©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Colchicine Binding by the Isolated -Monomer of Tubulin (*)

J. Wolff (§) , Leslie Knipling

From the (1)Laboratory of Biochemical Pharmacology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

At mole ratios of lactoperoxidase to tubulin monomers of 3-4, bovine lactoperoxidase forms 1:1 adducts with both - and -tubulin from rat brain, thereby separating the tubulin heterodimer into its monomers. This mixture binds colchicine normally, and we show here by direct photoaffinity labeling that the bulk of the [H]colchicine becomes attached to -tubulin under these conditions. When the -tubulin has been displaced by lactoperoxidase, the ratio of label in -tubulin to -tubulin is increased. The amount of label in -tubulin decreases with a corresponding appearance of label in lactoperoxidase. The rate of labeling of -tubulin remains slow. We conclude that -tubulin is not necessary for colchicine binding and propose a model wherein the A and C rings of colchicine bind to -tubulin, while the B ring faces -tubulin in the dimer.


INTRODUCTION

During experiments on the iodination of tubulin, we found previously that lactoperoxidase (LPO)()associated with tubulin in a ratio of two molecules of enzyme to one of heterodimer. This led to splitting of the dimer into reversible and salt-sensitive 1:1 adducts of either - or -monomer with one LPO molecule (1, 2) without a requirement for unfolding and refolding of tubulin. These adducts could be readily identified on the basis of their size by sucrose density gradient centrifugation or gel filtration or by a change in the Soret spectrum ascribable to an interaction of the LPO with one or more sulfhydryl groups of tubulin(1) . However, a heme-free lactoperoxidase can also bind tubulin, hence the necessity for a heme interaction is not established(3) . The tubulin/LPO association has also been observed morphologically(4) . The bulk affinity constant for the dimer + 2 LPO molecules was 2 10M(1) , and although low mole ratios of LPO copolymerized with microtubules, adduct formation reversibly prevented polymerization of microtubule protein to microtubules at higher mole ratios. Despite the complete separation of the - and -monomers into the 1:1 adducts, [H]colchicine binding stoichiometry was unaffected in the presence of excess LPO when no residual tubulin heterodimer could be demonstrated(2, 5) , i.e. the bound colchicine was displaced toward a larger species (consistent with a 1:1 adduct of LPO + tubulin monomer in sucrose density gradient centrifugation). This led to the suggestion that only one of the monomers was required for colchicine binding and that the binding site retained its native character, but did not reveal which monomer contained the binding site.

It has now been shown by direct photolabeling of tubulin dimers with [H]colchicine that the great bulk of the colchicine covalently bound to the dimer resides in -tubulin(6, 7) ; the distribution of label between - and -tubulin (the / ratio) is critically dependent on the size of the spacer (decreasing size increases the / ratio) and is sensitive to incubation and irradiation times, to urea, ionic strength, and other solution variables, and to the age of the tubulin. It was, therefore, important to employ the direct, zero length spacer, photolabeling method to determine whether or not one could covalently label -tubulin under conditions when no -tubulin was near, i.e. in the -tubulin/LPO adduct. We show here that this is feasible and that the -monomer is not required for colchicine binding to its site, although its presence in the dimer has major kinetic consequences for the binding reaction(8) .


EXPERIMENTAL PROCEDURES

Materials

[ring C,methoxy-H]Colchicine, 70 Ci/mmol, was obtained from DuPont NEN; lactoperoxidase (bovine) was obtained as a lyophilized powder from Sigma. Rat brain microtubule protein preparation, and its purification to tubulin was carried out as described previously(9) . The protein was drop-frozen and stored in liquid nitrogen, where it is stable for many months. All reactions were carried out in Mes assembly buffer (0.1 M Mes, pH 6.9, 1 mM MgCl, 1 mM EGTA). In some experiments this buffer was used at half-strength.

Methods

Because the interaction between tubulin and LPO is slow(1) , preincubations were carried out at room temperature (23 °C) for 30-40 min in a number of experiments. After preincubation, tubulin or tubulin/LPO mixtures were incubated in a Dubnoff shaker in the dark at 36 °C with [H]colchicine containing the indicated carrier concentrations. Incubation times are as indicated in the figures. Thereafter, colchicine-containing samples were irradiated at 4 °C under 2 cm of 20% CuSO5H0 (acting as a low UV and IR filter), with a high pressure Osram mercury lamp at 90 ± 4 W delivering 49 ± 3 mW/cm at the sample surface over a wavelength range of 320-380 nm as measured by a Spectroline DM-365N radiometer. Irradiation times are as indicated in the figure legends. After irradiation, samples were denatured by boiling for 3 min in loading buffer (0.01 M Tris-HCl, pH 6.8, 1% sodium dodecyl sulfate, 10% (v/v) glycerol, 5% mercaptoethanol, and 0.05% bromphenol blue). Samples were then subjected to gel electrophoresis in sodium dodecyl sulfate-8% polyacrylamide gels cast on GelBond films (FMC) and separated at 20 mA for 20 min past expulsion of the dye front. The stained gels were then photographed and either exposed to Kodak X-Omat AR films for autography after enhancement in 1.0 M sodium salicylate, or 5-mm bands were cut from the lanes and dissolved in 300 µl of 30% HO at 65 °C, cooled, mixed with 5 ml of Ultima Gold scintillation fluid (Packard), allowed to stand for 8-12 h at 4 °C to reduce chemiluminescence, and counted in a 2-18.6-keV window. For native gels samples in 10% glycerol and half-strength buffer were applied to horizontal 1% agarose gels cast on agarose-compatible GelBond films (FMC) and electrophoresed in an ice bath at 30 mA in half-strength buffer with frequent mixing of the compartments to maintain the starting pH, until expulsion of the dye front had occurred. Gels were rinsed with water, fixed in 50% methanol, 10% acetic acid for 30 min, flattened, air-dried, stained for 5 min with Coomassie Blue, destained, and redried.


RESULTS

Previous demonstration of adduct formation was accomplished by gel filtration and sucrose density gradient centrifugation(1) . Adduct formation can also be shown by electrophoresis on native agarose gels. As shown in Fig. 1A, LPO moves tubulin cathodally from the anodal compartment and tubulin reduces the cathodal mobility of LPO, presumably as a result of adduct formation. At a mole ratio of 1.0 (LPO/monomer) some residual tubulin can be seen as trailing material in the anodal compartment. At mole ratios of 2-3, this residual tubulin is no longer apparent and the tail is shorter. Although we do not know the reason for this trailing, it suggests an equilibrium between LPO and tubulin. In agreement with the above suggestion, note that, although the LPO is overloaded, tubulin is able to reduce its cathodal migration. At higher ionic strength there is no tubulin trail, and more residual tubulin can be demonstrated (data not shown). This is, perhaps, not surprising since the isoelectric point for the tubulin monomers averages 5.5, whereas that for the LPO isoforms is >9.0(10) . These results also confirm earlier results (1, 5) that at appropriate mole ratios of LPO to tubulin, no native tubulin dimer remains demonstrable.


Figure 1: Interaction of lactoperoxidase with rat brain tubulin. A, native electrophoresis on agarose of the lactoperoxidase-tubulin complex at different mole ratios. Lanes 1 and 8, 6 µg of tubulin alone; lanes 2, 4, and 6, lactoperoxidase + tubulin at mole ratios of 4.1, 2.1, and 1.3, respectively; lanes 3, 5, and 7, comparable concentrations of lactoperoxidase alone. All samples were preincubated at room temperature for 45 min before electrophoresis. B, photocross-linking of 3 µM [H]colchicine with tubulin or lactoperoxidase-tubulin monomer complex at a mole ratio of 8. Panel I, radioautograph. Note weak labeling of the faster moving portion of the lactoperoxidase band (circled). Panel II, Coomassie stain of the same gel. C = control; LPO = lactoperoxidase; MW = molecular weight markers. Samples were preincubated for 40 min at room temperature and incubated at 36 °C with label for 30 min and irradiated at 43 mW/cm and processed as described. The / ratios were 10.1 for control and 15.6 for lactoperoxidase-tubulin complexes.



Upon photolabeling of rat brain tubulin with colchicine in the presence of LPO under conditions where the - and -monomers are completely separated in the adduct, labeling of -tubulin occurs readily and, as will be shown below, with a generally increased selectivity for - vis vis -tubulin (Fig. 1B). The labeling pattern is a function of the mole ratio of LPO to tubulin. In Fig. 2are plotted the counts of H incorporated into protein bands cut out and dissolved from SDS-polyacrylamide gels (solid lines) as well as the calculated ratios of H in -tubulin with respect to -tubulin (/ ratios). Fig. 2A represents data carried out without preincubation of LPO with tubulin, whereas Fig. 2B depicts data collected after 40 min of preincubation at room temperature. At low mole ratios of LPO/monomer -tubulin radioactivity is increased or stays roughly constant, whereas at higher mole ratios labeling of -tubulin with colchicine is decreased. By contrast, label in -tubulin decreases markedly even at low mole ratios of LPO/monomer. Comparison of the two panels reveals that the long incubation period used here is sufficient to overcome the preincubation requirement noted previously(1) . Absorption of the incident UV light by the excess LPO decreases the counts incorporated. We found it difficult to compensate for this by longer irradiation times because of the rapid photoisomerization of colchicine to lumicolchicine. For this reason comparisons relied on the relativeH contents of the - and - monomers, called here the / ratio. It is a useful index of the selectivity of the labeling process for -tubulin. This ratio is frequently greater in the presence of LPO than with the native heterodimer alone, and this is due, in part, to a decrease in the amount of label in -tubulin. The decrease in -tubulin labeling in the presence of LPO was generally accompanied by an increase in label found in the LPO band, particularly its faster portion (no attempt was made to separate the different LPO isomers). When label in -tubulin was compared with the sum of H in -tubulin + LPO, the ratio of -tubulin to this sum remained nearly constant until high mole ratios of LPO were used. This finding suggests that part of the LPO (guest monomer) must have been near the binding site for colchicine on -tubulin. To check whether or not tubulin was necessary for this reaction, similar photolabeling experiments were carried out with LPO alone. Low level, but significant, labeling of LPO could be demonstrated but this was always enhanced by tubulin (average, 1.4-fold).


Figure 2: Covalent photolabeling of tubulin/lactoperoxidase mixtures with 3 µM [H]colchicine as a function of the mole ratio of lactoperoxidase to tubulin monomers. , dpm in -tubulin; , dpm in lactoperoxidase; , dpm in -tubulin; , / ratios. A, no preincubation, 45-min incubation with label at 36 °C followed by 5-min irradiation at 43 mW/cm. B, 40-min preincubation at room temperature followed by 30-min incubation with label at 36 °C and irradiation at 45 mW/cm for 5 min as described under ``Methods.''



Colchicine binding to tubulin is a slow reaction requiring a conformational change in tubulin and colchicine (Ref. 11 and references in Ref. 12). Part of this can be ascribed to the bulky B ring which acts as a kinetic barrier to binding(8) . Because the - and -monomers are separated by LPO, it was expected that the rate of binding to -tubulin might be faster under these conditions. It turned out, however, that the -tubulin/LPO adduct was labeled at approximately the same slow rate (or even more slowly in some experiments such as shown in Fig. 3) as the native tubulin dimer. Because binding was followed by 5 min of irradiation, these time estimates are only approximate. Nevertheless, it is clear that colchicine binding to the adduct is slow and that the kinetic obstacle to colchicine binding to -tubulin presumably presented by -tubulin (8, 11, 12) appeared to have been replaced by the kinetic obstacle of LPO in the adduct (Fig. 3). This suggests that part of the LPO in the adduct is near enough to the colchicine binding site to affect the kinetics of binding. Whether or not this LPO contributes to the binding energy remains to be determined.


Figure 3: The rate of binding of [H]colchicine in the absence (left panel) and presence (right panel) of lactoperoxidase at a mole ratio of 3. All samples were preincubated at room temperature for 40 min without label and were then added to tubes containing [H]colchicine (3 µM final concentration) and cooled immediately to 4 °C and irradiated (``zero time'') or incubated at 36 °C for the indicated times and then irradiated at 50 mW/cm. All curves begin at 5 min (the time for irradiation). Samples were processed as described under ``Methods.''




DISCUSSION

The present study was carried out under conditions in which the - and -monomers of rat brain tubulin (K for dimer dissociation 0.1-0.8 µM)(13, 14, 15, 16) were completely separated from each other by adduct formation with bovine LPO (mean K for adduct dissociation 0.5 µM)(1) . Under these conditions colchicine binds normally to the mixture of adducts(6) , and we show here that the bulk of the label is found on -tubulin as it is in native dimer, with / ratios normally 5 or greater increasing to 15-20 in some cases when LPO is present. This suggests strongly that the -monomer is not required for colchicine binding to -tubulin. The present method does not permit a reliable estimate of the affinity constant of colchicine for -tubulin in the adduct, and any contribution that the guest LPO may make to the affinity is not known at present.

The ready substitution of LPO for either tubulin monomer in the adduct suggested a search for homology between LPO and the monomers. Using the GCG GAP alignment program, there is an 18.8% sequence identity between lactoperoxidase and rat -tubulin and a 16.5% identity with -tubulin. This is, however, of doubtful significance, because identities between LPO and actin or bovine serum albumin are 18.8 and 16.2%, respectively. It should be pointed out that the isoelectric points of the two partners in the adduct are 5.5 for tubulin and >9.0 for the various isoforms of lactoperoxidase(10) . Thus, a charge-based interaction is likely to contribute to adduct formation, and the salt sensitivity (1) confirms this. Despite such charge contributions to adduct formation, there is a high degree of specificity for LPO, since other basic proteins do not disrupt the adduct (Ref. 1, and as also shown in native electrophoresis, see above). Whatever the nature of the LPO-tubulin contacts may be, it is clear that colchicine binding to -tubulin does not require the presence of -tubulin.

Recently, Shearwin and Timasheff (17) have also shown, on the basis of linkage arguments and the fluorescence of allocolchicine promoted by binding to tubulin that had been dissociated into monomers by dilution, that one of the monomers alone is sufficient for colchicicne binding. The strength of allocolchicine binding depends on the nucleotide present in the exchangeable site, with GTP > GDP. Because this site is located on -tubulin(18, 19, 20) , it was argued that the G-nucleotide effect indicated allocolchicine binding to -tubulin. Furthermore, a comparison with podophyllotoxin binding led to the conclusion that the G-nucleotide effect is exerted primarily on the C ring of colchicine or allocolchicine. The contribution of -tubulin to the binding energy of allocolchicine was calculated to be 10% of the total. Whether or not -tubulin makes an equally small contribution to the binding energy of colchicine remains to be determined.

We have previously suggested (6, 7) that colchicine binds to -tubulin near the interface between - and -tubulin because: (i) during direct photolabeling some [H]colchicine binds to -tubulin. This fraction can be increased by mild denaturation. Moreover, the proportion of label in -tubulin is a function of the length of the spacer, with zero length (unsubstituted) colchicine yielding the lowest proportion of -labeled tubulin. (ii) Colchicine induces photo-cross-linking of the tubulin monomers(21) . (iii) Low concentrations of urea (1.0 M) ``tighten'' the structure of the dimer as assessed by several functional criteria (9), and the yield of photo-cross-linked tubulin dimers is increased. By the same reasoning, we suggest that a part of LPO must be near the colchicine site, since the kinetic obstacle to colchicine binding presented by -tubulin in the native dimer appears to have been mimicked by LPO, as judged by the rate of binding, i.e. the rate is approximately as slow as in the native dimer. The nearness of LPO to the site is also suggested by the fact that LPO itself becomes labeled with colchicine, and this occurs in a roughly reciprocal relation to the amount of label found in -tubulin. This leads us to the following proposal. The bulk of the binding energy for colchicine derives from the binding of its A and C rings (plus a contribution from the cratic entropy connecting these two rings)(22) , and two ring analogues such as 2-methoxy-5-(2`,3`,4`-trimethoxyphenyl)tropone (MTPT) bind with similar affinity constants as does colchicine(12, 23, 24) . Clearly both of these rings must bind to -tubulin. However, the rate of binding of two ring analogues is orders of magnitude (6000-fold) faster than for colchicine. Addition of the B ring, and especially bulky substituents at position C7, progressively decreases the rate, but has only a small (20-fold) effect on the affinity constants(8) . Thus the B ring is primarily a kinetic barrier to binding. A possible explanation for this is that in the binding site the B ring faces -tubulin, whereas the A and C rings are in contact with -tubulin, both near the / contact surface. This would account for the labeling of -tubulin and the kinetic effect of bulky substituents at C7. Additional factors must be involved in the photosensitization of monomer/monomer cross-linking, because the two ring analogue of colchicine, MTPT (see above), also promotes such cross-linking(21) . Replacement of -tubulin by LPO does not alleviate the kinetic barrier as the rate of labeling remains slow, but does lead to LPO labeling. This proposal would predict that the isolated native -monomer present in dilute tubulin solutions would bind colchicine rapidly, and colchicine should bind as rapidly as the two ring analogues.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 301-496-2685; Fax: 301-402-0240.

The abbreviations used are: LPO, bovine lactoperoxidase; A ring, trimethoxyphenyl ring; C ring, tropolone ring; B ring, ring connecting the A and C rings; W, watt(s); Mes, 4-morpholineethanesulfonic acid; MTPT, 2-methoxy-5-(2`,3`,4`-trimethoxyphenyl)tropone.


ACKNOWLEDGEMENTS

We are indebted to Dr. Dale Graham of Division of Computer Research and Technology, National Institutes of Health, for the GCG GAP search and to Dr. Dan Sackett for an incisive critique.


REFERENCES
  1. Rousset, B., and Wolff, J. (1980) J. Biol. Chem.255, 2514-2523 [Abstract/Free Full Text]
  2. Rousset, B., and Wolff, J. (1980) J. Biol. Chem.255, 11677-11681 [Abstract/Free Full Text]
  3. Dumontet, C., and Rousset, B. (1983) J. Biol. Chem.258, 14166-14172 [Abstract/Free Full Text]
  4. Suzuki, T., Fujii, T., and Tanaka, R. (1984) Biochem. Med.31, 211-216 [Medline] [Order article via Infotrieve]
  5. Rousset, B., and Wolff, J. (1980) FEBS Lett.115, 235-238 [CrossRef][Medline] [Order article via Infotrieve]
  6. Wolff, J., Knipling, L., Cahnmann, H. J., and Palumbo, G. (1991) Proc. Natl. Acad. Sci. U. S. A.88, 2820-2824 [Abstract]
  7. Uppuluri, S., Knipling, L., Sackett, D. L., and Wolff, J. (1993) Proc. Natl Acad. Sci. U. S. A.90, 11598-11602 [Abstract]
  8. Bhattacharyya, B., Howard, R., Maity, S. N., Brossi, A., Sharma, P. N., and Wolff, J. (1986) Proc. Natl. Acad. Sci. U. S. A.83, 2052-2055 [Abstract]
  9. Sackett, D. L., Bhattacharyya, B., and Wolff, J. (1994) Biochemistry33, 12868-12878 [Medline] [Order article via Infotrieve]
  10. Righetti, P. G., and Caravaggio, T. (1976) J. Chromatogr.127, 1-28 [CrossRef][Medline] [Order article via Infotrieve]
  11. Garland, D. L. (1978) Biochemistry17, 4266-4272 [Medline] [Order article via Infotrieve]
  12. Hastie, S. B. (1991) Pharmacol. Ther.51, 377-401 [CrossRef][Medline] [Order article via Infotrieve]
  13. Detrich, H. W., III, Williams, R. C., Jr., and Wilson, L. (1982) Biochemistry21, 2392-2400 [Medline] [Order article via Infotrieve]
  14. Mejillano, M. R., and Himes, R. H. (1989) Biochemistry28, 6518-6524 [Medline] [Order article via Infotrieve]
  15. Sackett, D. L., and Lippoldt, R. E. (1991) Biochemistry30, 3511-3517 [Medline] [Order article via Infotrieve]
  16. Panda, D., Roy, S., and Bhattacharyya, B. (1992) Biochemistry31,9709-9716 [Medline] [Order article via Infotrieve]
  17. Shearwin, K. E., and Timasheff, S. N. (1994) Biochemistry33, 894-901 [Medline] [Order article via Infotrieve]
  18. Hesse, J., Thierauf, M., and Ponstingl, H. (1987) J. Biol. Chem.262, 15472-15475 [Abstract/Free Full Text]
  19. Shivanna, B. D., Mejillano, M. R., Williams, T. D., and Himes, R. H. (1993) J. Biol. Chem.268, 127-132 [Abstract/Free Full Text]
  20. Jayaram, B., and Haley, B. E. (1994) J. Biol. Chem.269, 3233-3242 [Abstract/Free Full Text]
  21. Wolff, J., Hwang, J., Sackett, D. L., and Knipling, L. (1992) Biochemistry31, 3935-3940 [Medline] [Order article via Infotrieve]
  22. Andreu, J. M., and Timasheff, S. N. (1982) Biochemistry21, 534-543 [Medline] [Order article via Infotrieve]
  23. Engelborghs, Y., and Fitzgerald, T. J. (1987) J. Biol. Chem.262, 5204-5209 [Abstract/Free Full Text]
  24. Bane, S., Puett, D., Macdonald, T. L., and Williams, R. C., Jr. (1984) J. Biol Chem.259, 7391-7398 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.