Stabilization of hepatic colchicine-binding activity by organic acids

Previous work has shown that the total hepatic tubulin pool and the hepatic microtubule-derived tubulin pool do not have identical [3H]colchicine binding properties. Rapid loss of colchicine-binding activity was noted in the microtubule-derived fractions of liver tubulin. Furthermore, quantitative determination of the total and polymerized tubulin in the liver by the [3H]colchicine-binding assay was hampered by rapid and unequal loss of binding sites under assay conditions. The organic acids, glutamate and glucose 1-phosphate, have been shown to stabilize calf brain tubulin against loss of colchicine-binding sites. Therefore, these compounds were tested as possible protecting agents against loss of colchicine binding activity of liver tubulin. It was found that these agents stabilized liver tubulin under [3H]colchicine-binding conditions. Additional experiments showed that these agents also prevented the rapid loss of colchicine-binding activity that occurred when purified brain tubulin was exposed to liver supernates. These results suggest that the inclusion of the organic acids, glutamate and glucose 1-phosphate, may modify the time decay properties of liver tubulin in solution. Further, these data suggest that these protecting agents may be of analytical value in [3H]colchicine-binding assay systems for liver tubulin.     

Stabilization of the colchicine-binding activity of tubulin by glutathione and by dietary selenium-deficiency

The lability of tubulin in supernatant fluids from rat brain was studied by measuring the loss of colchicine-binding activity. During incubation at 37 degrees C for 1 hr approximately 50% of the binding activity of rat brain supernatant fluids was lost. This loss was reduced by mercaptoethanol or reduced glutathione. Anaerobic conditions did not affect loss of binding, while oxygen markedly reduced it, especially in the presence of reduced glutathione. Supernatant fluids prepared from brains of animals deficient in selenium showed a lower loss of binding than those from selenium-supplemented controls.     

Characterization of colchicine-binding activity in Dictyostelium discoideum.

A colchicine-binding component was detected in vegetative amoebae of Dictyostelium discoideum by using a Millipore-filter assay. The colchicine-binding activity is temperature-and time-dependent, maximum binding occurring at 22-35 degrees C after 60 min incubation. Further increases in temperature are without effect on the extent of binding, but bound colchicine is released with increased time of incubation. Furthermore, colchicine-binding activity itself decreased in the high-speed supernatant from D. discoideum, with half the activity being lost in approx. 2.5h. Several lines of evidence, including the saturation kinetics of colchicine binding, enhancement of colchicine binding by tartrate, insensitivity to lumicolchicine, precipitation of the binding protein by vinblastine and behaviour of the binding protein on DEAE-cellulose and Sephadex resins, suggest that the colchicine-binding protein may be tubulin.     

Microtubule assembly in developing mammalian brain.

Microtubule protein was measured in mouse brain homogenates by quantitative colchicine binding. Neonatal animals contained more than twice the amount of brain tubulin as adult mice. The percentage of colchicine-binding protein which was polymerized was determined by extracting brain at room temperature into a medium designed to stabilize intact microtubules. Under identical conditions and tubulin concentrations, neonatal brain tubulin (colchicine-binding activity) had a greater proportion of the total extracted in an apparently polymerized state (pelletable by centrifugation) than did adult brain. A slight variation in the ratio of assembled to unassembled tubulin was observed with varying protein concentration (volume of extract), indicating that the values obtained may not reflect exactly the in vivo situation, because a rapid equilibration takes place upon homogenization. At all protein concentrations, the neonatal brain extracts contained a significantly greater proportion of assembled tubulin than did adult brain. This proportion began to fall at 5 days postnatal and reached the adult level at 30 days. The tubulin assembled/not assembled ratios were not altered by addition of nucleoside triphosphates, additional EGTA, or sulfhydryl protecting agents, and did not vary with preparation times of 30–90 min. The colchicine-binding reaction and decay of colchicine-binding activity with time were similar in extracts of different aged mouse brains, with neonatal slightly more stable than adult. Pools of tubulin from any age which were soluble at room temperature (unpolymerized) could not repolymerize well in vitro when incubated with GTP at 37 °C, whereas pools of tubulin which were sedimentable at room temperature (polymerized) could be redissolved at 0 °C and readily reassembled at 37 °C. The neonatal extract tubulin was thus more polymerization competent than the adult extracts; this correlates with a greater proportion of assembled tubulin in extracts at room temperature and possibly in vivo.     

Membrane-bound tubulin in brain and thyroid tissue.

Brain and thyroid tissue contain membrane-bound colchicine-binding activity that is not due to contamination by loosely bound cytoplasmic tubulin. This activity can be solubilized to the extent of 80 to 90% by treatment with 0.2% Nonidet P-40 with retention of colchicine binding. Extracts so obtained contain a prominent protein band in disc gel electrophoresis that co-migrates with tubulin. Membranes, and the solubilized protein therefrom, exhibit ligand binding properties like tubulin; for colchicine the KA is approximately 1 X 10(6) M-1 in brain and approximately 0.6 X 10(6) M-1 in thyroid; for vinblastine the KA is approximately 8 X 10(6) M-1 for both tissues; and for podophyllotoxin the Ki is approximately 2 X 10(-6) M for both tissues. Displacement by analogues of colchicine is of the same order as for soluble tubulin. Although membrane-bound colchicine-binding activity shows greater thermal stability and a higher optimum binding temperature (54 degrees versus 37 degrees) than soluble tubulin, this appears to be the result of the membrane environment since the solubilized binding activity behaves like the soluble tubulin. Antibody against soluble brain tubulin reacts with membranes and solubulized colchicine-binding activity from both brain and thyroid gland. We conclude that brain and thyroid membrane preparations contain firmly bound tubulin or a very similar protein.     

Insulin release and the microtubular system of the islets of Langerhans. Identification and characterization of tubulin-like protein.

1. Incubation of islets of Langerhans in vitro in the presence of colchicine produced a progressive inhibition of the insulin-secretory response to glucose, which was dependent on the time of incubation. 2. The uptake of [3-H]colchicine by islet cells was a rapid process, equilibrium being reached in less than 30 min. Part of the colchicine taken up was bound to protein material, which was recovered largely in a post-microsomal supernatant fraction prepared from the islets. In contrast with this rapid uptake, the binding of colchicine by islet-cell proteins in intact islets or in islet homogenates was a slow process, and equilibrium was not reached for 60-90 min. After an initial 30 min delay, the time-course of the binding of [3-H]colchicine to islet-cell proteins paralleled that for the inhibitory effect of colchicine on insulin release. 3. Some purification of the colchicine-binding material present in islet homogenates could be achieved by precipitation of the protein with 2mM-CaCl2 (2.8-fold). However, ion-exchange chromatography on DEAE-Sephadex produced a further 27-fold purification on elution with 0.6M-NaCl. 4. Colchicine-binding protein prepared from islets by ion-exchange chromatography showed an intrinsic association constant for colchicine of 1.4muM and an apparent molecular weight on gel filtration of 110000. 5. These results suggest that colchicine-binding protein in islet cells closely resembles tubulin extracted from the other tissues. The delayed effectiveness of colchicine in inhibiting insulin secretion is not due to poor penetration of colchicine into the cells but rather to slow binding of the alkaloid to islet-cell tubulin. It seems likely that, as in other tissues, this binding prevents polymerization of the tubulin into microtubules, and thus interferes with the release process.     

A modified colchicine-binding assay for the measurement of total and microtubule-derived tubulin in rat liver

The usual measurement of liver tubulin by the colchicine-binding assay does not take into account the accelerated decay of the colchicine-binding capacity of tubulin when liver supernatants, especially those containing microtubule-derived tubulin, are incubated at 37°C. This results in marked underestimations. Our findings indicate that this alteration is due to an inhibitor of colchicine-tubulin binding in liver supernatants that is probably extracted from particulate fractions. The inhibitory activity is decreased by dilution of the supernatants and by increasing the concentration of colchicine. However, the former modification decreases the sensitivity of the assay and the latter increases the nonspecific binding of colchicine to liver proteins other than tubulin. Assessment of the decay and correction for it by calculating the initial binding capacity results in complete recovery of brain tubulin from liver supernatants and values for microtubule-derived tubulin that closely correspond to those expected from simultaneous morphometric assessment of liver microtubules by electron microscopy. The modified method also indicates that the fraction of liver tubulin assembled in microtubules is greater than previously reported.     

Glutamine synthetase cascade: enrichment of uridylyltransferase in Escherichia coli carrying hybrid ColE1 plasmids

Colchicine-binding properties of the total cytoplasmic pool of tubulin from rat liver were evaluated in tubulin-stabilizing (TS) supernates. Microtubules were separated from free tubulin using a microtubule-stabilizing solution (MTS) and ultracentrifugation. [³H]Colchicine-binding properties of microtubule-derived tubulin were investigated in supernates prepared after resuspension of MTS pellets in TS. In TS buffer at 37 °C the colchicine-binding activity of the total cytoplasmic pool of tubulin decayed with $\text{T}\text{1}\text{2}$ of 3.39 h. Resuspended pellet tubulin decayed much more rapidly under the same conditions with a $\text{T}\text{1}\text{2}$ of 0.72 h. This rapid time decay of microtubule-derived tubulin was found to be at least partially attributable to prior microtubule-stabilizing solution exposure. Since tartrate has been reported to increase the rate of colchicine binding to tubulin, sodium tartrate (150 mm) was added to our colchicine-binding system. This addition increased the detectable [³H]colchicine binding by 10% in the total cytoplasmic preparation and by 85% in the resuspended pellet preparation. Addition of tartrate (150 mm) also resulted in a 105% increase in the $\text{T}\text{1}\text{2}$ for total cytoplasmic tubulin and a 412% increase for microtubule derived tubulin. Total cytoplasmic supernates of liver bound [³H]colchicine linearly over a wide range of tissue concentrations. However, resuspended microtubule-stabilizing solution pellet supernates in tubulin-stabilizing solution showed some increase in colchicine binding per tissue weight in the more dilute samples. Our data which demonstrate differences in colchicine-binding properties for total cytoplasmic and microtubule-derived pools of tubulin suggest that present assays for hepatic tubulin polymerization which assume identical binding properties should be interpreted with caution.     

Tubulin and microtubules from bovine kidney: purification, properties, and characterization of ligand binding.

Tubulin was purified from bovine renal medulla by in vitro assembly of microtubules in the presence of dimethyl sulfoxide and glycerol. Light scattering measurements of the polymerization process demonstrate that dimethyl sulfoxide and glycerol decrease the critical concentration of tubulin required for polymerization. The minimum concentration of tubulin from bovine renal medulla is about 1% of the total soluble protein. Assembly occurs in the absence of detectable amounts of high-molecular weight proteins or τ-protein. Microtubules polymerized in the absence and presence of 10% dimethyl sulfoxide and 4 m glycerol are similar morphologically as detected by electron microscopy. Molecular weights of α- and β-tubulin from bovine renal medulla are 54,000 ± 700 and 52,000 ± 800, respectively, as determined by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. Colchicine-binding activity of renal medullary tubulin decays in an apparent first-order process which is temperature dependent. The half-time of decay in buffer is 5.1 h and addition of 5 μm vinblastine sulfate increases the half-time of decay to 10.9 h at 37 °C. Calculations based on measurements of the rate of decay of colchicine-binding activity at different temperatures indicates that vinblastine sulfate stabilizes the binding activity by decreasing the entropy of activation of the decay process. Colchicine decreases the rate of decay about 3.5-fold both in the absence and presence of vinblastine sulfate at 37 °C. Values of the apparent colchicine-binding constant, K_A, of bovine renal medullary tubulin are 5.9 × 10⁶ and 7.8 × 10⁶m^?1 at 37 °C in the absence and presence of vinblastine sulfate. Vinblastine sulfate decreases the rate of decay and increases the apparent binding constant of colchicine binding. Lumicolchicine does not affect the binding of colchicine. Podophyllotoxin apparently competitively inhibits the binding of colchicine; the apparent K_i for podophyllotoxin is 4.0 × 10^?7m at 37 °C. Thus, tubulin from bovine renal medulla has ligand-binding characteristics which exhibit differences and similarities to the corresponding characteristics of the brain tubulin. These biochemical properties of the colchicine-binding activity of bovine renal medullary tubulin support previous physiologic studies which demonstrate that microtubules are required for the function of vasopressin in mammalian kidneys.     

Colchicine-binding protein of the liver. Its characterization and relation to microtubules

Colchicine-binding activity of mouse liver high-speed supernate has been investigated. It has been found to be time and temperature dependent. Two binding activities with different affinities for colchicine seem to be present in this high-speed supernate, of which only the high-affinity binding site (half maximal binding at 5 x 10(-6) M colchicine) can be attributed to microtubular protein by comparison with purified tubulin. Vinblastine interacted with this binding activity by precipitating it when used at high concentrations (2 x 10(- 3) M), and by stabilizing it at low concentrations (10(-5) M). Lumicolchicine was found not to compete with colchicine. The colchicine-binding activity was purified from liver and compared with that of microtubular protein from brain. The specific binding activity of the resulting preparation, its electrophoretic behavior, and the electron microscope appearance of the paracrystals obtained upon its precipitation with vinblastine permitted its identification as microtubular protein (tubulin). Electrophoretic analysis of the proteins from liver supernate that were precipitated by vinblastine indicated that this drug was not specific for liver tubulin. Preincubation of liver supernate with 5 mM EGTA resulted in a time- dependent decrease of colchicine-binding activity, which was partly reversed by the addition of Ca++. However, an in vitro formation of microtubules upon lowering the Ca++ concentration could not be detected. Finally, a method was developed enabling that portion of microtubular protein which was present as free tubulin to be measured and to be compared with the total amount of this protein in the tissue. This procedure permitted demonstration of the fact that, under normal conditions, only about 40% of the tubulin of the liver was assemled as microtubules. It is suggested that, in the liver, rapid polymerization and depolymerization of microtubules occur and may be an important facet of the functional role of the microtubular system.     

Aging of tubulin at neutral pH

The aging of calf brain tubulin in neutral solution has been investigated using the techniques of sedimentation velocity and equilibrium, microtubule assembly, fluorescence spectroscopy, circular dichroism and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The results indicate that tubulin incubated at 4 °C undergoes a slow association process leading to the generation of a 9 S component. The fraction of 9 S component increases progressively with incubation time and appears to follow mono-molecular kinetics. The generation of the 9 S species is paralleled closely by inhibition of microtubule assembly and loss of colchicine-binding ability. Fluorescence spectroscopy and circular dichroism spectra indicate that tryptophan moieties are perturbed during the aggregation process and that the tubulin dimer undergoes a conformational change. There is no protein degradation up to an incubation period of 50 hours. Sedimentation equilibrium experiments in 6 m-guanidine hydrochloride, both in the presence and absence of 2-mercaptoethanol, indicate that the aggregates are stabilized by disulfide bonds and hydrophobic interactions. At periods of incubation >50 hours, the protein starts to be degraded.     

Immunological detection of microtubule poison-induced conformational changes in tubulin

The interaction of tubulin-microtubule poison complexes with anti-tubulin antisera has been investigated using radioimmunoassay. The binding of the major antiserum used in this study to tubulin does not interfere with the binding of colchicine to the tubulin or affect the decay of the colchicine-binding activity of the tubulin. Conversely, if colchicine is incubated with the tubulin, forming tubulin-colchicine complexes, the tubulin-colchicine complexes are less efficient competitors for antibody-binding sites than tubulin alone. This is the result of the formation of specific colchicine-tubulin complexes, since tubulin, incubated with lumicolchicine or isocolchicine, behaves as if the tubulin were incubated alone in the radioimmunoassay. When tubulin is incubated with other microtubule poisons, podophyllotoxin or vinblastine, the tubulin-drug complexes have diminished ability to compete with tubulin as did the tubulin-colchicine complexes. These changes observed in the binding of tubulin-microtubule poison complexes to anti-tubulin antisera in a tubulin radioimmunoassay suggest that the binding of colchicine, podophyllotoxin, or vinblastine to tubulin induces subtle conformational changes on the surface of the tubulin dimer involving antigenic determinant sites.     

Colchicine binding activity of rat brain polysomes

In this communication, we report the presence of a unique colchicine-binding activity in the polysomes of rat brain. This drug-binding property, is somewhat similar to that of tubulin isolated from many sources; however, it differs in several bio-chemical characteristics such as (i) thermal stability of colchicine-binding site, (ii) protection of binding site by vinblastine and (iii) time required for binding equilibration. Such binding of colchicine to the polysomes is most probably due to the presence of a nascent peptide chain of tubulin in the polysome.     

Stabilization of the colchicine-binding activity of tubulin by organic acids

A number of carboxylic acids and organic phosphates were found to be highly effective in stabilizing the colchicine-binding activity of calf brain tubulin. The most active were glutamate, glutarate, delta-aminovalerate, glucose 1-phosphate, glucose 6-phosphate, fructose 1,6-(bis)phosphate, creatine phosphate and 6-phosphogluconate Maximum effects occurred at high concentrations. Combinations of agents were also examined, and the most effective mixture for stabilizing tubulin found thus far was the combination of 1.0 M glutamate, 100 mM glucose 1-phosphate, 1 mM GTP and 0.5 mg/ml of albumin. No loss of activity occurred over 48 h at 37 degrees C with tubulin was present at a concentration of 100 microgram/ml.     

Properties of tubulin in unfertilized sea urchin eggs. Quantitation and characterization by the colchicine-binding reaction

The colchicine-binding assay was used to quantitate the tubulin concentration in unfertilized Strongylocentrotus purpuratus eggs and to characterize pharmacological properties of this tubulin. Specificity of colchicine binding to tubulin was demonstrated by apparent first-order decay colchicine-binding activity with stabilization by vinblastine sulfate, time and temperature dependence of the reaction, competitive inhibition by podophyllotoxin, and lack of effect of lumicolchicine. The results demonstrate that the minimum tubulin concentration in the unfertilized egg is 2.71 mg per milliliter or 5.0% of the total soluble cell protein. Binding constants and decay rates were determined at six different temperatures between 8 degrees C and 37 degrees C, and the thermodynamic parameters of the reaction were calculated. delta H0=6.6 kcal/mol, delta S0=46.5 eu, and, at 13 degrees C, delta G=-6.7 kcal/mol. The association constants obtained were similar to those of isolated sea urchin egg vinblastine paracrystals (Bryan, J. 1972. Biochemistry. 11:2611-2616) but approximately 10 times lower than that obtained for purified chick embryo brain tubulin at 37 degrees C (Wilson, L.J.R. Bamburg, S.B. Mizel, L. Grisham, and K. Creswell. 1974. Fed Proc. 33:158-166). Therefore, the lower binding constants for colchicine in tubulin-vinblastine paracrystals are not due to the paracrystalline organization of the tubulin, but are properties of the sea urchin egg tubulin itself.     

A sensitive method for measuring polymerized and depolymerized forms of tubulin in tissues

A rapid method for measuring polymerized and depolymerized forms of tubulin in tissues has been developed. The procedure consists of homogenization and centrifugation of the tissue in a microtubule- stabilizing solution and depolymerization of the precipitated microtubules; polymerized and depolymerized forms of tubulin are quantitated by a colchicine-binding assay. The validity of the technique was assessed by electron microscopy and recovery studies with labeled and unlabeled preparations of polymerized and depolymerized forms of rat brain tubulin. The sensitivity of this technique allows quantitation of tubulin in 150 micrograms of tissue, wet weight. The method demonstrated that both the polymerized and depolymerized forms of tubulin were present in rat liver cells, and that in the fed state 31.3 +/-0.7% of the total tubulin pool was in the polymerized form.     

Colchicine binding in the free-living nematode Caenorhabditis elegans

The [3H]colchicine-binding activity of a crude supernatant of the free-living nematode Caenorhabditis elegans was resolved into a non-saturable component and a tubulin-specific component after partial purification of tubulin by polylysine affinity chromatography. The two fractions displayed opposing thermal dependencies of [3H]colchicine binding, with non-saturable binding increasing, and tubulin binding decreasing, at 4 degrees C. Binding of [3H]colchicine to C.elegans tubulin at 37 degrees C is a pseudo-first-order rate process with a long equilibration time. The affinity of C. elegans tubulin for [3H]colchicine is relatively low (Ka = 1.7 x 10(5) M(-1)) and is characteristic of the colchicine binding affinities observed for tubulins derived from parasitic nematodes. [3H]Colchicine binding to C. elegans tubulin was inhibited by unlabelled colchicine, podophyllotoxin and mebendazole, and was enhanced by vinblastine. The inhibition of [3H]colchicine binding by mebendazole was 10-fold greater for C. elegans tubulin than for ovine brain tubulin. The inhibition of [3H]colchicine binding to C. elegans tubulin by mebendazole is consistent with the recognised anthelmintic action of the benzimidazole carbamates. These data indicate that C. elegans is a useful model for examining the interactions between microtubule inhibitors and the colchicine binding site of nematode tubulin.     

Interaction of reduced glutathione with bovine brain tubulin

Incubation of phosphocellulose-purified tubulin with GSH at 30 degrees C results in an inhibition of colchicine binding activity. GSSG has a protective effect against the GSH-induced loss of colchicine-binding. Incubation of tubulin with GSH at 30 degrees C results in the formation of abnormal tubulin polymers which are insensitive to cold. Such aggregation is insensitive to antimicrotubular drugs. Aggregation is inhibited by GSSG but not by DTT or mercaptoethanol. GSH-induced aggregation is very sensitive to the ionic strength of the assembly medium; both the aggregation and colchicine binding inhibition induced by GSH are inhibited at higher ionic strength. These results indicate a very complex interaction of GSH with tubulin.     

Adenosine modifies colchicine-uptake into tissues from the mammalian brain

Colchicine is taken up by isolated cerebral tissues incubated in glucose-salt solutions and is maintained in the tissues when they are superfused with colchicine-free solutions. Most of the colchicine in such tissues remains uncombined after an hour's incubation; up to 20% is combined with tubulin of cytosolic and particulate fractions. Adenosine, and conditions of stimulation or depolarization which release adenosine, diminish the uptake of colchicine and less appears in combined forms. Uptake is diminished also by 2-chloroadenosine and by cyclic AMP; is increased by theophylline and by adenosine deaminase but unaffected by dipyridamole. Adenylate cyclase complexes, with their known tubulin association, are suggested as involved in a route of colchicine-entry and so could give a means whereby environmental changes modify colchicine toxicity, and colchicine-attachment to cerebral preparations.     

The assembly of tubulin into membranes

Tubulin in high-speed supernatants of brain undergoes an alternate form of polymerization into structures that resemble membranes rather than microtubules. The reaction required elevated temperature (37°C) and was prevented by 1mM CaCl₂ or 10^?4 M maytansine. The membraneous material was composed of tubulin (80%) and microtubule-associated proteins (8%) and contained phospholipids. The tubulin was identified on the basis of comigration in two-dimensional gel electrophoresis and colchicine-binding activity.