soluble forms of tubulin. The solubilized tubulin (mol wt, 115,000) bound 0.9 ± 0.2 mol of colchicine per mol of tubulin, with a binding constant of 6.3 x 105 liters/mol at 37°C. The colchicine binding reaction was both time and temperature dependent, and the binding of colchicine was prevented in a competitive manner by podophyllotoxin (Ki = 1.3 x 10-6 M). The first-order decay of colchicine binding activity was substantially decreased by the addition of the vinca alkaloids, vinblastine sulfate or vincristine sulfate, thus demonstrating the presence of a vinca alkaloid binding site(s) on the outer doublet tubulin. Tubulin contained within the assembled microtubules did not decay. Intact outer doublet microtubules bound less than 0.001 mol of colchicine per mol of tubulin contained in the microtubules, under conditions where soluble tubulin would have bound 1 mol of colchicine per mol of tubulin (saturating concentration of colchicine, no decay of colchicine binding activity). The presence of colchicine had no effect on the rate of solubilization of outer doublet microtubules during incubation at 37°C. Therefore, the colchicine binding site on tubulin is blocked (not available to bind colchicine) when the tubulin is in the assembled outer doublet microtubules. 相似文献
3.
Microtubule protein was measured in mouse brain homogenates by quantitative colchicine binding. Neonatal animals contained more than twice the amount of brain tubulin as adult mice. The percentage of colchicine-binding protein which was polymerized was determined by extracting brain at room temperature into a medium designed to stabilize intact microtubules. Under identical conditions and tubulin concentrations, neonatal brain tubulin (colchicine-binding activity) had a greater proportion of the total extracted in an apparently polymerized state (pelletable by centrifugation) than did adult brain. A slight variation in the ratio of assembled to unassembled tubulin was observed with varying protein concentration (volume of extract), indicating that the values obtained may not reflect exactly the in vivo situation, because a rapid equilibration takes place upon homogenization. At all protein concentrations, the neonatal brain extracts contained a significantly greater proportion of assembled tubulin than did adult brain. This proportion began to fall at 5 days postnatal and reached the adult level at 30 days. The tubulin assembled/not assembled ratios were not altered by addition of nucleoside triphosphates, additional EGTA, or sulfhydryl protecting agents, and did not vary with preparation times of 30–90 min. The colchicine-binding reaction and decay of colchicine-binding activity with time were similar in extracts of different aged mouse brains, with neonatal slightly more stable than adult. Pools of tubulin from any age which were soluble at room temperature (unpolymerized) could not repolymerize well in vitro when incubated with GTP at 37 °C, whereas pools of tubulin which were sedimentable at room temperature (polymerized) could be redissolved at 0 °C and readily reassembled at 37 °C. The neonatal extract tubulin was thus more polymerization competent than the adult extracts; this correlates with a greater proportion of assembled tubulin in extracts at room temperature and possibly in vivo. 相似文献
4.
Colchicine-binding protein of the liver. Its characterization and relation to microtubules 总被引:6,自引:2,他引:4
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《The Journal of cell biology》1975,66(3):609-620
Colchicine-binding activity of mouse liver high-speed supernate has been investigated. It has been found to be time and temperature dependent. Two binding activities with different affinities for colchicine seem to be present in this high-speed supernate, of which only the high-affinity binding site (half maximal binding at 5 x 10(-6) M colchicine) can be attributed to microtubular protein by comparison with purified tubulin. Vinblastine interacted with this binding activity by precipitating it when used at high concentrations (2 x 10(- 3) M), and by stabilizing it at low concentrations (10(-5) M). Lumicolchicine was found not to compete with colchicine. The colchicine-binding activity was purified from liver and compared with that of microtubular protein from brain. The specific binding activity of the resulting preparation, its electrophoretic behavior, and the electron microscope appearance of the paracrystals obtained upon its precipitation with vinblastine permitted its identification as microtubular protein (tubulin). Electrophoretic analysis of the proteins from liver supernate that were precipitated by vinblastine indicated that this drug was not specific for liver tubulin. Preincubation of liver supernate with 5 mM EGTA resulted in a time- dependent decrease of colchicine-binding activity, which was partly reversed by the addition of Ca++. However, an in vitro formation of microtubules upon lowering the Ca++ concentration could not be detected. Finally, a method was developed enabling that portion of microtubular protein which was present as free tubulin to be measured and to be compared with the total amount of this protein in the tissue. This procedure permitted demonstration of the fact that, under normal conditions, only about 40% of the tubulin of the liver was assemled as microtubules. It is suggested that, in the liver, rapid polymerization and depolymerization of microtubules occur and may be an important facet of the functional role of the microtubular system. 相似文献
5.
Membrane-bound tubulin in brain and thyroid tissue. 总被引:26,自引:0,他引:26
Brain and thyroid tissue contain membrane-bound colchicine-binding activity that is not due to contamination by loosely bound cytoplasmic tubulin. This activity can be solubilized to the extent of 80 to 90% by treatment with 0.2% Nonidet P-40 with retention of colchicine binding. Extracts so obtained contain a prominent protein band in disc gel electrophoresis that co-migrates with tubulin. Membranes, and the solubilized protein therefrom, exhibit ligand binding properties like tubulin; for colchicine the KA is approximately 1 X 10(6) M-1 in brain and approximately 0.6 X 10(6) M-1 in thyroid; for vinblastine the KA is approximately 8 X 10(6) M-1 for both tissues; and for podophyllotoxin the Ki is approximately 2 X 10(-6) M for both tissues. Displacement by analogues of colchicine is of the same order as for soluble tubulin. Although membrane-bound colchicine-binding activity shows greater thermal stability and a higher optimum binding temperature (54 degrees versus 37 degrees) than soluble tubulin, this appears to be the result of the membrane environment since the solubilized binding activity behaves like the soluble tubulin. Antibody against soluble brain tubulin reacts with membranes and solubulized colchicine-binding activity from both brain and thyroid gland. We conclude that brain and thyroid membrane preparations contain firmly bound tubulin or a very similar protein. 相似文献
6.
Enrique Baraona Frances Finkelman Yoshiro Matsuda Charles S. Lieber 《Analytical biochemistry》1983,130(2)
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. 相似文献
7.
Microtubules from the cow adrenal cortex and brain were purified by three cycles of the temperature-dependent polymerization-depolymerization procedure. Whereas tubulin comprised approximately 8--10% of soluble brain protein, it comprised only 0.5-1.0% of the soluble adrenocortical protein. The partially purified tubulin from both sources gave similar results in the following studies: (1) [3H]colchicine binding examined by Scatchard analysis revealed an apparent Ka of 1 . 10(6) M-1 and a colchicine/tubulin molar binding ratio of 0.4-0.6; (2) tyrosylation studies using a specific tubulin-tyrosine ligase (which adds a tyrosine residue to the C-terminal glutamate or glutamine of the alpha-chain) in conjunction with carboxypeptidase A (which recovers the tyrosine) and (3) amino acid analysis. Examination of protein bands, in addition to the tubulin doublet of 55 000 molecular weight, on sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a difference between the two tubulin preparations. The adrenocortical preparation had protein bands corresponding to apparent molecular weight of 36 000, 60 000, and 68 000. In contrast the brain preparation had only proteins of molecular weights greater than 200 000 (these bands were absent in all adrenal preparations). It would thus appear that if proteins which copurify with tubulin through repeated cycles of polymerization-depolymerization play a role in either microtubule formation or function there is a distinct difference between neural and non-neural tissue. 相似文献
8.
A M Chambaut-Guérin P Muller B Rossignol 《The Journal of biological chemistry》1979,254(21):10734-10739
The specific inhibitory effect of colchicine upon protein secretion by lacrimal glands could be related to the formation of a complex between colchicine and tubulin from the soluble fraction of the gland. By gel electrophoresis under nondissociating conditions, it is shown that this complex is similar to the colchicine . tubulin complex from brain. The complex isolated from lacrimal glands is highly inhibitory upon brain tubulin assembly since as low as 0.07 microM complex impedes the polymerization of 8 microM tubulin by 50%, compared to 3 microM for free colchicine. Therefore, a small percentage of complexed tubulin (0.9%) is enough for polymerization to be blocked. In lacrimal glands the complex might prevent the polymerization of tubulin, and colchicine shift the tubulin in equilibrium microtubules equilibrium to microtubules disassembly. The disorganization of the labile microtubular system could lead to a modification of the transport of the secretory granules and to a perturbation of secretion. 相似文献
9.
Identification with cellular microtubules of one of the co-assemlbing microtubule-associated proteins. 总被引:37,自引:0,他引:37
In this paper we describe a procedure for detecting proteins associated with cytoplasmic microtubules in vivo. Detergent-extracted cytoskeletons of NIL8 hamster cells are prepared under conditions which preserve the microtubules. The cytoskeletons are then extracted in the presence of calcium, which depolymerizes the microtubules and quantitatively extracted cytoskeletons are prepared from cells that have been incubated with colchicine. The cytoskeletons from these cells contain no microtubules or tubulin. Electrophoretic analysis of the calcium extracts of the colchicine-treated and untreated cells reveals several radioactively labeled polypeptides. There is, however, no apparent quantitative or qualitative difference between the two extracts other than the tubulin polypeptides. Each of the extracts is mixed with an excess of unlabeled calf brain microtubule protein and carried through cycles of temperature-dependent microtubule assembly. Distinct species from each extract co-assemble at a constant ratio, but only one polypeptide is uniquely derived from cells containing intact microtubules. The molecular weight of this polypeptide is similar to that proposed for the tau species detected in brain microtubule preparations. 相似文献
10.
Leonard F. Liebes Howard Fleit Dorothea Zucker-Franklin Robert Silber 《Biochimica et Biophysica Acta (BBA)/General Subjects》1980,633(2):245-257
Tubulin has been purified from human blood and tonsil lymphocytes. Using gel filtration, the molecular weight of human lymphocyte tubulin was estimated to be 119 000. The proteins was shown to consist of two subunits, with molecular weights of 61 000 and 58 000 comparable to the α and β polypeptides of human brain tubulin. A partial identity reaction was observed between lymphocyte tubulin and human tubulin when tested by double immunodiffusion against a rabbit anti-human brain tubulin antibody. In the presence of GTP, the purified protein polymerized to form microtubules. Tubulin was localized to the cell's juxtacentriolar region by immunofluorescence and electron microscopy. When assayed by a colchicine-binding assay corrected for time decay, the binding affinity was 1.50 ± 0.86 · 106M?1 and a level in normal lymphocytes of 1.21 · 10?2 ± 0.79 g/g of soluble protein was determined. Since chronic lymphocytic leukemia lymphocytes have an anomalous capping behavior as well as an unusual susceptibility to colchicine toxicity, the properties and levels of tubulin were determined in these cells. Similar values were obtained for the level, decay rate, molecular weight, and Ka for colchicine as for normal lymphocytes. Chronic lymphocytic leukemia lymphocyte tubulin polymerized in a normal fashion. It thus appears that a decrease in the quantity or function of tubulin does not account for these anomalies in the chronic lymphocytic leukemia lymphocyte. 相似文献
11.
Release of C-terminal tyrosine from tubulin and microtubules at steady state. 总被引:3,自引:0,他引:3
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Microtubule protein preparations purified by cycles of assembly-disassembly contain the enzyme tubulinyltyrosine carboxypeptidase (TTCPase). Using these preparations, containing tubulinyl[14C]tyrosine, we studied the release of [14C]tyrosine from assembled and non-assembled tubulin under steady-state conditions. It was found that both states of aggregation were detyrosinated at similar rates by the action of the endogenous TTCPase. However, practically no release of [14C]tyrosine from the non-assembled tubulin pool was found when microtubules were previously eliminated from the incubation mixture. These results indicated that non-assembled tubulin requires to interact with microtubules to be detyrosinated. This interaction seems to occur through the incorporation of dimers into microtubules, since when the capability of tubulin to incorporate into microtubules was diminished by binding of colchicine a concomitant decrease in the rate of release of tyrosine was observed. When detyrosination was accelerated by increasing the concentration of TTCPase relative to the microtubule protein concentration, microtubules were found to be detyrosinated faster than was non-assembled tubulin. Using exogenous TTCPase in an incubation system in which the formation of microtubules was not allowed, tubulinyl[14C]tyrosine and tubulinyl[14C]tyrosine-colchicine complex were shown to have similar capabilities to act as substrates for this enzyme. Free colchicine was shown not to affect the activity of TTCPase. 相似文献
12.
Quantitative analysis of tubulin and microtubule compartments in isolated rat hepatocytes 总被引:4,自引:4,他引:0
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《The Journal of cell biology》1977,75(3):731-742
A combined morphometric and biochemical approach has been used to identify and quantitate microtubules and tubulin in isolated hepatocytes. The total soluble pool of microtubule protein was estimated by specific high affinity binding to radiolabeled colchicine. Scatchard analysis of the data identified two populations of binding sites: high affinity-low capacity sites resembling tubulin and low affinity-high capacity sites believed to represent nonspecific colchicine-binding sites. Data from these studies indicate that tubulin represents 1% of the soluble protein of the cell, that 9.0 X 10(-14) dimers of tubulin are present per microgram soluble hepatocyte protein, and that the average hepatocyte contains 3.1 X 10(7) tubulin dimers. Our calculations suggest that this amount of tubulin would form a microtubule 1.9 cm in length if totally assembled. However, stereological measurements indicate that the actual length of microtubules in the cytosolic compartment of the average hepatocyte is only 0.28 cm. Thus, these experiments suggest that only 15% of the available tubulin in hepatocytes of postabsorptive rats is assembled in the form of microtubules. 相似文献
13.
Polymerization-competent extracts of suspension-cultured HeLa cells and porcine brain tissue were assayed for tubulin content. Five different methods were used to assay identically prepared extracts: two types of sodium dodecyl sulfate-containing acrylamide gels, a DEAE retention assay, a colchicine-binding assay, and a radioimmunoassay. The colchicine-binding and radioimmunoassay results were in close agreement and are therefore considered reliable assays for tubulin content in cell extracts. The DEAE retention assay gave slight overestimates, but the gel methods seriously overestimated tubulin content. Based on data from colchicine binding and radioimmunoassay, the proportion of soluble cell protein which is tubulin is 4.3% for HeLa cell extracts and 12.1% for brain tissue extracts. 相似文献
14.
THE MECHANISM OF ACTION OF COLCHICINE : Binding of Colchincine-3H to Cellular Protein 总被引:81,自引:41,他引:40
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The majority of the colchicine-3H bound by tissue culture cells (KB or Hela) was found to be present as a noncovalent complex with a macromolecule which appears in the soluble fraction after homogenization. Similar binding was demonstrated in vitro and was confined to a component of the soluble fraction. The binding-equilibrium constant and the kinetic constants were essentially the same in vivo and in vitro. Bound radioactivity was reisolated and shown to be present in a molecule with the same chromatographic behavior and specific antimitotic activity as colchicine. In vitro assay of binding activity of a variety of cells and tissues showed a correlation with the presence of microtubules. High binding activity was given by dividing cells, mitotic apparatus, cilia, sperm tails, and brain tissue. Binding to extracts of slime mold or to purified muscle proteins was very low or undetectable. The binding site had a sedimentation constant of 6S and it is suggested that the protein is a subunit of microtubules. 相似文献
15.
Elizabeth C. Raff 《Developmental biology》1977,58(1):56-75
Tubulin from eggs and embryos of the Mexican axolotl was characterized by electrophoresis and colchicine binding. In urea-polyacrylamide gel electrophoresis, soluble axolotl egg tubulin migrated as two bands, identical to tubulins from sea urchin sperm and Drosophila eggs. However, in SDS-containing gels, on which the α and β subunits of standard tubulins were well resolved, axolotl egg tubulin migrated as a single band with an apparent molecular weight of 53,500. The method of disruption of the eggs affected both yield of tubulin from vinblastine sulfate precipitates and stability of the colchicine binding activity. The colchicine binding activity of soluble tubulin from gently disrupted eggs was specific and of high affinity, with properties similar to those reported for other tubulins. The tubulin pool in unfertilized eggs was determined to be approximately 2 μg/egg; the level decreased 20% after initiation of cleavage and then remained constant through development to postneurula stages. The colchicine binding activity of soluble tubulin from embryos was much less stable than that of unfertilized eggs and decreased further during development. No differences were found in properties of tubulin from eggs of several strains of normally pigmented axolotls; however, tubulin from albino eggs showed slightly different properties in both electrophoresis and colchicine binding. The colchicine binding activity of soluble tubulin accounts for only half the total activity in axolotl eggs; they possess, in addition, a particulate nontubulin colchicine binding activity. 相似文献
16.
Perturbation of microtubule polymerization by quercetin through tubulin binding: a novel mechanism of its antiproliferative activity 总被引:3,自引:0,他引:3
The dietary flavonoid quercetin has a broad range of biological activities, including potent antitumor activity against several types of tumors. Recently, it has been shown that quercetin inhibits cancer cells proliferation by depleting cellular microtubules and perturbing cellular microtubule functions. However, the direct interactions of quercetin with tubulin and microtubules have not been examined so far. Here, we found that quercetin inhibited polymerization of microtubules and depolymerized microtubules made from purified tubulin in vitro. The binding of quercetin with tubulin was studied using quercetin fluorescence and intrinsic tryptophan fluorescence of tubulin. Quercetin bound to tubulin at a single site with a dissociation constant of 5-7 microM, and it specifically inhibited colchicine binding to tubulin but did not bind at the vinblastine site. In addition, quercetin perturbed the secondary structure of tubulin, and the binding of quercetin stimulated the intrinsic GTPase activity of soluble tubulin. Further, quercetin stabilized tubulin against decay and protected two cysteine residues of tubulin toward chemical modification by 5,5'-dithiobis-2-nitrobenzoic acid. Our data demonstrated that the binding of quercetin to tubulin induces conformational changes in tubulin and a mechanism through which quercetin could perturb microtubule polymerization dynamics has been proposed. The data suggest that quercetin inhibits cancer cells proliferation at least in part by perturbing microtubule functions through tubulin binding. 相似文献
17.
Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), a naphthoquinone isolated from the roots of Plumbaginaceae plants, has potential antiproliferative activity against several tumor types. We have examined the effects of plumbagin on cellular microtubules ex vivo as well as its binding with purified tubulin and microtubules in vitro. Cell viability experiments using human non-small lung epithelium carcinoma cells (A549) indicated that the IC 50 value for plumbagin is 14.6 microM. Immunofluorescence studies using an antitubulin FITC conjugated antibody showed a significant perturbation of the interphase microtubule network in a dose dependent manner. In vitro polymerization of purified tubulin into microtubules is inhibited by plumbagin with an IC 50 value of 38 +/- 0.5 microM. Its binding to tubulin quenches protein tryptophan fluorescence in a time and concentration dependent manner. Binding of plumbagin to tubulin is slow, taking 60 min for equilibration at 25 degrees C. The association reaction kinetics is biphasic in nature, and the association rate constants for fast and slow phases are 235.12 +/- 36 M (-1) s (-1) and 11.63 +/- 11 M (-1) s (-1) at 25 degrees C respectively. The stoichiometry of plumbagin binding to tubulin is 1:1 (mole:mole) with a dissociation constant of 0.936 +/- 0.71 microM at 25 degrees C. Plumbagin competes for the colchicine binding site with a K i of 7.5 microM as determined from a modified Dixon plot. Based on these data we conclude that plumbagin recognizes the colchicine binding site to tubulin. Further study is necessary to locate the pharmacophoric point of attachment of the inhibitor to the colchicine binding site of tubulin. 相似文献
18.
Regulation of the microtubule nucleating activity of centrosomes in Xenopus egg extracts: role of cyclin A-associated protein kinase 总被引:6,自引:2,他引:4
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Isolated centrosomes nucleate microtubules when incubated in pure tubulin solutions well below the critical concentration for spontaneous polymer assembly (approximately 15 microM instead of 60 microM). Treatment with urea (2-3 M) does not severely damage the centriole cylinders but inactivates their ability to nucleate microtubules even at high tubulin concentrations. Here we show that centrosomes inactivated by urea are functionally complemented in frog egg extracts. Centrosomes can then be reisolated on sucrose gradients and assayed in different concentrations of pure tubulin to quantify their nucleating activity. We show that the material that complements centrosomes is stored in a soluble form in the egg. Each frog egg contains enough material to complement greater than 6,000 urea-inactivated centrosomes. The material is heat inactivated above 56 degrees C. One can use this in vitro system to study how the microtubule nucleating activity of centrosomes is regulated. Native centrosomes require approximately 15 microM tubulin to begin nucleating microtubules, whereas centrosomes complemented in interphase extracts begin nucleating microtubules around 7-8 microM tubulin. Therefore, the critical tubulin concentrations for polymer assembly off native centrosomes is higher than that observed for the centrosomes first denatured and then complemented in egg extracts. In vivo, the microtubule nucleating activity of centrosomes seems to be regulated by phosphorylation at the onset of mitosis (Centonze, V. E., and G. G. Borisy. 1990. J. Cell Sci. 95:405-411). Since cyclins are major regulators of mitosis, we tested the effect of adding bacterially produced cyclins to interphase egg extracts. Both cyclin A and B activate an H1 kinase in the extracts. Cyclin A-associated kinase causes an increase in the microtubule nucleating activity of centrosomes complemented in the extract but cyclin B does not. The critical tubulin concentration for polymer assembly off centrosomes complemented in cyclin A-treated extracts is similar to that observed for centrosomes complemented in interphase extracts. However, centrosomes complemented in cyclin A treated extracts nucleate much more microtubules at high tubulin concentration. We define this as the "capacity" of centrosomes to nucleate microtubules. It seems that the microtubule nucleating activity of centrosomes can be defined by two distinct parameters: (a) the critical tubulin concentration at which they begin to nucleate microtubules and (b) their capacity to nucleate microtubules at high tubulin concentrations, the latter being modulated by phosphorylation. 相似文献
19.
The important characteristic of novel Mimosa pudica tubulin is its ability to bind colchicine only when dithiothreitol is included in the isolation buffer, indicating the involvement of sulfhydryl groups in colchicine binding. Modification of sulfhydryl groups by a sulfhydryl modifying agent also affects the normal assembly of tubulin into microtubules, as revealed by electron microscopic and spectrophotometric studies. The number of free sulfhydryl groups present in tubulin protein responsible for both colchicine binding and polymerization has been found to be 4, distributed in alpha and beta subunits, and is distinctly different from the number reported for animal tubulin. 相似文献
20.
A macromolecular structure favouring microtubule assembly in NGF-differentiated pheochromocytoma cells (PC12). 总被引:1,自引:0,他引:1
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Cellular extracts derived from pheochromocytoma cells (PC12-) inhibit the assembly of calf brain tubulin, while those derived from nerve growth factor-differentiated cells (PC12+) do not display this effect. Incubation with RNase abolishes the inhibition by PC12- extracts and reveals the presence of an activating effect exerted by PC12+ extracts. Activation of microtubule assembly is enhanced when extracts are prepared from PC12+ cells exposed for 1 day to 1.0 microM taxol and is abolished when PC12+ extracts are: (a) prepared from cells incubated for 1 day with 1 microM colchicine, (b) treated with the non-ionic detergent Nonidet P-40 or (c) centrifuged at 100 000 g instead of 80 000 g. 2D gel electrophoresis of the proteins of the 100 000 g pellet responsible for the activating effect (referred to as 100 K g pellet) reveals the presence of 100 K, 88 K and 32 K proteins which are markedly enriched in PC12+ extracts. The 88 K protein is further enriched in taxol-treated cells and markedly reduced in the same cells incubated with colchicine. A correlation between the differential protein composition of the 100 K g pellets and their effect on microtubule formation is postulated. 相似文献