Effect of experimental colchicine encephalopathy on brain protein synthesis and tubulin metabolism

Colchicine blocks axoplasmic flow and produces neurofibrillary degeneration. Brain slices from mice injected intracerebrally with colchicine incorporated more [¹⁴C]leucine into protein and had a decreased uptake of [¹⁴C]leucine into the perchloric acid-soluble pool than did their controls. Brain RNA content was decreased and free leucine increased by colchicine-induced encephalopathy. The specific activities of proteins from subcellular fractions of colchicine-injected brain were increased in the nuclear fraction, the 100,000-g supernatant, and its vinblastine-precipitable tubulin. The ratio of the specific activity of the crude mitochondrial fraction to that of the total homogenate was decreased, as would consistent with impaired movement of newly labeled protein into synaptosomes. Colchicine-injected brain extracts contained one or more cytosol fractions that stimulated ribosomal incorporation of [¹⁴C]leucine into protein in a cell-free system. Colchicine-binding-activity measurements indicated loss of soluble and particulate tubulin in colchicine-injected brains; the decrease of soluble tubulin was verified by its selective precipitation with vinblastine. Colchicine encephalopathy did not affect the rate of spontaneous breakdown of in vitro colchicine binding activity. Similarities of colchicine encephalopathy to the neuron's response to axonal damage suggest that colchicine-induced increase in protein synthesis may, in part, reflect a neuronal response to blockage of neuroplasmic transport.     

Polymerization and colchicine binding. Two independent properties of tubulin

When stored frozen in 1 M sucrose and 1 mM GTP, tubulin loses polymerizing ability exponentially. Since addition of diethiothreitol does not change the decay half-life, this decrease in activity can not be attributed to disulfide bond formation. When tubulin is stored frozen in dithiothreitol and GTP only, the decay half-life increases by a factor of four, indicating that sucrose destabilizes polymerizing ability. Frozen storage in sucrose has the opposite effect on colchicine binding, which remains at 100% for 40 days. This temporal divergence indicates that colchine binding and polymerization are two independent properties of tubulin.     

Microtubules and protein secretion in rat lacrimal glands. Inhibitory effect of the tubulin . colchicine complex isolated from lacrimal glands upon brain tubulin polymerization. Identification of the complex by gel electrophoresis.

The specific inhibitory effect of colchicine upon protein secretion by lacrimal glands could be related to the formation of a complex between colchicine and tubulin from the soluble fraction of the gland. By gel electrophoresis under nondissociating conditions, it is shown that this complex is similar to the colchicine . tubulin complex from brain. The complex isolated from lacrimal glands is highly inhibitory upon brain tubulin assembly since as low as 0.07 microM complex impedes the polymerization of 8 microM tubulin by 50%, compared to 3 microM for free colchicine. Therefore, a small percentage of complexed tubulin (0.9%) is enough for polymerization to be blocked. In lacrimal glands the complex might prevent the polymerization of tubulin, and colchicine shift the tubulin in equilibrium microtubules equilibrium to microtubules disassembly. The disorganization of the labile microtubular system could lead to a modification of the transport of the secretory granules and to a perturbation of secretion.     

Charge effect on the colchicine binding-site of tubulin

Poly(L-lysine) was found to enhance colchicine binding activity of brain tubulin to a several folds. Bases of biological interests that were tested and found to be inactive were spermine, spermidine and even L-lysine. Part of this enhance binding is due to the increase in the affinity of colchicine-tubulin interaction in the presence of poly(L-lysine). Moreover, poly(L-lysine) stabilized the colchicine binding site of tubulin against thermal denaturation.     

Luminescence studies of perturbation of tryptophan residues of tubulin in the complexes of tubulin with colchicine and colchicine analogues

Tubulin, a heterodimeric (alphabeta) protein, the main constituent of microtubules, binds efficiently with colchicine (consisting of a trimethoxybenzene ring, a seven-member ring and methoxy tropone moiety) and its analogues, viz., demecolcine and AC [2-methoxy-5-(2',3',4'-trimethoxyphenyl)tropone]. Tubulin contains eight tryptophan (Trp) residues at A21, A346, A388, A407, B21, B103, B346, and B407 in the two subunits. The role of these eight Trp residues in this interaction and also their perturbation due to binding have been explored via time-resolved fluorescence at room temperature and low-temperature (77 K) phosphorescence in a suitable cryosolvent. Both the time-resolved fluorescence data and 77 K phosphorescence spectra indicate that the emitting residues move toward a more hydrophobic and less polar environment after complex formation. The environment of emitting Trps in the complex also becomes slightly more heterogeneous. Our analysis using the experimental results, the calculation of the accessible surface area (ASA) of all the Trps in the wild type and tubulin-colchicine complex [Ravelli, R. B. G., et al. (2004) Nature 428, 198-202], the distance of the Trp residues from the different moieties of the colchicine molecule, the knowledge of the nature of the immediate residues (<5 A) present near each Trp residue, and the calculation of the intramolecular Trp-Trp energy transfer efficiencies indicate that Trp A346, Trp A407, Trp B21, and Trp B407 are the major contributors to the emission in the free protein, while Trp B21 and Trp B103 are mainly responsible for the emission of the complexes. A comparative account of the photophysical aspects of the drug molecules bound to protein in aqueous buffer and in buffer containing 40% ethylene glycol has been presented. The quantum yield and average lifetime of fluorescence in tubulin and its complexes with colchicine are used to predict the possible donors and the energy transfer (ET) efficiency in the ET process from Trps to colchicine in the complex. This study is a unique attempt to identify the Trp residues contributing to the emission in the free protein and in a complex of a multi-Trp protein with a drug molecule without performing the mutation of the protein.     

Effect of inhibition of protein synthesis on lipid metabolism in Lactobacillus plantarum.

In Lactobacillus plantarum 17-5, lipid synthesis appears to be correlated with protein synthesis. Inhibition of protein synthesis by chloramphenicol (50 mug/ml) caused the nearly simultaneous inhibition of incorporation of radioactive oleic acid into polar lipids before the cessation of growth. In addition, de novo fatty acid synthesis, as determined by the incorporation of radioactive acetate into cellular lipids, was also inhibited. Removal of the antibiotic resulted in the resumption of growth, protein synthesis, and polar lipid synthesis. Inhibition of protein synthesis by leucine deprivation also produced a marked reduction in the incorporation of radioactive oleic acid into the total polar lipids at about the same time that growth stopped (30 to 60 min after the removal of leucine). However, the different classes of lipids behaved differently. For example, the incorporation of oleic acid into cardiolipin was inhibited immediately upon removal of leucine from the cultures, whereas incorporation into phosphatidyl-glycerol was maintained at near normal rates for 60 min after the removal of leucine and then ceased. In contrast, the accumulation of radioactive oleic acid in a neutral lipid identified as diglyceride occurred to a much greater extent in leucine-deprived cultures than in control (+ leucine) cultures. Upon addition of leucine to leucine-deprived cultures, the rates of synthesis of phosphatidyl-glycerol and cardiolipin returned to normal; the amount of radioactivity in the diglyceride fraction decreased to normal levels concomitantly with increased phospholipid synthesis.     

Effect of tubulin binding and self-association on the near-ultraviolet circular dichroic spectra of colchicine and analogues

Near-UV circular dichroic (CD) spectra of three colchicine analogues that differ at the C-10 position have been obtained in the presence and absence of tubulin. All three colchicine analogues show dramatic alterations in the low-energy near-UV CD band upon tubulin binding that cannot be mimicked by solvent, but in no event does the rotational strength of the CD band decrease to nearly zero as in the case of colchicine [Detrich, H. W., III, Williams, R. C., Jr., Macdonald, T. L., & Puett, D. (1981) Biochemistry 20, 5999-6005]. The effect of self-association of colchicine and one of the C-10 analogues, thiocolchicine, on the near-UV CD band was also investigated. A qualitative similarity was seen between the near-UV CD spectra of colchicine and thiocolchicine dimers and the spectra of these molecules bound to tubulin. These observations support the previous suggestion that ligands bound to the colchicine site on tubulin may be interacting with an aromatic amino acid in the colchicine binding site [Hastie, S. B., & Rava, R. P. (1989) J. Am. Chem. Soc. 110, 6993-7001].     

Release and metabolism of brain noradrenaline during experimental ischemia. Effect of glycerol treatment

The release and catabolism of brain NA were studied after labelling of endogenous NA stores with 3H NA in rats submitted to cerebral ischemia by injecting calibrated microspheres into the internal carotid artery. Twenty four hours after embolization, both NA release and catabolism were found to be reduced. They were restored after treatment with hyperosmolar solution of glycerol.     

Anion-induced increases in the rate of colchicine binding to tubulin.

The rate of binding of colchicine to tubulin to tubulin is enhanced by certain anions. Among the inorganic anions tested, only sulfate was effective. The organic anions include mostly dicarboxylic acids, among which tartrate was the most effective. This effect occurs onlt at low concentrations of colchicine (less than 0.6 X 10(-5) M). The rate increase dor sulfate and L-(+)-tartrate is ca. 2.5-fold at 1.0 mM and plateaus at a limiting value of ca. 4-fold at 100mM. The overall dissociation rate of the colchicine from the complex, which includes both the true rate of dissociation and the rate of irreversible denaturation of tubulin, is not influenced by 1.0 mM tartrate. The affinity constants for colchicine determined from the rate constants are 8.7 X 10(6) and 2.1 X 10(7) M-1 in the absence and the presence of 1.0 mM L-(+)-tartrate. The limiting value is 3.2 X 10(7) M-1. The affinity constant calculated from steady-state measurements is 3.2 X 10(6) M-1 with or without anions. The binding of other ligands like podophyllotoxin, vinblastine, and 1 -anilino-8-naphthalenesulfonate to tubulin is not affected by tartrate. No major conformational changes resulting from anion treatment could be detected by circular dichroism or intrinsic fluorescence. However, the ability of tubulin to polymerize is inhibited by L-(+)-tartrate at concentrations that increase the rate of colchicine binding. We conclude that anions must have a local effect at or near the binding site which enhances the binding rate of colchicine and which may be related to inhibition of polymerization.     

Distinct colchicine binding kinetics of bovine brain tubulin lacking the type III isotype of beta-tubulin

In mammalian brain, beta-tubulin occurs as a mixture of four isotypes designated as types I, II, III, and IV. It has been speculated in recent years that the different tubulin isotypes may confer functional diversity to microtubules. In an effort to investigate whether different tubulin isotypes differ in their functional properties we have studied the colchicine binding kinetics of bovine brain tubulin upon removal of the beta III isotype. We found that the removal of the beta III isotype alters the binding kinetics from biphasic to monophasic with the disappearance of the slow phase. The kinetics become biphasic with the reappearance of the slow phase when the beta III-depleted tubulin was mixed with the beta III fraction eluted from the affinity column with 0.5 M NaCl. The analysis of the kinetic data reveals that the tubulin dimers containing beta III bind colchicine at an on-rate constant of 35 M-1 s-1 while those lacking beta III bind at 182 M-1 s-1. Our results strongly suggest that the beta-subunit plays a very important role in the interaction of tubulin with colchicine.     

Effect of colchicine on the carbohydrate metabolism of mycelial felts of cunninghamella Sp.

Summary An experiment is reported in which 5 days old Cunninghamella spec. mats were incubated at 25°C over Richard's medium alone or together with colchicine. The results show that, up to 20 p.p.m., colchicine had no effect on dry weight and soluble sugar content of the fungal mats but caused an acceleration in the rate of sugar absorption and utilisation and polysaccharide accumulation especially the glucosans. 10 p.p.m. concentration further induced an increase in CO₂ production and synthesis of mononucleotides and nucleoproteins as indicated by excessive accumulation of conjugated pentoses and the pentosan fraction of polysaccharides.     

Podophyllotoxin as a probe for the colchicine binding site of tubulin.

The binding of [3H]podophyllotoxin to tubulin, measured by a DEAE-cellulose filter paper method, occurs with an affinity constant of 1.8 X 10(6) M-1 (37 degrees at pH 6.7). Like colchicine, approximately 0.8 mol of podophyllotixin are bound per mol of tubulin dimer, and the reaction is entropy-driven (43 cal deg-1 mol-1). At 37 degrees the association rate constant for podophyllotoxin binding is 3.8 X 10(6) M-1 h-1, approximtaely 10 times higher than for colchicine; this is reflected in the activation energies for binding which are 14.7 kcal/mol for podophyllotoxin and 20.3 kcal/mol for colchicine. The dissociation rate constant for the tubulin-podophyllotoxin complex is 1.9 h-1, and the affinity constant calculated from the ratio of the rates is close to that obtained by equilibrium measurements. Podophyllotxin and colchicine are mutually competitive inhibitors. This can be ascribed to the fact that both compounds have a trimethoxyphenyl ring and analogues of either compound with bulky substituents in their trimethoxyphenyl moiety are unable to inhibit the the binding of either of the two ligands. Tropolone, which inhibits colchicine binding competitively, has no effect on the podophyllotoxin/tubulin reaction. Conversely, podophyllotoxin does not influence tropolone binding. Moreover, the tropolone binding site of tubulin does not show the temperature and pH lability of the colchicine and podophyllotoxin domains, hence this lability can be ascribed to the trimethoxyphenyl binding region of tubulin. Since podophyllotoxin analogues with a modified B ring do not bind, it is concluded that both podophyllotoxin and colchicine each have at least two points of attachment to tubulin and that they share one of them, the binding region of the trimethoxyphenyl moiety.