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Some features of the vinblastine-induced assembly of porcine tubulin
ARCHIVES
OF BIOCHEMISTRY
Some Features
AND
of Chemistry
154-162
171,
(1975)
of the Vinblastine-Induced Tubulin
MARTHA Departments
BIOPHYSICS
VENTILLA,’
and Biological
Assembly
CHARLES
Sciences,
Columbia
of Porcine
R. CANTOR, Unviersity,
New
York,
New
York,
10027
AND
MICHAEL Department
of Pathology,
Albert
L. SHELANSKP
Einstein Received
College March
of Medicine,
Bronx,
New
York,
10461
12, 1975
Porcine tubulin precipitated by 10e3 M vinblastine (VLB) contains approximately 0.50 molecule of VLB bound per llO,OOO-molecular-weight tubulin dimer. The amount of precipitate, followed by turbidity, is a linear function of the initial tubulin concentration. The rate of precipitation is roughly first order in protein concentration. Vindoline and velbanamine halves of VLB are ineffective separately or together in producing the tubular aggregates observed for VLB precipitates by electron microscopy. At 10e3 M concentrations no turbidity is observed nor is there any competition with VLB-induced turbidity. Removal of GTP from tubulin by dialysis or incubation of tubulin in the absence of added GTP blocks VLB-induced assembly. Readdition of GTP at room temperature or above restores sensitivity to VLB precipitation. The 6,~ methylene analog of GTP cannot substitute for GTP in this process. About 0.7 mol of added GTP is found bound per mole of tubulin dimer. During the course of VLB-induced assembly, roughly half of this GTP is displaced. These results show interesting similarities and differences in the VLB-induced assembly of tubulin and the normal in vitro assembly of microtubules. Further comparisons between both assembly processes should be useful.
Following the definition of the conditions for the in vitro assembly of tubulin into microtubules by Weisenberg (11, it has become possible to analyze directly the factors that regulate and initiate the assembly and disassembly of microtubules (2-4). However, one can also gain insight into the mechanism of tubulin selfassembly by the comparison of normal assembly with conditions, such as vinblastine-induced assembly, that produce alternate, ordered structures. When vinblastine-precipitated tubulin is examined by electron microscopy an array of open helical structures is seen (5). The individual helices in these 1 Current address: Department UCLA School of Medicine, Los
arrays are similar in appearance to the helices reported by Kirschner et al. (6) on the disassembly of normal microtubules, and the ringlike structures seen in such preparations are morphologically indistinguishable from those suggested by Borisy and Olmsted as possible nucleation centers for microbubule assembly (7). A vinblastine-induced assembly of tubulin has been observed both in viuo (8-10) and in vitro (5, 11, 12). The mechanism of the in vitro aggregation has been studied by Weisenberg and Timasheff (13) who, using ultracentrifugal techniques, found that increasing concentrations of vinblastine caused an increase in sedimentation velocity with the formation of 9s and ultimately 30s forms prior to complete precipitation of the protein. The reaction was found to be dependent on the presence of
of Psychiatry, Angeles, Calif.
90024.
2 Current ogy, Harvard
address: Department University, Boston,
of NeuropatholMass. 02115. 154
Copyright All rights
0 1975 by Academic Press, of reproduction in any form
Inc. reserved.
ASSEMBLY
OF
TUBULIN
magnesium ions but to differ in assembly characteristics and morphology from the precipitates induced by magnesium alone. A tentative binding of 2 mol of vinblastine per mol of tubulin dimer (110,000 M3 was found. More recently, vinblastine-induced aggregates have been isolated from sea urchin eggs (14) and 1 mol of vinblastine was reported to be bound per mole of IIO,OOO-molecular-weight tubulin. Other studies using tritiated vinblastine (15) have given a value of 0.5 mol of vinblastine (VLBj3 per mole of tubulin dimer. In this report we reexamine the interaction of VLB and tubulin principally by using turbidimetric measurements. MATERIALS
AND
METHODS
Purification of tub&n. Protein used for these experiments was purified from porcine brains by the method of Weisenberg et al. (161, modified as previously described (17). All preparations were greater than 90% pure tubulin as judged by acrylamide-gel electrophoresis and densitometry. Protein concentrations were determined by the method of Lowry et al. (18). A standard curve was prepared by using tubulin which had been extensively dialyzed against distilled water and then dried in a vacuum oven prior to weighing. All experiments were carried out within 24 h after the preparation of the protein. These preparations of tubulin were not capable of reassembly into normal microtubules under the conditions of Weisenberg (1). Nucleation of the mixture, however, with small amounts of the crude 100,OOOg supernatant fraction resulted in the formation of large numbers of tubules, suggesting that the subunits prepared were not severely denatured and that only a nucleation factor was lacking (7). No such nucleation factor was required for the vinblastine-induced assembly. The standard buffer (PM) used for protein purification and most experiments is 0.01 M sodium phosphate, pH 6.5, containing 0.01 M Mg*+ and 10m4 GTP, except where otherwise specified. Vinblastine and colchicine binding. Vinblastine sulfate was kindly provided by Dr. Norbert Neuss of the Eli Lilly Co. (Indianapolis, Ind.). Vinblastine removed from tubulin samples was identified chromatographically by its migration of silica-gel G thin-layer plates in an ethylacetate/ethanol(3:1) sol3 Abbreviation used: VLB, vinblastine; PM buffer, 0.01 M sodium phosphate, pH 6.5, containing 0.01 M Mg2+ and lo4 M GTP; DEAE, diethylaminoethyl; GMPPCP, the P,r methylene analog of GTP; PEI plates, polythylenimine cellulose thin layer plates.
BY
155
VINBLASTINE
vent with reference to pure standards. The two halves of the vinblastine molecule, vindoline and velbanamine, were also prepared and provided by Dr. Neuss. Colchicine was purchased from Sigma Chemical Company (St. Louis, MO.) and [3H]colchicine was purchased from New England Nuclear (Boston, Mass.) Colchicine-binding activity was measured by incubation of tubulin in PM buffer at 37°C for 1 h in the presence of a saturating lo-fold molar excess of the drug. Binding to tubulin was determined by using radioactive colchicine and DEAE-impregnated filter-paper sandwiches (16, 19) or by the optical density of bound colchicine at 350 nm after the removal of free colchicine on a column of Sephadex G-10. Colchicine-binding activities of freshly prepared preparations averaged 0.48 ? 0.16 mol of colchicine per mole of tubulin. Bound vinblastine was measured after perchloric acid precipitation of the protein and neutralization of the supernatant fluid with KOH, by the method of Jakovljevic (20). In this procedure an acetic anhydride/pyridine/H2S0, mixture is added to the vinblastine-containing solution and heated to obtain a rose color. This color is specific for vinblastine and has not been observed with any other vinca alkaloid. An additional advantage of the method is that the presence of tubulin or GTP does not interfere. The absorbance at 574 nm is linear (Fig. 1) over the range of 5-60 pg of vinblastine. For determinations below this limit a 5-pg aliquot of vinblastine was added to the sample and its contribution subtracted from the result of the assay. Turbidimetry. The appearance of turbidity was measured with a Gary 15 or Gilford 240 spectrophotometer with l-cm path-length cells at ambient temperature (approximately 21°C). Measurements were at either 450 or 500 nm, and protein concentrations were in the range of l-2 mgiml. Each determination was started by the addition of appropriate aliquots of VLB, GTP, or other ligands directly to the protein solution in the cuvette, followed by rapid and thorough mixing.
0.4 1 z 4" 0.2 t
--L-~
0 0
20
40 VLB
FIG.
VLB
1. Spectrophotometric by using the method
60
pi
SO
CONC (mcghl)
determination of Jakovljevic (20).
of
156
VENTILLA,
CANTOR
RESULTS
Binding
of Vinblastine
to Tubulin
Tubulin solutions in PM buffer with concentrations varying between 2 and 7 mg/ml were incubated with 10e3 M VLB for 30 min at room temperature and the precipitates collected by centrifugation. Over 90% of the protein was precipitated by this procedure. The precipitates were then washed three times by resuspension and centrifugation in PM buffer. This procedure removes all the free vinblastine with a loss of less than 15% of the protein. Any loosely bound VLB is likely to be removed also. Electron microscopic examination of the precipitate after washing revealed numerous aggregates of rings and spirals equivalent in appearance to those described by Marantz and Shelanski (21). We shall call such samples characteristic vinblastine aggregates. Pellets after the third wash were resuspended in distilled water and the protein precipitated with perchloric acid at a final concentration of 5%. The bound vinblastine was released in this step. After centrifugation, the supernatant was reserved and the pellet again washed in 5% perchloric acid and centrifuged again. The pooled supernatant fluids were then analyzed for vinblastine as described in Materials and Methods. In 16 determinations the VLB:tubulin ratio varied between 0.47 and 0.54 mol/mol with an average value of 0.50. This is the amount of VLB bound tightly enough to resist washing with buffer but not perchloric acid. Further extraction of the pellet with hot and cold trichloroacetic acid, perchloric acid and methanol resulted in no further release of vinblastine. The uv spectrum of redissolved protein from the pellet did not differ from that of tublin with no exposure to vinblastine which has otherwise been treated in the same manner. The material released from the VLBtubulin complex by perchloric acid treatment was concentrated and adjust to pH 6.5 prior to thin-layer chromatography. The mobility of the material recovered from the complex was identical to the mobility of unreacted vinblastine. The uv
AND
SHELANSKI
spectra of the reacted and unreacted materials were also identical. By these criteria, vinblastine, like colchicine, undergoes no detectable chemical change on binding to tubulin. Turbidimetric
Anulysis of Vinblastine-lnduced Aggregation
Vinblastine-induced formation of aggregates was monitored by measurements of absorbance in a narrow-slit spectrophotometer. This yields an estimate of the total scattering, the turbidity, which is relatively linear in the weight concentration of scattering material. Thus, the method can provide useful if somewhat limited information. Working conditions were determined by variation of the protein concentration. The amount of protein aggregation appears to be strongly concentration dependent (Fig. 21, consistent with the previous observations of Weisenberg and Timasheff (13). If the plateau absorbance values in Fig. 2 are replotted as a function of the initial protein concentration, it is apparent that the maximum turbidity is a linear function of initial protein concentration, at least to one mg/ml (Fig. 3). Nor-
T--+---O
FIG. 2. Development of turbidity in tubulin solutions treated with 10v3 M VLB at ambient temperature as a function of protein concentration: (A), 0.05 mg/ml; (B), 0.45 mglml; (0, 0.70 mglml; CD), 0.90 mg/ml.
ASSEMBLY
0-
OF
TUBULIN
BY
157
VINBLASTINE
In spite of these complications, at a constant 10e3 M VLB concentration the turbidity of the samples approximates a linear measure of the protein concentration of the precipitate. Thus, it is possible to analyze the kinetics of the VLB-induced assembly under these conditions. The results of Fig. 5 show that, qualitatively, assembly rates are not markedly affected by protein concentration. These results were analyzed by Guggenheim plots (24). From the two illustrative curves shown in Fig. 5, it is clear that the rate of appearance of turbidity is consistent with a first-order process. Within the limited range studied, the firstorder rate constant is not dependent on protein concentration. 0.2 MTP
0.6 CONC
I.0 mg/ml
FIG. 3. Final turbidity of VLB-induced tubulin aggregates as a function of protein concentration. All samples contained 10m3 M VLB. All these data were obtained by using a single tubulin preparation. Results on other preparations are in very good quantitative agreement.
mally, one would expect the turbidity to be a function of particle size as well as the total amount of assembled protein. The linear results of Fig. 3 suggest that at lop3 M VLB the aggregates produced are so large that turbidity depends only on the weight of assembled material and not on the detailed size distribution of the aggregates. This kind of behavior has been cbserved for the turbidity of in vitro assembled microtubules (22, 23). At lower vinblastine concentrations the aggregates are apparently smaller in overall size as seen by qualitative visible inspection in the electron microscope. The amount of protein that can be pelleted by centrifugation at 100,OOOgfor 30 min remains constant even at VLB concentrations as low as lop4 M. However, the turbidity at these low VLB concentrations is less, as shown in Fig. 4. Tubulin can be precipitated by either a single addition of VLB or by stepwise addition of lower concentrations. In either case the final turbidity is the same, and storage at the same temperature results in no further increase of turbidity (Fig. 4).
04
:: 2
0.2
0
0.5 TIME,
1.0 hrs
24
FIG. 4. Effect of stepwise VLB addition on the turbidity of tubulin aggregates. One sample (-0-l was brought to 1O-3 M VLB by a single addition (arrow 4). The second C-A-) was brought stepwise up to 2 x 10m4 M (arrow l), ‘7 X 10m4 M (arrow 2) and 10e3 M VLB (arrow 3). The tubulin concentration of the samples was 0.8 mg/ml.
1.0
.
TIME.min
FIG. 5. Analysis of the kinetics of assembly of samples B and D of Fig. 2. This Guggenheim plot (24) of the turbidity shows that the kinetics is approximately first order.
158
VENTILLA,
CANTOR
Vindoline
and Velbanamine Interaction with Tub&in Vindoline and velbanamine are approximate halves of the vinblastine structure (Fig. 6). They might be expected to mimic vinblastine action if the effect of vinblastine were simply a conformational change induced by binding to the protein. It had previously been argued (25) that vinblastine sulfate can act as a cation, precipitating acidic proteins with a mechanism similar to the action of calcium ions. Turbidimetric experiments were performed using lop3 M vindoline, lop3 M velbanamine and a one to one mixture of the two under the same conditions as were used for VLBinduced precipitation. Even after long incubations, no turbidity was seen with either of the monomers or with the mixture under these conditions. When lo-’ M concentrations of these agents were used there was a precipitate formed, but this precipitate was amorphous or fibrous and did not show the characteristic morphology of the VLB aggregates. Further experiments showed that velbanamine and vindoline were not effective competitors of VLB-induced tubuline precipitation. Effect of GTP on VLB-Induced Aggregation The addition of VLB at a concentration of 10e3 M to 100,OOOg supernatant fractions of brain homogenates in PM buffer results in the rapid formation of aggregates
AND
SHELANSKI
whether or not any GTP is present. Tubulin that has been purified in the presence of PM buffers containing lop4 M GTP is also rapidly precipitated whether or not there is any GTP present in the medium at the time of VLB addition. Some procedures of GTP removal eliminate the ability of 10e3 M VLB to cause precipitation of purified tubulin. These include overnight 4°C dialysis or elution on a Sephadex G100 column, with PM buffer but no GTP, followed by room temperature storage for 4 h. The addition of fresh 1O-3 M GTP at 4°C to these samples fails to restore VLB precipitability. When 10e3 M VLB was added first and then GTP was added at either room temperature or at 37”C, rapid precipitation was observed. Electron microscopic examination showed the characteristic morphology of VLB precipitates. As monitored by turbidity, the extent of precipitation is dependent on the quantity of GTP added (Fig. 7). With samples where GTP is required for VLB precipitation, the nonhydrolyzable GTP analog GMPPCP was ineffective in replacing GTP (Fig. 8). The ability of 0.05 M Mg2+ to induce precipitation of these GTP-dependent samples of tubulin was also examined. In the absence of VLB, 0.05 M Mg2+ did cause some precipitation in the presence of 10d3 M GMPPCP, but the resulting turbidity was much less than when GTP was present instead (Fig. 8). Precipitates formed by Mg2+ addition rather than VLB appear amorphous or fibrous in the electron microscope and do
VELBANAMINE
FIG.
VINDOLINE
6. Structures
of vinblastine.
velbanamine
and vindoline.
ASSEMBLY
OF
I
0.2 -
0
TUBULIN
I
2
TIME.
3
hrs
FIG. 7. Effect of stepwise addition of GTP on the turbidity of tubulin precipitates. One sample was brought up to 10e3 M GTP by a single addition (arrow 5) at ambient temperature. The second was brought up stepwise to 2 X 10e4 M (arrow 11, 4 X 10m4 hi (arrow 21, 6 x 10e4 M (arrow 3), and 10m3 M GTP (arrow 4). The protein concentration of the samples was 0.8 mg/ml.
0.2 :: 2 0
00 0
2 TIME,
hrs
FIG. 8. Effect of GMPPCP on MgZf and VLB-induced turbidity in tubulin solutions. The original samples contained 0.46 mg/ml of protein in PM buffer. The protein was chromatographed on a Sephadex G-25 column to remove any free guanosine nucleotides. Samples A and B were then incubated with 10m3 M GMPPCP for 30 min while samples C and D were incubated with GTP at the same concentration. At zero time, 10m3 M VLB was added to A and C at ambient temperature. Sample B was brought up to 0.05 M Mg*+ concentration; sample D was brought up to twice this Mg2+ concentration.
not resemble characteristic vinblastine aggregates. In a second set of experiments tubulin was prepared as above so that GTP is required for assembly. Samples were then incubated with GTP for 30 min at 37”C, followed by the removal of all free GTP on a Sephadex G-100 column eluted with PM buffer lacking GTP. Subsequent addition
BY
159
VINBLASTINE
of 10e3 M vinblastine causes rapid assembly with no apparent requirement for GTP (Fig. 9). The addition of lo-’ M GMPPCP or GDP neither increases or diminishes the VLB-inducible precipitation of these samples. This suggests that the original 37°C incubation results in a state of tubulin similar to that originally isolated from brain homogenates. GTP-incubated samples require several hours after GTP removal before VLB-induced precipitation again becomes GTP dependent. The nature of the nucleotides bound to tubulin was examined by treatment of protein samples with trichloroacetic acid. Nucleotides released were analyzed by thinlayer chromatography on PEI plates (26): Spots were scraped from the plate, eluted and quantitated by absorbance. Tubulin, immediately after preincubation with GTP and removal of free GTP on Sephadex G-100 contained an average of 1.4 moles of GTP bound per 110,000-M, dimer. No detectable GDP could be found. When such tubulin was allowed to stand at room temperature for 4 h before a second Sephadex chromatography and nucleotide analysis, only 0.7 mole of bound guanine nucleotide was found per dimer. All of it was GDP. After dialysis at 4°C against GTP-free DPM buffer, the nucleotide content of freshly incubated and chromatographed samples fell to a value of 0.7 mole per dimer of which 80% is GTP and the remain-
-
GMPPCP
-
GTP
FIG. 9. Effect of preincubation of tubulin samples on VLB precipitation. Tubulin was preincubated with 10m3 M GTP at 37°C. After removal of free GTP by Sephadex G-100 chromatography, the sample was divided into four aliquots, and nucleotides were added at 10m3 M as indicated. The VLB concentration was 10e3 M. The final protein concentration was 0.3 mg/ml
160
VENTILLA,
EFFECT
OF
VLB
PRECIPITATION
13H]GTP
CANTOR
AND
SHELANSKI
TABLE I ON THE AMOUNT OF BOUND Perchloric
OF GUANINE
acid treated
NUCLEOTIDE VLB
treated
Expi:ment .= Supernatant 1 2 3
Counts Percent Counts Percent Counts Percent
per of per of per of
minute totaP minute total minute total
(1 For each experiment 1.0 ml an additional hour in PM buffer column the 2.0-ml peak fraction with 5% perchloric acid. b Percents for VLB samples precipitation.
Supernatant
Precipitate
10,240 98 2,240 80 10,600 96
200 2 370 14 400 4
5,170 50 1,080 42 5,100 46
Precipitate 5,070 49 1,060 41 3,800 35
of tubulin was incubated at 37°C for 1 h in GTP-free PM buffer and then for containing lO+ M [3H]GTP. After elution from a 1 x 27-cm Sephadex G-100 was split into two l-ml aliquots. One was treated with low3 M VLB, the other shown
relative
to the total
der GDP. If a 4-h, 37°C incubation step precedes the dialysis, the molar binding is approximately the same but all the nucleotide is recovered as GDP. Preparation of tubulin in the presence of [3H]GTP presumably leads to 13HlGTP bound at the exchangeable nucleotidebinding site (16, 27). We repeated these results and found that after rapid Sephadex G-100 filtration of such material, an amount of 3H equivalent to about 0.7 molecule of GTP is bound per tubulin dimer. This stoichiometry is equimolar to the colchicine-binding activity in the same samples. When such tubulin containing bound [3H]GTP was precipitated by VLB and the precipitate pelleted, half of the previously bound radioactivity is found free in the supernatant fluid, while the other half is bound to the protein in the pellet (Table I). This is consistent with the notion that there is only 1 mole of exchangeable GTP bound per 240,000-M, tubulin (tetramer) in the precipitate. Resuspension of the pellet in PM buffer free of Mg2+ and VLB results in a loss of turbidity. Sucrose gradient analysis showed that the precipitate has been converted to a 28-308 form, possibly similar to one observed previously by Weisenberg and Timasheff (13). Readdition of vinblastine and magnesium to this form results in the reprecipitation of the protein but no further release of GTP. The 28-30s form retains the ability to rebind an
counts
per
minute
obtained
by perchloric
acid
amount of 13HlGTP equivalent to that released by the original VLB precipitation. These results show that in the VLB precipitate one of the two previously exchangeable sites is apparently sequestered. The other site still retains the ability to rebind VLB. DISCUSSION
The studies described here have enabled us to understand with a bit more clarity the mechanism of action of the vinca alkaloids. The vinblastine-induced assembly as viewed by turbidity is strongly dependent on protein concentration. This is in agreement with the earlier centrifugal studies by Weisenberg and Timasheff (13) who found that the formation of species with higher sedimentation velocity was dependent on protein concentration as well as vinblastine concentration. The final extent of turbidity is much more dependent on protein concentration than is the rate of assembly. In this respect the VLB-induced assembly resembles normal microtubule assembly (22). We were unable to acquire data over a sufficient range of protein and VLB concentrations to establish definitely a kinetic model for the vinblastine-induced aggregation. However, the apparent first-order kinetic dependence on protein concentration we observed at 10m3 M VLB is not consistent with a simple condensation polymeriza-
ASSEMBLY
tion reaction M + VLB
OF
TUBULIN
such as: fast
M*;
nM* slow M,
In this scheme-& binding of Vzto MTP is fast and the rate-limiting step is the growth of the polymer chain. If, instead, one considers the formation of M* to be the rate-limiting step and the growth of the chain to be fast, then one could expect first-order kinetics since VLB is always in great excess during our experiments. An alternative mechanism is a two-step reaction with nucleation and chain elongation. The first-order rate-limiting step could be the formation of nuclei. Possible nuclei could be tubulin subunits which have been “activated” by vinblastine binding, specific aggregates of tubulin, or specific structures such as the rings and coils which are seen in VLB-tubulin mixtures by electron micorscopy (11). This is similar to the mechanisms that have recently been proposed for normal microtubule assembly (4, 6,22), where similar rings or spirals are prime candidates for the nucleation centers. The binding levels which we have found for vinblastine are in agreement with those of Owellen et al. (15) but are lower than reported by Bryan (14). It is not possible for us to reconcile these differences. Possible sources of error could come from standards used in the determination of tubulin concentration or differences in the purity and activity of various protein preparations. The inability of either of the halves of vinblastine to cause assembly on their own is interesting. Neither of these half molecules is effective at inhibiting the vinblastine induced precipitation of tubulin. Therefore, if they bind to tubulin at all, their binding to the protein cannot be as strong as vinblastine itself. Wilson et al. mention that vindoline, the lower indole moiety of vinblastine, was totally ineffective in precipitating tubulin (28), which our experiments confirmed. They used catharantine as a model for the other half of VLB rather than velbanamine. Weak antimitotic activity was observed but no direct experiments on tubulin are reported. Clearlv.” I it will be of interest in the future
BY
161
VINBLASTINE
to compare catharantine and velbanamine in in vitro experiments. If a significant difference in activity is observed it could be an important clue to the mechanism of VLB action. The requirement for GTP in vinblastineinduced aggregation was not expected. However, as can be seen in Fig. 7, this is strictly required and the amount of assembly obtained to a certain degree can be regulated by the GTP concentration. If, however, the protein is preincubated with GTP and the free GTP is then removed by column chromatography, the protein will still be precipitable by vinblastine. GMPPCP was not effective in replacing the GTP. The release experiments are consistent with a mechanism in which one of the four nucleotide sites per tetramer is sequestered in the assembly while a second remains exposed to the medium. This is in marked contrast to normal assembly where all exchangeable sites are sequestered and opens a way to investigating the directionality of the subunit assembly in the VLB paracrystals. The vinblastine-induced assembly of tubulin resembles normal assembly in its likely nucleation, in the requirement for GTP and in the concentration dependence of the final amount of assembly obtained. These similarities together with the difference in GTP site sequestration, colchicinebinding-site sequestration and final morphology make it a useful system for further physicochemical studies. ACKNOWLEDGMENTS This work was supported by grants from the USPHS, No. GM 14825, NS 08180 and NS 11504. M.V. was supported by an NIH predoctoral fellowship, No. GM 46,899. We are grateful to Dr. Nobert Neuss for generous gifts of vinblastine and other alkaloids. REFERENCES 1. WEISENBERG, R. C. (1972) Science 177, 1104. 2. ERICKSON, H. P. (1974) J. Cell Biol. 60, 153. 3. BORISY, G. G., AND OLMSTED, J. B. (1972)Science 177, 1196. 4. SHELANSKI, M. L., GASKIN, F., AND CANTOR, C. R. (1973)Proc. Nat.Acad. Sci. USA 70,765. 5. BENSCH, K. G., MARANTZ, R., WISNIEWSKI, H., AND SHELANSKI, M. L. (1969) Science 195,495. 6. KIRSCHNER, M., WILLIAMS, R. C., WEINGARTEN,
162
7. 8. 9. 10. 11. 12. 13. 14. 15.
16. 17.
VENTILLA,
CANTOR
M., AND GERHART, J. (1974) Proc. Nat. Acad. Sci. USA 71, 1159. BORISY, G. G., OLMSTED, J. B., MARCIJM, J. M., AND ALLEN, C. (1974) Fed. Proc. 33, 167. BENSCH, K. G., AND MALAWISTA, S. E. (1969) J. Cell Biol. 40, 95. BENSCH, K. G., AND MALAWISTA, S. E. (1968) Nature (London) 218, 1176. BRYAN, J. (1971) Exp. Cell Res. 66, 129. MARANTZ, R., VENTILLA, M., AND SHELANSKI, M. L. (1969) Science 195, 498. VENTILLA, M., CANTOR, C. R., AND SHELANSKI, M. L. (1972) Biochemistry 11, 1554. WEISENBERG, R. C., AND TIMASHEFF, S. N. (1970) Biochemistry 9, 4110. BRYAN, J. (1972) Biochemistry 11, 2611. OWELLEN, R. J., OWENS, A. H., JR., AND DONNIGAN, D. W. (1972) Biochem. Biophys. Res. Commun. 47, 685. WEISENBERG, R. C., BORISY, G. G., AND TAYLOR, E. W. (1968) Biochemistry 7, 4466. SHELANSKI, M. L., AND WEISENBERG, R. C (1972) in Techniques of Biochemical and Biophysical
AND
18.
19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
SHELANSKI Morphology (Glick, D., and Rosenbaum, R. M., eds.), p. 25, Wiley, New York. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L. AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265. BORISY, G. G. (1972) Anal. Biochem. 50, 373. JAKOVLJEVIC, I. M. (1962) J. Pharm. Sci. 51, 187. MARANTZ, R., AND SHELANSKI, M. L. (1970) J. Cell Biol. 44, 234. GASKIN, F., CANTOR, C. R., AND SHELANSKI, M. L. (1974) J. Mol. Biol. 89, 737. BERNE, B. (1974) J. Mol. Biol. 89, 755. GUGGENHEIM, E. A. (1926) Phil. Mag. 2, 538. WILSON, L., BRYAN, J., RUBYN, A., AND MAZIA, D. (197O)Proc. Nat. Acad. Sci. USA 66,807. REMENCHIK, A. P., AND BERNSOHN, J. (1967) Anal. Biochem. 18, 1. BERRY, R. W., AND SHEANSKI, M. L. (1972) J. Mol. Biol. 71, 71. WILSON, L., BAMBURG, J. R., MIZEL, S. B., GRI& HAM, L. M., AND CRESWELL, K. M. (1974) Fed. Proc. 33, 158.
OF BIOCHEMISTRY
Some Features
AND
of Chemistry
154-162
171,
(1975)
of the Vinblastine-Induced Tubulin
MARTHA Departments
BIOPHYSICS
VENTILLA,’
and Biological
Assembly
CHARLES
Sciences,
Columbia
of Porcine
R. CANTOR, Unviersity,
New
York,
New
York,
10027
AND
MICHAEL Department
of Pathology,
Albert
L. SHELANSKP
Einstein Received
College March
of Medicine,
Bronx,
New
York,
10461
12, 1975
Porcine tubulin precipitated by 10e3 M vinblastine (VLB) contains approximately 0.50 molecule of VLB bound per llO,OOO-molecular-weight tubulin dimer. The amount of precipitate, followed by turbidity, is a linear function of the initial tubulin concentration. The rate of precipitation is roughly first order in protein concentration. Vindoline and velbanamine halves of VLB are ineffective separately or together in producing the tubular aggregates observed for VLB precipitates by electron microscopy. At 10e3 M concentrations no turbidity is observed nor is there any competition with VLB-induced turbidity. Removal of GTP from tubulin by dialysis or incubation of tubulin in the absence of added GTP blocks VLB-induced assembly. Readdition of GTP at room temperature or above restores sensitivity to VLB precipitation. The 6,~ methylene analog of GTP cannot substitute for GTP in this process. About 0.7 mol of added GTP is found bound per mole of tubulin dimer. During the course of VLB-induced assembly, roughly half of this GTP is displaced. These results show interesting similarities and differences in the VLB-induced assembly of tubulin and the normal in vitro assembly of microtubules. Further comparisons between both assembly processes should be useful.
Following the definition of the conditions for the in vitro assembly of tubulin into microtubules by Weisenberg (11, it has become possible to analyze directly the factors that regulate and initiate the assembly and disassembly of microtubules (2-4). However, one can also gain insight into the mechanism of tubulin selfassembly by the comparison of normal assembly with conditions, such as vinblastine-induced assembly, that produce alternate, ordered structures. When vinblastine-precipitated tubulin is examined by electron microscopy an array of open helical structures is seen (5). The individual helices in these 1 Current address: Department UCLA School of Medicine, Los
arrays are similar in appearance to the helices reported by Kirschner et al. (6) on the disassembly of normal microtubules, and the ringlike structures seen in such preparations are morphologically indistinguishable from those suggested by Borisy and Olmsted as possible nucleation centers for microbubule assembly (7). A vinblastine-induced assembly of tubulin has been observed both in viuo (8-10) and in vitro (5, 11, 12). The mechanism of the in vitro aggregation has been studied by Weisenberg and Timasheff (13) who, using ultracentrifugal techniques, found that increasing concentrations of vinblastine caused an increase in sedimentation velocity with the formation of 9s and ultimately 30s forms prior to complete precipitation of the protein. The reaction was found to be dependent on the presence of
of Psychiatry, Angeles, Calif.
90024.
2 Current ogy, Harvard
address: Department University, Boston,
of NeuropatholMass. 02115. 154
Copyright All rights
0 1975 by Academic Press, of reproduction in any form
Inc. reserved.
ASSEMBLY
OF
TUBULIN
magnesium ions but to differ in assembly characteristics and morphology from the precipitates induced by magnesium alone. A tentative binding of 2 mol of vinblastine per mol of tubulin dimer (110,000 M3 was found. More recently, vinblastine-induced aggregates have been isolated from sea urchin eggs (14) and 1 mol of vinblastine was reported to be bound per mole of IIO,OOO-molecular-weight tubulin. Other studies using tritiated vinblastine (15) have given a value of 0.5 mol of vinblastine (VLBj3 per mole of tubulin dimer. In this report we reexamine the interaction of VLB and tubulin principally by using turbidimetric measurements. MATERIALS
AND
METHODS
Purification of tub&n. Protein used for these experiments was purified from porcine brains by the method of Weisenberg et al. (161, modified as previously described (17). All preparations were greater than 90% pure tubulin as judged by acrylamide-gel electrophoresis and densitometry. Protein concentrations were determined by the method of Lowry et al. (18). A standard curve was prepared by using tubulin which had been extensively dialyzed against distilled water and then dried in a vacuum oven prior to weighing. All experiments were carried out within 24 h after the preparation of the protein. These preparations of tubulin were not capable of reassembly into normal microtubules under the conditions of Weisenberg (1). Nucleation of the mixture, however, with small amounts of the crude 100,OOOg supernatant fraction resulted in the formation of large numbers of tubules, suggesting that the subunits prepared were not severely denatured and that only a nucleation factor was lacking (7). No such nucleation factor was required for the vinblastine-induced assembly. The standard buffer (PM) used for protein purification and most experiments is 0.01 M sodium phosphate, pH 6.5, containing 0.01 M Mg*+ and 10m4 GTP, except where otherwise specified. Vinblastine and colchicine binding. Vinblastine sulfate was kindly provided by Dr. Norbert Neuss of the Eli Lilly Co. (Indianapolis, Ind.). Vinblastine removed from tubulin samples was identified chromatographically by its migration of silica-gel G thin-layer plates in an ethylacetate/ethanol(3:1) sol3 Abbreviation used: VLB, vinblastine; PM buffer, 0.01 M sodium phosphate, pH 6.5, containing 0.01 M Mg2+ and lo4 M GTP; DEAE, diethylaminoethyl; GMPPCP, the P,r methylene analog of GTP; PEI plates, polythylenimine cellulose thin layer plates.
BY
155
VINBLASTINE
vent with reference to pure standards. The two halves of the vinblastine molecule, vindoline and velbanamine, were also prepared and provided by Dr. Neuss. Colchicine was purchased from Sigma Chemical Company (St. Louis, MO.) and [3H]colchicine was purchased from New England Nuclear (Boston, Mass.) Colchicine-binding activity was measured by incubation of tubulin in PM buffer at 37°C for 1 h in the presence of a saturating lo-fold molar excess of the drug. Binding to tubulin was determined by using radioactive colchicine and DEAE-impregnated filter-paper sandwiches (16, 19) or by the optical density of bound colchicine at 350 nm after the removal of free colchicine on a column of Sephadex G-10. Colchicine-binding activities of freshly prepared preparations averaged 0.48 ? 0.16 mol of colchicine per mole of tubulin. Bound vinblastine was measured after perchloric acid precipitation of the protein and neutralization of the supernatant fluid with KOH, by the method of Jakovljevic (20). In this procedure an acetic anhydride/pyridine/H2S0, mixture is added to the vinblastine-containing solution and heated to obtain a rose color. This color is specific for vinblastine and has not been observed with any other vinca alkaloid. An additional advantage of the method is that the presence of tubulin or GTP does not interfere. The absorbance at 574 nm is linear (Fig. 1) over the range of 5-60 pg of vinblastine. For determinations below this limit a 5-pg aliquot of vinblastine was added to the sample and its contribution subtracted from the result of the assay. Turbidimetry. The appearance of turbidity was measured with a Gary 15 or Gilford 240 spectrophotometer with l-cm path-length cells at ambient temperature (approximately 21°C). Measurements were at either 450 or 500 nm, and protein concentrations were in the range of l-2 mgiml. Each determination was started by the addition of appropriate aliquots of VLB, GTP, or other ligands directly to the protein solution in the cuvette, followed by rapid and thorough mixing.
0.4 1 z 4" 0.2 t
--L-~
0 0
20
40 VLB
FIG.
VLB
1. Spectrophotometric by using the method
60
pi
SO
CONC (mcghl)
determination of Jakovljevic (20).
of
156
VENTILLA,
CANTOR
RESULTS
Binding
of Vinblastine
to Tubulin
Tubulin solutions in PM buffer with concentrations varying between 2 and 7 mg/ml were incubated with 10e3 M VLB for 30 min at room temperature and the precipitates collected by centrifugation. Over 90% of the protein was precipitated by this procedure. The precipitates were then washed three times by resuspension and centrifugation in PM buffer. This procedure removes all the free vinblastine with a loss of less than 15% of the protein. Any loosely bound VLB is likely to be removed also. Electron microscopic examination of the precipitate after washing revealed numerous aggregates of rings and spirals equivalent in appearance to those described by Marantz and Shelanski (21). We shall call such samples characteristic vinblastine aggregates. Pellets after the third wash were resuspended in distilled water and the protein precipitated with perchloric acid at a final concentration of 5%. The bound vinblastine was released in this step. After centrifugation, the supernatant was reserved and the pellet again washed in 5% perchloric acid and centrifuged again. The pooled supernatant fluids were then analyzed for vinblastine as described in Materials and Methods. In 16 determinations the VLB:tubulin ratio varied between 0.47 and 0.54 mol/mol with an average value of 0.50. This is the amount of VLB bound tightly enough to resist washing with buffer but not perchloric acid. Further extraction of the pellet with hot and cold trichloroacetic acid, perchloric acid and methanol resulted in no further release of vinblastine. The uv spectrum of redissolved protein from the pellet did not differ from that of tublin with no exposure to vinblastine which has otherwise been treated in the same manner. The material released from the VLBtubulin complex by perchloric acid treatment was concentrated and adjust to pH 6.5 prior to thin-layer chromatography. The mobility of the material recovered from the complex was identical to the mobility of unreacted vinblastine. The uv
AND
SHELANSKI
spectra of the reacted and unreacted materials were also identical. By these criteria, vinblastine, like colchicine, undergoes no detectable chemical change on binding to tubulin. Turbidimetric
Anulysis of Vinblastine-lnduced Aggregation
Vinblastine-induced formation of aggregates was monitored by measurements of absorbance in a narrow-slit spectrophotometer. This yields an estimate of the total scattering, the turbidity, which is relatively linear in the weight concentration of scattering material. Thus, the method can provide useful if somewhat limited information. Working conditions were determined by variation of the protein concentration. The amount of protein aggregation appears to be strongly concentration dependent (Fig. 21, consistent with the previous observations of Weisenberg and Timasheff (13). If the plateau absorbance values in Fig. 2 are replotted as a function of the initial protein concentration, it is apparent that the maximum turbidity is a linear function of initial protein concentration, at least to one mg/ml (Fig. 3). Nor-
T--+---O
FIG. 2. Development of turbidity in tubulin solutions treated with 10v3 M VLB at ambient temperature as a function of protein concentration: (A), 0.05 mg/ml; (B), 0.45 mglml; (0, 0.70 mglml; CD), 0.90 mg/ml.
ASSEMBLY
0-
OF
TUBULIN
BY
157
VINBLASTINE
In spite of these complications, at a constant 10e3 M VLB concentration the turbidity of the samples approximates a linear measure of the protein concentration of the precipitate. Thus, it is possible to analyze the kinetics of the VLB-induced assembly under these conditions. The results of Fig. 5 show that, qualitatively, assembly rates are not markedly affected by protein concentration. These results were analyzed by Guggenheim plots (24). From the two illustrative curves shown in Fig. 5, it is clear that the rate of appearance of turbidity is consistent with a first-order process. Within the limited range studied, the firstorder rate constant is not dependent on protein concentration. 0.2 MTP
0.6 CONC
I.0 mg/ml
FIG. 3. Final turbidity of VLB-induced tubulin aggregates as a function of protein concentration. All samples contained 10m3 M VLB. All these data were obtained by using a single tubulin preparation. Results on other preparations are in very good quantitative agreement.
mally, one would expect the turbidity to be a function of particle size as well as the total amount of assembled protein. The linear results of Fig. 3 suggest that at lop3 M VLB the aggregates produced are so large that turbidity depends only on the weight of assembled material and not on the detailed size distribution of the aggregates. This kind of behavior has been cbserved for the turbidity of in vitro assembled microtubules (22, 23). At lower vinblastine concentrations the aggregates are apparently smaller in overall size as seen by qualitative visible inspection in the electron microscope. The amount of protein that can be pelleted by centrifugation at 100,OOOgfor 30 min remains constant even at VLB concentrations as low as lop4 M. However, the turbidity at these low VLB concentrations is less, as shown in Fig. 4. Tubulin can be precipitated by either a single addition of VLB or by stepwise addition of lower concentrations. In either case the final turbidity is the same, and storage at the same temperature results in no further increase of turbidity (Fig. 4).
04
:: 2
0.2
0
0.5 TIME,
1.0 hrs
24
FIG. 4. Effect of stepwise VLB addition on the turbidity of tubulin aggregates. One sample (-0-l was brought to 1O-3 M VLB by a single addition (arrow 4). The second C-A-) was brought stepwise up to 2 x 10m4 M (arrow l), ‘7 X 10m4 M (arrow 2) and 10e3 M VLB (arrow 3). The tubulin concentration of the samples was 0.8 mg/ml.
1.0
.
TIME.min
FIG. 5. Analysis of the kinetics of assembly of samples B and D of Fig. 2. This Guggenheim plot (24) of the turbidity shows that the kinetics is approximately first order.
158
VENTILLA,
CANTOR
Vindoline
and Velbanamine Interaction with Tub&in Vindoline and velbanamine are approximate halves of the vinblastine structure (Fig. 6). They might be expected to mimic vinblastine action if the effect of vinblastine were simply a conformational change induced by binding to the protein. It had previously been argued (25) that vinblastine sulfate can act as a cation, precipitating acidic proteins with a mechanism similar to the action of calcium ions. Turbidimetric experiments were performed using lop3 M vindoline, lop3 M velbanamine and a one to one mixture of the two under the same conditions as were used for VLBinduced precipitation. Even after long incubations, no turbidity was seen with either of the monomers or with the mixture under these conditions. When lo-’ M concentrations of these agents were used there was a precipitate formed, but this precipitate was amorphous or fibrous and did not show the characteristic morphology of the VLB aggregates. Further experiments showed that velbanamine and vindoline were not effective competitors of VLB-induced tubuline precipitation. Effect of GTP on VLB-Induced Aggregation The addition of VLB at a concentration of 10e3 M to 100,OOOg supernatant fractions of brain homogenates in PM buffer results in the rapid formation of aggregates
AND
SHELANSKI
whether or not any GTP is present. Tubulin that has been purified in the presence of PM buffers containing lop4 M GTP is also rapidly precipitated whether or not there is any GTP present in the medium at the time of VLB addition. Some procedures of GTP removal eliminate the ability of 10e3 M VLB to cause precipitation of purified tubulin. These include overnight 4°C dialysis or elution on a Sephadex G100 column, with PM buffer but no GTP, followed by room temperature storage for 4 h. The addition of fresh 1O-3 M GTP at 4°C to these samples fails to restore VLB precipitability. When 10e3 M VLB was added first and then GTP was added at either room temperature or at 37”C, rapid precipitation was observed. Electron microscopic examination showed the characteristic morphology of VLB precipitates. As monitored by turbidity, the extent of precipitation is dependent on the quantity of GTP added (Fig. 7). With samples where GTP is required for VLB precipitation, the nonhydrolyzable GTP analog GMPPCP was ineffective in replacing GTP (Fig. 8). The ability of 0.05 M Mg2+ to induce precipitation of these GTP-dependent samples of tubulin was also examined. In the absence of VLB, 0.05 M Mg2+ did cause some precipitation in the presence of 10d3 M GMPPCP, but the resulting turbidity was much less than when GTP was present instead (Fig. 8). Precipitates formed by Mg2+ addition rather than VLB appear amorphous or fibrous in the electron microscope and do
VELBANAMINE
FIG.
VINDOLINE
6. Structures
of vinblastine.
velbanamine
and vindoline.
ASSEMBLY
OF
I
0.2 -
0
TUBULIN
I
2
TIME.
3
hrs
FIG. 7. Effect of stepwise addition of GTP on the turbidity of tubulin precipitates. One sample was brought up to 10e3 M GTP by a single addition (arrow 5) at ambient temperature. The second was brought up stepwise to 2 X 10e4 M (arrow 11, 4 X 10m4 hi (arrow 21, 6 x 10e4 M (arrow 3), and 10m3 M GTP (arrow 4). The protein concentration of the samples was 0.8 mg/ml.
0.2 :: 2 0
00 0
2 TIME,
hrs
FIG. 8. Effect of GMPPCP on MgZf and VLB-induced turbidity in tubulin solutions. The original samples contained 0.46 mg/ml of protein in PM buffer. The protein was chromatographed on a Sephadex G-25 column to remove any free guanosine nucleotides. Samples A and B were then incubated with 10m3 M GMPPCP for 30 min while samples C and D were incubated with GTP at the same concentration. At zero time, 10m3 M VLB was added to A and C at ambient temperature. Sample B was brought up to 0.05 M Mg*+ concentration; sample D was brought up to twice this Mg2+ concentration.
not resemble characteristic vinblastine aggregates. In a second set of experiments tubulin was prepared as above so that GTP is required for assembly. Samples were then incubated with GTP for 30 min at 37”C, followed by the removal of all free GTP on a Sephadex G-100 column eluted with PM buffer lacking GTP. Subsequent addition
BY
159
VINBLASTINE
of 10e3 M vinblastine causes rapid assembly with no apparent requirement for GTP (Fig. 9). The addition of lo-’ M GMPPCP or GDP neither increases or diminishes the VLB-inducible precipitation of these samples. This suggests that the original 37°C incubation results in a state of tubulin similar to that originally isolated from brain homogenates. GTP-incubated samples require several hours after GTP removal before VLB-induced precipitation again becomes GTP dependent. The nature of the nucleotides bound to tubulin was examined by treatment of protein samples with trichloroacetic acid. Nucleotides released were analyzed by thinlayer chromatography on PEI plates (26): Spots were scraped from the plate, eluted and quantitated by absorbance. Tubulin, immediately after preincubation with GTP and removal of free GTP on Sephadex G-100 contained an average of 1.4 moles of GTP bound per 110,000-M, dimer. No detectable GDP could be found. When such tubulin was allowed to stand at room temperature for 4 h before a second Sephadex chromatography and nucleotide analysis, only 0.7 mole of bound guanine nucleotide was found per dimer. All of it was GDP. After dialysis at 4°C against GTP-free DPM buffer, the nucleotide content of freshly incubated and chromatographed samples fell to a value of 0.7 mole per dimer of which 80% is GTP and the remain-
-
GMPPCP
-
GTP
FIG. 9. Effect of preincubation of tubulin samples on VLB precipitation. Tubulin was preincubated with 10m3 M GTP at 37°C. After removal of free GTP by Sephadex G-100 chromatography, the sample was divided into four aliquots, and nucleotides were added at 10m3 M as indicated. The VLB concentration was 10e3 M. The final protein concentration was 0.3 mg/ml
160
VENTILLA,
EFFECT
OF
VLB
PRECIPITATION
13H]GTP
CANTOR
AND
SHELANSKI
TABLE I ON THE AMOUNT OF BOUND Perchloric
OF GUANINE
acid treated
NUCLEOTIDE VLB
treated
Expi:ment .= Supernatant 1 2 3
Counts Percent Counts Percent Counts Percent
per of per of per of
minute totaP minute total minute total
(1 For each experiment 1.0 ml an additional hour in PM buffer column the 2.0-ml peak fraction with 5% perchloric acid. b Percents for VLB samples precipitation.
Supernatant
Precipitate
10,240 98 2,240 80 10,600 96
200 2 370 14 400 4
5,170 50 1,080 42 5,100 46
Precipitate 5,070 49 1,060 41 3,800 35
of tubulin was incubated at 37°C for 1 h in GTP-free PM buffer and then for containing lO+ M [3H]GTP. After elution from a 1 x 27-cm Sephadex G-100 was split into two l-ml aliquots. One was treated with low3 M VLB, the other shown
relative
to the total
der GDP. If a 4-h, 37°C incubation step precedes the dialysis, the molar binding is approximately the same but all the nucleotide is recovered as GDP. Preparation of tubulin in the presence of [3H]GTP presumably leads to 13HlGTP bound at the exchangeable nucleotidebinding site (16, 27). We repeated these results and found that after rapid Sephadex G-100 filtration of such material, an amount of 3H equivalent to about 0.7 molecule of GTP is bound per tubulin dimer. This stoichiometry is equimolar to the colchicine-binding activity in the same samples. When such tubulin containing bound [3H]GTP was precipitated by VLB and the precipitate pelleted, half of the previously bound radioactivity is found free in the supernatant fluid, while the other half is bound to the protein in the pellet (Table I). This is consistent with the notion that there is only 1 mole of exchangeable GTP bound per 240,000-M, tubulin (tetramer) in the precipitate. Resuspension of the pellet in PM buffer free of Mg2+ and VLB results in a loss of turbidity. Sucrose gradient analysis showed that the precipitate has been converted to a 28-308 form, possibly similar to one observed previously by Weisenberg and Timasheff (13). Readdition of vinblastine and magnesium to this form results in the reprecipitation of the protein but no further release of GTP. The 28-30s form retains the ability to rebind an
counts
per
minute
obtained
by perchloric
acid
amount of 13HlGTP equivalent to that released by the original VLB precipitation. These results show that in the VLB precipitate one of the two previously exchangeable sites is apparently sequestered. The other site still retains the ability to rebind VLB. DISCUSSION
The studies described here have enabled us to understand with a bit more clarity the mechanism of action of the vinca alkaloids. The vinblastine-induced assembly as viewed by turbidity is strongly dependent on protein concentration. This is in agreement with the earlier centrifugal studies by Weisenberg and Timasheff (13) who found that the formation of species with higher sedimentation velocity was dependent on protein concentration as well as vinblastine concentration. The final extent of turbidity is much more dependent on protein concentration than is the rate of assembly. In this respect the VLB-induced assembly resembles normal microtubule assembly (22). We were unable to acquire data over a sufficient range of protein and VLB concentrations to establish definitely a kinetic model for the vinblastine-induced aggregation. However, the apparent first-order kinetic dependence on protein concentration we observed at 10m3 M VLB is not consistent with a simple condensation polymeriza-
ASSEMBLY
tion reaction M + VLB
OF
TUBULIN
such as: fast
M*;
nM* slow M,
In this scheme-& binding of Vzto MTP is fast and the rate-limiting step is the growth of the polymer chain. If, instead, one considers the formation of M* to be the rate-limiting step and the growth of the chain to be fast, then one could expect first-order kinetics since VLB is always in great excess during our experiments. An alternative mechanism is a two-step reaction with nucleation and chain elongation. The first-order rate-limiting step could be the formation of nuclei. Possible nuclei could be tubulin subunits which have been “activated” by vinblastine binding, specific aggregates of tubulin, or specific structures such as the rings and coils which are seen in VLB-tubulin mixtures by electron micorscopy (11). This is similar to the mechanisms that have recently been proposed for normal microtubule assembly (4, 6,22), where similar rings or spirals are prime candidates for the nucleation centers. The binding levels which we have found for vinblastine are in agreement with those of Owellen et al. (15) but are lower than reported by Bryan (14). It is not possible for us to reconcile these differences. Possible sources of error could come from standards used in the determination of tubulin concentration or differences in the purity and activity of various protein preparations. The inability of either of the halves of vinblastine to cause assembly on their own is interesting. Neither of these half molecules is effective at inhibiting the vinblastine induced precipitation of tubulin. Therefore, if they bind to tubulin at all, their binding to the protein cannot be as strong as vinblastine itself. Wilson et al. mention that vindoline, the lower indole moiety of vinblastine, was totally ineffective in precipitating tubulin (28), which our experiments confirmed. They used catharantine as a model for the other half of VLB rather than velbanamine. Weak antimitotic activity was observed but no direct experiments on tubulin are reported. Clearlv.” I it will be of interest in the future
BY
161
VINBLASTINE
to compare catharantine and velbanamine in in vitro experiments. If a significant difference in activity is observed it could be an important clue to the mechanism of VLB action. The requirement for GTP in vinblastineinduced aggregation was not expected. However, as can be seen in Fig. 7, this is strictly required and the amount of assembly obtained to a certain degree can be regulated by the GTP concentration. If, however, the protein is preincubated with GTP and the free GTP is then removed by column chromatography, the protein will still be precipitable by vinblastine. GMPPCP was not effective in replacing the GTP. The release experiments are consistent with a mechanism in which one of the four nucleotide sites per tetramer is sequestered in the assembly while a second remains exposed to the medium. This is in marked contrast to normal assembly where all exchangeable sites are sequestered and opens a way to investigating the directionality of the subunit assembly in the VLB paracrystals. The vinblastine-induced assembly of tubulin resembles normal assembly in its likely nucleation, in the requirement for GTP and in the concentration dependence of the final amount of assembly obtained. These similarities together with the difference in GTP site sequestration, colchicinebinding-site sequestration and final morphology make it a useful system for further physicochemical studies. ACKNOWLEDGMENTS This work was supported by grants from the USPHS, No. GM 14825, NS 08180 and NS 11504. M.V. was supported by an NIH predoctoral fellowship, No. GM 46,899. We are grateful to Dr. Nobert Neuss for generous gifts of vinblastine and other alkaloids. REFERENCES 1. WEISENBERG, R. C. (1972) Science 177, 1104. 2. ERICKSON, H. P. (1974) J. Cell Biol. 60, 153. 3. BORISY, G. G., AND OLMSTED, J. B. (1972)Science 177, 1196. 4. SHELANSKI, M. L., GASKIN, F., AND CANTOR, C. R. (1973)Proc. Nat.Acad. Sci. USA 70,765. 5. BENSCH, K. G., MARANTZ, R., WISNIEWSKI, H., AND SHELANSKI, M. L. (1969) Science 195,495. 6. KIRSCHNER, M., WILLIAMS, R. C., WEINGARTEN,
162
7. 8. 9. 10. 11. 12. 13. 14. 15.
16. 17.
VENTILLA,
CANTOR
M., AND GERHART, J. (1974) Proc. Nat. Acad. Sci. USA 71, 1159. BORISY, G. G., OLMSTED, J. B., MARCIJM, J. M., AND ALLEN, C. (1974) Fed. Proc. 33, 167. BENSCH, K. G., AND MALAWISTA, S. E. (1969) J. Cell Biol. 40, 95. BENSCH, K. G., AND MALAWISTA, S. E. (1968) Nature (London) 218, 1176. BRYAN, J. (1971) Exp. Cell Res. 66, 129. MARANTZ, R., VENTILLA, M., AND SHELANSKI, M. L. (1969) Science 195, 498. VENTILLA, M., CANTOR, C. R., AND SHELANSKI, M. L. (1972) Biochemistry 11, 1554. WEISENBERG, R. C., AND TIMASHEFF, S. N. (1970) Biochemistry 9, 4110. BRYAN, J. (1972) Biochemistry 11, 2611. OWELLEN, R. J., OWENS, A. H., JR., AND DONNIGAN, D. W. (1972) Biochem. Biophys. Res. Commun. 47, 685. WEISENBERG, R. C., BORISY, G. G., AND TAYLOR, E. W. (1968) Biochemistry 7, 4466. SHELANSKI, M. L., AND WEISENBERG, R. C (1972) in Techniques of Biochemical and Biophysical
AND
18.
19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
SHELANSKI Morphology (Glick, D., and Rosenbaum, R. M., eds.), p. 25, Wiley, New York. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L. AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265. BORISY, G. G. (1972) Anal. Biochem. 50, 373. JAKOVLJEVIC, I. M. (1962) J. Pharm. Sci. 51, 187. MARANTZ, R., AND SHELANSKI, M. L. (1970) J. Cell Biol. 44, 234. GASKIN, F., CANTOR, C. R., AND SHELANSKI, M. L. (1974) J. Mol. Biol. 89, 737. BERNE, B. (1974) J. Mol. Biol. 89, 755. GUGGENHEIM, E. A. (1926) Phil. Mag. 2, 538. WILSON, L., BRYAN, J., RUBYN, A., AND MAZIA, D. (197O)Proc. Nat. Acad. Sci. USA 66,807. REMENCHIK, A. P., AND BERNSOHN, J. (1967) Anal. Biochem. 18, 1. BERRY, R. W., AND SHEANSKI, M. L. (1972) J. Mol. Biol. 71, 71. WILSON, L., BAMBURG, J. R., MIZEL, S. B., GRI& HAM, L. M., AND CRESWELL, K. M. (1974) Fed. Proc. 33, 158.