Microtubule Assembly in the Absence of Added Nucleotides (original) (raw)
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Biochemical Studies on the in Vitro Assembly and Disassembly of Microtubules
Annals of the New York Academy of Sciences, 1975
The mechanism of assembly and disassembly of cytoplasmic microtubules in vivo is not clearly understood, Kinetic and equilibrium studies in vitro are potentially useful in suggesting reasonable models of tubulin assembly, which can then be tested in vivo. Large quantities of pure tubulin are needed for reliable kinetic studies and other solution physicochemical techniques. We have developed a purification scheme for neurotubules, based on assembly and disassembly under the conditions defined by Weisenberg,' modified by the addition of glycerol as a stabilizing agent.' Several minor components present in these tubulin preparations have been characterized.3 One has similar physical properties to the ATPase known as dynein, found in the microtubules of the axonemes of flagella and cilia;'. except that the brain dynein has very low ATPase activity. The kinetics and equilibria of tubule assembly have been studied mainly by quantitative turbidimetry. The results of these studies are insufficient to establish a unique model for tubulin assembly, but they do place strong constraints on possible models of assembly in vitro.
Journal of Neurochemistry, 1986
Fast-performance liquid chromatography was used to purify assembly-competent tubulin from porcine brain microtubule protein prepared by two cycles of assembly-disassembly. Microtubule protein (1 -100 mg at 1.5-2.5 mg/ml) in buffer consisting of 0.1 M 2-(N-mor-pho1ino)ethanesulfonic acid, 0.5 mM MgCI2, 1 mM EGTA, 0.3 M KCI, and 0.02 mM GTP (pH 6.6) was applied to the Mono Q column (anion exchanger). The microtubule-associated proteins, GTP and GDP, eluted in the void volume. The tubulin fraction eluted at 0.45-0.50 M KC1 with 65-80% recovery. The tubulin fraction contained trace enzymatic activities when compared with the starting microtubule protein, i.e., < I versus 60 mU/mgl min of nucleoside diphosphate kinase, 0.2 versus 7.0 nmolimglmin of Mg-ATPase at pH 6.6, and 0.2 versus 88 mU/mg/min of adenylate kinase. Both the Mono Q-purified tubulin and the pelleted microtubules that were assembled in 0.5 mM [3H]GTP contained 0.77 mol of labeled nucleotideitubulin dimer. The Mono Q-purified tubulin fraction was competent to assemble, i.e., the critical concentration was 0.1 mgiml in the presence of 0.03 mM taxol and 1 mM GTP at 37°C. The Mono Q-purified tubulin fraction showed trace high-molecular-weight components, which were removed on Mono S (cation exchanger) columns. Alternatively, microtubule protein in buffer was applied to the Mono S column. Tubulin, trace nontubulin proteins, and several enzymatic activities came off in the void volume. A combination of Mono Q-Mono S or Mono S-Mono Q chromatography resulted in highly purified protein. However, it is possible to separate active tubulin-nucleotide rapidly from the major microtubule-associated proteins, enzyme contaminants, and unbound nucleotide with just Mono Q column chromatography. Key Words: Brain tubulin-Microtubule-Neurotubule-Microtubule-associated proteins-HPLC -Fast-performance liquid chromatography. Roychowdhury S. and Gaskin F. Separation of assembly-competent tubulin from brain microtubule protein preparations using a fast-performance liquid chromatography procedure.
Microtubule Assembly of Isotypically Purified Tubulin and Its Mixtures
Biophysical Journal, 2008
Numerous isotypes of the structural protein tubulin have now been characterized in various organisms and their expression offers a plausible explanation for observed differences affecting microtubule function in vivo. While this is an attractive hypothesis, there are only a handful of studies demonstrating a direct influence of tubulin isotype composition on the dynamic properties of microtubules. Here, we present the results of experimental assays on the assembly of microtubules from bovine brain tubulin using purified isotypes at various controlled relative concentrations. A novel data analysis is developed using recursive maps which are shown to be related to the master equation formalism. We have found striking similarities between the three isotypes of bovine tubulin studied in regard to their dynamic instability properties, except for subtle differences in their catastrophe frequencies. When mixtures of tubulin isotypes are analyzed, their nonlinear concentration dependence is modeled and interpreted in terms of lower affinities of tubulin dimers belonging to the same isotype than those that represent different isotypes indicating hitherto unsuspected influences of tubulin dimers on each other within a microtubule. Finally, we investigate the fluctuations in microtubule assembly and disassembly rates and conclude that the inherent rate variability may signify differences in the guanosine-5′-triphosphate composition of the growing and shortening microtubule tips. It is the main objective of this article to develop a quantitative model of tubulin polymerization for individual isotypes and their mixtures. The possible biological significance of the observed differences is addressed.
Association of Newly Synthesized Tubulin with Brain Microsomal Membranes
Journal of Neurochemistry, 1980
Tubulin has been found to be synthesized on both membrane-bound and free polyribosomes prepared from brain. Cell-free studies indicate that tubulin made on rough microsomes is incorporated into the endoplasmic reticulum membrane as it is synthesized. This tubulin remains associated with the membrane after sedimentation and washing. The tubulin is not removed from the membrane after stripping ribosomes from the membranes in KCIpuromycin, followed by repeated washing by either sedimentation or flotation in 0.5 M-KCI. The membrane tubulin is partially susceptible to proteolysis by trypsin and chymotrypsin: p-tubulin is more accessible to the proteases than is a-tubulin. Nonionic detergents extract mostly 0-tubulin from the microsomal membrane. Newly synthesized tubulin which has been extracted from micro-soma1 membranes in 0.5% Nonidet P-40, coassembles and disassembles with carrier microtubule protein. The insertion of newly synthesized tubulin into endoplasmic reticulum membrane may be the first step in the incorporation of tubulin into the plasma membrane. Key words: Rough microsomes-Cell-free protein synthesis-Microtubule protein-Membrane protein. Soifer D. and Czosnek H. Association of newly synthesized tubulin with brain microsomal membranes.
Opposite end assembly and disassembly of microtubules at steady state in vitro
Cell, 1978
Measurements of tubulin exchange into and from bovine brain microtubules at steady state in vitro were made with 3H-GTP as a marker for tubulin addition to or loss from microtubules. Tubulin has an exchangeable GTP binding site that becomes nonexchangeable in the microtubule. We found that tubulin addition to and loss from microtubules under steady state conditions occurred at equivalent rates, that loss and gain were linear, and that exchange rates (percentage of total tubulin in microtubules lost or gained per hour) were dependent upon microtubule length. Furthermore, we found that podophyllotoxin blocked steady state assembly, but did not alter the rate of steady state tubulin loss. When the assembling microtubule end was pulsed with 3H-GTP at steady state, the label was almost completely retained during a subsequent chase. We conclude that the microtubule assembly-disassembly "equilibrium" is a steady state summation of two different reactions which occur at opposite ends of the microtubule, and that assembly and disassembly occur predominantly and perhaps exclusively at the ofiposite ends under steady state conditions in vitro.
Association of brain g-tubulins with ab-tubulin dimers
Biochemical …, 2002
g-Tubulin is necessary for nucleation and polar orientation of microtubules in vivo. The molecular mechanism of microtubule nucleation by g-tubulin and the regulation of this process are not fully understood. Here we show that there are two g-tubulin forms in brain that are present in complexes of various sizes. Large complexes tend to dissociate in the presence of high-salt concentration. Both g-tubulins copolymerized with tubulin dimers and multiple g-tubulin bands were identified in microtubule protein preparations under conditions of non-denaturing electrophoresis. Immunoprecipitation experiments with monoclonal antibodies against g-tubulin and a-tubulin revealed interactions of both g-tubulin forms with tubulin dimers irrespective of the size of complexes. We suggest that besides small and large g-tubulin complexes, other molecular g-tubulin form(s) exist in brain extracts. Twodimensional electrophoresis revealed multiple charge variants of g-tubulin both in brain extracts and in microtubule protein preparations. Post-translational modification(s) of gtubulins might therefore play an important role in the regulation of microtubule nucleation in neuronal cells.
Proceedings of the National Academy of Sciences, 1974
Depolymerization products of purified microtubules from porcine brain were examined by sedimentation analysis and electron microscopy. The complete depolymerization mixture exhibited 36S and 6S components in concentration-dependent equilibrium, whether depolymerization was caused by low temperature or by calcium ion. These components were recognized by electron microscopy as spirals and rings, and small particles. Agarose column chromatography yielded two major fractions, a leading one comprising mostly 36S and some 6S material and a trailing one of solely 6S material. The latter had high specific colchicine-binding activity and no tendency to polymerize. For the leading peak material these properties were the converse. It is proposed that tubulin molecules (of mass 110,000 daltons) exist in two states, here called X and Y, with those of the X-state equivalent to the material found predominantly in the trailing fraction, and those of the Y-state equated with the material in the lead...
Separation of microtubule populations in rat brain homogenates by differential centrifugation
Brain Research, 1986
Subcellular fractions were separated from rat homogenates at 600 g for 5 min (P1), 15,000 g for 10 min (P2), 48,000 g for 60 min (P3), 100,000 g for 150 min (P4) and a final supernatant (S). They contained 5.0, 14.3, 14.7, 24.7 and 41.3% of the total tubulin in the homogenate measured by colchicine binding. The microtubules recovered in a pool of fractions P1 +P2+P3 underwent a faster depolymerization than those of P4 when the sediments were suspended in buffered solutions, poor in the stabilizing agent glycerol. As was observed with the electron microscope, P4 was richer than the other fractions in microtubules associated with membranous structures (MTA) by filamentous lateral connections. Subcellular distribution and lability properties may be the result of the connection of microtubules with membranous structures; the attached membranes would decrease the sedimentation velocity of microtubules, and lateral bridges could increase microtubular stability. Differences in length were not a cause of microtubule separation since this parameter did not vary among the microtubule of the different fractions or between MTA and non-associated microtubules of the same fraction.