Colchicine Binding Activity Of Tubulin Research Articles

SPECIFIC PROPERTIES OF TUBULIN IN THE PREFRONTAL CORTEX IN CONTROL, SCHIZOPHRENIA AND VASCULAR DEMENTIA

The aim of the study was to evaluate the rate of polymerization of tubulin into microtubules and determine the level of colchicine binding (colchicine-binding activity of tubulin) in the prefrontal cortex in schizophrenia, vascular dementia (VD) and control. Colchicine-binding activity of tubulin was determined by Sherlinе in tubulin-enriched extracts of proteins from the samples. Measurement of light scattering during the polymerization of the tubulin was carried out using the nephelometric method at a wavelength of 450-550 nm. There was a significant decrease in colchicine-binding activity and the rate of tubulin polymerization in the prefrontal cortex in both diseases, and in VD to a greater extent than in schizophrenia. The obtained results suggest that not only in Alzheimer's disease, but also in other mental diseases such as schizophrenia and VD, there is a decrease in the level of tubulin in the prefrontal cortex of the brain, although to a lesser extent than in Alzheimer's disease, and consequently the amount of microtubules.

Binding of Colchicine to Tubulin in the Brain Structures in Normal Conditions and in Schizophrenia

Abstract—One of the components of the cytoskeleton is microtubules (MT), a complex intracellular system, damage to which may cause disturbances that lead to the development of pathological processes in the cell. This gives rise to the study of tubulin, the main MT protein, in schizophrenia. To assess the relative amount of tubulin, its specific property to bind colchicine at an equimolar ratio was used. The aim of the study was to determine the level of colchicine binding (the colchicine-binding activity of tubulin) in various brain structures under normal conditions and in schizophrenia. The study was performed with samples of the structures of the postmortem brain of patients with schizophrenia (n = 7) and individuals in the control group (n = 15). The colchicine-binding activity of tubulin was determined using the Scherline method in tubulin-rich protein extracts (a single polymerization-depolymerization). In the prefrontal (area 10), limbic (area 23/24), and temporal (area 21) cerebral cortex of patients with schizophrenia, a significant decrease in the colchicine-binding activity of tubulin was observed in comparison with the control. In the thalamus, colchicine-binding activity in schizophrenia did not change relative to the control, but it was lower than in the areas of the cortex. In schizophrenia, we observed a decrease in the colchicine-binding activity of tubulin in the prefrontal (area 10), limbic (area 23/24), and temporal (area 21) cerebral cortex but not in the thalamus (subcortical structure of the brain).

Полимеризация тубулина и его колхицинсвязывающая активность в различных структурах головного мозга в норме и при шизофрении

Introduction: abnormalities of neuronal cytoskeleton in mental disorders require to study microtubules and their proteins. Tubulin (the main protein of microtubules) has specific properties: reversibly polymerize into microtubules and bind mitotic poison colchicine in equimolar quantities. Objective: to evaluate the process of tubulin polymerization by light scattering change and to determine the level of colchicine binding (colchicine binding activity of tubulin) in various brain structures in healthy and schizophrenia brains. Material and methods: autopsy brain samples from patients with schizophrenia (n = 6) and from the control group (n = 9) were studied. Samples of the prefrontal (area 10), temporal (area 21), cingulate cortex (area 23/24) and thalamus were isolated (Brodmann's areas). Measurements of light scattering during tubulin polymerization and colchicine-binding activity of tubulin were determined as described earlier. Results: tubulin polymerization was not disturbed in schizophrenia as compared to controls, except for the cingulate cortex that showed slight but significant decrease in light scattering. At the same time, the binding of colchicine in schizophrenia was reduced in all examined areas of the cortex. This decrease was not associated with age, sex, and postmortem interval since the groups were matched by these factors. The tubulin colchicine-binding activity in thalamus remained at the same level both in control and schizophrenia, but it was lower than in the areas of the cortex. Conclusion: Decreased activity and hence decreased amount of tubulin in the cerebral cortical areas without changes of the tubulin polymerization in microtubules have been shown in schizophrenia. The results confirm the literature data on the changes in the cytoskeleton in cortical areas in schizophrenia.

Nucleotide-dependent bisANS binding to tubulin

Non-covalent hydrophobic probes such as 5,5′-bis(8-anilino-1-naphthalenesulfonate) (bisANS) have become increasingly popular to gain information about protein structure and conformation. However, there are limitations as bisANS binds non-specifically at multiple sites of many proteins. Successful use of this probe depends upon the development of binding conditions where only specific dye-protein interaction will occur. In this report, we have shown that the binding of bisANS to tubulin occurs instantaneously, specifically at one high affinity site when 1 mM guanosine 5′-triphosphate (GTP) is included in the reaction medium. Substantial portions of protein secondary structure and colchicine binding activity of tubulin are lost upon bisANS binding in absence of GTP. BisANS binding increases with time and occurs at multiple sites in the absence of GTP. Like GTP, other analogs, guanosine 5′-diphosphate, guanosine 5′-monophosphate and adenosine 5′-triphosphate, also displace bisANS from the lower affinity sites of tubulin. We believe that these multiple binding sites are generated due to the bisANS-induced structural changes on tubulin and the presence of GTP and other nucleotides protect those structural changes.

Differential effects of active isomers, segments, and analogs of dolastatin 10 on ligand interactions with tubulin: Correlation with cytotoxicity

Dolastatin 10 is a potent antimitotic peptide isolated from the marine mollusk Dolabella auricularia. Four of its five residues are modified amino acids (in sequence, dolavaline, valine, dolaisoleuine, dolaproine, dolaphenine). Besides inhibiting tubulin polymerization, dolastatin 10 noncompetitively inhibits vinca alkaloid binding to tubulin, inhibits nucleotide exchange and formation of the β s cross-link, and stabilizes the colchicine binding activity of tubulin. To examine the mechanism of action of dolastatin 10 we prepared six chiral isomers, one tri- and one tetrapeptide segment, and one pentapeptide analog of dolastatin 10, all of which differ little from dolastatin 10 as inhibitors of tubulin polymerization. However, only two of the chiral isomers were similar to dolastatin 10 in their cytotoxicity for L1210 murine leukemia cells and in their effects on vinblastine binding, nucleotide exchange, β s cross-link formation, and colchicine binding. These were isomer 2, with reversal of configuration at position C(19a) in the dolaisoleuine moiety, and isomer 19, with reversal of configuration at position C(6) in the dolaphenine moiety. The pentapeptides with reduced cytotoxicity and reduced effects on tubulin interactions with other ligands were all modified in the dolaproine moiety at positions C(9) and/ or C(10). The tripeptide and tetrapeptide segments which inhibited polymerization but not ligand interactions were the amino terminal tripeptide (lacking dolaproine and dolaphenine) and the carboxyl terminal tetrapeptide (lacking dolavaline). We speculate that strong inhibition of other ligand interactions with tubulin requires stable peptide binding to tubulin (i.e. slow dissociation), but that inhibition of polymerization requires only rapid binding to tubulin.

Dolastatin 15, a potent antimitotic depsipeptide derived from Dolabella auricularia: Interaction with tubulin and effects on cellular microtubules

Dolastatin 15, a seven-subunit depsipeptide derived from Dolabella auricularia, is a potent antimitotic agent structurally related to the antitubulin agent dolastatin 10, a five-subunit peptide obtained from the same organism. We have compared dolastatin 15 with dolastatin 10 for its effects on cells grown in culture and on biochemical properties of tubulin. the IC 50 values for cell growth were obtained for dolastatin 15 with L1210 murine leukemia cells, human Burkitt lymphoma cells, and Chinese hamster ovary (CHO) cells (3,3, and 5 nM with the three cell lines, respectively). For dolastatin 10, IC 50 values of 0.4 and 0.5 nM were obtained with the L1210 and CHO cells, respectively. At toxic concentrations dolastatin 15 caused the leukemia and lymphoma cells to arrest in mitosis. In the CHO cells both dolastatin 15 and dolastatin 10 caused moderate loss of microtubules at the IC 50 values and complete disappearance of microtubules at concentrations 10-fold higher. Despite its potency and the loss of microtubules in treated cells, the interaction of dolastatin 15 with tubulin in vitro was weak. Its IC 50 value for inhibition of glutamate-induced polymerization of tubulin was 23 μM, as compared to values of 1.2 μM for dolastatin 10 and 1.5 μM for vinblastine, Dolastatin 10 noncompetitively inhibits the binding of vincristine to tubulin, inhibits nucleotide exchange, stabilizes the colchicine binding activity of tubulin, and inhibits tubulin-dependent GTP hydrolysis (Bai et al., Biochem Pharmacol 39: 1941–1949, 1990; Bai et al. J Biol Chem 265: 17141–17149, 1990). Only the latter reaction was inhibited by dolastatin 15. Nevertheless, its structural similarity to dolastatin 10 indicates that dolastatin 15 may bind weakiy in the “vinca domain” of tubulin (a region of the protein we postulate to be physically close to but not identical with the specific binding site of vinca alkaloids and maytansinoids), presumably in the same site as dolastatin 10 (the “peptide site”).

Binding of dolastatin 10 to tubulin at a distinct site for peptide antimitotic agents near the exchangeable nucleotide and vinca alkaloid sites.

Dolastatin 10, a potent antimitotic peptide from a marine animal, strongly inhibits microtubule assembly, tubulin-dependent GTP hydrolysis, and the binding of vinca alkaloids to tubulin. In studies of the binding of [3H]vincristine to the protein, with vinblastine as a control for competitive inhibition (Ki, 6.6 microM), we found that the macrolide antimitotic agents maytansine and rhizoxin were also competitive inhibitors (Ki values, 3.1 and 12 microM). Dolastatin 10 and an unrelated peptide antimitotic, phomopsin A, were more potent but noncompetitive inhibitors (Ki values, 1.4 and 2.8 microM). Since maytansine and, to a much lesser extent, vinblastine interfere with nucleotide exchange on tubulin, all drugs were examined for effects on nucleotide interactions at the exchangeable GTP site. Rhizoxin had effects intermediate between those of vinblastine and maytansine. Both peptides inhibited binding of radiolabeled GTP to tubulin even more strongly than did maytansine, but no drug displaced nucleotide from tubulin. The drugs were evaluated for stabilizing effects on the colchicine binding activity of tubulin. The peptides prevented loss of this activity, and vinblastine provided partial protection, while rhizoxin and maytansine did not stabilize tubulin. A tripeptide segment of dolastatin 10 also effectively inhibits tubulin polymerization and GTP hydrolysis. The tripeptide did not significantly inhibit either vincristine binding or nucleotide exchange, nor did it stabilize colchicine binding. These findings are rationalized in terms of a model with two distinct drug binding sites in close physical proximity to each other and to the exchangeable GTP site on beta-tubulin.

4‐Hydroxynonenal interacts with tubulin by reacting with its functional ‐SH groups

4-Hydroxynonenal, which is one of the most important products of lipid peroxidation, alters microtubular organization and structure in 3T3 fibroblasts. Changes in cell shape and the disappearance of microtubules are observed by immunofluorescence after incubation with the aldehyde, and the colchicine binding activity of tubulin from 3T3 cells is modified. Moreover, the aldehyde determines a decrease in the ability of purified tubulin to polymerize and to bind colchicine. These effects may be related to the interaction of the aldehyde with functional -SH groups of tubulin which are necessary for protein integrity and functions. Indeed, the addition of cysteine protects against the damaging effects of the aldehyde.

Colchicine-binding activity distinguishes sea urchin egg and outer doublet tubulins.

The colchicine-binding activity of tubulin has been utilized to distinguish the tubulins from two distinct microtubule systems of the same species, the sea urchin Strongylocentrotus purpuratus. We have analyzed the colchicine-binding affinities of highly purified tubulins from the unfertilized eggs and from the flagellar outer doublet microtubules by van't Hoff analysis, and have found significant differences in the free energy, enthalpy, and entropy changes characterizing the binding of colchicine to the two tubulins. The data indicate that significant chemical differences in the tubulins from the two functionally distinct microtubule systems exist, and that the differences are expressed in the native forms of the tubulins. Our findings are discussed in terms of the possibility that the colchicine-binding site may be an important regulatory site on the tubulin molecule.

Microtubule associated proteins stabilize the colchicine binding activity of tubulin

Microtubule associated proteins stabilize the colchicine binding activity of tubulin

A conditional cell division mutant of Chlamydomonas reinhardii having an increased level of colchicine resistance

Conditional cell division mutants were isolated from Chlamydomonas reinhardii. They were unable to form colonies at 34 °C but not at 23 °C. One of the mutants, TS-60, could neither divide at high nor at low (15 °C) temperature, and seemed to continue protein synthesis at restrictive temperatures. TS-60 also exhibited resistance to 6 mM colchicine which inhibited cell division of the wild-type. Observing that TS-60 flagella were highly resistant to colchicine in their regeneration, it is concluded that the mutational alteration has affected not only the mitotic apparatus but also the flagella. Thermolability of TS-60 was not detected in flagellation but in cell division, though colchicine resistance was expressed in both flagellation and cell division. This suggests that the stable formation of the flagellar microtubule mainly depends on the specific organization of its component. Both thermolability and colchicine resistance of TS-60 were inherited in a Mendelian fashion and unseparable from each other. Reversion tests indicated that the two characters were caused by a single mutation. It is inferred that the above-mentioned phenotypes of TS-60 are the consequence of a mutation in factor(s) involving the colchicine binding activity of tubulin and that this mutational change pleiotrophically leads to some impediment in microtubule formation at restrictive temperature.