The Experts below are selected from a list of 81 Experts worldwide ranked by ideXlab platform
J. Bordas - One of the best experts on this subject based on the ideXlab platform.
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X-ray solution scattering studies on vinblastine-induced polymers of Microtubule Protein: structural characterisation and effects of temperature.
Journal of structural biology, 1992Co-Authors: J.l. Hodgkinson, T. Hutton, F.j. Medrano, J. BordasAbstract:We report here on X-ray solution scattering and electron microscopy studies of Microtubule Protein in the presence of the antimitotic drug, vinblastine. In buffer conditions used for Microtubule assembly, vinblastine caused the formation of coil-like structures. The coils appeared to be made up of two protofilaments. Details of the structure and behaviour of coils in solution were obtained from interpretation of their solution scattering patterns. Upon increasing temperature from 4 to 37 degrees C the pitch of the coils increased from 25.92 to 26.96 nm. However, little change was observed in their mean diameters (38.46 and 38.45 nm, respectively). Increasing the temperature also favoured increased formation and/or elongation of the coils. The effect of temperature on the pitch was fully reversible. Vinblastine-induced assembly of pure tubulin also showed the formation of coils. However, these coils appeared to consist of only one protofilament. Their mean diameters (38.35 nm) were similar to those of the coils formed from Microtubule Protein.
R G Burns - One of the best experts on this subject based on the ideXlab platform.
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Kinetics of GTP hydrolysis during the assembly of chick brain MAP2-tubulin Microtubule Protein
Biochemical Journal, 1991Co-Authors: R G BurnsAbstract:The kinetics of GTP hydrolysis during Microtubule assembly have been examined using chick brain MAP2-tubulin Microtubule Protein in a NaCl-supplemented buffer. The elongating Microtubules terminate in a ‘GTP cap’, since the kinetics of GTP hydrolysis are slower than those of subunit addition. GTP hydrolysis is (a) stoichiometric, (b) occurs as a vectorial wave as the initial rate of hydrolysis is proportional to the molar concentration of Microtubule ends and not to the initial rate of subunit addition, and (c) either does not occur, or occurs only at a much lower rate, in the terminal subunits.
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Assembly of chick brain MAP2-tubulin Microtubule Protein. Analysis of tubulin subunit flux rates by immunofluorescence microscopy.
Biochemical Journal, 1991Co-Authors: M F Symmons, R G BurnsAbstract:A filter-based immunofluorescence-microscopy method for obtaining Microtubule lengths has been developed and evaluated. Kinetic constants and mean lengths obtained show close agreement with those obtained by complementary methods applied to chick brain MAP2-tubulin Microtubule Protein in NaCl-supplemented buffer. The filter-based method has been used to estimate tubulin subunit flux (Jon) resulting from isothermal dilution of Microtubule populations to various free tubulin concentrations, (c). This experimental Jon(c) plot is significantly different from that predicted by a variety of theoretical models, but is consistent with a ‘lateral cap’ model of dynamic instability [Bayley, Schilstra & Martin (1990) J. Cell. Sci. 95, 33-48] adapted to accommodate the observed vectorial GTP hydrolysis.
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Assembly of chick brain MAP2-tubulin Microtubule Protein. Characterization of the Protein and the MAP2-dependent addition of tubulin dimers.
The Biochemical journal, 1991Co-Authors: R G BurnsAbstract:The principle Proteins present in twice-cycled chick brain Microtubule Protein were characterized. The Protein consists of a stoichiometric mixture of MAP2 and tubulin, together with a number of minor components. Its composition remains unaltered after a third cycle of assembly in a buffer supplemented with 67 mM-NaCl, with the exception of the phosphorylation of MAP2 to a low level (congruent to 1 mol.mol-1). The inclusion of 67 mM-NaCl dissociates the MAP2-tubulin oligomers, and restricts the assembly to the MAP2-dependent addition and loss of tubulin dimers, such that the assembly kinetics approximate to a simple pseudo-first-order reaction. The assembled Microtubules exhibit dynamic instability, with no evidence for end-to-end annealing.
Daniel L. Purich - One of the best experts on this subject based on the ideXlab platform.
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Microtubule Protein ADP-ribosylation in vitro leads to assembly inhibition and rapid depolymerization
Biochemistry, 1992Co-Authors: Robin M. Scaife, Leslie Wilson, Daniel L. PurichAbstract:Bovine brain Microtubule Protein, containing both tubulin and Microtubule-associated Proteins, undergoes ADP-ribosylation in the presence of [14C]NAD+ and a turkey erythrocyte mono-ADP-ribosyltransferase in vitro. The modification reaction could be demonstrated in crude brain tissue extracts where selective ADP-ribosylation of both the alpha and beta chains of tubulin and of the high molecular weight Microtubule-associated Protein MAP-2 occurred. In experiments with purified Microtubule Protein, tubulin dimer, the high molecular weight Microtubule-associated Protein MAP-2, and another high molecular weight mirotubule-associated Protein which may be a MAP-1 species were heavily labeled. Tubulin and MAP-2 incorporated [14C]ADP-ribose to an average extent of approximately 2.4 and 30 mol of ADP-ribose/mol of Protein, respectively. Assembly of Microtubule Protein into Microtubules in vitro was inhibited by ADP-ribosylation, and incubation of assembled steady-state Microtubules with ADP-ribosyltransferase and NAD+ resulted in rapid depolymerization of the Microtubules. Thus, the eukaryotic enzyme can ADP-ribosylate tubulin and Microtubule-associated Proteins to much greater extents than previously observed with cholera and pertussis toxins, and the modification can significantly modulate Microtubule assembly and disassembly.
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Evidence against impaired brain Microtubule Protein polymerization at high glucose concentrations or during diabetes mellitus.
Journal of neurochemistry, 1991Co-Authors: Ervin Y. Eaker, Daniel L. Purich, James M. Angelastro, Charles A. SninskyAbstract:Previous studies suggest that brain Microtubule Protein exposed to high glucose levels or isolated from diabetic rats can become glucosylated and that this impairs GTP-induced Microtubule polymerization. We set out to extend that investigation to define the mechanistic basis for inhibition of Microtubule assembly during diabetes or on incubation at high glucose levels. Rat and bovine brain Microtubule Protein was purified by cycles of polymerization/depolymerization. When Microtubules were incubated for 1 h in either buffer or buffer containing glucose (up to 165 mM), there was no difference in polymerization, a finding contrary to the earlier study. Other rats were injected with vehicle or streptozotocin (90 mg/kg) to induce diabetes as evidenced by serum glucose in excess of 300 mg%, and at 4 weeks, brain Microtubule Protein was isolated by the polymerization cycling method. Again, there was no difference in the amount or purity of isolated Microtubule Protein between control or diabetic rats. We also observed no increase in Microtubule glucosylation, and GTP-induced polymerization in vitro was indistinguishable for Protein derived from brains of normal rats and rats with diabetes as measured by turbidity or electron microscopy. Our results suggest that in vitro incubation with glucose or in vivo elevation of glucose during diabetes fails to impair Microtubule polymerization, pointing to other mechanisms for the neuropathy associated with diabetes.
J.l. Hodgkinson - One of the best experts on this subject based on the ideXlab platform.
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X-ray solution scattering studies on vinblastine-induced polymers of Microtubule Protein: structural characterisation and effects of temperature.
Journal of structural biology, 1992Co-Authors: J.l. Hodgkinson, T. Hutton, F.j. Medrano, J. BordasAbstract:We report here on X-ray solution scattering and electron microscopy studies of Microtubule Protein in the presence of the antimitotic drug, vinblastine. In buffer conditions used for Microtubule assembly, vinblastine caused the formation of coil-like structures. The coils appeared to be made up of two protofilaments. Details of the structure and behaviour of coils in solution were obtained from interpretation of their solution scattering patterns. Upon increasing temperature from 4 to 37 degrees C the pitch of the coils increased from 25.92 to 26.96 nm. However, little change was observed in their mean diameters (38.46 and 38.45 nm, respectively). Increasing the temperature also favoured increased formation and/or elongation of the coils. The effect of temperature on the pitch was fully reversible. Vinblastine-induced assembly of pure tubulin also showed the formation of coils. However, these coils appeared to consist of only one protofilament. Their mean diameters (38.35 nm) were similar to those of the coils formed from Microtubule Protein.
Timothy L. Karr - One of the best experts on this subject based on the ideXlab platform.
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Biochemical characterization of related Microtubule Proteins in Drosophila melanogaster and adult rat brain
Brain Research, 1995Co-Authors: Shaila Srinivasan, Timothy L. KarrAbstract:We describe the biochemical characteristics of three Proteins isolated from Drosophila embryos and the rat brain. We refer to these Proteins as DMAPs (Drosophila Microtubule-associated Proteins) since they were identified by monoclonal antibodies generated against Microtubule Protein (MTP) purified from Drosophila melanogaster embryos. DMAP-45 is a 45 kDa Protein that binds Microtubules in an ATP dependent manner. Preliminary biochemical evidence suggests that DMAP-45 may be an actin-related Protein. DMAP-55 is a 55 kDa Protein and based on its molecular weight and isoelectric point, may be a novel isoform of tubulin. DMAP-66 is a 66 kDa Protein that binds strongly to Microtubules in vitro and has multiple isoforms. Analyses of Proteins in rat brain tissue extracts and purified rat brain MTP identified Proteins of similar molecular weight and isoelectric points and are designated DMAP-45R, -55R and -66R. The presence of Proteins with common biochemical properties in these widely divergent animal species suggests that they are related Proteins.