PRELIMINARY STUDIES ON AFFINITY LABELING OF THE TUBULIN-COLCHICINE BINDING SITE

PRELIMINARY STUDIES ON AFFINITY LABELING OF THE TUBULIN-COLCHICINE BINDING SITE *

Joseph Bryan

Biology Department University of Pennsylvania

Philadelphia, Pennsylvania 191 74

At least three chemically distinct types of drugs, colchicine, vinblastine, podophyllotoxin, and their various derivatives, are known to inhibit mitosis in dividing cells. The only known common mode of action of these agents is their interaction with tubulin. These interactions, particularly the binding parameters and stoichiometries, have now been studied in some detai1.l. This work indicates that tubulin has at least three “binding sites” for drugs: one for colchicine and podophyllotoxin, and at least one (and probably two) for vinblastine. A more detailed understanding of tubulin-drug interactions at a molecular level will require knowledge of the chemistry of the individual drugs and their derivatives, information on the stoichiometries and thermodynamic parameters that characterize the binding reactions between tubulin and each drug, and a thorough understanding of the chemistry of those regions of the tubulin polypeptides that serve as drug-binding sites. A great deal of information is available on the first two problems, particularly in the case of colchicine and its derivatives. Almost nothing, on the other hand, is known about the chemical nature of the colchicine binding site. We have previously attempted, without success, to inhibit colchicine binding in vinblastine-induced microtubule crystals, using several protein-modifying reagents in the hope of localizing the binding site. The present report deals with an alternative approach to the problem of localizing the colchicine binding site : the use of affinity labeling.

The usual method of affinity-labeling an enzyme involves the synthesis of a small molecule that closely resembles the substrate, but is substituted with some chemically reactive group that is able to react covalently with the amino acid residues at the enzyme active site. Since enzyme substrates are not normally irreversibly bound to the enzyme, labeling can often be detected by gel filtration or dialysis to remove unattached ligand. Since tubulin has a reasonably high affinity for colchicine, a somewhat different method was devised to detect the presumptive covalent labeling of tubulin with colchicine derivatives. Our approach was first to form the tubulin-colchicine derivative complex, then to irradiate it at 366 nm, in order to convert the C-ring of colchicine to the lumicolchicine configuration, which has a greatly reduced affinity for tubulin. Our rationale was that only those modified colchicine molecules that were covalently incorporated into tubulin should remain bound. The initial results obtained by this assay method indicated that a variety of derivatives, including chlorocyanoethylcolchicine, bromoacetylcolchicine, and diazonium salts, were incorporated into a macromolecular fraction and were not released by extrac- tions with perchloric acid. This incorporation was accompanied by a propor-

’Ic This work was supported by Grant GB-3287X from the National Science Foun- dation.

247

248 Annals New York Academy of Sciences

tionate loss of colchicine-binding sites, which suggested that the active site had been specifically labeled. Subsequently, however, it has been possible to demon- strate that the incorporation is directly dependent upon the irradiation step, that it will occur to a limited extent with unmodified colchicine, and that the irradiation product is a small molecule that can be extracted into chloroform- methanol. In short, colchicine and several of its derivatives are able to act as photolabeling reagents. In no case, however, have we been successful in labeling the tubulin polypeptides.

MATERIALS AND METHODS

Preparation of N-[meth~xy-~H]deacetylcolchicine

N-deacetylcolchicine labeled with tritium on the C-ring methoxy group was synthesized by methylation of trimethylcolchicinic acid with [3H]diazomethane (the reaction was carried out by the New England Nuclear Corporation). Trimethylcolchicinic acid was prepared from colchicine by hydrolysis in acidic methanol, according to the procedures outlined by Wilson and Friedkh4 Labeled iso-N-deacetylcolchicine was separated from labeled N-deacetylcolchicine by means of preparative thin layer chromatography (TLC) plates (silica gel G, layers 1 mm thick); ethanol was used as the solvent. The specific activity of the purified product was 1554 Ci/ mole.

Preparation of [methoxy-'H]colchicine

[Metho~y-~H]coIchicine was prepared from N-[metho~y-~H]deacetylcolchicine by acetylation with acetic anhydride, as detailed by Wilson and F~iedkin.~ Final purification was done by TLC, using preparative plates (silica gel G, 1 mm thick) with methanol as the solvent.

Derivatives

Several colchicine derivatives have been prepared from N-[3H]deacetylcolchicine. Since the results with each of these were qualitatively the same in this assay procedure, the synthesis of only one will be detailed here. It should be indicated that these derivatives are all ring-B nitrogen additions.

Preparation of N-[SH]chlorocyanoethylcolchicine

The chlorocyaaoethyl group is a potential alkylating reagent, which has been coupled with galactopyranosylmercaptide and used to label the lactose repressor.6 N-chlorocyanoethylcolchicine (CCE-colchicine) can be synthesized from N-deacetylcolchicine in one step by reaction with a-chloroacrylonitrile. Equimolar amounts of a-chloroacrylonitrile and N-[3H]deacetylcolchicine were reacted overnight in ethanol that contained 0.01 % NH,OH. [3H]CCE-colchicine was purified by preparative TLC on silica gel; ethanol was used as a solvent. R,s in methanol were: isodeacetylcolchicine, 0.5; deacetylcolchicine, 0.66; colchicine, 0.8; and CCE-colchicine, 0.92.

Bryan: The Tubulin-Colchicine Binding Site 249

Trrbulin Preparations

Crude brain extracts were prepared from 10-15 day chick embryo brains by homogenization and 100,000 X g centrifugation for 60 min. Tubulin was column-purified on DEAE-cellulose according to the method of Eipper or Bryan and W i l ~ o n . ~

Irradiation Conditions

Samples were irradiated with a mercury lamp and filter, to remove radiation below approximately 300 nm. The light source was a Blak Ray (Model B- 1OOA). Quartz cuvettes were placed 15 cm from the lamp face. Irradiations were done at 0-4" C. Alternatively, the cuvettes were placed in an NaNO, bath 15 cm from the mercury lamp (used without the filter). The available technical data indicate that the intensity of 366 nm radiation with this lamp is 8400 pW/cm2 at 18 inches (Blak Ray).

RESULTS

The specificity of CCE-colchicine was tested by means of two assays. Bio- logical activity was scored by measuring inhibition of mitosis in dividing sea urchin zygotes. CCE-colchicine was approximately 100 times as effective as colchicine in this assay, with one-half maximal inhibition at 1 pM. The chemical specificity was tested in competition experiments with [3H]colchicine, using the vinblastine microtubular crystal assay procedure described el~ewhere.~ CCE-colchicine competitively inhibited colchicine binding in this assay. The association constant is approximately the same as that of colchicine. Gel filtration experiments indicate that [3H]CCE-colchicine binds to the tubulin dimer.

Evidence for Incorporation

FIGURE 1 illustrates the progressive incorporation of either [3H]colchicine or [3H]CCE-colchicine into perchloric acid (PCA) -precipitable material in crude chick embryo brain preparations (100,000 X g, 60 min supernatants), prelabeled with equal concentrations of the drugs (0.15 pM) and irradiated at 366 nm for progressively longer times. Qualitatively similar results were obtained with 253 nm illumination. The ratio of the specific activity of incorporation for colchicine to that of CCE-colchicine is 0.26. In short, CCE-colchicine is approximately 4 times more effective.

FIGURE 2 demonstrates the initial binding of labeled drug to tubulin in the DEAE-disc assay.s Upon irradiation there is an initial loss of bound label, followed by an increase. The final level for the derivative usually equals but can sometimes exceed the initial binding activity. The incorporation has an absolute requirement for irradiation of the colchicine in the presence of the supernatant. Irradiation of the drug alone, followed by mixing, produces only background levels of incorporation. FIGURE 3 shows the results of a separate experiment under slightly different conditions; it indicates that bovine serum


z t-

0.4

a 0.3 2

< I

0.2

0.1

w A 0

1 0

0- 0

4 8 12 16 20

MINUTES

FIGURE 1 . Incorporation of tritiated colchicine (0) or tritiated CCE-colchicine ( A ) into perchloric acid-precipitable material as a function of time of irradiation (in min). Chick embryo brain supernatants (100,000 x g, for 60 min) homogenized in 0.1 M Mes and 1 mM MgCL at pH 6.5 were used at 1 mg protein/ml. Supernatants were prelabeled for 2 hours a t 37" C with 0.15 pM colchicine or CCE-colchicine, and irradiated at 350 nm as described under MATERIALS AND METHODS. Aliquots were removed and layered over 10% perchloric acid; the resulting precipitates were washed twice, dissolved, and counted. The ratio of incorporation into perchloric acid-precipitable material (CCE-colchicine/colchicine) after 20 min is 3.8.

z F

4 8 12 16 20 MINUTES

FIGURE 2. Incorporation of tritiated colchicine (0) or tritiated CCE-colchicine ( A ) into material retained by DEAE paper discs (diameter 2.5 cm, Reeve Angel Co.) as a function of irradiztion time (in min). Incubation conditions were identical to those given in FIGURE 1. The initial binding (0.36 pmole/g protein) represents the extent of complex formation at zero time, before irradiation. The ratio of incorporation (CCE-colchicine/colchicine) after 20 min is 4.1.

Bryan: The Tubulin-Colchicine Binding Site 25 1

albumin at 10 mg/ml is only labeled to approximately 10% of the level of the tubulin-containing preparations.

Evidence for Photosensitization and Loss of Binding Sites

TABLE 1 indicates the destruction of binding sites by irradiation of tubulin- colchicine complexes. Protein irradiated without colchicine present retains >95% of its binding activity. Approximately 40% of the binding activity is lost if irradiation is carried out after prelabeling with 100 pM colchicine, while

4 8 12 16 20 MINUTES

FIGURE 3. Comparison of the incorporation of tritiated colchicine into brain supernatants or bovine serum albumin solutions as a function of irradiation time. Here 12-day chick embryo brain supernatant (100,000 x g, 60 min) at 2 m g / d or bovine serum albumin at 10 mg/ml was incubated with 0.45 pM [*H]colchicine for 2 hours at 37" C, then irradiated as described under MATERIALS AND METHODS. Then 100 pl aliquots were assayed with DEAE discs or precipitated with 10% perchloric acid and washed twice. The buffer was 0.1 M MES and 1 mM MgCL (pH 6.5) . (O= Brain supernatant, DEAE disc assay; .=bovine serum albumin, DEAE disc assay; A =brain supernatant, PCA-precipitable radioactivity; A =bovine serum albumin, PCA-precipitable radioactivity.)

62% is lost if irradiation is carried out after prelabeling with 100 pM CCE- colchicine. Similar results have been reported by Amhreim and Filner9 in a more extensive characterization of this process for colchicine alone.

We have attempted to quantitate the number of sites lost during irradiation of the complex and to compare this directly with the number of molecules incorporated into the irradiation product. The complex was formed by incubation of a 100,000 X g, 60 min supernatant of chick embryo brain with 10 pM CCE-colchicine (either labeled or unlabeled) for 2 hours at 37" C. The complex was separated from unbound CCE-colchicine by gel filtration on Bio-Gel P-30 (from Bio Rad Labs). The complex was irradiated for increasing lengths


TABLE 1

EFFECTS OF IRRADIATION AT 350 NM ON THE COLCHICINE-BINDING SITE*

Amount Percentage of Conditions Incorporated Control

cpm ccg Protein, unirradiated 27 1 100 Protein, irradiated 260 96 Protein plus colchicine, irradiated 161 59 Protein plus CCE-colchicine, irradiated 104 38 Protein plus irradiated colchicine 249 92 Protein plus irradiated CCE-colchicine 252 93

* In these experiments, 12-day chick brain supernatant (100,000 x g for 60 min) in 0.1 M MES and 1 mM MgCl? (pH 6.5) was incubated for 2 hours at 37" C with 100 pM of the agents indicated, then irradiated for 30 min on ice and finally assayed for colchicine-binding activity by incubation with 1 pM [*H]colchicine for 2 hours at 37" C. Binding was measured by means of the DEAE-disc assay. The protein con- centration was 2 mg/ml.

of time. The experiment with [3H]CCE-colchicine was analyzed by the DEAE- disc procedure S or by PCA precipitation to measure the extent of incorporation. The complex with unlabeled CCE-colchicine was irradiated under identical conditions, and samples were removed and challenged to rebind [*~C]colchicine (33 pM for 60 min at 37" C), in order to assay their colchicine-binding activity. The results are shown in FIGURE 4. The incorporation curve indicates the initial complex present at zero time, and a sharp drop in the label retained by DEAE-discs, followed by a progressive incorporation into material that is retained by the DEAE paper and is also PCA-precipitable. The behavior is qualitatively equivalent to that shown in FIGURE 2, where excess CCE-colchicine is present. The results of the [14C]colchicine-rebinding assays are also shown in FIGURE 4. From 30 seconds on, there is a progressive decrease in the number of sites that are detected by the rebinding assay. The kinetics of site loss and incorporation are different, which suggests that the processes are not strictly dependent upon each other. The stoichiometries, however, are in reasonable agreement, with these figures: that 0.95 pmoles of derivative were incorporated/gm protein, and 1.05 pmoles of sites were lost/gm protein, after 20 min of irradiation.

Characterization of the Irradiation Product

Nondenaturing Conditions

Chick embryo brain supernatants (100,000 X g, 60 min) were prelabeled with CCE-colchicine at 37" C and irradiated on ice for 10 min. Equal volumes (0.5 ml) of irradiated supernatant and unirradiated supernatant prelabeled with [14C]CLC were mixed and chromatographed on agarose A-15 m (from BioRad Laboratories). The pattern at zero time (which was chromatographed immediately) is shown in FIGURE 5A. The majority of the irradiation product co-

Bryan: The Tubulin-Colchicine Binding Site 253

chromatographed with the [l~lClcolchicine-labeled tubulin dimer. The pattern (for protein at 20" C ) after 24 hours is shown in FIGURE 5B. There is essentially n o label in the dimer fraction, but the amount of label running in the void volume is now greatly increased. Calculation of the relative amounts of material indicate that 55% of the label that originally chromatographed with the dimer a t zero time runs in the void volume after 24 hours a t 20" C. Both the dimer and void volume fractions contain a- and p-tubulins in equal ratio when analyzed by SDS polyacrylamide gel electrophoresis.

Denaturing Conditions

T h e incorporated PCA-precipitable label can be retained through the usual procedures for reduction and acetylation in 1% SDS.lo FIGURE 6 indicates the electrophoretic behavior of the irradiation product from DEAE-cellulose- purified tubulin.G The label migrates somewhat behind the dye front in 5 %

4 8 12 16 20 MINUTES

FIGURE 4. Comparison of the number of colchicine-binding sites lost during irradiation of the colchicine-tubulin complex with the incorporation into irradiation product. Here CCE-colchicine-tubulin complex was formed by incubating 12-day chick embryo brain supernatants (3 mg/ml) with 10 pM CCE-colchicine for 2 hours at 37" C, then isolated by gel filtration on il Bio-Gel P-30 column (1x 15 cm). The complex was irradiated as described under MATERIALS AND METHODS. Tritiated CCE- colchicine was used to assay the extent of incorporation in the DEAE-disc assay (0, left abscissa). To assay the sites remaining after irradiation of complex, cold complex was formed, isolated, and irradiated. At each time point, aliquots were taken for a reassay of colchicine-binding sites by incubation with ["C]colchicine (33 pM) for 60 min at 37" C. The amount of ["C]colchicine-tubulin formed ( A , left abscissa) was determined by the DEAE-disc assay. A calculated value for the number of colchicine-binding sites lost as a function of irradiation time was calculated as follows: pmoles o€ colchicine-binding sites lost/g protein=pmoles of ["Clcolchicine bound at zero time/g protein - pmoles [lJClcolchicine bound/@; protein after irradiation. These values, pmoles sites lost/g, are plotted on the right abscissa (A),


acrylamide (0.2% bisacrylamide) gels that contained 0.1% SDS and 20 mM Tris-glycine at pH 8.2.” The labeled material has an apparent molecular weight of 16,500 when the gels are standardized with globular proteins. The irradiation product fixes poorly, if a t all, however, with 0.025% Coomassie Blue, 1% Amido Schwartz, or 1% fast green, with a variety of different concentrations of acetic acid and methanol. Typically 90-95% of the label is lost

z a 0

r)

I

W m

5 10 15 20 25 30 35 TUBE NUMBER

FIGURE 5. (A) Agarose A-15 m chromatography of irradiation product. Here 12- day chick embryo brain supernatant (100,000 x g, 60 min) at 2 mg/ml was incubated for 90 min at 37” C with either 0.3 p M CCE-colchicine or 15 pM [“C]colchicine. The “C-labeled sample was kept on ice, while the [sH]CCE-colchicine sample was irradiated for 20 min on ice. Aliquots of each (0.5 mi) were mixed and chromatographed on a 1 x 50 cm column of Agarose A-15 m, and 1.7 ml fractions were collected; 98% of the initial 11,000 cpm *H-label and 86% of the initial 23,250 cpm “C-label were recovered. FIGURE 5B shows the results for an equivalent preparation after “decay” of colchicine-binding activity for 24 hours at 20” C. Conditions were the same as for FIGURE 5A. There was 99% recovery of both labels (the initial 11,100 cpm ‘H and the initial 23,500 cpm “C). Approximately 5 0 4 5 % of the 3H- label previously in dimer is now in void volume. Vo=column void volume; VD= elution position of tubulin dimer; 0 = [“C]colchicine; A = [JH]CCE-colchicine after irradiation.

Bryan: The Tubulin-Colchicine Binding Site

I 255

0 1 2 3 4 5 6 CM

FIGURE 6. SDS gel electrophoresis of DEAE-purified CCE-colchicine-tubulin complex after irradiation, reduction, and acetylation. Gel conditions: 5 % Acrylamide, 0.2%bisacrylamide, 0.025 M Tris-glycine, and 0.1% SDS at pH 8.2. For scanning and subsequent tracing, gels were stained with 0.025% Coomassie Blue in 50% methanol, 7% acetic acid, and destained in 10% methanol, 5% acetic acid. For slicing and counting, gels were frozen immediately on powdered dry ice and sliced into 1 mm pieces which were then digested with 30% hydrogen peroxide at 45" C before the counting. O=Radioactivity per 1 mm slice. Some background points have been omitted for clarity. The arrows indicate the region of the dye front.

from gels when we use our normal procedures for staining and destaining tubulin-containing gels.', 1') Less than 5% of the label is associated with either of the two known tubulin polypeptides (FIGURE 6) .

Extraction and Dialysis Experiments

The results of experiments with SDS gel electrophoresis suggested that the irradiation product might be a low molecular weight polypeptide. Further experiments indicated, however, that although the label was not dialyzable against a variety of denaturing solvents (TABLE 2) , it was completely removed by dialysis against chloroform-methanol (2: 1) ; this argues against a molecular weight as large as 16,000 (TABLE 2 ) . These results suggested that the irradiation product could be extracted by organic solvents. Several have been tried. Ether alone does not remove significant label from PCA precipitates, but chloroform-ether ( 1 : 1 ) removes approximately 30% of the label, and chloroform-methanol (2: 1 ) removes > 90% of the PCA-precipitable radioactivity.

The partitioning of irradiation product during a Folch extraction l 2 for phospholipids is indicated in TABLE 3. Approximately 80% of the PCA-


TABLE 2 SUMMARY OF DIALYSIS EXPERIMENTS ''

Percentage Conditions Initial Count Final Count Retained

cpm/mL cpm/mL Dialysis versus 1 % SDS

Protein plus CCE-colchicine, 192,700 440 0.2 unirradiated

irradiated

colchicine

Protein plus CCE-colchicine, 192,700 95,620 t 49.6

Protein plus irradiated CCE- 192,700 430 0.2

Irradiated CCE-colchicine 192,700 410 0.2

Dialysis versus 5 M guanidine hydrochloride Protein plus CCE-colchicine 192,700 25,000 13

Irradiated CCE-colchicine 192,700 400 0.2 irradiated

Dialysis versus chloroform-methanol (2: 1 )

Protein plus CCE-colchicine, 192,700 956 0.5 irradiated

Irradiated CCE-colchicine 192,700 450 0.2

* In these experiments, 12-day brain supernatant was labeled with tritiated CCE- colchicine at 0.2 p M for 2 hours at 37" C, irradiated (if indicated), then dialyzed against 4 changes (500 ml each) of the solvent indicated.

i This figure is 98% of the perchloric acid precipitable radioactivity in this sample.

TABLE 3 SUMMARY OF DATA ON A FOLCH EXTRACTION OF IRRADIATION PRODUCT

FROM PERCHLORIC ACID PRECIPITATES * __

Percentage Fraction dPm of Total

(1) Perchloric acid precipitate 878,500 100 (2) First chloroform-methanol extract 712,300 81 (3) Second chloroform-methanol extract 89,100 10 (4) Residual protein pellet 77,100 8.8 (5) Aqueous phase 104,565 11.9 (6) Organic phase 736,681 83.9

104.6 Total recovery (4 + 5 + 6)

&' Twice-washed 10% perchloric acid precipitate of irradiated CCE-colchicine-brain supernatant was extracted twice with 5 ml chloroform-methanol (2: 1). The extracts were packed and 0.2 volumes of 50 m M CaCll were added; they were next emulsified by vortexing then centrifuged to give an aqueous phase and an organic phase. Further washing of the organic phase results in less than 5% further loss in radioactivity.

Bryan : The Tubulin-Colchicine Binding Site 25 7

precipitable radioactivity was recoverable in the organic phase. When the solvent was removed, the residue was negative for protein.

Thin-Layer Chromatography

Our preliminary results indicate that the irradiation product chromatographs as two major spots on Silica gel G in chloroform-methanol-H?O (65:25:4). The two components are distinct from CCE-colchicine, colchicine and their lumi-analogues. We have not yet established the chemical identity of these compounds, although their solubility properties suggest that they contain lipids or phospholipids.

DISCUSSION

The present results show that [:lH]colchicine or a chlorocyanoethyl derivative of [3H]colchicine (N-CH,-CHCl-CN-colchicine) can be bound to tubulin and will, upon irradiation, become incorporated into a PCA-precipitable, chloroform-methanol soluble material, presumably a lipid- or phospholipid-drug complex. Under the present conditions, CCE-colchicine is approximately 3-4 times as effective as colchicine in this incorporation. In aqueous solvents this material, which we have referred to as irradiation product, is aggregated and appears in a macromolecular form. Gel filtration experiments indicate that the irradiation product initially cochromatographs with the tubulin dimer, but aggregates to a higher molecular weight as a function of time. Both the dimer and the higher molecular weight component(s) contain a- and 8-tubulins.

The irradiation product can be separated from the tubulin polypeptides on SDS polyacrylamide gels. The label runs somewhat behind the dye front when a 5% gel system is used. The apparent molecular weight of this material, which is retained during dialysis against 1% SDS or 5 M guanidine HCL, is 16,500. We have been unable, however, to label the irradiation product in vivo with amino acids or in vitro with iodoacetic acid or N-ethylmaleimide. This failure to incorporate amino acids, the poor fixation on gels, and the lack of Lowry-positive material in the chloroform-methanol extracts all argue strongly that the irradiation product is not protein and will not give correct molecular weights on SDS gels. The belief that the irradiation product is not protein is further reinforced by the solubility properties of the product in organic solvents. The results cannot be explained simply as arising from irradiation-induced changes in the drugs alone, since irradiation of either colchicine or CCE- colchicine alone does not produce PCA-precipitable, nondialyzable material. Similarly, the mixing of lumicolchicine or iumi-CCE-colchicine with tubulin does not produce incorporation into irradiation product.

The present data also confirm the results of Amhrein and Filner,O and establish that irradiation of tubulin-colchicine complexes (but not of tubulin alone) results in a decrease in colchicine-binding activity. In short, colchicine- binding sites are altered or destroyed if they are irradiated with uv light while complexed with colchicine.

The two major conclusions that we would like to draw from these experiments are first, that colchicine-binding sites are lost if colchicine-tubulin complexes are irradiated at 366 nm, and second, that a lipid or phospholipid-


lumicolchicine complex irradiation product is formed under essentially the same irradiation conditions. The question that remains to be answered is whether a causal relationship exists between these two phenomena. In brief, does the formation of irradiation product result in the site loss?

Two interpretations or possible answers to this question are consistent with the present information, which is not sufficient to distinguish rigorously between them. One scheme would hold that lipid components (presumably unsaturated) are present in our tubulin preparations as contaminants, and can act as “traps” for the activated intermediates generated when colchicine or colchicine derivatives are converted to the lumi-forms by irradiation. The alternative interpretation would be that the lipid components were actually associated with tubulin, at or close to the colchicine-binding site.

The two hypotheses are indicated in SCHEME 1. A colchicine-tubulin complex (CT) is irradiated to produce a transient complex (C*T) between tubulin (T) and a reactive intermediate (C*). C* is presumably generated when colchicine is irradiated and then converted to lumicolchicine. The CCE- group could be more efficient, because it stabilizes C* rather than being reactive

PATH 2

C * + T i PATH 1

L I k+2

SCHEME 1 , Hypotheses on the relationship between formation of irradiation product and site loss. C=colchicine; C* =activated intermediate in transition from colchicine to lumicolchicine; Tztubulin; TI =inactivated tubulin; L=lipid or phospholipid; LCL=lipid-lumicolchicine complex.

itself. In Scheme 1, the first model is given in path 1, in which C* leaves tubulin (k+ l ) and inactivates the binding site. C* is then free to react with a contaminant lipid (L) component and form the irradiation product, which consists of lipid with a covalently attached lumicolchicine (,CL). In this scheme the inactivation of the site takes place when the complex is irradiated, and proceeds independently of the formation of irradiation product. The ob- servation that the number of sites lost is approximately equal to the number of molecules incorporated is perhaps fortuitous, since in path 1 the loss of sites will depend upon the efficiency of the inactivation step, while formation of ,CL will depend upon a variety of factors, including the availability of L and the loss of C* through competing reactions such as conversion to lumicolchicine or reaction with the solvent. Path 1 agrees nicely with the kinetics of incorporation as assayed by the DEAE-disc procedure, if we assume that all the intermediates but C* will bind to DEAE groups.

The alternative path (2) is also shown in SCHEME 1. Here C*T undergoes an internal reaction on tubulin, in which the activated intermediate C‘: reacts

Bryan : The Tubulin-Colchicine Binding Site 259

with a lipid molecule at or near the active site to form CT'. Presumably this complex (CT') is stable enough at 0-4" C to give the gel filtration results shown in FIGURE 5. This complex then dissociates to ,CL and inactivated tubulin (T,). Path 2 does not agree so well with the results of kinetic experiments unless we make further assumptions; on the other hand, it easily explains the gel filtration results. In short, neither of the two simple pathways is totally adequate to explain the data.

Other Evidence that Lipids May Associate with Tubulin

As indicated in SCHEME 1, one interpretation of the data would be that some type of lipid is associated with tubulin. Two other pieces of evidence suggest a similar conclusion. Eipper 13 has shown that chloroform-methanol extractable phosphate (presumably phospholipids) is isolated with the tubulin fraction during purification on DEAE cellulose. This material can subsequently be separated from the dimer by gel filtration. The aggregated lipids that elute in the void volume have a higher apparent molecular weight than the dimer. We have repeated these experiments with similar results,'.' using chick embry- onic brain and CHO cells labeled with ;jzP.

We have also attempted to enzymatically remove or alter phospholipids in crude brain extracts (supernatants from 100,000 X g for 60 min or 35,000 X g for 60 min) that are competent to assemble microtubules. The addition of phospholipase A (bee venom) at 5-10 pg/ml completely inhibits microtubule assembly when the supernatants are warmed to 37" C. Similar results are obtained with phospholipase C from Clostridium. These results, the copurifi- cation of phospholipids with tubulin and the inhibition of assembly in crude extracts, argue (but do not prove) that tubulin may associate with phospholipids.

SUMMARY

The present results argue that irradiation of a colchicine-tubulin complex at 366 nm results in destruction of the binding site and the labeling of a small molecule soluble in chloroform-methanol.

REFERENCES

1. WILSON, L. & J. BRYAN. 1974. Biochemical and pharmacological properties of microtubules. In Advances in Cell and Molecular Biology. E. J. DuPraw, Ed. Vol. 111. Academic Press, Inc. New York, N.Y.

2. WILSON, L. This monograph. 3. BRYAN, J. 1972. Biochemistry 11: 2611. 4. WILSON, L. & M. FRIEDKIN. 1966. Biochemistry 5: 2463. 5. RANDO, R. 1971. Nature New Biol. 234: 183. 6. EIPPER, B. A. 1972. Proc. Nat. Acad. Sci. U.S. 69: 2283. 7. BRYAN, J. & L. WILSON. 1971. Proc. Nat. Acad. Sci. U.S. 68: 1762. 8. WILSON, L. 1970. Biochemistry 9: 4999. 9. AMHREIN, N. & P. FILNER. 1973. FEBS Letters. 33: 139.

10. BRYAN, J . 1972. J. Mol. Biol. 66: 157. 11 . BRYAN, J. 1974. Federation Proc. 33: 152. 12. FOLCH, J. , I. ASCALI, M. LEES, J. A. MEATH & F. N. LEBARON. 1951. J. Biol.

13. EIPPER, B. A. 1974. J. Biol. Chem. 249: 1407. 14. NAGLE, B. & J . BRYAN. Unpublished data.

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PRELIMINARY STUDIES ON AFFINITY LABELING OF THE TUBULIN-COLCHICINE BINDING SITE

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