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In this paper, we report on the effect of brain microtubule-associated proteins (MAPs) on the dynamic instability of microtubules as well as on the nucleation activity of purified centrosomes. Under our experimental conditions, tau and MAP2 have similar effects on microtubule nucleation and dynamic instability. Tau increases the apparent elongation rate of microtubules in proportion to its molar ratio to tubulin, and we present evidence indicating that this is due to a reduction of microtubule instability rather than to an increase of the on rate of tubulin subunits at the end of growing microtubules. Increasing the molar ratio of tau over tubulin leads also to an increase in the average number of microtubules nucleated per centrosome. This number remains constant with time. This suggests that the number of centrosome-nucleated microtubules at steady state can be determined by factors that are not necessarily irreversibly bound to centrosomes but, rather, affect the dynamic properties of microtubules.  相似文献   

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E Hamel  C M Lin 《Biochemistry》1984,23(18):4173-4184
A new method for separating microtubule-associated proteins (MAPs) and tubulin, appropriate for relatively large-scale preparations, was developed. Most of the active tubulin was separated from the MAPs by centrifugation after selective polymerization of the tubulin was induced with 1.6 M 2-(N-morpholino)ethanesulfonate (Mes) and GTP. The MAPs-enriched supernatant was concentrated and subsequently clarified by prolonged centrifugation. The supernatant (total soluble MAPs) contained almost no tubulin, most of the nucleosidediphosphate kinase activity of the microtubule protein, good activity in promoting microtubule assembly in 0.1 M Mes, and proteins with the electrophoretic mobility of MAP-1, MAP-2, and tau factor. The pellet, inactive in supporting microtubule assembly, contained denatured tubulin, most of the ATPase activity of the microtubule protein, and significant amounts of protein with the electrophoretic mobility of MAP-2. Insoluble material at this and all previous stages, including the preparation of the microtubule protein, could be heat extracted to yield soluble protein active in promoting microtubule assembly and containing MAP-2 as a major constituent. The total soluble MAPs were further purified by DEAE-cellulose chromatography into bound and unbound components, both of which induced microtubule assembly. The bound component (DEAE-MAPs) contained proteins with the electrophoretic mobility of MAP-1, MAP-2, and tau factor. The polymerization reaction induced by the unbound component (flow-through MAPs) produced very high turbidity readings. This was caused by the formation of bundles of microtubules. Although the flow-through MAPs contained significantly more ATPase, tubulin-independent GTPase, and, especially, nucleosidediphosphate kinase activity than the DEAE-MAPs, preparation of a MAPs fraction without these enzymes required heat treatment.  相似文献   

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Tubulin is a major substrate for endogenous Ca2+-calmodulin-dependent phosphorylation in synaptic cytoplasm. The present study details the purification to apparent homogeneity and characterization of a brain cytosolic Ca2+-calmodulin-dependent kinase which phosphorylates tubulin and microtubule-associated proteins as major substrates. The cytosolic kinase system, purified by sequential chromatography on phosphocellulose resin, calmodulin-affinity resin, and Fractogel TSK HW-55, chromatographs as a homogeneous complex of approximately 600,000 Da on Sephacryl S-300. This calmodulin-dependent kinase possesses a group of properties which specifically characterize this enzyme system: 1) the enzyme contains two calmodulin-binding doublets, rho and sigma, of approximately 52,000 and 63,000 Da, respectively; 2) both the rho and the sigma subunits demonstrate isoelectric points between 6.7 and 7.2; 3) both the rho and sigma subunits demonstrate autophosphorylation; 4) both the rho and sigma subunits show significant homologies as assessed by tryptic peptide fingerprints; 5) in the absence of substrate, both the rho and sigma subunits manifest lower mobility autophosphorylated species; 6) the kinase phosphorylates beta-tubulin equally on threonine and serine residues. Substrate specificity, kinetic parameters, calmodulin-binding properties, subunit composition, and subunit isoelectric points clearly differentiate this enzyme from other previously reported calmodulin-dependent kinases.  相似文献   

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Tubulin, the major constituent protein of microtubules, is a heterodimer of alpha and beta subunits. Both alpha and beta exist in multiple isotypic forms. It is not clear if different isotypes perform different functions. In order to approach this question, we have made a monoclonal antibody specific for the beta III isotype of tubulin. This particular isotype is neuron-specific and appears to be phosphorylated near the C terminus. We have used immunoaffinity depletion chromatography to prepare tubulin lacking the beta III subunit. We find that removal of the beta III isotype results in a tubulin mixture able to assemble much more rapidly than is unfractionated tubulin when reconstituted with either of the two microtubule-associated proteins (MAPs), tau or MAP 2. Our results suggest that the different isotypes of tubulin differ from each other in their ability to polymerize into microtubules. We have also found that the anti-beta III antibody can stimulate microtubule assembly when reconstituted with tubulin and either tau or MAP 2. When reconstituted with tubulin lacking the beta III isotype, the antibody causes the tubulin to polymerize into a polymer that is a microtubule in the presence of MAP 2 and a ribbon in the presence of tau.  相似文献   

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Two different proteins, tau and microtubule-associated protein 2 (MAP 2), are able to stimulate tubulin polymerization into microtubules in vitro, but it is not certain if both proteins act by the same mechanism. We have examined the effects of tau and MAP 2 on the vinblastine-induced polymerization of tubulin into spiral filaments. In the presence of tau, vinblastine induced extensive aggregation of tubulin as shown by a large increase in turbidity. The increase in turbidity was accompanied by the formation of large numbers of spirals composed of a filament 40-60 A in diameter. The rate and extent of this aggregation into spirals were dependent on the concentrations of tubulin, tau, and vinblastine. Unlike normal microtubule assembly, this type of aggregation was not inhibited by colchicine or podophyllotoxin. In contrast, MAP 2, even at high concentrations, was less effective than tau at promoting the vinblastine-induced increase in turbidity of tubulin. In fact, MAP 2 strongly inhibited the effect of tau. These results indicate that tau and MAP 2 interact differently with the tubulin molecule in the presence of vinblastine and suggest that the two proteins may play different roles in regulating or promoting microtubule assembly. Vinblastine may thus be a useful probe in analyzing the modes of interactions of tau and MAP 2 with tubulin.  相似文献   

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T Fujii  R Tanaka 《Life sciences》1979,24(18):1683-1690
The interaction between actomyosin from rabbit skeletal muscle and microtubule proteins or 6S tubulin from rat brain was investigated with respect to the change in ATPase activity and physicochemical properties. Myosin bound to both microtubule proteins and 6S tubulin at low ionic strength. In the aggregates the molar ratio of microtubule proteins or 6S tubulin to myosin was 0.5–1.5 or 1.5–2.5. The superprecipitation of actomyosin was inhibited by 6S tubulin. The degree of superprecipitation inhibition was dependent on the mixing order of myosin, actin, 6S tubulin, and ATP. When myosin was preincubated first with 6S tubulin, the inhibition was most marked. The actin activation of myosin Mg-ATPase was inhibited by both microtubule proteins and 6S tubulin with stronger effects by the latter. The preincubation of myosin with 6S tubulin prior to the addition of actin induced not only greater inhibition of ATPase but also the binding of a larger quantity of 6S tubulin to myosin than the preincubation of myosin with actin. The similar results were obtained with microtubule proteins.  相似文献   

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We have prepared microtubules from brain tissue by stabilizing the cellular microtubules in 6.7 M glycerol buffer, instead of the usual procedure which extracts the solubilized protein and then reassembles microtubules in vitro at some later time. There are substantial differences in the microtubule associated proteins obtained by the two methods, and brain spectrin is a major component of the stabilized microtubules. We have now modified the buffer used for the isolation of stabilized microtubules to minimize their tendency to aggregate. When the stabilized microtubules were further purified by sucrose density gradient centrifugation, we were able to distinguish previously unidentified polypeptides at 49, 74 (doublet), and 100 kilodaltons (doublet). These bands maintained staining intensity in the same proportion to tubulin as in the original homogenate, whereas background proteins were diminished in staining intensity. We now report the identification of the 74-kilodalton doublet polypeptides as synapsin-1 by peptide mapping. Synapsin-1 is a protein known to bind to brain spectrin and also to microtubules, and may thus serve as a linker between these cytoskeletal components.  相似文献   

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Cyclic-AMP stimulates phosphorylation of Hf1 and Hf2b histone fractions and of protamine by tubulin preparations. Lyophilization of the tubulin removes the requirement for cyclic-AMP; the kinase is fully active in the absence of cyclic nucleotide. The casein kinase activity of tubulin is independent of cyclic-AMP. Only cyclic-IMP can replace cyclic-AMP at the low concentrations at which the cyclic-AMP is effective. 5′-AMP inhibits basal levels of Hf 1 histone phosphorylation. Several attempts have been made to separate the kinase activity from tubulin. Both tubulin and kinase activity are precipitated by vinblastine. Mg2+ precipitation of tubulin inactivates the kinase; there is no evidence that the precipitation step removes the kinase from tubulin. Tubulin which has been assembled into microtubules, collected by centrifugation and disassembled, retains its kinase activity. Kinase activity and tubulin co-elute from DEAE-cellulose. Taken together, these experiments demonstrate that tubulin, prepared by fractionation with ammonium sulfate followed by elution from DEAE Sephadex with 0-8 M KC1 and concentration by reprecipitation with ammonium sulfate has a closely associated, cyclic-AMP dependent protein kinase activity, which may be intrinsic to the tubulin peptides themselves.  相似文献   

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This study provides a comprehensive, high-resolution structural analysis of the central-pair microtubule apparatus of sperm flagella. It describes the arrangement of several microtubule-associated sheath components and suggests, contrary to previous thinking, that microtubules are structurally asymmetric. The two microtubules of the central pair are different in several respects: the C2 tubule bears a single row of 18-nm-long sheath projections with an axial periodicity of 16 nm, whereas the C1 tubule possesses rows of 9-nm globular sheath components with an axial repeat of 32 nm. The lumen of the C2 tubule always appears completely filled with electron-dense material; that of the C1 tubule is frequently hollow. The C2 tubule also possesses a series of beaded chains arranged around the microtubule; the beaded chains are composed of globular subunits 7.5-10 nm in diameter and appear to function in the pairing of the C1 and C2 tubules. These findings indicate: that the beaded chains are not helical, but assume the form of lock washers arranged with a 16-nm axial periodicity on the microtubule; and that the lattice of tubulin dimers in the C2 tubule is not helically symmetric, but that there are seams between certain pairs of protofilaments. Proposed lattice models predict that, because of these seams, central pair and perhaps all singlet microtubules may contain a ribbon of 2-5 protofilaments that are resistant to solubilization; these models are supported by the results of the accompanying paper (R. W. Linck, and G. L. Langevin. 1981. J. Cell Biol. 89: 323-337.  相似文献   

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Following induction of hemopoiesis, poly(A)-rich RNA was prepared from the heart of the tarantula, Eurypelma californicum, and translated in rabbit reticulocyte lysates. In vitro translation products were immunoprecipitated with antiserum against whole dissociated Eurypelma hemocyanin. Analysis of the immunoprecipitate by sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed a set of polypeptides comigrating with authentic Eurypelma hemocyanin. The mRNA was transcribed into cDNA, clones were constructed using the pUC9 vector and probed with a synthetic 17-mer oligonucleotide probe complementary to the amino acid sequence of the 'copper A' binding site of chelicerate hemocyanins. One clone, pHC4, contained a 1.62-kb cDNA insert, which was subcloned into phage M13. Sequence analysis by the dideoxynucleotide chain-termination method yielded a nucleotide sequence coding for 526 amino acids of Eurypelma hemocyanin subunit e.  相似文献   

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Bovine brain tau protein (tau) consists of four closely related phosphoproteins named tau 1, tau 2, tau 3 and tau 4, that range in size from 55 to 68 kDa (as determined by gel electrophoresis). Here we report an improved large-scale purification method for tau protein and the separation of the four individual tau protein species. The separation of the individual tau protein was accomplished by two chromatographic techniques: hydroxyapatite chromatography allowed the separation of two pairs of tau protein (tau 1 and tau 3) and (tau 2 and tau 4); fast protein liquid chromatography on a Mono Q column at basic pH achieved the resolution of the individual tau protein species in each pair derived from hydroxyapatite columns. Chromatography on the Mono Q column revealed that tau protein possesses previously unrecognized, highly reactive sulfhydryl groups that may oxidize to form intermolecular disulfide bridges. The isolation of individual species of tau in substantial quantities permitted an improved amino acid analysis that demonstrated the occurrence of cysteine and tryptophan in the protein. The availability of individual tau protein species greatly simplified the analysis for mode II phosphorylation of tau, which was found to be catalyzed by the calcium/phospholipid-dependent protein kinase C. The mode II phosphorylation of tau by protein kinase C was not associated with a mobility shift for tau protein in SDS-polyacrylamide gel electrophoresis, in contrast to mode I phosphorylation of tau by the Ca2+/calmodulin-dependent kinase, which produces a substantial shift in mobility.  相似文献   

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We have recently reported a neural variant of microtubule-associated protein 4 with a short pro-rich region (MAP4-SP). Here, we show that the neural MAP4 has reduced microtubule-stabilizing activity, compared to the ubiquitous MAP4 with a long pro-rich region (MAP4-LP), both in vitro and in vivo. Fluorescence recovery after photobleaching analyses revealed that the interaction of MAP4-SP with the microtubules is very rapid, with a half-time of fluorescence recovery of 7 +/- 2.36 s, compared to 19.5 +/- 3.03 s in case of MAP4-LP. The dynamic interaction of MAP4-SP with microtubules in neural cells may contribute to the dynamic behaviors of extending neurites.  相似文献   

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R B Maccioni  C I Rivas    J C Vera 《The EMBO journal》1988,7(7):1957-1963
In previous studies we have demonstrated that a 4-kd tubulin fragment, including amino acid residues from Phe418 to Glu450 in alpha-subunit and Phe408-Ala445 of the beta-sequence, plays a major role in controlling tubulin interactions leading to microtubule assembly. The 4-kd carboxyl-terminal domain also constitutes an essential domain for the interaction of microtubule-associated proteins (MAPs). Removal of the 4-kd fragment facilitates tubulin self-association and renders the assembly MAP-independent. In order to define the substructure of the tubulin domain for MAP interaction, we have examined the binding of 3H-acetylated C-terminal peptides to MAP-2 and tau. Two synthetic peptides from the low-homology region within the 4-kd domain alpha (430-441) and beta (422-434) and the peptide, alpha (401-410) of the high-homology region adjacent to the 4-kd domain, were analyzed with respect to MAP interaction. The binding data showed a relatively strong interaction of MAP-2 with the beta (422-434) peptide and a weaker interaction of both MAPs components with alpha (430-441) tubulin peptide as analyzed by Airfuge ultracentrifugation and zone filtration chromatography. The homologous alpha (401-410) peptide did not bind to either MAP-2 or tau. Equilibrium dialysis experiments showed a co-operative binding of beta (422-434) peptide to multiple sites in tau. The alpha (430-441) peptide exhibited a stronger interaction for tau as compared with MAP-2.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

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Bacterial LPS is a potent stimulator of immune cells, but its mechanisms are unknown. A possible role for microtubules in LPS actions has been indicated by previous findings that the microtubule-active agent, taxol, can mimic some effects of LPS in macrophages from normal strains of mice, but not from genetically LPS-hyporesponsive strains. In this report we demonstrate that isolated microtubules from mouse brain can bind LPS in vitro. LPS and tubulin coeluted through a gel filtration column, and LPS was cross-linked to microtubule proteins with an iodinatable, photoreactive agent, sulfosuccinimidyl 2-(p-azidosalicylamido) ethyl-1,3'-dithiopropionate. beta-Tubulin and microtubule-associated protein-2 (MAP), a predominant MAP in the brain, bound LPS specifically. Cross-linking was inhibited by an excess of unlabeled LPS or partially by unlabeled lipid A, but not by 2 M NaCl. Under the same conditions, neither myosin nor soybean trypsin inhibitor was labeled by the photoaffinity LPS probe, nor did these proteins compete for binding of LPS to beta-tubulin. These findings support the hypothesis that the microtubule network could be an intracellular target for LPS, and suggest further that a beta-tubulin-associated MAP could have an important role in LPS actions.  相似文献   

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Tubulin and Microtubule-Associated Proteins (MAPs) isolated from chick brain by cycles of assembly and disassembly in vitro have been stoichiometrically labelled with the fluorogenic compound fluorescamine. Under the conditions employed, tubulin can be labelled with up to 2 moles of fluorescamine/mole of dimer while the MAPs accept up to 8 moles/mole of protein, assuming an average molecular weight of 300 000 D. After the labelling procedure, both the tubulin and MAPs retain their native conformations as judged by several criteria: (a) the labelled proteins remain competent to participate in further rounds of the assembly-disassembly procedure in vitro, and the kinetics of this assembly are identical to those seen with an unlabelled control sample; (b) incubation of the fluorescent microtubule proteins with the antimitotic agent vinblastine sulfate results in the formation of birefringent vinblastine-tubulin paracrystals, indicating the binding site for this ligand is unaltered; (c) the mobilities of the fluorescent tubulin and MAPs on SDS-polyacrylamide gels are unaltered when compared to the mobilities of the respective unlabelled control proteins. The results are discussed in relation to the use of these fluorescent cytoskeletal proteins as easily detectable biochemical and histochemical probes.  相似文献   

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Microtubules were reconstituted from homogeneous brain tubulin and homogeneous preparations of two different microtubule associated proteins, the high molecular weight MAP 2 proteins or the tau proteins. The resulting microtubules were characterized by three electron microscopical procedures: Thin sectional analysis of embeded material, negative staining analysis using a STEM microscope and high resolution metal-shadowing analysis. By all three procedures MAP 2 microtubules have a much rougher surface morphology than tau microtubules, in agreement with the much higher molecular weight of the MAP 2 proteins. Tau microtubules, however, do not show the very smooth surface of microtubules assembled from pure tubulin in the absence of any microtubule associated proteins. In the case of MAP 2 microtubules thin sectional analysis as well as metal shadowing reveals that the globular protrusions seen in negative staining analysis appear as linear side arms which may extend by as much as 30 nm on both sides from the microtubular wall proper, giving rise to an overall structure with a diameter close to 100 nm. The possible implication of such structures for in vivo situations is briefly discussed as is the possibility that the "halo-effect" around microtubules seen in vivo may be due to a structural organization similar to that of MAP 2 tubules in vitro.  相似文献   

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