Tubulin and microtubules from bovine kidney: Purification, properties, and characterization of ligand binding

ARCHIVES

OF BWHEMISTRY

AND BIOPHYSICS

Vol. 196, No. 2, September, pp. 511-524, 1979

Tubulin

and Microtubules from Bovine Kidney: and Characterization of Ligand LARRY

D. BARNES

Department of Biochemistry,

AND GLENDA

Purification, Binding

Properties,

M. ROBERSON

The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284

Received December 21, 1978; revised March 27, 1979 Tubulin was purified from bovine renal medulla by in vitro assembly of microtubules in the presence of dimethyl sulfoxide and glycerol. Light scattering measurements of the polymerization process demonstrate that dimethyl sulfoxide and glycerol decrease the critical concentration of tubulin required for polymerization. The minimum concentration of tubulin from bovine renal medulla is about 1% of the total soluble protein. Assembly occurs in the absence of detectable amounts of high-molecular weight proteins or T-protein. Microtubules polymerized in the absence and presence of 10% dimethyl sulfoxide and 4 M glycerol are similar morphologically as detected by electron microscopy. Molecular weights of cc-and /3-tubulin from bovine renal medulla are 54,000 2 700 and 52,000 k 800, respectively, as determined by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. Colchicine-binding activity of renal medullary tubulin decays in an apparent first-order process which is temperature dependent. The half-time of decay in buffer is 5.1 h and addition of 5 pM vinblastine sulfate increases the half-time of decay to 10.9 h at 37°C. Calculations based on measurements of the rate of decay of colchicine-binding activity at different temperatures indicates that vinblastine sulfate stabilizes the binding activity by decreasing the entropy of activation of the decay process. Colchicine decreases the rate of decay about 3.5fold both in the absence and presence of vinblastine sulfate at 37°C. Values of the apparent colchicine-binding constant, KA, of bovine renal medullary tubulin are 5.9 x lo6 and 7.8 x lo6 M-l at 37°C in the absence and presence of vinblastine sulfate. Vinblastine sulfate decreases the rate of decay and increases the apparent binding constant of colchicine binding. Lumicolchicine does not affect the binding of colchicine. Podophyllotoxin apparently competitively inhibits the binding of colchicine; the apparent Ki for podophyllotoxin is 4.0 x 10e7 M at 37°C. Thus, tubulin from bovine renal medulla has ligand-binding characteristics which exhibit differences and similarities to the corresponding characteristics of the brain tubulin. These biochemical properties of the colchicine-binding activity of bovine renal medullary tubulin support previous physiologic studies which demonstrate that microtubules are requiredfor the functionof vasopressin in mammalian kidneys.

Microtubules are required for the cellular action of antidiuretic hormone, vasopressin, in toad urinary bladder (1,2) and mammalian renal medulla (3, 4), but how microtubules ai-e involved is unknown. BioEhemicai and

morphologic characterizations of tubulin and microtubules from mammalian renal medulla are necessary to understand the physiologic function of microtubules in the action of vasopressin. We initiated the present studies to purify and characterize tubulin from mammalian renal medulla as an initial step in determining the role of microtubules in the action of vasopressin.

The colchicine-binding activity of tubulin may be useful for indirectly measuring levels of tubulin in renal medullary tissue in different states of diuresis and antidiuresis. However, colchicine can affect cellular processes unrelated to its disruption of microtubules (5, 6). Consequently, a prerequisite for quantitation of tubulin by binding of colchicine is characterization of the parameters of the colchicine-tubulin interaction as described by Wilson and Bryan (5). Substances which bind colchicine are present in bovine renal medullary tissue (3, 7), but these components have 511

0003-9861/79/100511-14$02.00/O Copyright 8 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

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not been biochemically characterized. The present results characterize the colchicinebinding activity in bovine renal medulla and demonstrate that the binding activity observed is due to the interaction of colchicine with tubulin. We pm&d tubuhn from bovine renal medulla by polymerization of tubulin in the presence of 10% (v/v> dimethyl sulfoxide plus 4 M glycerol. These solvents lower the critical concentration of tubulin required for polymerization and, thus, enable one to purify tubulin from a tissue containing a small amount of tubulin. A preliminary account of part of this work has been reported (8, 9). EXPERIMENTAL

PROCEDURES

Purijkation of tubulin. Medullary tissue was dissected from bovine kidneys of freshly slaughtered animals. One gram wet weight of tissue per 0.9 ml of buffer A (0.1 M Mes,’ 1 mM EGTA, 0.5 mM MgCl,, and 0.1 mM GTP, pH 6.7) was homogenized in a Waring Blendor (2 x lo4 rpm; 2 min) at 4°C. The homogenate was centrifuged at 2.8 x 10’9 for 30 min at 4°C and the supernatant fraction was recentrifuged at 4.4 x 104g for 30 min at 4°C. GTP, glycerol, and dimethyl sulfoxide (Me,SO) were added to the crude supernatant fraction to final concentrations of 1 mM, 4 M, and 10% (v/v), respectively. This preparation was incubated at 37°C for 30 min and then centrifuged at 4.4 x 104g at 30°C for 30 min. The pellet of microtubules was resuspended in buffer A (Ysoof the volume of the supernatant fraction after polymerization) and depolymerized by incubation on ice for 30 min. Tubulin, termed first-cycle tubulin, was obtained by centrifugation at 4.4 x 10’9 for 20 min at 4°C. This cycle of polymerization and depolymerization was repeated to yield second-cycle tubulin. The third cycle of polymerization was initiated by adding GTP and glycerol to tubulin to final concentrations of 2 mM and 4 M, respectively, and incubating at 37°C for 30 min. Centrifugation at 4.4 X 10’s at 30°C for 30 min yielded a pellet of microtubules which was depolymerized in buffer A as described above. Glycerol was added to a final concentration of 4 M to the tubulin

1 Abbreviations used: Me$O, dimethyl sulfoxide; Mes, 2-(N-morpholino)ethanesulfonic acid; EGTA, ethylene glycol bi@-aminoethylether)N,N ‘-tetraacetic acid; SDS, sodium dodecyl sulfate; k, the rate constant for an apparent first-order process; tllz, the half-time for an apparent first-order process; K,, the apparent binding constant for colchicine. buffer A, 0.1 M Mes, 1 mM EGTA, 0.5 mM MgCl,, and 0.1 mM GTP; buffer B, 0.1 M Mes, 1 mM EGTA, 0.5 mM MgCl*, and 1 mM GTP, pH 6.7.

obtained after three cycles and the preparation was stored at -20°C for 10 to 12 h. The fourth cycle of polymerization was initiated by warming the tubulinglycerol solution to 25”C, adding GTP to a final concentration of 2 mM, and incubating the preparation at 37°C for 30 min. Microtubules were sedimented by centrifugation at 4.4 x 1049 at 30°C for 30 min. The pellet was resuspended in buffer A and depolymerized as described above and tubulin was stored at -75°C. Purified tubulin was stored for a maximum of 2 months without loss of colchicine-binding activity. All tubulin used for the reported experiments was purified by four cycles of polymerization and depolymerization. Assay for cokhicine-binding activity. The colchicinebinding activity of tubulin was measured by retention of the colchicine-tubulin complex on DEAE-cellulose paper discs as described by Weisenberg et al. (10). To measure the activity of tubulin during purification, we added [4-3H]colchicine (approximately 5 x 105 dpm) to a final concentration of 5 pM to an assay tube containing 50 mM sodium phosphate, 1 mM MgSO,, 0.1 mM EGTA, 0.1 mM GTP, pH 6.8, and 20-400 pg of protein in a final volume of 200 ~1. Assay tubes were incubated for 90 min at 37°C in a covered water bath. The temperature, duration of binding, the addition of other components, such as vinblastine sulfate, and the concentration of colchicine used to characterize the binding activity of purified tubulin are described in the figure legends. The reaction was stopped by adding 1 ml of cold 10 mM sodium phosphate, pH 6.8, and the solutions were filtered through two Whatman DE-81 paper discs without suction on a Millipore Model 1225 sampling manifold. The discs were washed with four IO-ml portions of 10 mM sodium phosphate, pH 6.8, with low suction applied and were placed in 10 ml of tT-21 scintillation cocktail (11). Radioactivity of the colchicine-tubulin complex was measured after the complex had desorbed from the discs. The efficiency of counting was 36-38% as determined by the channels-ratio method with quenched standards. Background binding of r3H]colchicine to the discs in absence of tubulin was 0.1-O. 15% of the total radioactivity in the assay. With [3HJcolchicine of low specific activity (0.10 Ci/mmol) the binding activity was linear with 4902400 pg of crude supernatant protein and with 47-240 pg of purified tubulin. With [3H]colchicine of high specific activity (0.52 Ci/mmol) the binding activity was linear with 100-500 pg of crude supernatant protein and with 12-60 pg of purified tubulin. Stock [3H]colchicine was stored at -20°C in propanol: H,O (l:l), and the solvent was evaporated under a stream of N, gas prior to dilution with unlabeled colchicine for use in the assay. Concentrations of colchicine solutions were calculated from the absorbancy at 350 nm (12). Equilibrium binding of colchicine to tubulin can be determined using this method because

BOVINE

RENAL

MEDULLARY

TUBULIN

LIGAND

BINDING

513

the separation of free and bound colchicine is rapid were performed at least twice. Representative data relative to the rates of association and dissociation of individual experiments are presented unless noted of colchicine with tubulin. otherwise. Slopes and intercepts of lines expressing Light scattering measurement of in vitro polymerlinear relationships were determined by linear ization. Polymerization of purified tubulin was regression analysis. measured by the scattering of light at 350 nm as [4-3H]Colchicine (specific activity, 7.6 Ci/mmol) was described by Gaskin et al. (13). Turbidity was purchased from Amersham/Searle and unlabeled detected with a Beckman Model 25 recording spectro- colchicine from Aldrich Chemical Company. Purity of photometer thermostatically regulated at 37°C with an both colchicine preparations was at least 98% as internal cuvette heater. The reference cuvette contained determined by thin-layer chromatography on silica the polymerization solvent without added tubulin. gel (EM Laboratories, Inc.) in chloroform:acetone: Cuvettes were cooled on ice to depolymerize micro- diethylamine (5:4:1) (22). Lumicolchicine was prepared tubles. The actual temperature of the microtubuleby irradiation of colchicine in ethanol at 366 nm (6). tubulin system in the cooled cuvette was measured Measurement of the absorbancy at 350 nm and thinwith a thermistor (Yellow Springs Instrument Co.; layer chromatography on silica gel in benzene:ethanol YSI Model 42SC Tele-Thermometer). (3:l) (R, of lumicolchicine:R, of colchicine, 1.6:l) Polyacrylamide disc gel electrophoresis. Electroindicated that at least 97% of the colchicine was phoresis on polyacrylamide gels containing 0.1% converted to lumicolchicine. Concentrations of lumicolsodium dodecyl sulfate was performed as described by chicine were calculated from values of absorbancy at Studier (14) with minor modifications. The concentra267 nm (6). Podophyllotoxin (Aldrich Chemical Co.) tions of Tris and glycine in the electrode buffer were was dissolved in 1% (v/v) ethanol and its concentration one-half the concentrations specified by Studier. This calculated from the absorbancy at 290 nm (12). A final modification yielded a better separation of a- and concentration of 0.1% (v/v) ethanol, which corresponded p-tubulin because the relative electrophoretic mobility to the ethanol concentration from the podophyllotoxin of a-tubulin is anomalously, but reproducibly, dependent solution, in the binding reaction solution had no upon ionic strength (15). Tubulin fractions and standard effect on the colchicine-binding activity of tubulin. proteins were dissociated in 1% (w/v) sodium dodecyl Vinblastine sulfate was kindly donated by Eli Lilly sulfate and 1% (v/v) 2-mercaptoethanol at 85°C for and Company. GTP (Type II, 95-98% purity) and 15 min prior to electrophoresis. Proteins used as standard proteins for electrophoresis were purchased molecular-weight standards were carbonic anhydrase, from Sigma Chemical Company. Acrylamide, N,N’glyceraldehyde 3-phosphate dehydrogenase, glutamate methylene-bisacrylamide, and N, N, N’, N’-tetramethyldehydrogenase, pyruvate kinase, and bovine serum ethylenediamine were electrophoresis-grade reagents albumin. Gels were stained with Coomassie blue R-250 from Eastman Organic Chemicals. Sodium dodecyl and destained according to Fairbanks et al. (16). sulfate (99% purity) was obtained from Accurate Densitometric scans of stained gels were obtained Chemical and Scientific Corp. Glycerol and Me,SO using a Gilford Model 250 recording spectrophotomwere obtained from Fisher Chemical Company. eter equipped with a linear transport accessory. Electron microscopy. Preparations of microtubules RESULTS were negatively stained with uranyl acetate for electron microscopic examination. A 5+1 sample was Purification of Tubulin from Bovine Renal placed on a 200-mesh copper grid coated with Medulla Parlodion and carbon, and treated with cytochrome c Levels of tubulin in bovine renal medulla and 1% uranyl acetate as described by Olmsted and are too low for polymerization to occur in Borisy (17). The air-dried samples were examined with a Siemens 1A microscope operated at 80 kV. the presence of buffer, GTP, and EGTA Additional methods and materials. Amounts of as originally described by Weisenberg for proteins were determined according to Lowry et al. brain tubulin (23). Polymerization occurs (18) after dissolving samples in 1% sodium dodecyl when glycerol is added to the buffer, but sulfate. Crystalline bovine albumin was used as the the degree of polymerization is too low to standard protein. Samples containing glycerol were allow purification to apparent homogeneity diluted to eliminate possible interference in the (7). Addition of glycerol and Me2S0 to protein determination (19). Me,SO in concentrations up final concentrations of 4 M and 10% (v/v), to 10% (v/v) shows no interference with the Lowry respectively, in the polymerization solution protein assay (20). for the first two cycles enhances the degree Data were statistically evaluated using Student’s of tubulin from bovine t test for group comparison, and statistical significance of polymerization After is expressed in terms of two-tailed analysis (21). All renal medulla to allow purification. assays were done in duplicate; and all experiments two cycles of polymerization-depolymeri-

514

BARNES

AND ROBERSON TABLE

I

PURIFICATION OFTUBULINFROMBOVINERENALMEDULLA"

Fraction Crude supernatant (44,OOOg) 1st Cycle tubulin 2nd Cycle tubulin 3rd Cycle tubulin 4th Cycle tubulin

Volume (ml)

Total protein bg)

Total activity (pm01 PH]colchicine bound/90 min)

Specific activity (pmol [3H]colchicine bound/mg protein/90 min)

Yield (8)

435 28 4.7 2.6 1.8

21,490 209 35.3 29.8 15.1

352,430 113,000 59,440 57,566 27,835

16.4 540 1685 1932 1843

100 32 17 16 7.9

“.Tubulin from bovine renal medulla was purified by four cycles of in vitro polymerization and depolymerization as described under Experimental Procedure. The data represent a typical purification starting with 40’7 g wet wt of bovine renal medulla.

partially purified tubulin will polymerize in the absence of Me,SO. Maintenance of glycerol in the last two cycles of polymerization increases the yield of tubulin in comparison to polymerization in the absence of glycerol. Table I expresses the purification in terms of the colchicinebinding activity of tubulin fractions. Based on these data the minimum concentration of tubulin in the crude supernatant fraction is 0.32 5 0.14 mg/ml or 7.8 + 3.0 pg tubulin/mg of total soluble protein. The yields of colchicine-binding activity and protein are 9.9 + 4.4% and 12.7 rf: 5.6 mg tubulin from 400 g wet wt of bovine renal medulla. (Values are means f SD for 14 preparations of tubulin.) These are minimum values because the colchicine-binding capacity of tubulin fractions decays during the isolation and binding assay (vide infra). The purity of bovine renal medullary tubulin is illustrated in Fig.1. The two major peaks detected by densitometric scan of SDS-polyacrylamide gels represent (Y-and p-tubulin. On gels loaded with 20 pg or more of protein, minor bands can be detected. The minor bands represent less than 5% of the total protein stained with Coomassie blue. No specific microtubuleassociated proteins such as the T-protein (24) or the high-molecular weight proteins (25, 26) were electrophoretically detected. The molecular weights of a-tubulin and p-tubulin were 54,000 + 700 and 52,000 2 800 (mean + SD for five preparations), respectively based on their relative mobilities

zation,

on SDS-polyacrylamide gels in comparison to standard proteins. These molecular weights of a+ and /3-tubulin from bovine renal medulla are comparable to those reported for tubulin subunits from several different sources (27). The relative intensities of staining of CY- and p-tubulin on gels suggest that their stoichiometry is one-to-one (Fig. 1). Thus, bovine renal medullary tubulin is probably structurally similar to tubulin from other sources. In Vitro Polymerization Polymerization of purified tubulin under four different solvent conditions was measured by the scattering of light at 350 nm. 2or

” I-1

/I

It)

FIG. 1. Densitometric scan of SDS-gel electrophoretic pattern of bovine renal medullary tubulin. Tubulm purified by four cycles of polymerization and depolymerization was dissociated by heating in 1% sodium dodecyl sulfate and 1% 2-mercaptoethanol. Tubulin (10 pg) was electrophoresed on polyacrylamide gels in the presence of SDS in a discontinuous buffer system. Proteins were stained with Coomassie blue.

BOVINE

RENAL

MEDULLARY

TUBULIN

The inset of Fig. 2 illustrates the polymerization of tubulin in buffer B (0.1 M Mes, 1 InM EGTA, 0.5 mM MgC&, and 1 mM GTP, pH 6.7) as a function of time with different concentrations of tubulin. Similar experiments were done with 4 M glycerol, 10% Me$O, or 4 M plus 10% Me,SO present in buffer B. The results are expressed as the maximal change in absorbance under the different solvent conditions as a function of the tubulin concentration (Fig. 2). Extrapolations of the data to the abscissa yield the critical concentration, C I‘, of tubulin required for polymerization under each solvent condition. The requirement of a critical concentration of tubulin for polymerization suggests that the process occurs by propagation in which the monomeric unit of tubulin adds to the polymeric unit of microtubules (23). The reciprocal of the critical concentration is approximately equal to the apparent association constant for addition of tubulin to a microtubule. The apparent free energy of binding at 37°C was calculated from the apparent

0

0.4

BINDING

515

association constant. Values for these parameters as determined for the four different solvent conditions are given in Table II. The C,, 0.52 2 0.0’7mg tubulin/ml, determined in buffer is above the concentration of tubulin (0.32 k 0.14 mg/ml) estimated to be present in the crude supernatant fraction. Addition of glycerol decreases the C, to a value approximately equal to the tubulin concentration in the crude supernatant fraction. Addition of Me,SO alone or in combination with glycerol decreases the critical concentration to a value less than the tubulin concentration in the crude supernatant fraction. These results indicate, in part, why tubulin does not detectably ptilymerize in buffer alone and why the degree of polymerization in the presence of glycerol is low (7). If the concentration of tubulin in the crude supernatant fraction of bovine renal medulla could be increased by approximately 50%, tubulin may polymerize in buffer alone. This would require increasing the total soluble protein concentration of the crude supernatant

OS Tubulin

LIGAND

1.2

1.6

(mg/ml)

FIG. 2. Effect of solvent conditions on turbidity at different concentrations of tubulin. Tubulin was polymerized at 37°C in: (A) buffer (0.1 M Mes, 1 mM EGTA, 0.5 mM MgC&, and 1 mM GTP, pH 6.7); (B) buffer plus 4 M glycerol; (C) buffer plus 10% Me,SO; and (D) buffer plus 10% Me,SO and 4 M glycerol. Values on the ordinate are the maximal changes in absorbance at 350 nm at the concentration of tubulin noted on the abscissa. Inset: Light scattering at different concentrations of tubulin as a function of time at 37°C. The solvent was 0.1 M Mes, 1 mM EGTA, 0.5 mM MgCl*, and 1 mM GTP, pH 6.7. The protein concentrations were: (A) 1.3 mg/ml; (B) 2.6 mg/ml; (C) 3.4 mg/ml; and (D) 5.1 mg/ml.

516

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AND ROBERSON TABLE

II

PARAMETERSOFIN VITRO POLYMERIZATIONOFTUBULIN

K, x 10-Z' (liters/mol)

-AG"d (kcal/moll

0.52 k 0.07’ 0.32 k 0.03* 0.18 ‘- o.otx*

2.13 _t 0.30 3.47 k 0.38* 6.79 f_ 2.W

7.52 2 0.08 7.83 2 0.06* 8.21 A 0.25*

0.27 + 0.08*

4.36 2 1.43*

7.94 + 0.19*

cr6 Solvent conditions Buffer alone Buffer + 4 M glycerol Buffer + 10% Me,SO Buffer + 10% Me,SO + 4 M glycerol

FROM BOVINE RENALMEDULLA~

(mg tubulin/ml)

n Tubulin purified by four cycles of polymerization-depolymerization was used to measure the effect of solvents on in vitro polymerization. Polymerization was measured by the increase in absorbance at 350 nM at 37°C in buffer (0.1 M Mes, 1 mM EGTA, 0.5 mM MgC&, and 1 mM GTP, pH 6.7) with the indicated solvent. * C, is the critical concentration of tubulin required for in. vitro polymerization under the specified solvent conditions. c K, is the apparent association constant for addition of a monomeric tubulin unit to a polymeric microtubule unit. d AG” is the apparent free energy of microtubule propagation. e Values are mean + SD; N = 5 preparations of tubulin. * Data were analyzed by the group t test, and all values determined for buffer plus solvent were significantly different than values determined for buffer alone; P < 0.01 or less.

fraction from approximately 42 mg/ml to at least 63 mg/ml. Such a high protein concentration is difficult to achieve in a practical purification procedure. Microtubules assembled under different solvent conditions show different labilities with respect to low temperatures. Polymerization of tubulin at 37°C in buffer plus 1 mM GTP is a reversible process; cooling to 8°C causes an 85% decrease in turbidity (data not shown). Polymerization of tubulin in the presence of 10% Me$O and 4 M glycerol yields a microtubule preparation which is maximally depolymerized by 25% upon cooling (data not shown). Assembly of microtubules from tubulin polymerized under different solvent conditions was ascertained by electron microscopy. Purified tubulin was polymerized in buffer (0.1 M Mes, 1 MM EGTA, 0.5 mM MgSO,, and 1 mM GTP, pH 6.7) and in buffer containing 10% MezSO and 4 M glycerol. Figure 3 illustrates that the microtubules formed in the presence of 10% Me,SO and 4 M glycerol (Fig. 3B) are morphologically similar, as viewed in longitudinal orientation, to microtubules formed in the absence of these solvents (Fig. 3A).

Decay of Colchicine-Binding

Activity

The rate of decay of colchicine-binding activity of brain tubulin is dependent upon several parameters: tubulin concentration, pH, ionic strength, temperature, and the concentrations of ligands such as colchicine, GTP, and vinca alkaloids (5, 29). These parameters must be considered if the colchicine-binding activity of renal tubulin is used to study the role of microtubules in the action of vasopressin. The same concentration of renal medullary tubulin, 0.1 mg/ml, was used in all of the present binding studies to prevent changes in the rate of decay caused by variations in tubulin concentrations. The effect of GTP and vinblastine sulfate on the decay rate of colchicine binding was determined at 37°C. Tubulin was preincubated at 37°C in the absence and presence of GTP and vinblastine sulfate for various times and subsequently incubated with [3H]colchicine. Binding activity decreased with apparent first-order kinetics (Fig. 4). The rate constants, k, for decay of the unbound colchicine binding site are 0.136 h-l in buffer alone, 0.127 h-l in the presence of 0.1 mM GTP, and 0.064 h-l in

BOVINE

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LIGAND

BINDING

517

FIG. 3. Electron micrographs of renal medullary tubulin assembled in the absence and presence of 10% Me&SO and 4 M glycerol. (A) Longitudinal view of microtubules assembled in buffer (0.1 M Mes, 1 mM EGTA, 0.5 mM MgSO,, and 1 ItIM GTP, pH 6.7) alone. (B) Longitudinal view of microtubules assembled in buffer containing 10% Me,SO and 4 M glycerol. The magnification of both micrographs is 120,000 x and the length of the bar lines represents 0.1 pm.

the presence of 5 PM vinblastine sulfate as determined from the slopes of the lines (Fig. 4). The corresponding values for the half-time of decay are 5.1, 5.5, and 10.9 h, respectively. Extrapolations of the lines to include the 1.5-h incubation with colchicine yield the initial colchicine binding activities. The decay rate of binding activity was determined as a function of temperature. Tubulin samples were preincubated at 20, 25,30, and 3’7°C in the absence or presence of vinblastine sulfate for different periods of time. Samples were subsequently incubated with [3H]colchicine at the preincubation temperatures. Values for the rate constants of decay were calculated from apparent first-order graphs, and these values as a function of temperature are depicted in Arrhenius plots (Fig. 5). The energies of activation for the decay in the absence and presence of vinblastine sulfate were calculated from the slopes of the Arrhenius plots. The thermodynamic parameters of the activation process, AH+, AS, and AP, for decay were calculated from the

Arrhenius equation and equations from transition-state theory (30). Values of these parameters at 300°K are given in Table III. The 95% confidence limits for the slopes and ordinates of the Arrhenius plots and the significance of the differences between the slopes and between the ordinate intercepts were calculated (21). The slopes of the Arrhenius plots are not significantly different. Thus, the energies and enthalpies of activation of decay are not significantly different in the absence and presence of vinblastine sulfate. The difference in the intercepts on the ordinate was significant at the 0.005 level for a two-sided t test. The entropy of activation of decay is calculated from this intercept, which is the logarithm of the preexponential or Arrhenius factor. Thus, the entropies of activation of decay in the absence and presence of vinblastine sulfate are significantly different. The difference in the decay of colchicinebinding activity in the absence and presence of vinblastine sulfate is due to the difference in the entropy of activation.

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AND ROBERSON

0.017 and 0.015 h-l in the presence of 0.1 and 5 PM colchicine, respectively, and the corresponding values of tllz are 40.0 and 46.3 h, respectively (data not shown). Equilibrium Tub&in

PREINCUBATION

TIME (hr.1

FIG. 4. Effect of GTP and vinblastine sulfate on the decay rate of colchicine-binding activity at 37°C. Renal medullsry tubulin samples, 0.1 mg/ml, were preincubated at 3’7°C in buffer (50 mM sodium phosphate, 1 mM MgSOI, and 0.1 mM EGTA, pH 6.8) alone (O), in buffer plus 0.1 mM GTP (O), and in buffer plus 5 ELM vinblastine sulfate (0) for the times indicated. Samples were subsequently incubated with 5 pM [3H]colchicine at 3’7°C for 1.5 h. Extrapolation of the lines to include the 1.5 h incubation with colchicine yield the initial colchicine-binding activities.

Values for the half-times of decay were calculated from the rate constants, and these values are expressed as a function of temperature in the inset in Fig. 5. Binding activity is partially stabilized by decreasing the temperature and is further stabilized by the presence of vinblastine sulfate. The rate of decay of the binding activity is also affected by the presence of colchicine. Tubulin samples were incubated with 0.1 or 5 PM colchicine (the lowest and highest concentrations used in determination of the apparent binding constant) in the absence or presence of vinblastine sulfate for different periods of time to a maximum of 24 h at 37°C. Samples incubated with either 0.1 or 5 PM colchicine were incubated for an additional 5 h to reach a steady state of binding. The rate constant for decay of the bound colchicine site is 0.042 h-l in the presence of either 0.1 or 5 FM colchicine in the absence of vinblastine sulfate, and the corresponding half-time of decay is 16.3 h. In the presence of 5 PM vinblastine sulfate the rate constants of decay are

Binding of Colchicine

to

The rate of equilibration of the colchicinetubulin complex is slow, especially at low concentrations of tubulin and colchicine and at low temperatures (29, 31, 32). The incubation times required to reach maximal binding of 0.1 and 1.0 PM colchicine to renal medullary tubulin are shown in Fig. 6. At 37°C approximately 5.5 and 8 h are required to reach equilibrium of binding with 1.0 and 0.1 PM colchicine, respectively, in both the absence and presence of vinblastine sulfate. Values for the amount of colchicine bound were corrected for decay using t 1,2 values of 16.3 and 40.0 h in the absence and presence of vinblastine sulfate. If corrections for decay are not made, the maximal amounts of colchicine are bound between 5.5 and 8 h and then decrease with increasing time of incubation. Approximately 24 h is required to reach maximal binding at 20°C with 0.1 PM colchicine in the presence of 5 PM vinblastine sulfate (data not shown). The equilibrium constants for the binding of colchicine were determined at 37°C in the absence and presence of 5 PM vinblastine sulfate. Tubulin samples were incubated with different concentrations of [3H]colchicine for 9 h. Amounts of bound colchicine were corrected for decay of binding activity which occurred during incubation. The data were analyzed as Scatchard plots (Fig. 7). The values of the apparent binding constants, K,, were determined from the slopes of the lines, and the stoichiometry of binding was determined from the intercepts on the abscissa. Values of the apparent binding constants were 5.9 2 0.7 x lo6 and 7.8 +- 0.3 x lo6 M-’ in the absence and presence of vinblastine sulfate, respectively. (Values are the means and standard deviations of three determinations; P < 0.02.) About 0.33 and 0.38 mol of colchicine are bound per mole of tubulin in the absence and presence of vinblastine sulfate, respectively

BOVINE

I

RENAL

MEDULLARY

I

I

3.20

3.25 f

TUBULIN

LIGAND

I

I

3.30

3.35

(103x

BINDING

I 3.40

519

I

OK-‘)

FIG. 5. Arrhenius plots of the rates of decay of colchicine-binding activity as a function of temperature in the absence and presence of vinblastine sulfate. Renal medullary tubulin samples, 0.1 mg/ml, were preincubated at 20,25,30, and 37°C in the absence (0) or presence (0) of 5 pM vinblastine sulfate. Samples were subsequently incubated with 5 pM [3H]colchicine for 4.5 h at 20 and at 25”C, 3.0 h at 30°C and 1.5 h at 37°C. The rate constants were determined from slopes of the apparent first-order decay curves (See Fig. 4). Inset: Half-time of decay of colchicine-binding activity as a function of temperature. Renal medullary tubulin samples, 0.1 mg/ml, were preincubated at 20, 25, 30, and 37°C in the absence (0) or presence (0) of 5 FM vinblastine sulfate. Samples were subsequently incubated with 5 pM [3H]colchicine for 4.5 h at 20 and at 25°C; 3.0 h at 30°C; and 1.5 h at 37°C. Values of the half-time of decay at each temperature were determined from the corresponding rate constants for decay.

(not statistically different). The method of isolation of the colchicine-tubulin complex (5, 10, 33), nonoptimal assay conditions, e.g., the small amount (20 pg per assay) of renal tubulin used, and loss of binding capacity during purifkation probably account for the low stoichiometry. Similar stoichiometric values for tubulin from other sources have been reported (34, 35). Effects of Lumicolchicine and Podophyllotoxin on the Binding of Colchicine

The binding of different concentrations of colchicine was measured in the presence of 50 PM lumicolchicine. This concentration of lumicolchicine was lo-fold larger than the

highest concentration of colchicine. An apparent dissociation constant, Kd, of ‘2.9 x lo-’ M was determined for colchicine binding at 37°C for 3 h, and the apparent competitive inhibition constant, Ki, for lumicolchicine was 4.5 x 10e4 M (Fig. 8). Podophyllotoxin appears to competitively inhibit the binding of colchicine to renal medullary tubiilin (Fig. 8). Both the maximal amount of colchicine bound and the apparent binding constant are decreased in the presence of podophyllotoxin. The effect on the amount of colchicine bound is probably related to the decay of colchicine-binding activity rather than a reflection of mixed type of inhibition. Since colchicine decreases the rate of decay, displacement of colchicine by podophyllotoxin may increase the rate of

520

BARNES

AND ROBERSON TABLE

III

THERMODYNAMIC PARAMETERS OF THE ACTIVATION OF DECAY OF THE COLCHICINE-BINDING ACTIVITY” Solvent conditions

E,* (kcal/mol)

AHfC (kcaYmo1)

ASd (cal/mol deg)

AGZe (kcal/mol)

Buffer (50 mM sodium phosphate, 1 mM MgS04, and 0.1 mM EGTA, pH 6.8)

15.3

14.7

31.6

24.1

Buffer + 5 PM vinblastine sulfate

13.2

12.6

39.6*

24.5

a The experimental conditions for measurement of the decay of the colchicine-binding activity in the absence and presence of vinblastine sulfate are described in the legend for Fig. 5. The values of AH+, AS, and AG+ were calculated at a temperature of 300°K. b E, was calculated from the slopes of the Arrhenius plots (Fig. 5). = AH+ = E, - RT. d AW2.303R = log A - log EkTIH, where A is the preexponential factor determined from the intercept on the ordinate of the Arrhenius plots (Fig. 5). e AG+ = AH+ - TAS. * Significantly different from AS+ in buffer, P < 0.005. See text for statistical analysis.

decay and decrease the apparent binding capacity. The apparently competitive inhibition constant, KI, for podophyllotoxin is 4.0 x 10e7 M at 3’7°C (not significantly different from the Kd for colchicine). DISCUSSION

Bovine renal medulla has one of the lowest tissue levels of tubulin for which purification by in vitro polymerization and depolymerization has been achieved. Both Me,SO and glycerol are necessary to enhance polymerization at the concentration of tubulin, a minimum of approximately 1% of the total soluble protein, present in bovine renal medulla. These solvents may permit purification of tubulin from other non-neural tissues which have a low content of tubulin. Glycerol has been used in the isolation of tubulin from non-neural sources such as Drosophilia (36), porcine platelets (37), bovine anterior pituitary (38), Ehrlich ascites tumor cells (39), and cultured 3T3 cells (40). Generally the amount of tubulin is between 2 and 5% of the soluble protein in these different sources. Dimethylsulfoxide and glycerol increased the extent of polymerization of tubulin in the crude supernatant fraction of bovine renal medulla by decreasing the apparent free energy for polymerization which is reflected by a decrease in C,. Margolis

and Wilson (41, 42) have proposed that assembly and disassembly of microtubules occur at opposite ends of the microtubule. The interaction of tubulin with a microtubule is not a true equilibrium in their model. Thus, calculation of the free energy of nolvmerization under different solvent conditions yields apparent values. Although

i

30

e

2.5

I 811 2

1.0

f E o.5 0

4

8

12

TIME hl

FIG. 6. Time course of binding of colchicine. Renal medullary tubulin, 0.1 mg/ml, samples were incubated at 37°C in the absence (closed symbols) or presence (open symbols) of 5 PM vinblastine sulfate and in the presence of 0.1 PM [3H]colchicine (round symbols) or 1.0 PM [3H]colchicine (square symbols) for the indicated periods of time. The amounts of colchicine bound were corrected for decay using half-times of 16.3 and 40.0 h in the absence and presence of vinblastine sulfate, respectively.

BOVINE RENAL MEDULLARY

FIG. 7. Scatchard plots of the binding of colchicine. Renal medullary tubulin samples, 0.1 mg/ml, were incubated with different concentrations of labeled colchicine for 9 h at 3’7°Cin the absence (closed circles) and in the presence (open circles) of 5 pM vinblastine sulfate. Data were corrected for the decay of binding activity using t% values of 16.3 and 40.0 h in the absence and presence of vinblastine sulfate, respectively.

the changes in C, under different solvent conditions are small, the values reflect the fact that crude tubulin does not polymerize in buffer alone and minimally polymerizes in the presence of glycerol. Relatively high concentrations of Me,SO are required to increase polymerization which indicates that it may act analogously to glycerol (43). Values of C, for brain tubulin range from 0.2 mg tubuWml(44) to 8 k 1 mg tubulin/ml (43) depending upon the solvent conditions used for polymerization. Values of C, for renal tubulin (Table II) are similar to the lower values of C, for brain tubulin. However, the decrease in C, is probably not the entire basis for the increased polymerization in the presence of Me&SO and glycerol. The lowest value of C, was obtained with purified tubulin in 10% MezSOalone, yet tubulin will not detectably polymerize from the crude supernatant fraction in the presence of 10% Me&SO without glycerol. Himes et al. (45) demonstrated that purified bovine brain tubulin will also polymerize in 10% Me&O. Renal medullary extracts may contain endogenous inhibitors of tubulin polymerization as has been demonstrated for other non-neural tissues (46, 47). Nagle et al. (48) found that several different hinds of cultured cells contain endogenous inhibitors of tubulin polymerization, and that glycerol decreased the effects of these inhibitors. Together

TUBULIN

LIGAND BINDING

521

FIG. 8. Effect of podophyllotoxin and lumicolchicine on the binding of colchicine. Renal medullary tubulin samples, 0.1 mg/ml, were incubated with different concentrations of [3H]colchicine at 37°C for 3 h in the absence (0) or in the presence of 50 PM lumicolchicine (X); 0.25 pM podophyllotoxin (O), or 0.50 pM podophyllotoxin (0).

Me&SO and glycerol may decrease the interaction between tubulin and possible endogenous inhibitors in the renal medulla. This idea is supported by the observation that podophyllotoxin does not inhibit in vitro polymerization of purified renal medullary tubulin in the presence of these solvents, but completely inhibits polymerization in their absence.* Polymerization of bovine renal medullary tubulin in Me,SO and glycerol occurs in the absence of specific microtubule-associated proteins. Some minor proteins are present after four cycles of polymerization-depolymerization of medullary tubulin, but we have not observed a consistent pattern of these proteins from preparation to preparation. Multiple, minor proteins have been reported with preparations of brain tubulin (49,50). Purified brain tubulin will polymerize under different solvent conditions in the absence of microtubule-associated proteins (45,51,52). Clearly, microtubule-associated proteins are not absolutely required for in vitro polymerization of tubulin from kidney and brain. Purified renal medullary tubulin polymerized in buffer plus GTP yields microtubules which have morphologic characteristics similar to cytoplasmic microtubules * G. M. Roberson and L. D. Barnes, unpublished results.

522

BARNESANDROBERSON

(27, 53). Thus, purification of renal tubulin in the presence of Me,SO and glycerol does not deleteriously affect polymerization in the absenceof these solvents. Polymerization in the presence of Me,SO and glycerol also yields intact, normal microtubules. Under these conditions we also observed a few microtubules with regions of protofilaments unrolled into sheets. Electron micrographs of tubulin from different sources polymerized in the presence of glycerol often depict normal and abnormal forms (54, 55), and the number of protofilaments per microtubule generally increases with increasing numbers of polymerizationdepolymerization cycles (55). Binding of colchicine to tubulin purified from bovine renal medulla is temperature dependent, time dependent, decays with apparent first-order kinetics, is apparently competitively inhibited by podophyllotoxin, and is negligibly affected by lumicolchicine. The rate of decay is decreased by vinblastine sulfate and colchicine itself. These characteristics of colchicine binding, described by Wilson and Bryan (5), demonstrate that complex formation occurs between colchicine and tubulin rather than another protein of bovine renal medulla. The half-time of decay of colchicinebinding activity of bovine renal medullary tubulin, 5.1 h, is similar to values for tubulin from chick embryo brain (12), sea urchin egg (32), Droso@iZia embryo (36), rat brain (56), and sea urchin outer doublet (57) which range from 2.8 to 6.6 h. Vinblastine sulfate at 5 pM caused a twofold increase in stabilization of binding activity of renal medullary tubulin. Wilson found a threefold increase in stabilization of binding activity of chick embryo brain tubulin in the presence of 310 pM vinblastine sulfate, and the degree of stabilization was dependent upon the concentration of vinblastine sulfate (12). In contrast to reports (10, 12) that GTP causesa two- to threefold increase in stability of binding activity of brain tubulin, we have found that 0.1 mM GTP does not significantly stabilize the binding of activity of renal medullary tubulin. The energies of activation, E, and the enthalpies of activation, AH*, for the decay of the unbound colchicine-binding site are

similar in the absence and presence of vinblastine sulfate. The entropy of activation, AS, in the presence of vinblastine sulfate is 8 cal/mol deg more negative than the entropy of activation in the absence of vinblastine sulfate. In the presence of vinblastine sulfate a stabilizing conformational change has occurred in the tubulin or the solvent is more ordered. Either of these more ordered states must be traversed to reach the transition state for the decay process. The larger negative entropy of activation in the presence of vinblastine sulfate than in its absence is the basis for the lower rate of decay in the presence of vinblastine sulfate. The rate of decay of activity of the colchicine-bound tubulin is about 3.5-fold less than the rate of decay of the unbound colchicine binding site at 37°C. The degree of stabilization of colchicine is about the same in the absence and presence of vinblastine sulfate. In the absence of vinblastine sulfate, the rate of decay of the bound site is constant over a 50-fold concentration range of colchicine. In the presence of vinblastine sulfate the rate of decay was slightly less with 5 pM colchicine than with 0.1 pM colchicine. Colchicine increases the stability of binding activity of tubulin from chick embryo brain (12) and rat brain (56) by about 1.5- and 2.5-fold, respectively. Values of the apparent colchicine binding constant, K.,,, obtained at an equilibration time of 9 h were 7.8 ? 0.3 x lo6 and 5.9 * 0.7 x lo6 M-' at 37°C in the presence and absence of 5 pM vinblastine sulfate, respectively. These values are slightly greater than other values, which range from 0.25 to 2 x lo6 M-', reported for tubulin from rat brain (56, 58), chick embryo brain (29), sea urchin egg (32), bovine thyroid (59), and porcine brain (60). Observed differences in values of KA probably reflect intrinsic differences in tubulins and differences in conditions and methodology of the binding reaction (61). Vinblastine sulfate increased the apparent colchicine binding constant and decreased the rate of decay of the colchicine-binding activity of renal tubulin. In contrast studies with tubulin from other sources suggest that vinblastine sulfate only affects the rate of decay and has no effect

BOVINE

RENAL

MEDULLARY

on the apparent affinity of colchicine binding (12, 32). The difference in results may be related to the source of tubulin or changes in the tubulin dimer which may occur during isolation in the presence of Me,SO and glycerol. Colchicine inhibits the action of vasopressin in rats when administered at 0.4 to 0.5 mg/kg body wt (3, 62). This dosage corresponds to a concentration of about 1.5-2.5 PM colchicine assuming distribution throughout the total body water. In acute (62) and chronic (3) experiments involving injection of colchicine into rats for 3 h and 3 days, respectively, the initiation of inhibition of vasopressin action is time dependent. Assuming that the colchicine-binding properties of rat renal medullary tubulin are similar to those of bovine renal medullary tubulin, the time dependence and K, of colchicine binding determined in vitro support the results obtained in vivo. The present biochemical studies complement the physiologic evidence that microtubules are required for the function of vasopressin in mammalian kidneys. We have identified and characterized the ligand-binding properties of tubulin in renal medulla. The colchicine-binding activity of renal medullary tubulin could now be used to determine indirectly if changes occur in the degree of microtubule polymerization in mammalian renal medulla during the response to vasopressin provided conditions which affect the rate of decay are considered. ACKNOWLEDGMENTS We appreciate the critical comments by Dr. Robert F. WiUiams and Preston N. Garrison. Mrs. Kathleen H. Doyle kindly performed the electron microscopy and Dr. Phillip Serwer prepared the grids for electron microscopy. Support was provided to Dr. Larry D. Barnes from National Institutes of Health General Research Support Grant 069000172-11 to The University of Texas Health Science Center at San Antonio. Dr. Jay H. Stein generously provided support from Grant AM 17387-04 from the National Institute of Arthritis, Metabolism, and Digestive Diseases. REFERENCES 1. TAYLOR, A., MAFFLY, R., WILSON, L., AND REAVEN, E. (1975) Ann. N.Y. Acad Sci. 253, 723-737.

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BINDING

523

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