Of the 20 cysteines of rat brain tubulin, some react rapidly with sulfhydryl reagents, and some react slowly. The fast reacting cysteines cannot be distinguished with [ 14 C]iodoacetamide, N-[ 2-iodoacetyl)amino)ethyl)amino) naphthalene-1-sulfonic acid]), since modification to mole ratios < <1 cysteine/dimer always leads to labeling of 6 -7 cysteine residues. These have been identified as Cys-305␣, Cys-315␣, Cys-316␣, Cys-347␣, Cys-376␣, Cys-241, and Cys-356 by mass spectroscopy and sequencing. This lack of specificity can be ascribed to reagents that are too reactive; only with the relatively inactive chloroacetamide could we identify Cys-347␣ as the most reactive cysteine of tubulin. Using the 3.5-Å electron diffraction structure, it could be shown that the reactive cysteines were within 6.5 Å of positively charged arginines and lysines or the positive edges of aromatic rings, presumably promoting dissociation of the thiol to the thiolate anion. By the same reasoning the inactivity of a number of less reactive cysteines could be ascribed to inhibition of modification by negatively charged local environments, even with some surface-exposed cysteines. We conclude that the local electrostatic environment of cysteine is an important, although not necessarily the only, determinant of its reactivity. C]ethylmaleimide, or IAE-DANS ([5-((((The 20 SH groups of the tubulin dimer have long led to speculation as to their function. Requirements for a few of the SH groups have been identified. Thus, Cys-12 is near the binding site of the exchangeable GTP of -tubulin (1), and a C12S mutation is lethal in haploid yeast, although a C12A mutation is survivable (2). Cys-241 1 and Cys-356 are near or are part of the binding site for colchicine and other agents (3)(4)(5). What the precise role of the involvement of these cysteines may be is for the most part not clear. Some of the SH groups of tubulin form thioesters with palmitic acid both in vivo and in vitro; these may be responsible for membrane localization of tubulin (6 -9). One of these has been located as Cys-376␣ (10). Except for this palmitoylation site, no specific functions have been identified for the 12 SH groups of ␣-tubulin, and the order of reactivities of the SH groups has not been definitively established. It has been repeatedly demonstrated (11) that reaction of an equivalent of 1 or 2 SH groups with the usual alkylating agents abolished polymerization competence, but their location in the sequence has not been unambiguously determined. Loss of colchicine binding requires modification of additional SH groups by these nonspecific SH reagents. For this reason we have approached the reactivity of tubulin SH groups in a more general sense, comparing the effects of thioether, disulfide, and thioester formation as well as their location in ␣-tubulin and -tubulin.Protein sulfhydryl groups can be involved in numerous reactions such as oxidation, disulfide interchange, thioether, and thioester formation. For the purpose of this discussion, we shall exclude oxid...
We have previously shown that rat brain tubulin, a heterodimer consisting of an a and fi monomer, can be covalently labeled with [3H]colchicine by near UV irradiation. Most of the label appears in (&tubulin. We show here that (3-tubulin can be separated and purified from SDS preparative gels and analyzed by proteolysis. Chymotrypsin yielded a labeled -4-kDa band that contained two peptides. Tryptic digestion also yielded an =4-kDa band containing two peptides. Sequence analysis revealed a peptide ofresidues 1-36 and 213-242 for chymotrypsin and a peptide of residues 1-46 and 214-241 for trypsin. To identify which peptide carried the label, limited hydrolysis of -tubulin was done with trypsin; this procedure yielded a labeled 16-kDa N-terminal peptide and a 35-kDa C-terminal peptide, as identified by antibodies. Isolation of these peptides and extensive digestion with trypsin yielded two labeled peptides corresponding to residues 1-46 from the 16-kDa N-terminal fragment and residues 214-241 from the 35-kDa C-terminal fragment. These results show that at least two regions in P-tubulin are specifically involved in colchicine binding and that the span of the colchicine molecule, s11 A, bridges these two regions in the native /3 monomer.Colchicine is the most studied of all antimitotic agents. It binds to tubulin with a stoichiometry of one and inhibits microtubule assembly substoichiometrically. Colchicine binding to tubulin exhibits pseudoirreversible kinetics; it displays a fast step followed by a slow step involving conformational changes of both ligand and tubulin. The latter, in turn, promote fluorescence characteristic of the tropolone moiety of colchicine. Many analogs have been studied, resulting in the proposal that the A and C rings of colchicine both bind to specific loci, whereas the B ring is primarily a regulator of binding kinetics (see refs. 1 and 2 and the references therein).Attempts to localize the binding site(s) in the tubulin dimer have led to conflicting results. Studies with bromacetylcolchicine indicated binding to a-tubulin (3), but these results have been criticized on the basis of the nonspecificity of the alkylation reaction. Proteolytic studies of the colchicinetubulin complex yielded a-tubulin-derived, colchicinebearing peptides of 16-18 kDa; because z90% of the label bound to the protein was lost during processing, conclusions are uncertain (4). Others (5) have also shown colchicine localization to a-tubulin by using a photoaffinity label with a long spacer arm. A shorter spacer arm led to colchicine binding on both a-and ,B-tubulins (6), suggesting that localization was a function ofthe spacer length. On the other hand, Luduena and Roach (7) [3H]colchicine that the label is localized almost exclusively on 3-tubulin. In late stages of irradiation, or with "damaged" tubulin, label also appeared in a-tubulin (9). These results suggested the possibility that the colchicine-binding domain on 3-tubulin might be near a-tubulin. That proposal was strengthened by the finding that co...
Chelidonine, sanguinarine, and chelerythrine are natural benzophenanthridine alkaloids that inhibit taxol-mediated polymerization of rat brain tubulin in the micromolar range. Chelidonine is a weak, competitive inhibitor of colchicine binding to tubulin but does not inhibit podophyllotoxin binding. On the other hand, sanguinarine inhibits both colchicine and podophyllotoxin binding to tubulin with I50 values of 32 and 46 microM, respectively, and chelerythrine inhibits with I50 values of 55 and 60 microM, respectively. The inhibition by these two agents is of the mixed type. Tubulin forms an acid-reversible pseudobase with the imminium ion of sanguinarine, probably through several of its sulfhydryl groups, as shown by the loss of the yellow color of sanguinarine and its 596-nm fluorescence emission peak. Chelidonine, on the other hand, cannot undergo such pseudobase formation, and we conclude that it acts by a different mechanism. A number of previously described pharmacologic effects of these agents may be due to their inhibition of microtubule function.
All 20 cysteine residues are accessible to disulphide reagents in the tubulin dimer, whereas only four are accessible in taxol-stabilized microtubules. Reaction rates with disulphide reagents are a function of the reagent, are decreased by G nucleotides, and increased with increase in pH and urea. With transient (stop-flow) kinetics, DTNB [5,5'-dithiobis-(2-nitrobenzoic acid)] and 2,2'-dithiodipyridine progress curves cannot be fitted by the sum of exponential terms based only on classes of cysteines. The mixed disulphide products react further to form both intra- and intermonomer disulphide bonds that can be reversed by reducing agents. With MMTS (methyl methanethiosulphonate) or ODNB (n-octyl-dithio-2-nitrobenzoate), virtually no protein-protein disulphide bonds are formed and the ODNB reaction can be given as the sum of three exponential terms with pseudo-first-order rate constants of 0.206, 0.069 and 0.010 s(-1) at pH 6.5, suggesting three classes of thiol reactivities. Limited cysteine substitution leads to only small changes in tryptophan or CD spectra, whereas complete substitution leads to loss of the helix content. MMTS-induced loss of SH groups leads to progressive increases in the critical concentration and loss of polymerization competence that can be reversed by assembly promoters such as higher protein concentration, taxol or high ionic strength. Under such conditions, the substituted tubulin forms protofilament-based structures such as microtubules, open tubules, sheets and/or bundles.
A role for charge-based interactions in protein stability at the monomer or dimer level is well known. We show here that such interactions can also be important for the higher-order structures of microtubule assembly. Alkali metal chlorides increase the rate of polymerization of pure tubulin driven by either taxol or dimethyl sulfoxide. The effect is cation selective, exhibiting a sequence Na+ > K+ > Lit > Cs+, with optimal concentrations for Na' at -160 mM.Hofmeister anion effects are additive with these rate stimulations. Sodium is less potent than guanidinium ion stimulation reported previously, but produces a larger fraction of normal microtubules. Alkali metal cations lower the critical concentration by a factor of -2, produce cold reversible polymers whose formation is sensitive to podophyllotoxin inhibition, increase the fraction of polymers present as microtubules from -0.9 to 0.99, and reverse or prevent urea-induced depolymerization of microtubules. In the presence of microtubule-associated proteins, the promotion of polymerization is no longer cation selective. In the polymerization of tubulin S, in which the acidic C termini of both monomers have been cleaved, the cation enhancement is markedly decreased, although selectivity persists. Because the selectivity sequence is similar to that of the coillhelix transition of polyglutamic acid, we suggest that a major part, although not all, of the cation selective enhancement of polymerization results from shielding of the glutamate-rich C termini of the tubulin monomers.
Ultraviolet irradiation of the [3Hjcolchicine-tubulin complex leads to direct photolabeling of tubulin with low but practicable efficiency. The bulk (70% to >90%) of the labeling occurs on (8-tubulin and appears early after irradiation, whereas a-tubulin is labeled later. The labeling ratio of .8-tubulin to a-tubulin (13/a ratio) is reduced by prolonged incubation, prolonged irradiation, urea, high ionic strength, the use of aged tubulin, dilution of tubulin, or large concentrations of colchicine or podophyllotoxin. Glycerol increases the (3/a ratio. Limited data with (3Hlpodophyllotoxin show that it covalently bound with a similar fl/a distribution.Vinblastine, on the other hand, exhibits preferential attachment to a-tubulin. The possibilities that colchicine binds at the interface between a-tubulin and j3-tubulin, that the drug spans this interface, and that both subunits may contribute to the binding site are suggested.Despite the fact that colchicine has been used as an antimicrotubule agent for many years, there is no unanimity regarding the location of the high-affinity binding site for the drug in the tubulin dimer, which is formed by the noncovalent association ofthe similar but not identical q and (3 monomers.Several studies have assigned the site to the a-subunit, but uncertainties exist regarding the specificity of the' reactions used. Thus, N-bromoacetyldesacetylcolchicine showed nonspecific alkylation (1), photoaffinity labels used long spacer arms (2, 3), and studies with limited proteolysis could have been affected by rearrangements during proteolysis in the damaged protein (4). Colchicine binding to a site on ,f-tubulin has been proposed on the basis of indirect experiments dealing with the reactivity ofcysteine residues in ,3-tubulin (5) and by findings that most tubulin mutations that confer colchicine resistance occur in 83-tubulin genes (6-8).The excitation maximum of colchicine occurs at a higher wavelength than that of the tryptophan residues of tubulin; hence, direct photolabeling of tubulin with colchicine, without irradiating the protein, appeared to be feasible. However, stoichiometric covalent binding would not be expected for such a reaction because the efficiency of direct photolabeling tends to be <25% (9) because of the short colchicine fluorescence lifetime (of 1.14 ns) (10) with little intersystem crossing to the triplet state or long lifetimes (11), and because of the powerfully competing photoisomerization reaction to form lumicolchicines from excited-state colchicine, which causes dissociation of the ligand (12-14). Nevertheless, such a reaction might be less subject to the specificity problems noted above and thus increases the probability that colchicine will cross-link to the "correct" site. The following study explores the conditions for the direct photolabeling reaction, the localization of the covalently bound colchicine, and the factors influencing the distribution of the drug on tubulin. A portion of this material has been presented (15). MATERIALS AND METHODST...
Low concentrations of guanidine hydrochloride (GuHCl) increase the rate (and to a lesser degree, the extent) of tubulin polymerization as assessed by light scattering. Maximum enhancement occurs at 120-160 mM GuHCl followed by decreases at higher GuHCl. The latent period is decreased, and there is a 3-4 fold reduction in the critical concentration of polymerization. Electronmicrographs reveal microtubules in the controls and an increasing fraction of total polymers present as aberrant microtubules as the GuHCl concentration is increased from 20 to 100 mM. The GuHCl effect is markedly reduced, but not abolished, in tubulin S (in which the anionic C termini of both monomers have been removed). The GuHCl-induced polymerization has an absolute requirement for GTP and taxol or DMSO, is very sensitive to podophyllotoxin inhibition, and can overcome urea-mediated inhibition of polymerization. Guanidinium analogues mimic the GuHCl effect roughly as a function of the number of potential hydrogen bonds. The anions of the guanidine salts superimpose their inhibitory action on the guanidinium cation effect according to the lyotropic series. At higher GuHCl concentrations (peak effect 500-700 mM), a different polymer (type II) is formed that is GTP and taxol independent, but whose polymerization is retarded but not prevented by podophyllotoxin. Its structure resembles the fibrillar network seen in unfolding intermediates of other proteins. We conclude that both charge and hydrogen-bonding ability are major contributors to the GuHCl-induced promotion of tubulin polymerization, and that charge-shielding is likely to be the basis for this effect.
Exposure of Chinese hamster ovary, mouse adrenal cortex tumor (Y-I), THP-1 and U-937 cells and human erythrocytes to adenylate-cyclase-containing urea extracts of Bordetellu pertussis (strain 114) organisms promotes the formation of large concentrations of intracellular CAMP. Accumulation is dependent on dose and temperature, with significant accumulation occurring at 4"C, and is virtually instantaneous, with a doubling at 1 min. There is an absolute Ca2 + requirement but external calmodulin (the activator of cyclase activity) has no effect except in erythrocytes and U-937 cells, where it reduces cAMP accumulation. However, calmodulin antagonists inhibit cAMP accumulation. In Y-1 adrenal cells the urea-extract adenylate cyclase stimulates steroidogenesis.Anti-(B. pertussis) antibodies inhibit cyclase activity and prevent further cAMP accumulation after 10 min in cells previously exposed to urea extract. The same effect is obtained by washing. This suggests that a portion of the cyclase is associated with cells in a form not accessible to antibody or washing but accessible to substrate, which we interpret as internalized enzyme with a short lifetime. Continuing cAMP accumulation thus appears to need a continuing source of external cyclase.Inhibitors of the effect of diphtheria toxin, such as NH,Cl, methylamine, chloroquin or monensin, have no inhibitory effect on the accumulation of intracelluar cAMP promoted by the internalized adenylate cyclase of urea extracts of B. pertussis organisms. We conclude that entry of the cyclase into cells is not by receptor-mediated endocytosis.
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