Brain Microtubule Protein Research Articles

Actin filaments modulate electrical activity of brain microtubule protein two-dimensional sheets.

The cytoskeleton of eukaryotic cells contains networks of actin filaments and microtubules (MTs) that are jointly implicated in various cell functions, including cell division, morphogenesis, and migration. In neurons, this synergistic activity drives both the formation of axons during development and synaptic activity in mature neurons. Both actin filaments and MTs also are highly charged polyelectrolytes that generate and conduct electrical signals. However, no information is presently available on a potential electrical crosstalk between these two cytoskeletal networks. Herein we tested the effect of actin polymerization on the electrical oscillations generated by two-dimensional sheets of bovine brain microtubule protein (2D-MT). The voltage-clamped 2D-MT sheets displayed spontaneous electrical oscillations representing a synchronous 224% change in conductance, and a fundamental frequency of 38 Hz. At 60 mV, a 4.15 nC of integrated charge transferred per second increased by 72.3% (7.15 nC) after addition of monomeric (G)-actin. This phenomenon had a 2-min lag time, and was prevented by the presence of the G-actin-binding protein DNAse I. Addition of prepolymerized F-actin, however, had a rapid onset (<10 s) and a higher effect on the tubulin sheets (~100% increase, 8.25 nC). The data are consistent with an interaction between the actin cytoskeleton and tubulin structures, in what seems to be an electrostatic effect. Because actin filaments and MTs interact with each other in neurons, it is possible for this phenomenon to be present, and of relevance in the processing of intracellular signaling, including the gating and activation of actin cytoskeleton-regulated excitable ion channels in neurons.

Effect of Aluminum, Iron, and Zinc Ions on the Assembly of Microtubules from Brain Microtubule Proteins.

Al(3+), Fe(3+), and Zn(2+) ions can disturb microtubule assembly from tubulin and microtubuleassociated proteins in rat brain. The main structural forms of these microtubules are rings and tangled bundles. These structures are formed only in the presence of Al(3+) and Fe(3+) ions. Therefore, Zn(2+) ions can be excluded from possible causes of structural abnormalities in microtubules during Alzheimer's disease. Al(3+) ions are the most probable etiological cause of Alzheimer's disease. The concentration of Al(3+) ions affecting the structure of microtubules is one order of magnitude lower than that of Fe(3+) ions (10 and 100 μM, respectively), which corresponds to their brain concentration reported in Alzheimer's disease.

A factor in the serum of diabetic patients inhibits microtubule formation.

The effect of serum from non-diabetic patients, diabetics without neuropathy and diabetics with significant neuropathy on the ability of pig brain microtubule proteins to polymerize in vitro to microtubules was observed. The extent of polymerisation on the second and third cycles of polymerisation was significantly reduced in the presence of diabetics' serum compared with non-diabetics' serum. No significant difference between the serum samples from diabetics with and without neuropathy could be found. The finding suggests the presence of a factor in the serum of diabetics, whether or not they have neuropathy, which will impair microtubule formation in vitro.

Tau – an inhibitor of deacetylase HDAC6 function

Analysis of brain microtubule protein from patients with Alzheimer's disease showed decreased alpha tubulin levels along with increased acetylation of the alpha tubulin subunit, mainly in those microtubules from neurons containing neurofibrillary tau pathology. To determine the relationship of tau protein and increased tubulin acetylation, we studied the effect of tau on the acetylation-deacetylation of tubulin. Our results indicate that tau binds to the tubulin-deacetylase, histone deacetylase 6 (HDAC6), decreasing its activity with a consequent increase in tubulin acetylation. As expected, increased acetylation was also found in tubulin from wild-type mice compared with tubulin from mice lacking tau because of the tau-mediated inhibition of the deacetylase. In addition, we found that an excess of tau protein, as a HDAC6 inhibitor, prevents induction of autophagy by inhibiting proteasome function.

Minimal Plus-end Tracking Unit of the Cytoplasmic Linker Protein CLIP-170

Cytoplasmic linker protein 170 (CLIP-170) is the prototype microtubule (MT) plus-end tracking protein (+TIP) and is involved in regulating MT dynamics. A comprehensive understanding of the process by which CLIP-170 tracks MT plus ends would provide insight into its function. However, the precise molecular mechanism of CLIP-170 +TIP behavior is unknown, and many potential models have been presented. Here, by separating the two CLIP-170 CAP-Gly domains and their adjacent serine-rich regions into fragments of varied size, we have characterized the minimal plus-end tracking unit of CLIP-170 in vivo. Each CLIP-170 fragment was also characterized for its tubulin polymerization activity in vitro. We found that the two CAP-Gly domains have different activities, whereas CAP-Gly-1 appears incompetent to mediate either +TIP behavior or MT nucleation, a CLIP-170 fragment consisting of the second CAP-Gly domain and its adjacent serine-rich region can both track MT plus ends in vivo and induce tubulin polymerization in vitro. These observations complement recent work on CLIP-170 fragments, demonstrate that CAP-Gly motifs do not require dimerization for +TIP and polymerization-promoting activities, and provide insight into CLIP-170 function and mechanism.

Nickel (Ni 2+) enhancement of microtubule assembly in vitrois dependent on GTP function

Microtubule (MT) assembly in vitro is accompanied by hydrolysis of tubulin-bound GTP at E-site. Ni 2+, a human carcinogen, has been shown to markedly perturb the MT system in cultured cells and enhance MT assembly in vitro. To further probe the mechanisms of such multiple Ni 2+ damaging actions on MT, we have focused on dissecting the role of the Ni 2+/GTP interaction in influencing MT assembly in vitro as monitored by a turbidity assay at A 350 at 27°C using purified bovine brain MT proteins containing 162 μM each of Mg 2+ and EGTA. MT assembly was initiated by addition of GTP and progressed in a GTP dose-dependent manner. The minimal and optimal exogenous [GTP] required for MT assembly were 15.6 and 500 μM, respectively. Replacement of GTP (25–87%) with increasing [NiCl 2] while keeping the sum of [GTP] and [Ni 2+] constant at 500 μM enabled MT assembly to proceed with shortened “lags” but reaching the same maximum plateau levels or elongation rates as with 500 μM GTP only. However, in reactions with Ni 2+ replacing >94% of GTP, marked inhibition of MT assembly (lower plateaus) occurred. Electron microscopic (EM) examinations showed that MT formed with high Ni 2+ substitutions for GTP appeared shorter, more numerous, and resistant to Ca 2+ disruption than those assembled with 500 μM GTP only. Notably, in the presence of 500 μM Ni 2+ with no GTP added, no typical MT were observed under EM, despite increases in turbidity of the reaction. In addition, the critical concentration of MT proteins required for assembly was also considerably decreased under conditions of Ni 2+ replacements of GTP. These results point to an important role of GTP/Ni 2+ interaction in modulating the Ni 2+ enhancement of MT assembly in vitro.

Peroxynitrite Oxidation of Tubulin Sulfhydryls Inhibits Microtubule Polymerization

Considerable evidence both in vitro and in vivo implicates protein damage by peroxynitrite as a probable mechanism of cell death. Herein, we report that treatment of bovine brain microtubule protein, composed of tubulin and microtubule-associated proteins, with peroxynitrite led to a dose-dependent inhibition of microtubule polymerization. The extent of cysteine oxidation induced by peroxynitrite correlated well with inhibition of microtubule polymerization. Disulfide bonds between the subunits of the tubulin heterodimer were detected by Western blot as a result of peroxynitrite-induced cysteine oxidation. Addition of disulfide reducing agents including dithiothreitol and β-mercaptoethanol restored a significant portion of the polymerization activity that was lost following peroxynitrite addition. Thus, peroxynitrite-induced disulfide bonds are at least partially responsible for the observed inhibition of polymerization. Sodium bicarbonate protected microtubule protein from the peroxynitrite-induced inhibition of polymerization. Tyrosine nitration of microtubule protein by 1 mM peroxynitrite increased approximately twofold when sodium bicarbonate was present whereas the extent of cysteine oxidation decreased from 7.5 to 6.3 mol cysteine/mol tubulin. These results indicate that cysteine oxidation of tubulin by peroxynitrite, rather than tyrosine nitration, is the primary mechanism of inhibition of microtubule polymerization.

Activity of a novel antitumor agent, TZT-1027

We studied the antitumor activity of newly synthesized dolastatin 10 analogs. TZT-1027 showed remarkable activity and was selected for further development. TZT-1027 was found to inhibit the assembly of porcine brain microtubule proteins and to depolymerize the polymerized microtubule proteins. Therefore, its mechanism of antitumor activity seems to be at least partially ascribed to the inhibition of microtubule assembly. We further studied the binding site of TZT-1027 on tubulin. Scatchard analysis of 3H-TZT-1027 binding data suggested two binding sites including a high affinity site and a low affinity site. TZT-1027 affected the binding of vinblastine (VBL) on tubulin but its binding site isn't identical to the VBL binding site. TZT-1027 induced apoptosis within 24 h, not only in human leukemia cells such as HL-60, but also in solid tumors such as human prostate carcinoma cells DU145 and human mammary carcinoma cells MCF-7. TZT-1027 showed good antitumor activity against human xenografts (MX-1 and LX-1) without causing serious body weight reduction, which resulted in tumor regression. We examined the effect of TZT-1027 on the established tumor vasculature using the dye perfusion into tumor. TZT-1027 exhibited considerable antivascular activity in tumor sections in addition to excellent cytotoxic effect.

Assessment of microtubule stabilizers by semiautomated in vitro microtubule protein polymerization and mitotic block assays.

Paclitaxel (Taxol) a clinically active anticancer agent, exerts its cytotoxicity by inducing tubulin polymerization, leading to cellular mitotic block. In contrast, other antimitotic drugs, such as colchicine, podophyllotoxin, and vinblastine, act by depolymerizing microtubules. We report here (a) a semiautomated assay which measures the tubulin-polymerizing activity of paclitaxel analogs and (b) a cellular assay to measure the potential of these compounds to block cells in mitosis. The microtubule-polymerizing assay measured the turbidity of bovine brain microtubule protein (MTP) polymerized by the test compound in a 96-well plate. We maximized the sensitivity of this assay by conducting the polymerization reaction at 20 degrees C, at which temperature the baseline reaction, i.e. the basic ability of the untreated MTP control to polymerize, was minimal. At 20 degrees C, the effect of 0.05 microg/ml of paclitaxel on MTP could be detected, whereas at 37 degrees C, > 1 microg/ml of paclitaxel was required to detect a significant effect relative to untreated MTP. We describe the analysis of the complex curves of MTP polymerization with varying concentrations of test compounds. The polymerization of microtubules leads to cells being blocked in mitosis. This mitotic blocking effect in intact cells was determined using a cell settling chamber which allowed eight samples to be deposited on a slide. This method required a smaller number of cells (10(3) - 10[5]), maintained cell morphology, and allowed for rapid screening of samples. The activity of several new paclitaxel analogs is reported.

Inhibition of mitosis and microtubule function through direct tubulin binding by a novel antiproliferative naphthopyran LY290181.

The mechanism of action of a novel antiproliferative compound LY290181 [2-amino-4-(3-pyridyl)-4H-naphtho(1,2-b)pyran-3-carbonitrile] was characterized. LY290181 is a potent inhibitor of cell proliferation, producing 50% inhibition of vascular smooth muscle, endothelial, Chinese hamster ovary, HeLa, and human erythroleukemia cells at concentrations of 8-40 nM. Cell cycle analysis showed that LY290181 caused accumulation of smooth muscle cells at the G2/M phase and induced mitotic arrest in Chinese hamster ovary cells and HeLa cells. At low concentrations (3-30 nM), LY290181 blocked transition of cells from metaphase to anaphase and disrupted mitotic spindle organization. At high concentrations (>/=100 nM), LY290181 produced a concentration-dependent loss of cytoplasmic and spindle microtubules. LY290181 inhibited the polymerization of purified bovine brain microtubule protein into microtubules, and it depolymerized preformed microtubules. Using tubulin-1-anilino-8-naphthalene sulfonate complex fluorescence, we have shown that LY290181 directly interacted with tubulin in a unique manner. These studies show that LY290181 induces cell growth arrest in prometaphase/metaphase, and tubulin appears to be its molecular target.

Protein complexes containing gamma-tubulin are present in mammalian brain microtubule protein preparations.

The presence of gamma-tubulin in microtubule preparations, obtained by disassembly/ assembly cycles at 0degreesC/37degreesC from the brain of several mammals, is demonstrated by immunoblotting with specific antibodies directed against three distinct regions of the protein. In contrast gamma-tubulin was absent from pure tubulin obtained by chromatography on phosphocellulose, but was retained on the column with the other microtubule-associated proteins. A large part of the gamma-tubulin was present in cold stable material remaining after microtubule disassembly at OdegreesC and was partially solubilized using high salt, thus preventing its purification by the usual assembly/disassembly procedure used for alpha/beta-tubulin heterodimers. Brain gamma-tubulin was purified by affinity chromatography with gamma-tubulin antibodies raised against its carboxyl terminal region. Purified gamma-tubulin consisted of at least two polypeptides present in equal quantities and exhibiting a pI of 6.5 and 6.6, respectively. It was associated with the alpha/beta-tubulin heterodimer and with at least five other polypeptides of 75, 105, 130, 195, and 250 kDa. With the exception of the 250 kDa polypeptide, all of these proteins seem to be present in gamma-tubulin complexes isolated from Xenopus eggs. But, in contrast with Xenopus egg complexes, brain complexes exhibited a considerable heterogeneity of their apparent masses and composition in sucrose gradient centrifugation, in agreement with the absence of an homogeneous structure in electron microscopy. Despite this heterogeneity, gamma-tubulin complexes bind quantitatively to microtubule extremities. The possibility to further use mammalian brain gamma-tubulin and some of its associated proteins in biochemical and pharmacological experiments is of interest since brain microtubule protein preparations have been extensively used for studying both microtubule dynamics and the activity of microtubule poisons.

In vitro assembly of microtubule protein with GTP and 2'dGTP: kinetic evidence for a preassembly conformational change.

The assembly of chick brain microtubule protein in a NaCl-supplemented buffer has been examined with respect to nucleation and the subsequent elongation as a function of the nucleotide (GTP vs 2'dGTP), and the protein and nucleotide concentrations. The kinetics suggest that unassembled tubulin can exist in two conformational states (termed Tu1,GTP and Tu2,GIP when GTP is bound to the exchangeable site), with Tu1,GTP contributing to nucleation and Tu2,GTP participating in elongation. The extent of self-nucleation is proposed to be determined, in part, by the rate constant governing this conformational change. This analysis contrasts with that of earlier studies, which concluded that the number of subunits interacting to form an effective nucleus could be estimated from the dependency of self-nucleation on the initial concentration of unassembled tubulin.

Site-directed mutagenesis of alpha-tubulin. Reductive methylation studies of the Lys 394 region

Previous studies have implicated at least two regions in alpha-tubulin that are important for the regulation of microtubule assembly. These regions include a cluster of basic residues consisting of Arg 390, His 393, and Lys 394 and the highly acidic carboxyl terminus. Lys 394 is highly reactive to HCHO and NaCNBH3. The reductive methylation of Lys 394 by these reagents is thought to be responsible for the profound inhibitory effects of low concentrations of HCHO on microtubule assembly (cf. Szasz J., M. B. Yaffe, M. Elzinga, G. S. Blank, and H. Sternlicht. 1986. Biochemistry. 25:4572-4582). In this study we reexamined the basis for this inhibition. Lys 394 in a human keratinocyte alpha-tubulin (k alpha 1) was replaced by a glutamic acid residue using site-directed mutagenesis. The mutant K394E was synthesized in vitro using rabbit reticulocyte lysates, and its ability to coassemble with bovine brain microtubule protein (MTP) before and after reaction with HCHO and NaCNBH3 was compared with that of wild-type. No differences in the coassemblies of the unmethylated proteins were detected suggesting that Lys 394 is not essential for microtubule assembly. However, methylated K394E prepared at low HCHO concentrations (< 1 mM) incorporated into microtubules to a greater extent (approximately 30-40%) than methylated wild-type. This result is consistent with the hypothesis that methylation of Lys 394 interferes with microtubule assembly. However, the extent of protection afforded by the replacement of Lys 394 with Glu 394 was less than half as large as that predicted from the earlier studies. We tentatively conclude that another residue(s) besides Lys 394 contributes significantly to the assembly-inhibition observed with low concentrations of HCHO. Since this residue(s) is less reactive than Lys 394, it would have to inhibit assembly substoichiometrically when methylated. Potential candidates for this residue include bulk lysyl residue(s), a lysyl residue(s) with intermediate reactivity toward HCHO, and the NH2-termini. The NH2-termini are especially attractive candidates since they appear to have a structural role in microtubule assembly.

Sulphonate buffers affect the recovery of microtubule-associated proteins MAP1 and MAP2: evidence that MAP1A promotes microtubule assembly.

The influence of two commonly used sulphonate buffers, PIPES and MES, on the in vitro assembly of bovine brain microtubule protein was examined. Microtubule assembly was monitored by turbimetry and, after centrifugation, the polymerised protein was analysed by SDS-PAGE and western blotting. Assembly in MES when compared with PIPES resulted in a higher recovery of microtubule proteins at both pH 6.4 and pH 6.9 and in an altered protein composition. The buffer pH affected the total amount of protein polymerised but did not significantly affect the protein composition. At both pH conditions the recovery of HMW-MAPs was markedly increased in MES buffer and this increase was mostly due to an increase in the amount of MAP1.

Posttranslational modifications of nerve cytoskeletal proteins in experimental diabetes.

Axonal transport is known to be impaired in peripheral nerve of experimentally diabetic rats. As axonal transport is dependent on the integrity of the neuronal cytoskeleton, we have studied the way in which rat brain and nerve cytoskeletal proteins are altered in experimental diabetes. Rats were made diabetic by injection of streptozotocin (STZ). Up to six weeks later, sciatic nerves, spinal cords, and brains were removed and used to prepare neurofilaments, microtubules, and a crude preparation of cytoskeletal proteins. The extent of nonenzymatic glycation of brain microtubule proteins and peripheral nerve tubulin was assessed by incubation with 3H-sodium borohydride followed by separation on two-dimensional polyacrylamide gels and affinity chromatography of the separated proteins. There was no difference in the nonenzymatic glycation of brain microtubule proteins from two-week diabetic and nondiabetic rats. Nor was the assembly of microtubule proteins into microtubules affected by the diabetic state. On the other hand, there was a significant increase in nonenzymatic glycation of sciatic nerve tubulin after 2 weeks of diabetes. We also identified an altered electrophoretic mobility of brain actin from a cytoskeletal protein preparation from brain of 2 week and 6 week diabetic rats. An additional novel polypeptide was demonstrated with a slightly more acidic isoelectric point than actin that could be immunostained with anti-actin antibodies. The same polypeptide could be produced by incubation of purified actin with glucose in vitro, thus identifying it as a product of nonenzymatic glycation. These results are discussed in relation to data from a clinical study of diabetic patients in which we identified increased glycation of platelet actin. STZ-diabetes also led to an increase in the phosphorylation of spinal cord neurofilament proteins in vivo during 6 weeks of diabetes. This hyperphosphorylation along with a reduced activity of a neurofilament-associated protein kinase led to a reduced incorporation of 32P into purified neurofilament proteins when they were incubated with 32P-ATP in vitro. Our combined data show a number of posttranslation modifications of neuronal cytoskeletal proteins that may contribute to the altered axonal transport and subsequent nerve dysfunction in experimental diabetes.

Biochemical changes in sciatic nerve of hens treated with tri- o-cresyl phosphate: increased phosphorylation of cytoskeletal proteins

Calcium- and calmodulin-regulated protein phosphorylation has been suggested to play a role in the pathogenesis of organophosphorus compound-induced delayed neurotoxicity (OPIDN). This condition is characterized by ataxia that progresses to paralysis concurrent with a central-peripheral distal axonopathy after a delay period of 1–2 weeks following exposure to an organophosphorus compound causing delayed neurotoxicity, such as tri- o-cresyl phosphate (TOCP). Calcium/calmodulin (CaM) kinase II is involved in the increased phosphorylation of brain microtubule and spinal cord neurofilament triplet proteins following treatment of animals with organophosphorus compounds that are capable of producing OPIDN. In this study, chickens were given a single oral neurotoxic dose of 750 mg TOCP/kg body weight and killed after 1, 6, 14 or 21 days following treatment. Protein kinase-mediated phosphorylation of cytoskeletal proteins was studied in proximal and distal parts of sciatic nerves of control and treated hens. Peripheral nerve proteins were phosphorylated in vitro using [ γ- 32P]ATP as a phosphoryl group donor. Phosphorylated proteins were separated by one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Protein phosphorylation was detected by autoradiography and quantified by laser microdensitometry. The extent of Ca 2+-calmodulin dependent phosphorylation of five cytoskeletal proteins was significantly increased in TOCP treated animals, particularly at 1 and 6 days after treatment, in both the proximal and distal portion of the nerve. The identity of these proteins was confirmed by 2-D PAGE as tubulin, the neurofilament triplet proteins and microtubule associated protein-2 (MAP-2). These results confirm earlier observation of the close temporal relationship between increased cytoskeletal protein phosphorylation and the development and OPIDN.

Microtubule protein ADP-ribosylation in vitro leads to assembly inhibition and rapid depolymerization

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.

High affinity DNA-microtubule interactions: evidence for a conserved DNA-MAP interaction involving unusual high CsCl density repetitious DNA families.

We have examined high affinity interactions of chick brain microtubule proteins with 35S labelled tracer DNAs from chick, mouse and D. melanogaster under equilibrium conditions by the nitrocellulose filter binding technique. Ternary reaction mixtures of the above two components and a third component, an excess of unlabelled competitor DNA from either E. coli., mouse, D. melanogaster or chick, were used to measure small fractions of DNA in each case (1-4%) bound to microtubule protein under high stringency- large competitor DNA concentration and 0.5 M NaCl. As seen in part previously (Marx, K.A. and Denial, T. (1985) in The Molecular Basis of Cancer, 172B, 65-75 (Rein, ed), A. Liss, N.Y.) the measured order of competitor DNA strengths was identical for all three tracer DNAs. That is: chick > mouse > D. melanogaster > E. coli competitor DNA. Since the homologous interaction, chick competitor DNA with chick brain microtubule protein, is always the strongest interaction measured, we interpret this as evidence for a conserved protein-DNA sequence interaction. 35S chick DNA tracer sequences, isolated from nitrocellulose filters following the stringent binding in the presence of 0.9 mM-1 E. coli. competitor DNA, was used in driven reassociation reactions with total chick driver DNA. This fraction was found to be significantly enriched in repetitive chick DNA sequences. Since we have observed a similar phenomenon in mouse, we then compared the stringent binding mouse sequences and showed that the bulk of these sequences did not cross-hybridize with total chick DNA. Finally, all three 35S tracer DNAs binding to nitrocellulose were isolated and sedimented to equilibrium on CsCl density gradients. The CsCl density distributions from all three DNAs showed significant (100-fold) enrichment in classical satellite DNAs as well as higher enrichment in two very unusual high CsCl density families of DNA (1.720-1.740 g/cm3; 1.750-1.765 g/cm3). These families are never observed as distinct bands in total DNA CsCl gradients, nor could we isolate them in purified tubulin control binding experiments. This apparently general phenomena may be identifying some of the sequence families involved in the high affinity microtubule interaction, which appears to be conserved in evolution.

Assembly of chick brain MAP2-tubulin microtubule protein. Characterization of the protein and the MAP2-dependent addition of tubulin dimers

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.

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.