ANALYTICAL

69, 92-99 (1975)

BIOCHEMISTRY

Charcoal

Adsorption

Colchicine

Binding Crude

ELIEZER The John Collins Hospital of Harvard

Assay and Tissue

for Measurement Tubulin

Content

of of

Extracts

RAPAPORT, PATRICIA D. BERKLEY, AND NANCY L. R. BUCHER Warren Laboratories of the Huntington Memorial University at the Massachusetts General Hospital, Boston, Massachusetts 02114

Received January 2, 1975; accepted May 27, 1975 A rapid, reproducible, and quantitative colchicine-binding assay for tubulin content of crude tissue extracts is described and applied to the high speed supernatant fraction of rat liver homogenates. Utilizing Scatchard plots, the colchicinetubuhn association constant is found to be in general agreement with values reported for purified tubulin from other sources.

The alkaloid, colchicine, binds specifically and stoichiometrically to tubulin, the subunit protein of microtubules (1). This colchicine-tubulin interaction has been widely employed to explore the role of microtubules in mitosis, morphogenesis, intracellular transport, secretion, and It has also served as a convenient means of numerous other functions. measuring the tubulin content of various cells and tissues. Tubulin preparations are commonly assayed by incubation with [3H] colchicine to promote interaction, followed by determination of the Eliminaradioactivity bound to protein, which is assumed to be tubulin. tion of the unbound colchicine is accomplished either by adsorption on DEAE-cellulose filter disks (2) or TEAE-cellulose (3) or DEAESephadex (4) or by separation from bound colchicine by gel filtration (5). Recently, the tubulin-colchicine complex has been measured directly, by fluorescence, without separation from the free colchicine (6). The respective disadvantages of these techniques are imprecision, laboriousness, necessity of prior purification of the tubulin, or requirement for specialized equipment. We now report a procedure based on adsorption of the unbound [3H]colchicine on charcoal, which is removed by centrifugation. It is easy, rapid, reproducible, requires no highly specialized equipment, and is satisfactory for assaying crude tissue extracts. Because of the simplicity of processing multiple samples, a wide range of [3H]colchicine concentrations can be employed in each assay and the resulting data ana92 Copyright All tights

@ 1975 by Academic Press, Inc. of reproduction in any form reserved.

TUBULIN-COLCHICINE

lyzed by means of Scatchard plots, yielding than relative values for tubulin content. MATERIALS

93

ASSOCIATION

AND

absolute

amounts

rather

METHODS

[ Methoxy-3H] colchicine was purchased from New England Nuclear, Boston, Mass. Unlabeled colchicine, recrystallized from ethyl acetate, was added to adjust the specific activity to 0.9 Cilmmole. Activated charcoal (Norit A from Fisher) was washed with 6 N HCl followed by water. until washings were neutral and the “fines” removed, then suspended in water (approx 100 mglml). Male Sprague-Dawley rats (Holtzman Co., Madison, Wis.), weighing about 200 g, were anesthetized with ether; livers were perfused with 10 ml of ice-cold P-G buffer (0.02 M sodium phosphate, 0.1 M sodium glutamate, pH 6.75) (1) containing 0.25 M sucrose, rinsed, blotted, weighed, and homogenized in a glass tissue grinder with a motor-driven Teflon pestle for about 5 min in 9 ml of medium (P-G-sucrose containing lop4 M GTP) per gram of liver. After centrifugation for 1 hr at lOO,OOOg, the clear supernatant was collected from beneath the fatty layer at the top of the tube. The procedure to this point was carried out at 4°C. Duplicate 200-4 portions of the supernatant fluid were added to tubes containing [3H] colchicine (requisite amounts of [3H] colchicine in a 9 : I benzene-ethanol solution had been previously added and the solvent blown off with nitrogen). The tubes were incubated in darkness at 37°C in a shaker for 1 hr, then immersed in ice, and 5 ~1 of unlabeled colchicine (0.25 mM), 100 ~1 of P-G buffer, and 100 ~1 of the charcoal suspension added. The mixture was thoroughly agitated (Vortex mixer) for about 10 set, allowed to stand for several minutes in ice, and centrifuged at 900g for 10 min. The supernatant fluid was transferred to clean tubes and recentrifuged at 12,500g for an additional 10 min. A 250~~1 portion of the final supernatant was mixed with 5 ml of Aquasol (New England Nuclear) and assayed for radioactivity in a Packard TriCarb liquid scintillation spectrometer. Samples of identical composition were immersed in ice immediately, without prior incubation, and treated as above, to serve as blanks. RESULTS

AND

DISCUSSION

The amount of charcoal used to adsorb the unbound colchicine and the time allowed for adsorption to occur are both sufficient for removal of many times the amount of free colchicine actually added. Accordingly, the radioactivity remaining in the final supernatant fraction, after subtraction of the blank, represents the amount of colchicine bound to protein and, because of the known specificity of the binding, it is assumed to be proportional to the tubulin content of the sample. The

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FIG. 1. Time curves of colchicine binding to normal (-----) and 36-hr regenerating (-) liver supernatants. Duplicate 200-4 portions of supernatant fluid were incubated with 8.0 X 1O-4 pmoles of [3H]colchicine at 37°C. Uppermost curve shows effects of preinFor control point, plotted at zero time, samples from regenerating cubation ( .-----.). liver were incubated for 1 hr with [3H] colchicine; for 1 hr point they were incubated for 1 hr without, followed by 1 hr with [3H]co1chicine; for 2.5 hr point, 2.5 hr without, and 1 hr with [3H]colchine.

agreement between duplicate determinations fell in most instances within 10% of the mean. The small amount of charcoal-resistant radioactivity present in the blanks is presumably due to low molecular weight radioactive compounds present as impurities in the [3H]colchicine preparations. This background is consistent and approximates 2-4% of the bound colchicine. The complex formation with rat liver tubulin, under the conditions employed here, is completed to a large extent within the first hour or less (Fig. 1). A rapid decay of the binding capacity in an apparent first-order manner has been emphasized in studies of chick brain tubulin (1). We found no evidence of such decay in a crude preparation: Preincubation of portions of supernatant fluid for an additional l-2.5 hr, before incubating for 1 hr with [3H]colchicine, caused no loss of activity (Fig. 1). In view of this relative stability of hepatic tubulin, it appeared unlikely that decay of its colchicine-binding capacity would be a significant source of error during the I-hr incubation period routinely used in our assays. Supernatants from normal rat livers (protein content approx 18 mglml) were incubated, as described, with varying amounts of colchicine. The resulting curve (Fig. 2) indicates that saturation of binding sites is

TUBLJLIN-COLCHICINE

25

50 +I-

’ /A-‘-L.

75

95

ASSOCIATION

loo 150200250

COL CHIC/NE

500

1000

1500

f M x f 0’1

FIG. 2. Colchicine concentration curve. Portions of supernatant fluid (100 ~1) from normal adult rat livers were incubated with the concentrations of [:‘H]colchicine indicated for 1 hr at 37°C.

reached at about 2.5 X lo-” M. Further increases in colchicine concentration are without effect until excessive amounts (above 2.5 X lo-” M) cause a further rise due to nonspecific (low affinity) binding. We previously compared the relative colchicine-binding activity of supernatants from four normal and four 48-hr regenerating livers at a [“Hlcolchicine concentration of 1.3 x lo-” M (i.e., at saturation levels, but below the amount resulting in nonspecific binding). The unbound colchicine was removed by passage through a column of Sephadex G-100 (5). The mean colchicine binding activities were 9600 I 1100 dpm/mg of protein for the normal and 20.200 k 1700 for the 4%hr regenerating livers, or a ratio of regenerating to normal of 2.1 to 1. The same ratio (2.1 to 1) is yielded by the data in Table 1, obtained by the charcoal adsorption TABLE HIGH-AFFINITY

Supernatant Normal Normal 36-Hr regenerated 48-Hr regenerated

I

BINDING OF COLCHICINE TO LIVER SUPERNATANT AT 37°C

Association constant (Mm’) 5.9 6.0 5.9 3.3

x x x x

1cF lo” 105 10s

100.000~

RAT

Amount of tubulin (mg of tubulinig of liver) 0.198 0.191 0.273 0.413

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BUCHER

procedure and Scatchard plot analysis, described in Materials and Methods. The good agreement supports the validity of the charcoal method. The colchicine-binding activity of liver extracts determined at saturation levels appears to be constant over a wide range of ligand concentrations (2.5 X 10m6-2.5 x lop5 M), as shown in Fig. 2. Conceivably, lowaffinity binding, which is obvious at concentrations in excess of 2.5 X 1O-5 M (Fig. 2), may also occur to a lesser extent within the saturating range. Possible errors, arising from this binding that is nonspecific and not due to tubulin-colchicine interaction, are obviaied by the use of Scatchard plots which employ a variety of [3H]colchicine concentrations well below the saturation range (Figs. 3 and 4). The range of ligand concentrations optimal for Scatchard plots varies; for example Figs. 3 and 4 show data for the same tissue (liver) in different physiological states (normal and regenerating). Both plots approach the useful lower and upper limits of ligand concentration, yet the ranges employed in the two figures differ by nearly a whole order of magnitude. Analysis of the colchicine-binding data by the method of Scatchard (7) is shown in Figs. 3 and 4. In this procedure aliquots of each supernatant are incubated with [“HIcolchicine over a wide range of concentrations, all below saturation levels, as noted, so that measurement of only specific, high-affinity binding is assured. From the slope of the lines in the Scatchard plots we determined the association constants for tubulins from normal and regenerating rat livers to be 3.3-6.0 x 105 liters/mole (Table 1). They are in reasonable agreement with reported values from

0

20

40

60

Bound FIG.

tion.

3. Scatchard plot of colchicine binding Saturation curve is shown in the inset.

60

100

Cokhicine to normal

120

140

160

160

f~Wx/O~l liver

lOO.OOOg

supernatant

frac-

TUBULIN-COLCHICINE

Free

\

Oo

1

IO

97

ASSOCIATION

Colchicine

lMx107)

/

I 20

Bound

30

40

Colchicine

50

60

70

00

(IV x IO 8,

FIG. 4. Scatchard plot of colchicine binding to 100,OOOg supernatant regenerating rat liver. Saturation curve is shown in the inset.

fraction

from

4%hr

Scatchard plots for tubulins from other sources: 2 x lo6 liters/mole for purified tubulin from chick embryo brain in the presence of vinblastine sulfate (1) and 6.3 X 105 liters/mole for purified outer doublet tubulin from sea urchin sperm tails (8). On the basis of a kinetic method, a value of 2.3 x 10fi liters/mole was found for tubulin from crude extracts of unfertilized sea urchin eggs (9) and 1.1 X lo6 for similar extracts from human cells in culture (KB strain) (10). That the charcoal method is satisfactory for measurement of colchicine-binding activity in crude tissue extracts is evidenced by the demonstration that Scatchard plots of data obtained from 100,OOOg supernatants of rat liver homogenates, assayed in this fashion, yielded values for colchicine-tubulin association constants that are in general agreement with those reported for both crude and purified tubulins from other sources. The use of the Scatchard plot enables tubulin values to be stated in absolute units of measurement, which is more meaningful than the relative colchicine-binding activity levels generally presented. The tubulin content of the livers was calculated from the Scatchard plots using the intercept with the abscissa and assuming the molecular weight of tubulin to be 110,000 and the number of colchicine-binding sites per tubulin molecule to be 1.0 (8). [By SDS-polyacrylamide gel electrophoresis (11) we have found the molecular weight of the tubulin monomeric subunits from rat liver to be 52,000 % 2000 (12).] Identical values are reported for tubulins from sea urchin eggs when determined by this technique (13), which yields estimates of molecular weight slightly below the gen-

98

RAPAF’ORT,

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BUCHER

erally accepted range of 1 lO,OOO-120,000 for the native dimer of various tubulins as determined by other means (14). The colchicine-binding determinations reported here were carried out on the high speed supernatant, or “soluble,” fraction of liver homogenates. The extensive binding to liver cell particulates and membranes reported by others, and also noted by us, now appears to be largely of the low-affinity and nonspecific type: it is considered that in rat liver, at least, only a minute fraction of the total tubulin could possibly constitute a membrane component (15). [In brain the situation is somewhat different (16).] Accordingly, the tubulin content of liver recorded in Table 1 is based only upon the high-affinity colchicine-binding activity of the soluble extract; for normal liver the value is approximately 0.2 mg of tubulin/g of liver, which is about 0.1% of the total liver protein, or slightly less that 1% of the protein in the soluble fraction. For comparison, in sea urchin eggs, the values are similar; the soluble tubulin comprises 0.37% of the total protein or 1.1% of the soluble protein. In chick brain, tubulin concentrations are much higher: 20-40% of the soluble protein, dropping to 25% in the adult, in keeping with the early report that extracts of rabbit brain contained over 15 times as much colchicine binding-activity as liver extracts (IO). After initial submission of this paper, Sherline et al. (17) reported a slightly different version of a charcoal adsorption procedure, which, in accord with our observations, shows excellent agreement between charcoal adsorption and Sephadex G- 100 chromatography in separating free from protein-bound colchicine. A problem that these investigators encountered was adsorption or disruption of the tubulin-colchicine complex by the preparation of activated charcoal (Matheson, Coleman, and Bell) which they employed without pretreatment, so that only very limited amounts could be used. On the other hand the gentler-acting acid-washed Norit A can be employed in large excess to ensure complete separation of bound and free colchicine, without this hazard. It has been widely used in many systems for measuring protein binding of small molecules; Rousseau et al. (18) found that even 300 mg/ml did not interfere with the analysis of steroid receptors in liver extracts, whereas gel filtration caused sufficient denaturation of the receptors to interfere with the assay. Our procedure, using acid-washed Norit A, employs nonincubated blanks as assurance that adsorption of free [“Hlcolchicine is complete. It depends upon colchicine concentration curves that must yield linear Scatchard plots for each sample, as assurance both that charcoal absorption of unbound colchicine is complete and that only high-affinity binding is being measured. It is further supported by the agreement between association constants obtained from our crude liver extracts and from purified tubulins reported by other investigators. For

TUBULIN-COLCHICINE

ASSOCIATION

99

these reasons our method has advantages over the recently reported procedure (17) and many others, where only a single concentration of [3H]colchicine is employed without indication as to where this concentration lies with respect to saturation; if below saturation the quantitative assessment will be too low, and if well beyond saturation it will be too high because of nonspecific binding. The ease and rapidity of the charcoal adsorption procedure makes the processing of multiple aliquots of each sample readily feasible for routine assays; Scatchard plots can then be employed both to check on the validity of data obtained from crude extracts (by linearity) and to determine even low amounts of tubulin accurately in terms of absolute units. ACKNOWLEDGMENTS We are indebted to the Medical Foundation. Inc., of Boston, Mass., for a fellowship granted to one of us (E.R.) and support from NIH Grant CA 02146-18, USPHS. This is publication 1492 of the Cancer Commission of Harvard University.

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7. 8. 9. 10. Il. 12. 13. 14. 15. 16. 17. 18.

Bhattacharyya, B., and Wolf, J. (1974) Proc. Nat. Acad. Sci. USA 71, 2627-2631. Scatchard, G. (1949) Ann. N.Y. Acud. Sci. 51, 660-672. Wilson, L., and Meza. I. (1973) J. Cell Biol. 58, 709-719. Borisy. G. G., and Taylor, E. W. (1967) J. Cell Biol. 34, 535-548. Borisy, G. G., and Taylor, E. W. (1967) J. Cell Biol. 34, 525-533. Weber, K., and Osborn, M. ( 1969) J. Biol. Chem. 244, 4406-4412. Bucher, N. L. R., and Berkley, P., unpublished results. Ratf, R. A., and Kaumeyer. J. F. (1973) Develop. Biol. 32, 309-320. Bryan, J. (1974) Fed. Proc. 33, 152-157. Stadler, J., and Franke, W. W. (1974) J. Cell Binl. 60, 297-303. Bamburg, J. R., Shooter, E. M., and Wilson, L. (1973) Biochemistry 12, 1476-1482. Sherline, P., Bodwin, C. K., and Kipnis, D. M. (1974) Anal. Biochem. 62, 400-407. Rousseau, G. G., Baxter, J. D., and Tomkins, G. M. (1972) J. Mol. Biol. 67, 99-l 15.