Regulatory aspects of the colchicine interactions with tubulin

Molecular and Cellular Biochemistry 73 :29 -36 (1987) © Martinus Nijhoff Publishers, Boston. Printed in the Netherlands

Original Article

Regulatory aspects of the colchicine interactions with tubulin

Jesfis Avila 1, Luis Serrano I and Ricardo B. Maccioni 2 1Centro de Biologia Molecular, Universidad Aut6noma, Canto Blanco 28049, Madrid, Spain," 2Department of Biochemistry, Biophysics and Genetics, University of Colorado Health Sciences Center, Denver, CO 80262, USA

Keywords: tubulin, S-tubulin, domain for colchicine binding, regulation colchicine interaction

Abstract

Limited proteolysis of tubulin with subtilisin results in the cleavage of both tubulin subunits yielding S- tubulin heterodimer and 4 kDa peptide fragments containing the carboxyl-terminal domains of ~- and/3- polypeptide chains. S-tubulin binds colchicine and the characterization of the binding of colchicine to S- tubulin molecules showed a decreased rate of decay of colchicine binding activity as compared to that of undigested tubulin. However, S-tubulin exhibited a lower colchicine binding constant than tubulin. Peptide fragments resulting from the controlled tryptic proteolysis of both pure tubulin and S-tubulin were purified by filtration chromatography and presented a strong colchicine binding activity with association constants of 4.5 × 106 and 2.7 x 106 M -l, respectively. Furthermore, these studies support our initial findings on the localization of the tubulin site for colchicine (Serrano L, Avila J, Maccioni RB: J Biol Chem 259:6607-6611, 1984) and define the colchicine binding domain in a domain of ot-subunit from the point of limited tryptic cleavage to the site of subtilisin controlled proteolysis of that tubulin subunit. On the basis of these alterations in the interaction of colchicine upon removal of the C-terminal moiety of tubulin and since no change in the number of binding sites was found after subtilisin digestion, we suggest that the carboxyl-terminal region of tubulin subunits modulates the binding of colchicine.

Introduction

Colchicine, a compound isolated from Colchi- cum autumnale binds specifically and with a high affinity to tubulin, the main component of microtubules (1, 2). Since colchicine binds to the native tubulin molecule, the analysis of colchicine binding has been complicated by the fact that colchicine binding activity of tubulin is not constant during the time and it decays following first order inactivation kinetics (2). The rate of decay of tubulin depends on different parameters such as the buffer conditions, temperature, the presence of different molecules like vinca drugs and GTP, and

also depends on tubulin and colchicine concentrations (3, 4). Recently, the effect of microtubule associated proteins (MAPs) on such rate of decay has been reported. MAPs stabilize the colchicine binding activity of tubulin (5) but these proteins also appear to compete with the drug for the binding of tubulin (6). A possible interpretation of the above features is that MAPs could interact directly with the colchicine binding site of tubulin molecules and thereby protect it from decay. However recent studies point to a localization of the binding sites for colchicine (7) and for MAPs (8, 9) in different tubulin domains, on the basis of limited proteolysis and binding studies. Colchicine binds to a domain

Address for offprints and correspondence." Ricardo B. Maccioni, Department of Biochemistry, Biophysics and Genetics, University of Colorado Health Sciences Center, 4200 E. 9th Avenue, B-121, Denver, CO 80262, USA.

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located in the one-third segment from the C- terminus end tubulin c~-subunit which is contained in the 16 kDa fragment resulting from controlled tryptic proteolysis of tubulin (7, 10). The MAP-2 binding site (8, 11) as well as the sites for MAP-1 and tau interaction (9) appear to be located on the 4 kDa fragment of the C-terminal moiety of tubulin resulting from subtilisin proteolysis. Another possible interpretation is that the MAP molecule could sterically hinder the interaction of colchicine with its binding site in tubulin on the basis of topo- graphic proximity. Since the 16 kDa peptide contains the 4 kDa fragment of c~-subunit we have examined whether the colchicine binding site is also present in this smaller C-terminal fragment. Our results indicate that after removal of the 4 kDa peptide tubulin retains its colchicine binding activity, indicating that the 4 kDa fragment is not involved in the colchicine site. Furthermore, a 12 kDa peptide, containing the domain from the tryptic to the subtilisin limited cleavage sites of tubulin subunit, presented a strong colchicine binding activity. The removal of the 4 kDa segment resulted in a stabilization of the colchicine binding activity of tubulin but with a decreased binding constant. These results suggest a regulatory role for the carboxy-terminal domain in the tubulin interaction with colchicine.

Experimental procedures

[3H]-colchicine (6.5 Ci/mmol) was purchased from the Radiochemical Centre Ltd., Amersham, England. Subtulisin and trypsin were obtained from Sigma Chemical, Co., St. Louis, MO.

Tubulin from porcine or lamb brains brain was prepared by in v i tro polymerization- depolymerization cycles by the procedure of Shelanski et al. (12), followed by phosphocellulose chromatography, to remove MAPs (13). Controlled proteolysis of tubulin (2 mg/ml) with 1.25% w/w subtilisin was performed by incubation at 30 °C for 30 min. To remove the protease after digestion, subtilisin-digested tubulin (S-tubulin) was subjected to additional cycles of assembly-disassembly as described by Serrano et al. (11). This procedure also allows separation of S-tubulin from the resulting 4 kDa fragments. Trypsin digestion was done as previously reported (7). Separation of the tryptic

peptides from both tubulin and S-tubulin, was done on a Sephadex G-100 fine column (56 × 1.4 cm) equilibrated in 20 mM MES, pH 6.4; 1 mM MgC12. Fractions of 1 ml were collected and assayed for colchicine binding as described below (see Fig. 2). Purification of the 16 C-terminal peptides from ~-tubulin was performed by chromotog- raphy of the tryptic cleavage product on Sephadex G-100 fine under the same condition described above except that 3-ml fractions were collected (see Fig. 3) followed by Sephadex G-100 rechromotography on the pooled fractions containing this peptide. The 12 kDa fragment from S-tubulin was purified under the same conditions. Rechromotography on Sephadex G-100 of the fractions containing either the 16 or the 12 kDa peptides was performed as a routine procedure of purification. Purity of both peptides was 94°7o as analyzed by electrophoresis. Electrophoresis experiments were performed in acrylamide SDS gels according to the procedure of Laemmli (14). The gels were stained with Coomas- sie blue using the method of Fairbanks et al. (15). In the experiment of Fig. 3, tubulin was labeled in the carboxyl-terminus of the a-subunit with [3H]- tyrosine as described previously (7, 8). The [3H] tyrosyl tubulin was obtained after two polimeriza- tion cycles with a specific radioactivity of 270 cpm//~g protein.

The assay of colchicine binding was performed as described by Sherline et al. (16) with some modifications. Briefly, 0.1 ml of brain tubulin (0.25 mg/ml) in buffer A (0.1 M MES, 2 mM EGTA, 0.5 mM MgC12, pH 6.4) were mixed with 7/A of an incubation mixture containing 1 mM GTP and 0.2 mM (or the concentration indicated in each case) colchicine, containing 100 ~Ci [3H]- colchicine//~mol. Tubulin or S-tubulin samples were incubated at 35 °C for 60 min in the presence of [3H]-colchicine and the reaction terminated by addition of 1 ml of a charcoal suspension (3 mg/ml) to the incubation samples. After 10 min at 0 °C the mixture was centrifuged and the radioactivity in the supernatant was counted. The bound colchicine remains in the supernatant while the unbound drug is adsorbed to charcoal. Back- grounds of <5°70 of uncomplexed colchicine re- mained in solution after charcoal adsorption, at the different concentration assayed in the binding experiments. For the time course analysis of colchicine binding decay, samples of either tubulin of S-

tubulin were withdrawn at different time intervals (as shown in Results) right after purification of both proteins was completed and assayed immediately for colchicine binding as indicated. Scatchard plot analysis (17) was performed after measuring the colchicine binding at different concentrations of the drug (from 8 X I 0 -4 to 5 × 1 0 .5 M). Assays

were run in triplicate and an average value was calculated for each point. Moles of protein in the colchicine binding analyses were calculated on the basis of a molecular weight of 92 kDa for S-tubulin (18) and 100 kDa for the tubulin heterodimer (19). The moles of C-terminal peptides used in the colchicine binding analyses of these fragments were calculated on the basis of molecular weight of 12 kDa for S-tubulin tryptic C-terminal peptide and 16 kDa for the peptide fragment from tubulin molecule.

Results

Tubulin domain for the binding of colchicine

Subtilisin proteolysis of tubulin depleted of MAPs results in the removal of a 4 kDa fragment from the carboxyl-terminal moiety of tubulin subunits (11). The colchicine binding behavior of both tubulin and the proteolytic derivative, S- tubulin was ,examined. A slightly higher colchicine binding actMty of tubulin (0.60_+0.05 moles colchicine/mol tubulin) as compared with subtilisin digested tubulin (0.55_+0.04 moles/mole) was found at a colchicine concentration of 1 × 10 -4 M and 37°C. This result indicated clearly that S- tubulin retained the colchicine binding site.

Our previous studies have provided evidence that the tubulin site for colchicine binding is located in the one-third segment including the C-terminus of tubulin c~-subunit, resulting from trypsin digestion of the tubulin molecule (7). The present observa- tion that the removal of a 4 kDa segment from the carboxyl-terminal end did not result in the loss of colchicine binding to tubulin indicate that the colchicine site is located within the a-tubulin domain from the tryptic cleavage site to the site of subtilisin limited proteolysis (Fig. 1A). Double digestion of tubulin with subtilisin and trypsin resulted in the formation of a 12 kDa fragment, originated from the tryptic cleavage of S-tubulin a-subunit which

31

Fig. 1. Scheme of proteolysis of tubulin by subtilisin and trypsin. (A) a-tubulin subunit is cleaved in the site T by digestion with trypsin generating fragments of 36 and 16 kDa (dashed line shows an additional minor cleavage), while digestion of tubulin with subtilisin resulted in the cleavage at site S in both tubulin subunits, with removal of a 4 kDa fragment located at the carboxyl-terminal moiety of tubulin subunits. Double digestion with subtilisin followed by trypsin resulted in the appearance of a 12 kDa fragment. (B) Tubulin (2 mg/ml) was digested with trypsin (1.25°70 w/w respect to tubulin) (a) or digested with subtilisin (1070 w/w) and afterwards with 1.25070 trypsin (b) and the samples were subjected to electrophoresis in a 10070 acrylamide slab gel in the presence of 0.1070 SDS. An undigested tubulin control is shown in (c).

lack the 4 kDa peptide fragment as seen in Fig. 1. Figure 1B shows the electrophoretic patterns with the tryptic digestion products from tubulin (a) and S-tubulin (b). Both the 16 and 12 kDa fragments were isolated separately by gel filtration from either tubulin or S-tubulin tryptic products as indicated in Methods, and the colchicine binding capacities of the purified fragments examined comparatively. Figure 2 shows the separation by Sephadex G-100 chromatography of the tryptic cleavage products from both tubulin and S-tubulin proteolysis. The first peak in both 2A and 2B correspond to undigested fractions of tubulin and S-tubulin respectively, which exhibited colchicine binding activities. The second peak, eluting in fractions 36-44 of the Sephadex chromatography in 2A contained the 32 kDa fragment resulting from the removal of the C-terminal one-third segment of tubulin c~-subunit (7) and a fraction of undigested /3-subunit as as- sessed by SDS-acrylamide electrophoresis. The peak in 2B also contained a 32 kDa fragment, a 48 kDa polypeptide from uncleaved S-tubulin /3- subunit and a minor fraction of undigested S- tubulin a-subunit. As in a previous report (7) was confirmed that, using [3H]-tyrosine tubulin (labeled in C-terminus a-subunit) as a substrate for tryptic limited cleavage, this second peak did not

32

u

.io o

T ] T r r

~o A

O8

0.6

0.4

0.2

0 - -

10 B

08 06

02

0 0 20 40 60 80 100

FracHon N °

08

06

o4 ~=

0.2

o .~ 1.0

:3E

08 .~

o o6 i-"

0.4 02

Fig. 2. Sephadex G-100 separation of tubulin and S-tubulin after limited proteolysis with trypsin and colchicine binding patterns of the eluted protein. (A) One milliliter tubulin sample (3.5 mg) was treated with 1.2% w/w trypsin for 5 min at 30°C. The digestion was terminated by the addition of a 5 : 1 w/w ex- cess soybean trypsin inhibitor. Immediately after proteolysis the sample was passed through a column of Sephadex G-100 fine (56 × 1.4 cm) pre-equilibrated in buffer 20 mM MES containing 1 mM Mg2+. Fractions of 1.0 ml were collected and the protein determined by absorbance at 280 n m ( o ). Aliquots of 50 ~1 were withdrawn from tubes indicated in the figure and the colchicine binding activity assayed in duplicate aliquots as described in Experimental procedures ( • ). (B) One milliliter S- tubulin sample (3.5 mg) was subjected to limited proteolysis with trypsin and the digestion products passed through a Sepha- dex G-100 column. The experimental conditions were the same as described above. The protein profile ( [] ) as well as the colchicine binding of aliquots (in duplicate) of samples indicated ( • ) were determined.

contain the radiolabeled C-terminal fragment. In both cases, 2A and 2B, the fractions contained in this second peak did not present a colchicine binding activity. The third peak, eluting in fractions 62-85 contained the C-terminal 16 kDa fragment in 2A and the 12 kDa peptide in 2B. The peptide fragments contained in this broader peak showed capacity to bind colchicine and the Fig. 2 also

shows that the 12 kDa peptide has a higher binding activity (2B) than the 16 kDa peptide fragment (2A). Since such differences could be due to an increase in stability of colchicine binding activity upon removal of a 4 kDa fragment we have compared the rates of the decay of the binding of colchicine of tubulin and S-tubulin.

Figure 3 shows a chromatographic separation of the tryptic digestion products from tubulin which has been tyrosinated in the a-subunit with [3H]- tyrosine. The first radiolabeled peak correspond to

Fig. 3. Separation of the 16 kDa fragment from other digestion products obtained by tryptic cleavage of [3H]-tyrosinated tubulin. The electrophoresis of eluted fragments. A labeled tubulin sample (2 mg) was treated with trypsin (1.2°70 w/w) for 5 min at 30 °C and the proteolysis terminated by addition of 5 : 1 w/w ex- cess soybean trypsin inhibitor. Labeled [3H]-tyrosyl tubulin was prepared as indicated in Experimental procedures. The cleavage sample was chromatographed on Sephadex G-100 fine as indicated in Fig. 2, fractions of 3.0 ml were collected and the radioactivity measured in 100/A aliquots. The inserts show two electrophoresis gels of sample from the first and second major radioactivity peaks. Fraction 12 contains uncleaved a-tubulin subunit, ~subunit (a), and fragments of 41 kDa (b), 36 kDa (c) and 31 kDa (d). Fraction 26 contains the 16 kDa peptide (e).

uncleaved [3Hl-o~-subunit. Slabs gel electrophoresis of a sample from fraction 12 of this peak shows the presence of intact unlabeled ~-subunit (a) as well as the uncleaved 41 kDa (band b) and 36 kDa fragments (band c). These fragments result from tryptic digestion of tubulin which has been preincubated with colchicine. A fragment of 31 kDa (d) results from further cleavage of the 36 kDa fragment. The insert of Fig. 3 also shows the electrophoresis of the fraction 26 included in the second radiolabeled peak (equivalent to peak 3 of Fig. 2) containing the 16 kDa C-terminal fragment (e) and the electrophoresis dye front (f). It is interesting to point out that in contrast to the peptides purified by the Sephadex filtration procedure, the 16 and 12 kDa peptides obtained after cutting the peptide bands from SDS-acrylamide slab gels did not present sig- nificant colchicine binding activities.

Rate of decay of the colchicine binding activity

The time course decay of the colchicine binding was measured for tubulin and S-tubulin under the same buffer conditions and at the same protein and colchicine concentrations. Figure 4A shows the rate of decay of tubulin and the S-tubulin derivative which followed pseudo-first order inactivation kinetics. The results indicate a lower rate for the decay of colchicine binding of S-tubulin (k o =

33

2.81+0.5x10 -3 min -I, n = 4) as compared to that of native tubulin (k o = 5 .2+8x10 -3 min-1). The increased stability of S-tubulin with respect to colchicine interaction is similar to that of tubulin in the presence of MAPs (5) or polylysine (20). In order to assess the effects of subtilisin cleavage on ageing tubulin the proteolytic enzyme was added to tubulin after 3 - 5 h incubation and the binding of colchicine was examined. A slight drop in colchicine binding followed subtilisin proteolysis, but decay of the cleavage product was less pronounced than that of the intact tubulin molecule (Fig. 4B). A difference between the experiments shown in Figs 4A and 4B is that in 4A S-tubulin was isolated by the cycling procedure but in 4B S-tubulin was generated after addition of subtilisin without further manipulation and assayed for colchicine bind- [ng.

Colchicine binding to tubulin and S-tubulin at different colchicine concentrations

An analysis by Scatchard plot of the initial binding activities of tubulin and S-tubulin, obtained over a range of colchicine concentrations from 8 × 1 0 - 4 - 5 x 1 0 -5 M is shown in Fig. 5. Plots from both protein samples differ in their slope but they shared a common intersection with the abcisal axis. The results indicate a different association constant

10

o 6

~ 4

5~ o ~ 2

c

_ A

I I r

10

8

~ ~ 2

0

E

1 2 3 o

hours

I I

Subtilisin

I L L ; L I

2 4 6 hours

Fig. 4. Decay of colchicine binding activities of tubulin and S-tubulin. (A) Tubulin ( o ) and S-tubulin ( • ) were prepared as indicated in Experimental procedures, samples withdrawn at times indicated, and assayed immediately for colchicine binding at a final protein concentration of 0.2 mg/ml , and at a colchicine concentration of 1 x l 0 -5 M. The values correspond to the average of six experiments. (B) A tubulin sample (1.2 ml) ( o ) was incubated at 30 °C and samples obtained at different t ime intervals were assayed for colchicine binding. An aliquot (0.5/zl) was recovered at 3.5 h, digested with 1% w/w subtilisin for 30 min, the reaction stopped by adding 1 m M PMSF and the colchicine binding activity was tested on fractions obtained at the times indicated ( • ) .

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0.2 O.Z,

moles co l ch i c i ne bound

mol protein

I 0.6

Fig. 5. Effect of colchicine concentration on colchicine binding to tubulin and S-tubulin. Purified tubulin ( o ) and S-tubulin ( • ) at 0.3 mg/ml were incubated as indicated in Methods with various concentrations of colchicine (8 × 10 -4 M to 5 × 10 -5 M) and the results obtained were represented in a Scatchard plot.

of colchicine to tubulin and to S-tubulin, but an identical number of binding sites per protein dimer. On the basis of the results of Fig. 5 the calculated association constants (Ka) for tubulin and S- tubulin were 6.4+0.6x106 M -t and 2.1 +0 .2x 106 M -1, respectively. To examine whether this difference in colchicine affinity exists also in the proteolytic fragments from tubulin and S- tubulin containing the colchicine domain we car- ried out a similar experiment using the Sephadex G-100 isolated tryptic 16 kDa fragment and the subtilisin-trypsin 12 kDa fragment, respectively. Figure 6 shows the colchicine binding activity of both fragments at different colchicine concentrations, analyzed by Scatchard plots. The data shows that the 16 kDa tubulin fragment has a slightly higher association constant (Ka = 4.5+0.5>(106 M -1) than the 12 kDa S-tubulin peptide (2.7 +0 .4x 106 M-I) . Similar results were also obtained with lamb brain tubulin preparations. Those values were also in the same range of the association constants for colchicine binding to their respective parent molecules, but the s toichiometry of colchicine binding to the peptides from the Scatchard analysis (0.1 + 0.02 moles colchicine/mol protein) was significantly lower than the value o f 0.5 +0.05 moles colchicine/mol protein for the parent molecules.

cO I C3 T--

X

==

X

C

"G o

r

c

0.05 0.1 0.15

moles c o l c h i c i n e b o u n d / m o l protein

Fig. 6. Effect of colchicine concentration on colchicine binding to 16 and 12 kDa tubulin fragments. The 16 kDa fragments ( o ) obtained by cleavage with trypsin of tubulin a-subunit and isolated by gel chromatography as described in Experimental procedures (see Fig. 2A) and 12 kDa fragment ( • ), obtained by double digestion with subtilisin and trypsin, and isolated as described (see Fig. 2B) were incubated with various concentrations of colchicine (8)<10 -4 M to 5)<10 5 M) and the results obtained were represented by a Scatchard plot.

Discussion

Comparison of the colchicine binding behavior of tubulin and S-tubulin have indicated that the lat- ter retained the colchicine binding site even though the characteristics of such binding suffered some alterations upon removal of the C-terminal domain of the tubulin molecule. A lower rate of decay of the colchicine binding activity is indicative of a higher stability of S-tubulin toward time dependent colchicine inactivation. However, S-tubulin exhibited a lower affinity than the native tubulin for the binding of colchicine.

Subtilisin selective proteolysis results in the

removal of a 4 kDa fragment from the carboxyl- terminal moiety of both tubulin subunits. This fragment contains the binding site for MAP-2 (8) and other MAPs (9). The characteristics of the colchicine binding to tubulin in the presence of MAPs (6) resembled those of tubulin lacking the 4 kDa C- terminal fragment indicated in these studies, e.g., decreased affinity for colchicine as compared with the intact tubulin. These observations support the idea that the 4 kDa fragment could play a role in a similar fashion as it regulates tubulin self- assembly into microtubules (11, 21). The function of the C-terminal domain in modulating the interactions responsible for tubulin self-association appear to be determined by both electrostatic and conformational effects (18). Thus, blockage of this acidic C-terminal tubulin domain by addition of MAPs (8) or its proteolytic removal from tubulin molecules appears to result in a stabilization of the colchicine binding activity. In other words, the presence of the 4 kDa domain in the tubulin molecule may decrease the structural stability of tubulin with respect to its colchicine binding capacity. On the other side, the presence of the C-terminal domain in the tubulin molecule increase affinity of tubulin by colchicine. This functional alteration could be related to the change in protein conformation that we have observed upon removal of the 4 kDa fragment from tubulin (18).

One of the main features of tubulin is the insta- bility of its active conformation. Evidence for such characteristics are its increased susceptibility to proteases during ageing (unpublished observations), increased capacity to reaggregate (22), reduced microtubule assembly (23) and a selectively fast decay in colchicine binding activity (2). Howev- er, S-tubulin exhibits an increased capacity to self associate (11) and a higher resistance to losing its adopted conformation as measured by the CD after incubation for several hours at 27 °C. The tubulin molecule exhibits a high degree of conformational mobility around its C-terminal domain as revealed by NMR (24) and circular dichroism (18), which may decrease its stability and self-association capacity. Thus, MAPs could increase stability and trigger tubulin assembly by restraining flexibility in the C-terminal moiety of tubulin upon binding to this domain (8).

The difference in the binding behavior between tubulin and S-tubulin are not due to changes in the

35

number of colchicine binding sites rather in the stability of colchicine interaction. In a previous report we indicated the localization of the colchicine binding site in the C-terminal one third of c~-subunit and that the domain where the 16 kDa and the 41 kDa fragments overlap appear to be involved in colchicine interaction (7). The present study pro- vides further information for the localization of colchicine binding site in a domain of c~-subunit from the tryptic cleavage site and the site for subtilisin limited proteolysis (see Fig. 1). This domain corresponding to the 12 kDa peptide binds colchicine tightly and stably. The above results also indicate that the domain containing the 4 kDa peptide from the C-terminal moiety of c~-subunit is not topographically involved in the binding of colchicine and it rather modulates the interaction.

Recently Little and Luduefia (25) have suggested that the colchicine binding site could be close to the c~/~3 interface to explain possible conflicting results suggesting the presence of colchicine binding sites not only in ~-subunit (7, 26-28) but also in /3- subunit (29-31). Our results provide evidences that the entire colchicine binding site could be on c~- subunit but does not preclude the probability of spatial proximity to ~-subunit, since the colchicine binding region in c~-subunit (between trypsin and subtilisin cleavage sites) could be close to a domain involved in the interaction of both tubulin subunits (25, 32).

Finally, our results could help to interpret the differences found for the colchicine binding affinity or the rate of decay from tubulin of different ori- gins (2, 4, 33-36). Since the main structural differences found for tubulin showing different affinities for colchicine binding, i.e., yeast (36) and pig (5) were found at the carboxy terminal regions (19, 37), we can suggest from the results shown in this work that the carboxy terminal region of tubulin molecule from those organisms could modulate the binding of colchicine to the protein.

Abbreviations

GTP, guanosine 5 '-triphosphate; EGTA, ethylene glycol bis 03-amino ethyl ether)-N,N,N ',N '- tetracetic acid; MES, 2-(N-morpholino) ethane- sulfonic acid; MAPs, microtubule associated proteins; MAP-2, one of the high molecular weight

36

MAPs; SDS, sodium dodecyl sulfate; S-tubulin, subtilisin cleaved tubulin heterodimer which lacks the 4 kDa carboxyl-terminal moiety of each tubulin subunit; kDa, kilodalton.

Acknowledgments

This research has been supported in part by the European Molecular Biology Organization (EMBO), American Cancer Society Institutional Grant IN-/X, Council Tobacco Research - U.S.A. Grant 1913. Grants BRSG-05357 and GM-28793 from the National Institutes of Health and a grant from Comision Asesora, Spain. L.S. was supported by Fondo de Investigaciones Sanitarias, Spain.

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Received 17 June 1986.

Regulatory aspects of the colchicine interactions with tubulin

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