Separation of active tubulin and microtubule-associated proteins by ultracentrifugation and isolation of a component causing the formation of microtubule bundles

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.     

Ionic and nucleotide requirements for microtubule polymerization in vitro.

The ionic and nucleotide requirements for the in vitro polymerization of microtubules from purified brain tubulin have been characterized by viscometry. Protein was purified by successive cycles of a temperature dependent assembly-diassembly scheme. Maximal polymerization occurred at a concentration of 0.1 M Pipes (piperazine-N,N'-bis(2-ethanesulfonic acid)); increasing ionic strength by addition of NaCl to samples prepared in lower buffer concentrations did not result in an equivalent level of polymerization. Both Na-+ and K-+ inhibited microtubule formation at levels greater than 240 mM, withmaximal assembly occurring at physiological concentrations of 150 mM. Maximal extent of assembly occurred at pH 6.8 and optimal rate at pH 6.6. Inhibition of polymerization was half-maximal at added calcium concentrations of 1.0 mM and magnesium concentrations of 10.0 mM. EGTA (ethylene glycol bis(beta-aminoethyl ether)tetraacetic acid), which chelates Ca-2+, had no effect on polymerization over a concentration range of 0.01-10.0 mM. In contrast, EDTA (ethylenediaminetetraacetic acid), which chelates both Mg-2+ and Ca-2+, inhibited assemble half-maximally at 0.25 mM and totally at 2.0 mM. As determined from experiments using Mg-2+-EDTA buffers, magnesium was required for polymerization. Magnesium promoted the maximal extent of assembly at substoichiometric levels relative to tubulin, but was maximal for both rate and extent at stoichiometric concentrations. Elemental analyses indicated that approximately 1 mol of magnesium was tightly bound/mol of tubulin dimer. Viscosity development was dependent upon hydrolyzable nucleoside triphosphate, and stoichiometric levels of GTP were sufficient for maximal polymerization. The effect of magnesium in increasing the rate of GTP-dependent polymerization suggests that a Mg-2+-GTP complex is the substrate required for a step in assembly.     

Bundling of microtubules in vitro by a high molecular weight protein prepared from the squid axon

A high molecular weight protein has been partially purified from sheaths of squid giant axons. This protein fraction was capable of restoring the membrane excitability of the squid axon which had been destroyed by internal perfusion of microtubule poison, when perfused along with microtubule proteins (Matsumoto et al. (1979) J. Biochem. 86, 1155-1158). This protein, designated as 260 K protein, was purified by gel filtration and Con A-Sepharose affinity chromatography. The apparent molecular weight of the axonal protein was estimated to be 260,000 by electrophoresis in the presence of sodium dodecylsulfate. This protein was revealed to be a glycoprotein. When phosphocellulose-purified tubulin was incubated with 260 K protein at 36 degrees C in the presence of dimethylsulfoxide, turbidity of the solution was much increased. 260 K protein co-sedimented with microtubles assembled from purified tubulin. Light microscopic and electron microscopic observations revealed that the high turbidity was due to bundling of microtubules which was caused by 260 K protein. On the other hand, the effect of this protein on the turbidity increase was not so prominent when microtubules were assembled from microtubule proteins consisting of tubulin and microtubule-associated proteins. High shear and low shear viscometry and co-sedimentation experiments revealed that 260 K protein had little effect on actin polymerization under the same medium conditions as used in tubulin polymerization.     

Enhancement of tubulin assembly as monitored by a rapid filtration assay

The early kinetics of microtubule formation from lamb brain tubulin isolated by affinity chromatography can be followed by a newly developed filter assay. The rapid collection of microtubules on glass fiber filters permits the calculation of the moles of tubulin polymerized. The filter assay gives both a rate and extent of polymerization that are identical to those obtained by turbidity or sedimentation analysis, respectively. The microtubules trapped by the filter are readily depolymerized by cold (t12= 3 min) and slowly by colchicine (t1/2= 32min). Tubulin purified by affinity chromatography requires a high protein concentration (>4 mg/ml) for polymerization. Although 5m glycerol allows polymerization to occur at tubulin concentrations below 2 mg/ml, the maximum amount of microtubule formation is observed at low tubulin concentration when microtubule-associated proteins are present. These proteins are not retained by the affinity resin; however, they can be eluted from diethylaminoethyl-Sephadex by solutions containing 0.3m KCl. Microtubule-associated proteins enhance both the rate of polymerization and the total amount of tubulin polymerized as assessed by the filter assay, suggesting that they are involved in both initiation and elongation of microtubules.     

Purification, characterization, and assembly properties of tubulin from unfertilized eggs of the sea urchin Strongylocentrotus purpuratus

Tubulin was purified from unfertilized eggs of the sea urchin Strongylocentrotus purpuratus by chromatography of an egg supernatant fraction on DEAE-Sephacel or DEAE-cellulose followed by cycles of temperature-dependent microtubule assembly and disassembly in vitro. After two assembly cycles, the microtubule protein consisted of the alpha- and beta-tubulins (greater than 98% of the protein) and trace quantities of seven proteins with molecular weights less than 55 000; no associated proteins with molecular weights greater than tubulin were observed. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on urea-polyacrylamide gradient gels, the alpha- and beta-tubulins did not precisely comigrate with their counterparts from bovine brain. Two-dimensional electrophoresis revealed that urchin egg tubulin contained two major alpha-tubulins and a single major beta species. No oligomeric structures were observed in tubulin preparations maintained at 0 degrees C. Purified egg tubulin assembled efficiently into microtubules when warmed to 37 degrees C in a glycerol-free polymerization buffer containing guanosine 5'-triphosphate. The critical concentration for assembly of once- or twice-cycled egg tubulin was 0.12-0.15 mg/mL. Morphologically normal microtubules were observed by electron microscopy, and these microtubules were depolymerized by exposure to low temperature or to podophyllotoxin. Chromatography of a twice-cycled egg tubulin preparation on phosphocellulose did not alter its protein composition and did not affect its subsequent assembly into microtubules. At concentrations above 0.5-0.6 mg/mL, a concentration-dependent "overshoot" in turbidity was observed during the assembly reaction. These results suggest that egg tubulin assembles into microtubules in the absence of the ring-shaped oligomers and microtubule-associated proteins that characterize microtubule protein from vertebrate brain.     

Analysis of microtubule polymerization inhibitors in sea urchin egg extracts: Evidence for a protease

Inhibitors of microtubule polymerization have been found in extracts of unfertilized sea urchin eggs using neural tubulin polymerization assays without glycerol. The inhibitory activity is partially destroyed by boiling or by reduction and carboxymethylation and is nondialyzable. When chromatographed on DEAE-cellulose, the inhibitory activity is eluted over a broad NaCl gradient and is in association with several peaks. This partially purified inhibitor is not destroyed by incubation with RNase A. When the partially purified inhibitor is incubated with brain microtubule protein under conditions which support microtubule polymerization, both high molecular weight-microtubule associated proteins and tubulin appear to be digested when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteolytic digestion as well as inhibition of microtubule polymerization depend upon similar concentrations of partially purified inhibitor present in the polymerization reaction. It appears as though at least part of the microtubule polymerization inhibitory activity present in unfertilized sea urchin eggs is due to this protease.     

The stabilization of microtubules in isolated spindles by tubulin-colchicine complex

We have analyzed the effect of colchicine and tubulin dimer-colchicine complex (T-C) on microtubule assembly in mitotic spindles. Cold- and calcium-labile mitotic spindles were isolated from embryos of the sea urchin Lytechinus variegatus employing EGTA/glycerol stabilization buffers. Polarization microscopy and measurements of spindle birefringent retardation (BR) were used to record the kinetics of microtubule assembly-disassembly in single spindles. When isolated spindles were perfused out of glycerol stabilizing buffer into a standard in vitro microtubule reassembly buffer (0.1 M Pipes, pH 6.8, 1 mM EGTA, 0.5 mM MgCl2, and 0.5 mM GTP) lacking glycerol, spindle BR decreased with a half-time of 120 s. Colchicine at 1 mM in this buffer had no effect on the rate of spindle microtubule disassembly. Inclusion of 20 microM tubulin or microtubule protein, purified from porcine brain, in this buffer resulted in an augmentation of spindle BR. Interestingly, in the presence of 20 microM T-C, spindle BR did not increase, but was reversibly stabilized; subsequent perfusion with reassembly buffer without T-C resulted in depolymerization. This behavior is striking in contrast to the rapid depolymerization of spindle microtubules induced by colchicine and T-C in vivo. These results support the current view that colchicine does not directly promote microtubule depolymerization. Rather, it is T-C complex that alters microtubule assembly, by reversibly binding to microtubules and inhibiting elongation. In vivo, colchicine can induce depolymerization of nonkinetochore spindle microtubules within 20 s. In vitro, colchicine blocks further microtubule assembly, but does not induce rapid disassembly.(ABSTRACT TRUNCATED AT 250 WORDS)     

Turbidity as a probe of tubulin polymerization kinetics: a theoretical and experimental re-examination

We report here an examination of the validity of the experimental practice of using solution turbidity to study the polymerization kinetics of microtubule formation. The investigative approach proceeds via numerical solution of model rate equations to yield the time dependence of each microtubule species, followed by the calculation of the time- and wavelength-dependent turbidity generated by the calculated distribution of rod lengths. The wavelength dependence of the turbidity along the time course is analyzed to search for generalized kinetic regimes that satisfy a constant proportionality relationship between the observed turbidity and the weight concentration of polymerized tubulin. An empirical analysis, which permits valid interpretation of turbidity data for distributions of microtubules that are not long relative to the wavelength of incident light, is proposed. The basic correctness of the simulation work is shown by the analysis of the experimental time dependence of the turbidity wavelength exponent for microtubule formation in taxol-supplemented 0.1 M Pipes buffer (1 mM GTP, 1 mM EGTA, 1 mM MgSO4, pH 6.4). We believe that the general findings and principles outlined here are applicable to studies of other fibril-forming systems that use turbidity as a marker of polymerization progress.     

Contrasting roles of tau and microtubule-associated protein 2 in the vinblastine-induced aggregation of brain tubulin

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.     

A porcine brain protein (35 K protein) which bundles microtubules and its identification as glyceraldehyde 3-phosphate dehydrogenase

A protein which binds to both tubulin and tubulin polymer was isolated from porcine brains. This protein has a molecular weight of 35,000 on SDS-polyacrylamide gel electrophoresis (designated as 35 K protein). The 35 K protein was purified through several steps of purification including ammonium sulfate fractionation, Sephadex G-100 gel filtration column chromatography, microtubule protein-agarose gel affinity column chromatography and phosphocellulose column chromatography. The 35 K protein caused pronounced enhancement of the turbidity increase produced by tubulin polymerization in the presence of DMSO, but did not have the ability to initiate polymerization of pure tubulin in the absence of DMSO. It was demonstrated that 35 K protein co-sediments with tubulin polymer in a concentration-dependent manner. Electron microscopic observation revealed the formation of bundles of tubulin polymer. Since the effect of 35 K protein was coupled with tubulin polymerization, 35 K protein did not cause the turbidity increase under conditions where tubulin polymerization was inhibited by Ca2+ or colchicine. The 35 K protein adsorbed on tubulin-Sepharose 4B was eluted by the addition of 2 mM ATP. ATP was shown to inhibit the interaction of 35 K protein with tubulin dimer or polymer. The 35 K protein was finally identified as glyceraldehyde 3-phosphate dehydrogenase from properties such as mobility on SDS-polyacrylamide gel electrophoresis, cleavage pattern on limited proteolysis, ability to bind to tubulin, and so on.     

Evidence Against Impaired Brain Microtubule Protein Polymerization at High Glucose Concentrations or During Diabetes Mellitus

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.     

Interference by Mes [2-(4-morpholino)ethanesulfonic acid] and related buffers with phenolic oxidation by peroxidase

While characterizing the kinetic parameters of apoplastic phenolic oxidation by peroxidase, we found anomalies caused by the Mes [2-(4-morpholino)ethanesulfonic acid] buffer being used. In the presence of Mes, certain phenolics appeared not to be oxidized by peroxidase, yet the oxidant, H(2)O(2), was utilized. This anomaly seems to be due to the recycling of the phenolic substrate. The reaction is relatively inefficient, but at buffer concentrations of 10 mM or greater the recycling effect is nearly 100% with substrate concentrations less than 100 microM. The recycling effect is dependent on substrate structure, occurring with 4'-hydroxyacetophenone but not with 3',5'-dimethoxy-4'-hydroxyacetophenone (acetosyringone). Characterization of the reaction parameters suggests that the phenoxyl radical from the peroxidase reaction interacts with Mes, causing the reduction and regeneration of the phenol. Similar responses occurred with related buffers such as Hepes [4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid] and Pipes [piperazine-1,4-bis(2-ethanesulfonic acid)]. Results from this work and other reports in the literature indicate that great care is required in interpreting any results involving these buffers under oxidizing conditions.     

Dependency of microtubule-associated proteins (MAPS) for tubulin stability and assembly; use of estramustine phosphate in the study of microtubules

Summary Microtubule-associated proteins (MAPS) were separated from tubulin with several different methods. The ability of the isolated MAPs to reinduce assembly of phosphocellulose purified tubulin differed markedly between the different methods. MAPs isolated by addition of 0.35 M NaCl to taxol-stabilized microtubules stimulated tubulin assembly most effectively, while addition of 0.6M NaCl produced MAPs with a substantially lower ability to stimulate tubulin assembly. The second best preparation was achieved with phosphocellulose chromatographic separation of MAPs with 0.6 M NaCl elution.The addition of estramustine phosphate to microtubules reconstituted of MAPS prepared by 0.35 M NaCl or phosphocellulose chromatography, induced less disassembly than for microtubules assembled from unseparated proteins, and was almost without effect on microtubules reconstituted from MAPs prepared by taxol and 0.6 M NaCl. Estramustine phosphate binds to the tubulin binding part of the MAPs, and the results do therefore indicate that the MAPs are altered by the separation methods. Since the MAPs are regarded as highly stable molecules, one probable alteration could be aggregation of the MAPs, as also indicated by the results. The purified tubulin itself seemed not to be affected by the phosphocellulose purification, since the microtubule proteins were unchanged by the low buffer strenght used during the cromatography. However, the assembly competence after a prolonged incubation of the microtubule proteins at 4° C was dependent on intact bindings between the tubulin and MAPs.Abbreviations Pipes 1,4-Piperazinediethanesulfonic acid - EDTA Ethylenedinitrilo Tetraacetic Acid - MAPs Microtubule-Associated Proteins - SDS-PAGE SDS-Polyacrylamide Gel Electrophoresis     

Purification of tau,a microtubule-associated protein that induces assembly of microtubules from purified tubulin

Tau, a microtubule-associated protein which copurifies with tubulin through successive cycles of polymerization and depolymerization, has been isolated from tubulin by phosphocellulose chromatography and purified to near homogeneity. The purified protein is seen to migrate during electrophoresis on acrylamide gels as four closely spaced bands of apparent molecular weights between 55,000 and 62,000. Specific activity for induction of microtubule formation from purified tubulin has been assayed by quantitative electron microscopy and is seen to be enhanced three- to fourfold in the purified tau when compared with the unfractionated microtubule-associated proteins. Nearly 90% of available tubulin at 1 mg/ml is found to be polymerizable into microtubules with elevated levels of tau. Moreover, the critical concentration for polymerization of the reconstituted tau + tubulin system is seen to be a function of tau concentration and may be lowered to as little as 30 μg of tubulin per ml. Under depolymerizing conditions, 50% of the tubulin at only 1 mg/ml may be driven into ring structures. A separate purification procedure for isolation of tau directly from cell extracts has been developed and data from this purification suggest that tau is present in the extract in roughly the same proportion to tubulin as is found in microtubules purified by cycles of assembly and disassembly. Tau is sufficient for both nucleation and elongation of microtubules from purified tubulin and hence the reconstituted tau + tubulin system defines a complete microtubule assembly system under standard buffer conditions. In an accompanying paper (Cleveland et al., 1977) the physical and chemical properties of tau are discussed and a model by which tau may function in microtubule assembly is presented.     

Biochemical and electron microscopy analysis of the endotoxin binding to microtubulesin vitro

The mechanisms involved in cellular activation and damage by bacterial endotoxins are not completely defined. In particular, there is little information about possible intracellular targets of endotoxins. Recently, the participation of a microtubule associated protein in endotoxin actions on macrophages has been suggested. In the present work, we have studied the effect ofE. coli lipopolysaccharide on the polymerization of microtubular proteinin vitro. Electrophoretic analysis of the polymerization mixtures showed that the endotoxin inhibited the polymerization when present at high concentrations. At lower concentrations, LPS selectively displaced the microtubule associated protein MAP-2 from the polymerized microtubules. Electron microscopy showed that LPS binds to microtubules of tubulin+MAPs and to microtubules of purified tubulin (without MAPs) polymerized with taxol. Gel filtration experiments confirmed the binding of LPS to tubulin, and by ligand blot assays an interaction LPS — MAP-2 was detected. The ability of LPS to interact with microtubular proteins suggests a possible participation of microtubules on the cellular effects of endotoxins.     

Zinc(II) complexation by some biologically relevant pH buffers

The isothermal titration calorimetry (ITC) technique supported by potentiometric titration data was used to study the interaction of zinc ions with pH buffer substances, namely 2‐(N‐morpholino)ethanesulfonic acid (Mes), piperazine‐N,N′‐bis(2‐ethanesulfonic acid) (Pipes), and dimethylarsenic acid (Caco). The displacement ITC titration method with nitrilotriacetic acid as a strong, competitive ligand was applied to determine conditional–independent thermodynamic parameters for the binding of Zn(II) to Mes, Pipes, and Caco. Furthermore, the relationship between the proposed coordination mode of the buffers and the binding enthalpy has been discussed. Copyright © 2014 John Wiley & Sons, Ltd.     

Stathmin family protein SCG10 differentially regulates the plus and minus end dynamics of microtubules at steady state in vitro: implications for its role in neurite outgrowth

SCG10 (superior cervical ganglia neural-specific 10 protein) is a neuron specific member of the stathmin family of microtubule regulatory proteins that like stathmin can bind to soluble tubulin and depolymerize microtubules. The direct actions of SCG10 on microtubules themselves and on their dynamics have not been investigated previously. Here, we analyzed the effects of SCG10 on the dynamic instability behavior of microtubules in vitro, both at steady state and early during microtubule polymerization. In contrast to stathmin, whose major action on dynamics is to destabilize microtubules by increasing the switching frequency from growth to shortening (the catastrophe frequency) at microtubule ends, SCG10 stabilized the plus ends both at steady state and early during polymerization by increasing the rate and extent of growth. For example, early during polymerization at high initial tubulin concentrations (20 microM), a low molar ratio of SCG10 to tubulin of 1:30 increased the growth rate by approximately 50%. In contrast to its effects at plus ends, SCG10 destabilized minus ends by increasing the shortening rate, the length shortened during shortening events, and the catastrophe frequency. Consistent with its ability to modulate microtubule dynamics at steady state, SCG10 bound to purified microtubules along their lengths. The dual activity of SCG10 at opposite microtubule ends may be important for its role in regulating growth cone microtubule dynamics. SCG10's ability to promote plus end growth may facilitate microtubule extension into filopodia, and its ability to destabilize minus ends could provide soluble tubulin for net plus end elongation.     

Polymerization of Antarctic fish tubulins at low temperatures: energetic aspects

Tubulins were purified from the brain tissues of three Antarctic fishes, Notothenia gibberifrons, Notothenia coriiceps neglecta, and Chaenocephalus aceratus, by ion-exchange chromatography and one cycle of temperature-dependent microtubule assembly and disassembly in vitro, and the functional properties of the protein were examined. The preparations contained the alpha- and beta-tubulins and were free of microtubule-associated proteins. At temperatures between 0 and 24 degrees C, the purified tubulins polymerized readily and reversibly to yield both microtubules and microtubule polymorphs (e.g., "hooked" microtubules and protofilament sheets). Critical concentrations for polymerization of the tubulins ranged from 0.87 mg/mL at 0 degrees C to 0.02 mg/mL at 18 degrees C. The van't Hoff plot of the apparent equilibrium constant for microtubule elongation at temperatures between 0 and 18 degrees C was linear and gave a standard enthalpy change (delta H degree) of +26.9 kcal/mol and a standard entropy change (delta S degree) of +123 eu. At 10 degrees C, tubulin from N. gibberifrons polymerized efficiently at high ionic strength; the critical concentration increased monotonically from 0.041 to 0.34 mg/mL as the concentration of NaCl added to the assembly buffer was increased from 0 to 0.4 M. Together, the results indicate that the polymerization of tubulins from the Antarctic fishes is entropically driven and suggest that an increased reliance on hydrophobic interactions underlies the energetics of microtubule formation at low temperatures. Thus, evolutionary modification to increase the proportion of hydrophobic interactions (relative to other bond types) at sites of interdimer contact may be one adaptive mechanism that enables the tubulins of cold-living poikilotherms to polymerize efficiently at low temperatures.     

Purification of microtubule protein from beef brain and comparison of the assembly requirements for neuronal microtubules isolated from beef and hog.

The polymerization of microtubule protein from beef brain is inefficient under the same conditions which are optimal for the assembly of microtubules isolated from hog brain (0.1 m piperazine-N,N′-bis(2-ethanesulfonic acid) buffer at pH 6.94). In examining the conditions required for microtubule polymerization in both beef brain extract and purified microtuble protein, it was determined that the pH optimum was pH 6.62 or 0.3 pH unit lower than the reported optimum for hog. Other assembly requirements (ionic strength, Mg²⁺ and nucleotide concentration, temperature) remained essentially the same as for hog. By separating and recombining fractions of tubulin and nontubulin components prepared from beef and hog microtubule protein, the requirement for the reduction in pH was found to be due to the tubulin and not to the microtubule-associated proteins. It was also determined that the efficiency of beef tubulin assembly, as measured by the yield of microtubule polymer, decreased rapidly after slaughter with a half-time of 19 min. Furthermore, when the overall efficiency of polymerization was reduced, the extent of assembly at each cycle of purification by disassembly and assembly was also observed to be depressed. The variations in the requirements for neuronal tubulin assembly in two closely related mammals suggest that the conditions required for assembly of microtubule protein in other tissues and cell types may also be different.     

Differential effects of magnesium on tubulin-nucleotide interactions

Magnesium-depleted 2-(N-morpholino)ethanesulfonate (Mes), glutamate, tubulin and microtubule-associated proteins were prepared and used to study the effects of exogenously added MgCl2 on tubulin-nucleotide interactions in 0.1 M Mes with microtubule-associated proteins and in 1.0 M glutamate. Endogenous levels of Mg2+ in the systems studied were approximately stoichiometric with the tubulin concentrations and largely derived from the tubulin. We examined the effects of added Mg2+ on tubulin polymerization, GDP inhibition of polymerization, binding of GDP and GTP to tubulin, and GTP hydrolysis. Exogenously added Mg2+ had markedly different effects on these reactions. The order of their sensitivity for a requirement for added Mg2+ was as follows: GTP binding greater than GTP hydrolysis greater than polymerization greater than GDP binding. Inhibition of polymerization by GDP varied inversely with the Mg2+ concentration and was greatest in the absence of the cation. These results indicate that GDP and GDP-Mg2+ interact with similar affinity at the exchangeable site, while GTP-Mg2+ has a higher affinity for tubulin than does free GTP. Nevertheless, under appropriate conditions, free GTP can interact sufficiently well with tubulin to permit both nucleation and elongation reactions.