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Sulfhydryl groups of Mimosa pudica tubulin implicated in colchicine binding and polymerization in vitro
ARCHIVES
Vol.
OF BIOCHEMISTRY
294, No. 2, May
AND
BIOPHYSICS
1, pp. 353-360,1992
Sulfhydryl Groups of Mmosa pudica Tubulin Implicated in Colchicine Binding and Polymerization in vitro Asis
Roychaudhuri
Department
Received
of Biochemistry,
August
and Susweta Bose
Institute,
8, 1991, and in revised
form
Biswasl Centenary
December
Building,
700 054.
Zndia
26, 1991
The important characteristic of novel Mimosa pudica tubulin is its ability to bind colchicine only when dithiothreitol is included in the isolation buffer, indicating the involvement of sulfhydryl groups in colchicine binding. Modification of sulfhydryl groups by a sulfhydryl modifying agent also affects the normal assembly of tubulin into microtubules, as revealed by electron microscopic and spectrophotometric studies. The number of free sulfhydryl groups present in tubulin protein responsible for both colchicine binding and polymerization has been found to be 4, distributed in a! and /I subunits, and is distinctly different from the number reported for animal tubulin. 0 1992 Academic Press, Inc.
Microtubules (MTs)’ from brain and flagellar outer fibers contain free sulfhydryl groups which help the polymerization and colchicine binding to tubulin of microtubular structure (l-3). In fact, several reports have been published proposing the importance of sulfhydryl groups for maintaining the stability and conformation of MTs also (2,4-6). Organic sulfhydryl blocking agents like Nethylmaleimide (NEM), p-chloromercuribenzenesulfonic acid (PCMS), and some other mercury compounds are known to inhibit the polymerization (1, 2, 7) as well as colchicine binding of animal tubulin (2). The inhibitory effect of oxidation of free sulfhydryl groups of tubulin from brain and flagella on polymerization and colchicine binding was studied (1, 2, 7, 8). As in animal cells, microtubules, are believed to fulfill several important functions in plant cells. But studies on the tubulin-microtui To whom correspondence should be addressed. * Abbreviations used: MTs, microtubules; NEM, N-ethylmaleimide; PCMS, p-chloromercuribenesulfonic acid; Pipes, 1,4-piperazinediethanesulfonic acid; PMSF, phenylmethylsulfonyl fluoride; EGTA, ethylene glycol bis(B-aminoethyl ether) NJ’-tetraacetic acid; DTNB, 5,5’-diothiobis-(2nitrobenzoic acid, SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis; DTT, dithiothreitol.
0993-9861/92 53.09 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Calcutta
bule system of higher plants appear to be difficult due to the low content of tubulin and the low affinity to colchitine. Mimosa pudica, both leaves and callus cells raised from pulvini, contains an appreciable amount of this protein which is distinctly different from that of other sources (9, 10). None of the available data concerning the involvement of -SH groups in the assembly and colchicine binding of plant tubulin, however, have so far been presented. Since M. pudica tubulin has been found to be different immunologically not only from animal tubulin but also from tubulin from different plant sources, the purpose of the present study is to ascertain the number and role of sulfhydryl groups on polymerization and colchicine binding of M. pudica tubulin. MATERIALS
AND METHODS
Chemicals. Pipes, GTP (type IIS), 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB), fluorescine-5-maleimide, N-ethylmaleimide, urea, dithiothreitol, [3H]colchicine, N-[‘4C]ethylmaleimide, and colcemid were purchased from Amersham International plc. Purification of tubulin protein from M. pudica pulvinar callus tissue. Pulvinar callus tissues generated from second and third leaves of M. pudicu plants were used throughout the experiment (10). Tubulin was prepared essentially by extracting 50 g of callus cells with a 1:l ratio of isolation buffer (50 mM Pipes-NaOH, pH 6.9, 1 mM MgC&, 1 mM dithiothreitol, 0.5 mM GTP, 1 mM PMSF, 1 PM pepstain A, 1 mM EGTA) according to the method described (10). The homogenate was centrifuged at 100,OOOg for 1 h. The suparnatant was treated with DEAESephadex A-50, in the ratio of 3 mg protein/ml of DEAE-Sephadex. Unabsorbed washings were subjected to O-55% ammonium sulfate saturation and desalted by passage through a Sephadex G-200 column (1.4 X 45 cm), previously equilibrated in the isolation buffer without DTT, and proteins were eluted by the same buffer, 2 ml/tube at a flow rate of 6.5 ml/h. Tubulin-enriched fractions were eluted in tubes 15-17 (volume 30-34 ml). Protein concentration was determined according to Bradford’s dye binding method (11) after precipitation of tub&n by 100% acetone. Bovine serum albumin was used as standard. Determination of free sulfhydryl groups. DTNB was used as a tool to determine the number of sulfhydryl groups of tubulin quantitatively (12). A stock solution of 10 mM DTNB in 50 mM Pipes buffer, pH 7.0, was prepared and the protein was taken in 50 mM Pipes buffer, pH 8.0, containing 1 mM MgCIZ and 1 mM EGTA. A 20-fold excess concentration of DTNB was added to 10m5 M tubulin and the mixture was incubated
353
ROYCHAUDHURI
354 TABLE
I
RESULTS
Effect of Dithiothreitol and Sulfhydryl Modifying Agent DTNB on M. pudica Tubulin-Colchicine Binding Reaction 1.
Tubulin
solution containing colcbicine but
+ Pipes and colcbicine 3. Tubulin + Pipes + DTNB
Effect of dithiothreitol and DTNB. M. pudica tubulin has been shown to bind colchicine appreciably in the presence of sulfhydryl reducing agents such as dithiothreitol. Results of the [3H]colchicine tubulin binding assay using GF/C filter disks are presented in Table I. This binding assay was done before and after modification of the free sulfhydryl groups of tub&n with the specific reagent DTNB. Colchicine binding affinity to M. pudica tubulin is found to be lowered in the absence of DTT in isolation buffer, suggesting the presence of free sulfhydryl group or groups taking part in colchicine tubulin binding.
cpm mg of protein-’ 0.22 0.28 2.44 2.50 0.35 0.37
no DTT
2. Tubulin
buffer
containing
buffer
+ DTT
DTT + colcbicine
x x X X x x
10’ 10’ 10’ 10’ 10’ 10’
Note. 0.5 ml of M. pudica tubulin (5 PM) solution in 50 mM Pipes buffer, pH 6.9, (1) containing no dithiothreitol in the isolation buffer, (2) containing 1 mM ditbiothreitol in the isolation buffer, or (3) preincubated with 20 X lo-’ M DTNB at O’C for 1 h was incubated with [3H]colchicine (1 PM) containing 25,000 cpm at 37°C for 1 h and assayed by the GF/C filter disk binding method.
at O’C for 1 h. The absorbance of the solution was measured at 412 nm against a blank without protein. An extinction coefficient of 13,600 M-’ cm-’ was used to calculate the concentration of reacting sulfhydryl groups. Fluorescine-5-maleimide was used to determine the number of sulfhydryl groups of tubulin quantitatively (13). A stock solution of 13 mM fluorescine-5-maleimide in 50 mu Pipes, pH 7.0, containing 1 mM MgClx and 1 mM EGTA was prepared and a 20-fold excess concentration was added to 10m5 M tubulin; the mixture was incubated at O’C for 1 h and dialyzed extensively against 50 mM Pipes, pH 7.0, and the optical density at 492 nm was measured. An extinction coefficient of 75,000 M-’ cm-’ was used to calculate the concentration of sulfhydryl groups present. Colchicine binding. tubulin were assayed
AND BISWAS
The colchicine binding reactions of M. pudica by the GF/C filter disk method (14).
Polymerization assay. Microtubule assembly was monitored turbidometrically at 37°C using Shimadxu ultraviolet visible recording spectrophotometer UV-160 as described by Gaskin et al. (15). Electron microscopy. Specimens for electron microscopy were made as described by Banejee et al. (16).
Presence of sulfhydryl group. The sulfhydryl groups of plant tubulin isolated from M. pudica were determined by using Ellman’s reagent (DTNB) and fluorescine-5maleimide (Table IIA). From absorption studies at 412 nm, E = 1.36 X lo4 M-l for DTNB and 492 nm, E = 75,000 M-’ cm-’ for fluorescine-5-maleimide, we recorded the presence of four -SH groups in M. pudica tubulin. Time-dependent modification of thiol groups of M. pudica tub&in by Ellman’s method. The kinetic study of the modification of thiol groups of M. pudica tubulin (Fig. 1) showed that one thiol group was completely modified within 5 min, and the remaining three thiol groups were modified sequentially with an increase of time. The number of thiol groups was determined by using the standard molar extinction coefficient for Ellman’s reaction (E = 1.36 X lo4 M-l cm-‘). This was shown categorically also (in the inset) where the number of thiol groups blocked was plotted against time. It is apparent from the data thus far presented that one thiol group is more highly exposed to the outer environment than the other thiol
TABLE Determination
of Free
Sulfhydryl
Groups
of
M. pudica
Tubulin
IIA by Ellman’s
Method
and
the Fluorescine Molar
Optical Reaction 1. 2. 3. 4. 5.
50 mM Pipes buffer, 50 mM Pipes buffer, 50 mM Pipes buffer, 50 mM Pipes buffer, 50 mM Pipes buffer, + fluorescine-5-maleimide
pH pH pH pH pH
8.0 8.0 8.0 7.0 7.0
+ + + + +
DTNB tubulin tubulin tubulin tubulin
+ DTNB
density
412 nm
492 nm
0.21 0.04 0.82 -
0.125
Resultant optical density
Labeling
Method
extinction
coe5cient
(E) in
(lo-‘M)
412 nm
492 nm
412 nm
492 nm
Number of SH groups
0.57
3.299
0.136
0.75
4
3.424
Note. 10 pM tubulin in 50 mM Pipes buffer, pH 8.0, was incubated with an excess concentration of DTNB (20 X lo-’ M) at O’C for 1 h, and maximum absorbance at 412 nm was measured against a blank lacking protein and DTNB. An extinction coefficient of 13,600 M-’ cm-’ was used to calculate the concentration of sulfhydryl groups reacted. For determining the sulfhydryl groups by the fluorescine-5-maleimide method, 10 pM tubulin in 50 mM Pipes buffer, pH 7.0, was incubated with an excess concentration of fluorescine-5-maleimide (20 X lo-’ M) at 0°C for 1 h and dialyzed extensively with three changes; the absorbance at 492 nm was measured against a blank lacking fluorescine-5-maleimide. An extinction coefficient of 75,000 M-’ cm-’ was used to calculate the number of sulthydryl groups reacted.
-SH
GROUPS
IN
Mimosa
pdca
355
TUBULIN
1
1
I
1
I
I
I
I
4
I
5
IO
15
20
25
30
35
CO
45
50
TIME lmin
15
20
25
30
35
40
45
I
55
I
50
55
60
Timelmin) FIG. 1. M. ptdica tubulin (10 pM or 10e5 M) in 50 mM Pipes buffer, pH 8.0, was incubated with an excess concentration of DTNB (20 X 10m5 M) at 25°C and the change in absorbance at 412 nm against time was monitored. The number of thiol groups was determined by using the standard molar extinction coefficient of DTNB at lo-’ M (E = 1.36 X 10’ M-l cm-‘). Inset: The number of thiol groups calculated from the main figure was plotted against time to show the time-dependent modification categorically.
groups, as modification of one thiol group occurs within 5 min. The sulfhydryl groups of this tubulin in the denatured state using 6 M urea were also determined with the same method. The results are shown in Table IIB. We have found that four sulfhydryl groups of tubulin are present also in the denatured state. The results suggest that all four sulfhydryl groups of M. pudica tubulin are exposed and not buried. Selective modification of -SH groups of M. pudica tubulin by Ellman’s reagent (DTNB) was done for [3H]colchicine-tubulin binding. The results depicted in Fig. 2 show that approximately 50-55% of colchicinetubulin binding is lowered after modification of one sulfhydryl group of tubulin compared with unmodified tubulin-colchicine binding activity. Further modification of
TABLE Determination
of Sulfhydryl Groups
Reactions 50 mM Pipes, pH 8.0 + urea + DTNB 50 mM Pipes + tubulin 50 mM Pipes + tubulin + urea + DTNB
of M.
Optical density at 412 nm 1.16 0.05 1.75
-SH groups leads to a decrease in tubulin-colchicine binding. From these results it is confirmed that free sulfhydryl groups of M. pudica tubulin are responsible for colchicine binding. The sulfhydryl modifying agent, fluorescine-5-maleimide, was used to determine the distribution of free SH groups between (Yand fl subunits. After modification of four free -SH groups (determined through absorption at 492 nm and E = 75,000 M-l cm-‘) of tubulin by fluorescine-5-maleimide, it has been found that on SDSPAGE, both subunits (Yand p fluoresce (Fig. 3), showing modification of the four -SH groups present in both subunits; however, greater fluorescence is exhibited by the fl subunit. Next, an experiment was performed to show the distribution of sulfhydryl groups present in (Yand /? subunits of tubulin using N- [ 14C]ethylmaleimide. The results
IIB
pudica Tubulin in the Denatured State by Ellman’s Method Resultant
optical density at 412 nm
0.54
Molar extinction coefficient, z (10m5 M)
Number of SH groups
0.136
4
Note. 10 pM M. pudica tubulin in 50 mM Pipes buffer, pH 8.0, was dialyzed against the same buffer containing 6 M urea at 4’C for l-2 h followed by incubation with an excess concentration of DTNB (20 X lo-’ M) at 0°C for 1 h; absorbance at 412 nm was measured against a blank lacking protein and DTNB. An extinction coefficient of 13,600 M-l cm-’ was used to calculate the number of sulfhydryl groups reacted.
356
ROYCHAUDHURI
AND
BISWAS
E :3 1.6Jz Y ; 1.2 “x s 0.8E HO.& 0.01
I 1SH
I 2SH
I 3SH
I LSH
No. of free -SH groups modified --c FIG. 2. Inhibition of tubulin colchicine binding by DTNB. M. pudica tubulin (10 PM) was preincubated with 22,45,92, or 135 pM DTNB at 0°C for 1 h. At 22 pM DTNB, the one -SH group per tubulin molecule was blocked by DTNB. The number of -SH groups was measured by Ellman’s method and the solution was incubated with [‘Hlcolchicine (1 PM) at 37’C for 1 h, and tubulin colchicine binding was assayed by the GF/C filter disk binding method.
depicted in Table III show the unequal distribution of sulfhydryl groups which support the earlier observation using fluorescine-5-maleimide. These results taken together indicate the presence of sulfhydryl groups in (Yand /3 subunits apparently in the ratio 1:3. The colcemid or colchicine binding site in tubulin was determined by binding lop5 M tubulin with 10e6 M colcemid at 37°C for 1 h, labeling first with 20 X low5 M unlabeled N-ethylmaleimide to modify the unmasked (not masked by colcemid molecule) thiol groups (if present), and then dialyzing against 50 mM Pipes buffer, pH 7.0, containing 6 M guanidine-HCl to dissociate the colcemid molecule from tubulin. Colcemid-masked thiol groups were now exposed to the outer environment. Fluorescine5maleimide (20 X lop5 M) was then added to modify the exposed -SH groups. From SDS-PAGE (Fig. 4) it has been found that both subunits ((u and p) fluoresce, indicating that both (Y and 6 subunits are responsible for colchicine binding. As a control, the native tubulin is treated with unlabeled NEM in the absence of colcemid and the tubulin solution is dialyzed against 50 mM Pipes buffer to denature the tubulin. containing 6 M guanidine-HCl The denatured protein is treated with fluorescine-5-maleimide and run on SDS-PAGE. No labeling of tubulin with fluorescine-5-maleimide occurs. It was also found that after colcemid binds with the tubulin molecule, no SH groups could be detected by Ellman’s reaction. So it is apparent all four -SH groups are involved in colcemid-
FIG. 3.
Modification of four sulfhydryl groups of tubulin by fluorestine-5-maleimide. Tubulin (10 PM) was incubated with 200 pM fluorestine-5maleimide at O’C for 1 h, followed by extensive dialysis with three changes against 50 mM Pipes buffer, pH 7.0. The dialysate was loaded on 10% SDS-PAGE, pH 8.8. After the run, the gel was excited at 280 nm and fluorescence recorded. Lane 1: 40 pg of -SH-modified M. pudka tubulin. Lane 2: 30 pg of Coomassie brilliant blue-stained M. pudica tubulin.
tubulin binding. Here, the concentration of tubulin and colcemid used was 10 PM. Inhibition of tubulin polymerization. The inhibition of tubulin polymerization by addition of the sulfhydryl modifying agent N-ethylmaleimide showed the sequential decrease in turbidity of tubulin polymerization (Figs. 5BE) compared with unmodified tubulin assembly (Fig. 5A). It was further supported by electron microscopic observation (Figs. 6A and B) in the absence and presence of
TABLE Distribution in
M
III
of N- [ “C]Ethylmaleimide pudica LY- and &tubulin
Concentration of M. pudica tubulin Concentration of IV-[i4C]ethylmaleimide cm Blcpm a Number of -SH groups (Y subunit fl subunit
~PM 200 pM 2.8 1
3 bwrox)
Note. Mimosa pudica tubulin was incubated with N-[“Clethylmaleimide in 50 mM Pipes buffer, pH 6.9, containing 1 mM MgClx and 1 mM EGTA for 1 h at 4°C followed by extensive dialysis against the same buffer to remove the free N-[i4C]ethylmaleimide. (r and fl subunits of M. pdca tubulin were separated in SDS-PAGE and each subunit was eluted separately.
-SH
GROUPS
IN
Mimosa
=“p
FIG. 4. Determination of colchicine or colcemid binding sites in M. p&co. Tubulm (10 pM) was preincubated with cold colcemid (colchicine analog, 1 pM) at 37’C for 1 h, followed by addition of an excess amount of unlabeled N-ethylmaleimide (20 X 10m5 M) and incubation at 0°C for 1 h. The solution was dialyzed extensively against 50 mM Pipes buffer, pH 7.0, containing 6 M guanidine-HCl. Finally, an excess concentration of fluorescine-5-maleimide (20 X 10m5 M) was added and the solution was incubated at 0°C for 30 min. The reaction mixture was loaded on 10% SDS-PAGE (pH 8.8). After the run, the gel was excited at 280 nm, and the fluorescence of both the a and fl subunits was recorded. Lane 1: Control. Lane 2: 18 pg of fluorescine-5-maleimide-labeled M. pudica tubulin.
an excess concentration of N-ethylmaleimide, respectively. of the specific sulfhydryl group in M. pudIdentification ica tubulin that determines the high affinity of the tubulincolchicine complex. To identify the location of the specific sulfhydryl group of M. pudica tubulin that determines the high affinity of the tubulin-colchicine complex and polymerization, 1O-5 M or 10 PM tubulin solution was reacted with an excess concentration of fluorescine-5-maleimide, (20 X lOA M), at 0°C for 2 min, and the reaction was stopped immediately by addition of an excess conAfter that, the recentration of dithiothreitol (1 mM). action mixture was loaded and run on 10% SDS-PAGE. After a complete run, (Yand /3labeled tubulin were eluted and protein concentration was measured by the Bradford method. It has been found fluorometrically that the p subunit of M. pudica tubulin gives maximum fluorescence at 520 nm compared with control and a-tubulin (Fig. 7). From this result, it is apparent that the high-affinity sulfhydryl group that participates in tubulin-colchicine binding is present in the fl subunit of M. pudica tubulin. DISCUSSION
The unique property of tubulin isolated from M. pudica callus is that it is basic in nature and binds appreciably with 10e6M colchicine or its analogs (10).
pudica
357
TUBULIN
If tubulin is isolated in the absence of the sulfhydryl reducing agent dithiothreitol, its affinity for binding to colchicine is reduced compared with tubulin isolated in the presence of dithiothreitol (Table I). The same observation was reported by Mizuno et al. (17). In the case of plant tubulin, it might be due to the presence of many aromatic secondary metabolites which, during isolation, oxidize to form quinone-like structures and bind with free sulfhydryl groups of cysteine residues present in tubulin protein. Dithiothreitol is therefore added to protect the -SH groups from secondary metabolites. Moreover, in the tubulin purification step, the binding affinity is not decreased when DTT is not added during the ammonium sulfate precipitation and gel filtration step because of the earlier removal of secondary metabolites during extractions. To support this initial finding, the purified tubulin is pretreated with the -SH modifying agent DTNB and the colchicine-tubulin binding reaction is allowed to proceed. It has been found that the colchicine binding reaction with modified tubulin is reduced compared with unmodified tubulin-colchicine binding (Table I). Therefore, it may be suggested that free -SH groups are responsible for tubulin-colchicine binding. The results of the determination of free -SH groups of M. pudica tubulin (Table IIA) by the Ellman and fluorescine+maleimide labeling methods showed that the tubulin molecule (with cx and /3 subunits) contains four free -SH groups that are exposed to the outer environment and not buried. This has also been proved by denaturing the tubulin with 6 M urea followed by DTNB-tubulin reaction as shown in Table IIB. Figure 1 also shows that
E c 0.300 zt
TIME(minute) FIG. 5. Inhibition of tubulin polymerization by N-ethylmaleimide. Tubulin solution (10 pM) in glycerol assembly buffer containing 2 mM GTP was quickly mixed with 20 pM (B), 40 pM (C), 80 pM (D), or 120 pM (E) N-ethylmaleimide and incubated at 0°C for 1 h followed by incubation at 37°C; turbidity at 400 nm was measured against time. Curve A represents assembly of M. put&a tubulin in the absence of NEM. In (B), one thiol group per tubulin molecule was blocked. It was determined by measuring the absorbance at 302 nm using E = 620 M-’ cm-‘.
358
ROYCHAUDHURI
AND
BISWAS
FIG. 6. Electron micrograph of negatively stained M. pudica microtubules assembled in vitro. For in vitro assembly 0.2 ml of tubulin solution (1 mgprotein/ml) containing 2 mM GTP was divided into two aliquots, one of which was incubated at 37°C for 1 h to permit microtubule assembly; the other was preincubated with an excess concentration of N-ethylmaleimide (20 X lo-’ M) at 0°C for 1 h followed by incubation at 37°C for 1 h and centrifugation at 100,OOOg in a Beckman airfuge for 1 h at 25°C. Both pellets were resuspended in isolation buffer containing glycerol and grids were prepared for electron microscopic study (1% uranyl acetate was used to stain). (A) Electron microscopic photograph of self-assembled microtubular structure in the presence of glycerol. (B) Electron microscopic photograph of -SH-modified microtubular structure in the presence of glycerol.
-SH
GROUPS
IN
Mimosa
Wave lengthtnm) FIG. 7.
Identification of the specific group of M. pudica tubulin that determines the high affinity of tubulin-colchicine binding. M. pzdica tubulin solution (10 pM) was reached with 20 X lo- M fluorescine-5maleimide at 0°C for 2 min and the reaction was stopped immediately by adding 1 mM dithiothreitol. The reaction mixture was then dialyzed extensively against 50 mM Pipes buffer, pH 6.9, and loaded on 10% SDS-PAGE. After the complete run, the gel containing labeled tubulin was cut out and eluted with 50 mM Pipes buffer, pH 7.0, containing 0.1% SDS. The protein concentrations of elutants (a- and /3-tubulin) were measured by the Bradford method; fluorescence of the elutants (protein concentration of each 01 and /3, 400 pg) was measured at 520 nm. ‘C’ is the control of 50 mM Pipes buffer, pH 7.0, containing 0.1% SDS.
one thiol group is more highly exposed to the environment than other thiols. The results of the selective modification of -SH groups of tubulin and the [3H]colchicine-tubulin binding assay (Fig. 2) show that of colchicine-tubulin binding is reduced approximately 50-55% after modification of one -SH group, compared with unmodified tubulin-colchicine binding activity. According to Fig. 3, all the -SH groups are distributed between (Yand fl subunits and the modification is unequal between a! and /l sulfhydryl groups as far as the intensity of the band is concerned, which has also been proved by the experiment using N[14C]ethylmaleimide. Sulfhydryl groups distribute between the (Y and /3 subunits apparently in the ratio of 1:3, respectively. The results of colchicine or colcemid binding site studies on the tubulin molecule (Fig. 4) show that both (Yand p subunits of M. pudica bind with colchicine or colcemid, whereas in animal tubulin mainly the fl subunit is responsible for colchicine binding (U-20). This uniqueness of M. pudica tubulin is due to scanty resemblance between CYand /3 subunits of brain and Mimosa tubulin as shown earlier (10).
pudica
359
TUBULIN
Inhibition of polymerization by the sulfhydryl modifying agent N-ethylmaleimide raised the possibility that though 4 mol of -SH groups could be associated with the binding sites of tubulin molecules for microtubule assembly, modification of only one sulfhydryl group markedly decreased microtubule assembly, which was also consistent with the colchicine binding reaction (Fig. 1). In case of animal brain tubulin, although there are seven -SH groups per subunit, mainly two -SH groups are responsible for microtubule assembly, but seven -SH groups are responsible for the colchicine binding reaction (2). It is evident from the electron micrographs that not only very few microtubule filaments are present in the N-ethylmaleimide-treated fraction, in comparison to the untreated one, but also the filaments are somehow crumpled and distorted showing abnormality in their appearance. Moreover, CY-and ,&tubulin of M. pudica have greater homology with the /3 subunit than with the a! subunit of brain tubulin. This is concluded from both peptide mapping and Western blotting results (10). Lastly, in identification of the high-affinity sulfhydryl group in (Yand ,6 subunits of M. pudica tubulin, it has been found from Fig. 7 that the ,6 subunit of M. pudica gives maximum fluorescence at 520 nm, which supports the conclusion that the high-affinity sulfhydryl group taking part in both colchicine-tubulin binding and polymerization is present in the p subunit of M. pudica tubulin. Further, when M. pudica tubulin is polymerized, the MT shows resistance to dissociation at O”C, unlike animal MTs. That this is perhaps due to properties of M. pudica tubulin has recently been elucidated in our laboratory (data not shown). Though the assembly of animal tubulin into MTs has been worked out in detail (21), the involvement of -SH groups in the polymerization of plant tubulin has not yet been worked out. The structural differences between brain tubulin and M. pudica tubulin can be resolved only by cloning and sequencing tubulin cDNA from M. pudica. This work is now in progress in our laboratory. ACKNOWLEDGMENTS We are much indebted to Dr. Sabita Mazumder and Mr. Sailen Das for their help in electron microscopic studies. We also thank CSIR for financial support. We are thankful to Professor B. B. Biswas and Professor B. Bhattacharyya, Biochemistry Department, for encouragement and help during the progress of this work.
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Vol.
OF BIOCHEMISTRY
294, No. 2, May
AND
BIOPHYSICS
1, pp. 353-360,1992
Sulfhydryl Groups of Mmosa pudica Tubulin Implicated in Colchicine Binding and Polymerization in vitro Asis
Roychaudhuri
Department
Received
of Biochemistry,
August
and Susweta Bose
Institute,
8, 1991, and in revised
form
Biswasl Centenary
December
Building,
700 054.
Zndia
26, 1991
The important characteristic of novel Mimosa pudica tubulin is its ability to bind colchicine only when dithiothreitol is included in the isolation buffer, indicating the involvement of sulfhydryl groups in colchicine binding. Modification of sulfhydryl groups by a sulfhydryl modifying agent also affects the normal assembly of tubulin into microtubules, as revealed by electron microscopic and spectrophotometric studies. The number of free sulfhydryl groups present in tubulin protein responsible for both colchicine binding and polymerization has been found to be 4, distributed in a! and /I subunits, and is distinctly different from the number reported for animal tubulin. 0 1992 Academic Press, Inc.
Microtubules (MTs)’ from brain and flagellar outer fibers contain free sulfhydryl groups which help the polymerization and colchicine binding to tubulin of microtubular structure (l-3). In fact, several reports have been published proposing the importance of sulfhydryl groups for maintaining the stability and conformation of MTs also (2,4-6). Organic sulfhydryl blocking agents like Nethylmaleimide (NEM), p-chloromercuribenzenesulfonic acid (PCMS), and some other mercury compounds are known to inhibit the polymerization (1, 2, 7) as well as colchicine binding of animal tubulin (2). The inhibitory effect of oxidation of free sulfhydryl groups of tubulin from brain and flagella on polymerization and colchicine binding was studied (1, 2, 7, 8). As in animal cells, microtubules, are believed to fulfill several important functions in plant cells. But studies on the tubulin-microtui To whom correspondence should be addressed. * Abbreviations used: MTs, microtubules; NEM, N-ethylmaleimide; PCMS, p-chloromercuribenesulfonic acid; Pipes, 1,4-piperazinediethanesulfonic acid; PMSF, phenylmethylsulfonyl fluoride; EGTA, ethylene glycol bis(B-aminoethyl ether) NJ’-tetraacetic acid; DTNB, 5,5’-diothiobis-(2nitrobenzoic acid, SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis; DTT, dithiothreitol.
0993-9861/92 53.09 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Calcutta
bule system of higher plants appear to be difficult due to the low content of tubulin and the low affinity to colchitine. Mimosa pudica, both leaves and callus cells raised from pulvini, contains an appreciable amount of this protein which is distinctly different from that of other sources (9, 10). None of the available data concerning the involvement of -SH groups in the assembly and colchicine binding of plant tubulin, however, have so far been presented. Since M. pudica tubulin has been found to be different immunologically not only from animal tubulin but also from tubulin from different plant sources, the purpose of the present study is to ascertain the number and role of sulfhydryl groups on polymerization and colchicine binding of M. pudica tubulin. MATERIALS
AND METHODS
Chemicals. Pipes, GTP (type IIS), 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB), fluorescine-5-maleimide, N-ethylmaleimide, urea, dithiothreitol, [3H]colchicine, N-[‘4C]ethylmaleimide, and colcemid were purchased from Amersham International plc. Purification of tubulin protein from M. pudica pulvinar callus tissue. Pulvinar callus tissues generated from second and third leaves of M. pudicu plants were used throughout the experiment (10). Tubulin was prepared essentially by extracting 50 g of callus cells with a 1:l ratio of isolation buffer (50 mM Pipes-NaOH, pH 6.9, 1 mM MgC&, 1 mM dithiothreitol, 0.5 mM GTP, 1 mM PMSF, 1 PM pepstain A, 1 mM EGTA) according to the method described (10). The homogenate was centrifuged at 100,OOOg for 1 h. The suparnatant was treated with DEAESephadex A-50, in the ratio of 3 mg protein/ml of DEAE-Sephadex. Unabsorbed washings were subjected to O-55% ammonium sulfate saturation and desalted by passage through a Sephadex G-200 column (1.4 X 45 cm), previously equilibrated in the isolation buffer without DTT, and proteins were eluted by the same buffer, 2 ml/tube at a flow rate of 6.5 ml/h. Tubulin-enriched fractions were eluted in tubes 15-17 (volume 30-34 ml). Protein concentration was determined according to Bradford’s dye binding method (11) after precipitation of tub&n by 100% acetone. Bovine serum albumin was used as standard. Determination of free sulfhydryl groups. DTNB was used as a tool to determine the number of sulfhydryl groups of tubulin quantitatively (12). A stock solution of 10 mM DTNB in 50 mM Pipes buffer, pH 7.0, was prepared and the protein was taken in 50 mM Pipes buffer, pH 8.0, containing 1 mM MgCIZ and 1 mM EGTA. A 20-fold excess concentration of DTNB was added to 10m5 M tubulin and the mixture was incubated
353
ROYCHAUDHURI
354 TABLE
I
RESULTS
Effect of Dithiothreitol and Sulfhydryl Modifying Agent DTNB on M. pudica Tubulin-Colchicine Binding Reaction 1.
Tubulin
solution containing colcbicine but
+ Pipes and colcbicine 3. Tubulin + Pipes + DTNB
Effect of dithiothreitol and DTNB. M. pudica tubulin has been shown to bind colchicine appreciably in the presence of sulfhydryl reducing agents such as dithiothreitol. Results of the [3H]colchicine tubulin binding assay using GF/C filter disks are presented in Table I. This binding assay was done before and after modification of the free sulfhydryl groups of tub&n with the specific reagent DTNB. Colchicine binding affinity to M. pudica tubulin is found to be lowered in the absence of DTT in isolation buffer, suggesting the presence of free sulfhydryl group or groups taking part in colchicine tubulin binding.
cpm mg of protein-’ 0.22 0.28 2.44 2.50 0.35 0.37
no DTT
2. Tubulin
buffer
containing
buffer
+ DTT
DTT + colcbicine
x x X X x x
10’ 10’ 10’ 10’ 10’ 10’
Note. 0.5 ml of M. pudica tubulin (5 PM) solution in 50 mM Pipes buffer, pH 6.9, (1) containing no dithiothreitol in the isolation buffer, (2) containing 1 mM ditbiothreitol in the isolation buffer, or (3) preincubated with 20 X lo-’ M DTNB at O’C for 1 h was incubated with [3H]colchicine (1 PM) containing 25,000 cpm at 37°C for 1 h and assayed by the GF/C filter disk binding method.
at O’C for 1 h. The absorbance of the solution was measured at 412 nm against a blank without protein. An extinction coefficient of 13,600 M-’ cm-’ was used to calculate the concentration of reacting sulfhydryl groups. Fluorescine-5-maleimide was used to determine the number of sulfhydryl groups of tubulin quantitatively (13). A stock solution of 13 mM fluorescine-5-maleimide in 50 mu Pipes, pH 7.0, containing 1 mM MgClx and 1 mM EGTA was prepared and a 20-fold excess concentration was added to 10m5 M tubulin; the mixture was incubated at O’C for 1 h and dialyzed extensively against 50 mM Pipes, pH 7.0, and the optical density at 492 nm was measured. An extinction coefficient of 75,000 M-’ cm-’ was used to calculate the concentration of sulfhydryl groups present. Colchicine binding. tubulin were assayed
AND BISWAS
The colchicine binding reactions of M. pudica by the GF/C filter disk method (14).
Polymerization assay. Microtubule assembly was monitored turbidometrically at 37°C using Shimadxu ultraviolet visible recording spectrophotometer UV-160 as described by Gaskin et al. (15). Electron microscopy. Specimens for electron microscopy were made as described by Banejee et al. (16).
Presence of sulfhydryl group. The sulfhydryl groups of plant tubulin isolated from M. pudica were determined by using Ellman’s reagent (DTNB) and fluorescine-5maleimide (Table IIA). From absorption studies at 412 nm, E = 1.36 X lo4 M-l for DTNB and 492 nm, E = 75,000 M-’ cm-’ for fluorescine-5-maleimide, we recorded the presence of four -SH groups in M. pudica tubulin. Time-dependent modification of thiol groups of M. pudica tub&in by Ellman’s method. The kinetic study of the modification of thiol groups of M. pudica tubulin (Fig. 1) showed that one thiol group was completely modified within 5 min, and the remaining three thiol groups were modified sequentially with an increase of time. The number of thiol groups was determined by using the standard molar extinction coefficient for Ellman’s reaction (E = 1.36 X lo4 M-l cm-‘). This was shown categorically also (in the inset) where the number of thiol groups blocked was plotted against time. It is apparent from the data thus far presented that one thiol group is more highly exposed to the outer environment than the other thiol
TABLE Determination
of Free
Sulfhydryl
Groups
of
M. pudica
Tubulin
IIA by Ellman’s
Method
and
the Fluorescine Molar
Optical Reaction 1. 2. 3. 4. 5.
50 mM Pipes buffer, 50 mM Pipes buffer, 50 mM Pipes buffer, 50 mM Pipes buffer, 50 mM Pipes buffer, + fluorescine-5-maleimide
pH pH pH pH pH
8.0 8.0 8.0 7.0 7.0
+ + + + +
DTNB tubulin tubulin tubulin tubulin
+ DTNB
density
412 nm
492 nm
0.21 0.04 0.82 -
0.125
Resultant optical density
Labeling
Method
extinction
coe5cient
(E) in
(lo-‘M)
412 nm
492 nm
412 nm
492 nm
Number of SH groups
0.57
3.299
0.136
0.75
4
3.424
Note. 10 pM tubulin in 50 mM Pipes buffer, pH 8.0, was incubated with an excess concentration of DTNB (20 X lo-’ M) at O’C for 1 h, and maximum absorbance at 412 nm was measured against a blank lacking protein and DTNB. An extinction coefficient of 13,600 M-’ cm-’ was used to calculate the concentration of sulfhydryl groups reacted. For determining the sulfhydryl groups by the fluorescine-5-maleimide method, 10 pM tubulin in 50 mM Pipes buffer, pH 7.0, was incubated with an excess concentration of fluorescine-5-maleimide (20 X lo-’ M) at 0°C for 1 h and dialyzed extensively with three changes; the absorbance at 492 nm was measured against a blank lacking fluorescine-5-maleimide. An extinction coefficient of 75,000 M-’ cm-’ was used to calculate the number of sulthydryl groups reacted.
-SH
GROUPS
IN
Mimosa
pdca
355
TUBULIN
1
1
I
1
I
I
I
I
4
I
5
IO
15
20
25
30
35
CO
45
50
TIME lmin
15
20
25
30
35
40
45
I
55
I
50
55
60
Timelmin) FIG. 1. M. ptdica tubulin (10 pM or 10e5 M) in 50 mM Pipes buffer, pH 8.0, was incubated with an excess concentration of DTNB (20 X 10m5 M) at 25°C and the change in absorbance at 412 nm against time was monitored. The number of thiol groups was determined by using the standard molar extinction coefficient of DTNB at lo-’ M (E = 1.36 X 10’ M-l cm-‘). Inset: The number of thiol groups calculated from the main figure was plotted against time to show the time-dependent modification categorically.
groups, as modification of one thiol group occurs within 5 min. The sulfhydryl groups of this tubulin in the denatured state using 6 M urea were also determined with the same method. The results are shown in Table IIB. We have found that four sulfhydryl groups of tubulin are present also in the denatured state. The results suggest that all four sulfhydryl groups of M. pudica tubulin are exposed and not buried. Selective modification of -SH groups of M. pudica tubulin by Ellman’s reagent (DTNB) was done for [3H]colchicine-tubulin binding. The results depicted in Fig. 2 show that approximately 50-55% of colchicinetubulin binding is lowered after modification of one sulfhydryl group of tubulin compared with unmodified tubulin-colchicine binding activity. Further modification of
TABLE Determination
of Sulfhydryl Groups
Reactions 50 mM Pipes, pH 8.0 + urea + DTNB 50 mM Pipes + tubulin 50 mM Pipes + tubulin + urea + DTNB
of M.
Optical density at 412 nm 1.16 0.05 1.75
-SH groups leads to a decrease in tubulin-colchicine binding. From these results it is confirmed that free sulfhydryl groups of M. pudica tubulin are responsible for colchicine binding. The sulfhydryl modifying agent, fluorescine-5-maleimide, was used to determine the distribution of free SH groups between (Yand fl subunits. After modification of four free -SH groups (determined through absorption at 492 nm and E = 75,000 M-l cm-‘) of tubulin by fluorescine-5-maleimide, it has been found that on SDSPAGE, both subunits (Yand p fluoresce (Fig. 3), showing modification of the four -SH groups present in both subunits; however, greater fluorescence is exhibited by the fl subunit. Next, an experiment was performed to show the distribution of sulfhydryl groups present in (Yand /? subunits of tubulin using N- [ 14C]ethylmaleimide. The results
IIB
pudica Tubulin in the Denatured State by Ellman’s Method Resultant
optical density at 412 nm
0.54
Molar extinction coefficient, z (10m5 M)
Number of SH groups
0.136
4
Note. 10 pM M. pudica tubulin in 50 mM Pipes buffer, pH 8.0, was dialyzed against the same buffer containing 6 M urea at 4’C for l-2 h followed by incubation with an excess concentration of DTNB (20 X lo-’ M) at 0°C for 1 h; absorbance at 412 nm was measured against a blank lacking protein and DTNB. An extinction coefficient of 13,600 M-l cm-’ was used to calculate the number of sulfhydryl groups reacted.
356
ROYCHAUDHURI
AND
BISWAS
E :3 1.6Jz Y ; 1.2 “x s 0.8E HO.& 0.01
I 1SH
I 2SH
I 3SH
I LSH
No. of free -SH groups modified --c FIG. 2. Inhibition of tubulin colchicine binding by DTNB. M. pudica tubulin (10 PM) was preincubated with 22,45,92, or 135 pM DTNB at 0°C for 1 h. At 22 pM DTNB, the one -SH group per tubulin molecule was blocked by DTNB. The number of -SH groups was measured by Ellman’s method and the solution was incubated with [‘Hlcolchicine (1 PM) at 37’C for 1 h, and tubulin colchicine binding was assayed by the GF/C filter disk binding method.
depicted in Table III show the unequal distribution of sulfhydryl groups which support the earlier observation using fluorescine-5-maleimide. These results taken together indicate the presence of sulfhydryl groups in (Yand /3 subunits apparently in the ratio 1:3. The colcemid or colchicine binding site in tubulin was determined by binding lop5 M tubulin with 10e6 M colcemid at 37°C for 1 h, labeling first with 20 X low5 M unlabeled N-ethylmaleimide to modify the unmasked (not masked by colcemid molecule) thiol groups (if present), and then dialyzing against 50 mM Pipes buffer, pH 7.0, containing 6 M guanidine-HCl to dissociate the colcemid molecule from tubulin. Colcemid-masked thiol groups were now exposed to the outer environment. Fluorescine5maleimide (20 X lop5 M) was then added to modify the exposed -SH groups. From SDS-PAGE (Fig. 4) it has been found that both subunits ((u and p) fluoresce, indicating that both (Y and 6 subunits are responsible for colchicine binding. As a control, the native tubulin is treated with unlabeled NEM in the absence of colcemid and the tubulin solution is dialyzed against 50 mM Pipes buffer to denature the tubulin. containing 6 M guanidine-HCl The denatured protein is treated with fluorescine-5-maleimide and run on SDS-PAGE. No labeling of tubulin with fluorescine-5-maleimide occurs. It was also found that after colcemid binds with the tubulin molecule, no SH groups could be detected by Ellman’s reaction. So it is apparent all four -SH groups are involved in colcemid-
FIG. 3.
Modification of four sulfhydryl groups of tubulin by fluorestine-5-maleimide. Tubulin (10 PM) was incubated with 200 pM fluorestine-5maleimide at O’C for 1 h, followed by extensive dialysis with three changes against 50 mM Pipes buffer, pH 7.0. The dialysate was loaded on 10% SDS-PAGE, pH 8.8. After the run, the gel was excited at 280 nm and fluorescence recorded. Lane 1: 40 pg of -SH-modified M. pudka tubulin. Lane 2: 30 pg of Coomassie brilliant blue-stained M. pudica tubulin.
tubulin binding. Here, the concentration of tubulin and colcemid used was 10 PM. Inhibition of tubulin polymerization. The inhibition of tubulin polymerization by addition of the sulfhydryl modifying agent N-ethylmaleimide showed the sequential decrease in turbidity of tubulin polymerization (Figs. 5BE) compared with unmodified tubulin assembly (Fig. 5A). It was further supported by electron microscopic observation (Figs. 6A and B) in the absence and presence of
TABLE Distribution in
M
III
of N- [ “C]Ethylmaleimide pudica LY- and &tubulin
Concentration of M. pudica tubulin Concentration of IV-[i4C]ethylmaleimide cm Blcpm a Number of -SH groups (Y subunit fl subunit
~PM 200 pM 2.8 1
3 bwrox)
Note. Mimosa pudica tubulin was incubated with N-[“Clethylmaleimide in 50 mM Pipes buffer, pH 6.9, containing 1 mM MgClx and 1 mM EGTA for 1 h at 4°C followed by extensive dialysis against the same buffer to remove the free N-[i4C]ethylmaleimide. (r and fl subunits of M. pdca tubulin were separated in SDS-PAGE and each subunit was eluted separately.
-SH
GROUPS
IN
Mimosa
=“p
FIG. 4. Determination of colchicine or colcemid binding sites in M. p&co. Tubulm (10 pM) was preincubated with cold colcemid (colchicine analog, 1 pM) at 37’C for 1 h, followed by addition of an excess amount of unlabeled N-ethylmaleimide (20 X 10m5 M) and incubation at 0°C for 1 h. The solution was dialyzed extensively against 50 mM Pipes buffer, pH 7.0, containing 6 M guanidine-HCl. Finally, an excess concentration of fluorescine-5-maleimide (20 X 10m5 M) was added and the solution was incubated at 0°C for 30 min. The reaction mixture was loaded on 10% SDS-PAGE (pH 8.8). After the run, the gel was excited at 280 nm, and the fluorescence of both the a and fl subunits was recorded. Lane 1: Control. Lane 2: 18 pg of fluorescine-5-maleimide-labeled M. pudica tubulin.
an excess concentration of N-ethylmaleimide, respectively. of the specific sulfhydryl group in M. pudIdentification ica tubulin that determines the high affinity of the tubulincolchicine complex. To identify the location of the specific sulfhydryl group of M. pudica tubulin that determines the high affinity of the tubulin-colchicine complex and polymerization, 1O-5 M or 10 PM tubulin solution was reacted with an excess concentration of fluorescine-5-maleimide, (20 X lOA M), at 0°C for 2 min, and the reaction was stopped immediately by addition of an excess conAfter that, the recentration of dithiothreitol (1 mM). action mixture was loaded and run on 10% SDS-PAGE. After a complete run, (Yand /3labeled tubulin were eluted and protein concentration was measured by the Bradford method. It has been found fluorometrically that the p subunit of M. pudica tubulin gives maximum fluorescence at 520 nm compared with control and a-tubulin (Fig. 7). From this result, it is apparent that the high-affinity sulfhydryl group that participates in tubulin-colchicine binding is present in the fl subunit of M. pudica tubulin. DISCUSSION
The unique property of tubulin isolated from M. pudica callus is that it is basic in nature and binds appreciably with 10e6M colchicine or its analogs (10).
pudica
357
TUBULIN
If tubulin is isolated in the absence of the sulfhydryl reducing agent dithiothreitol, its affinity for binding to colchicine is reduced compared with tubulin isolated in the presence of dithiothreitol (Table I). The same observation was reported by Mizuno et al. (17). In the case of plant tubulin, it might be due to the presence of many aromatic secondary metabolites which, during isolation, oxidize to form quinone-like structures and bind with free sulfhydryl groups of cysteine residues present in tubulin protein. Dithiothreitol is therefore added to protect the -SH groups from secondary metabolites. Moreover, in the tubulin purification step, the binding affinity is not decreased when DTT is not added during the ammonium sulfate precipitation and gel filtration step because of the earlier removal of secondary metabolites during extractions. To support this initial finding, the purified tubulin is pretreated with the -SH modifying agent DTNB and the colchicine-tubulin binding reaction is allowed to proceed. It has been found that the colchicine binding reaction with modified tubulin is reduced compared with unmodified tubulin-colchicine binding (Table I). Therefore, it may be suggested that free -SH groups are responsible for tubulin-colchicine binding. The results of the determination of free -SH groups of M. pudica tubulin (Table IIA) by the Ellman and fluorescine+maleimide labeling methods showed that the tubulin molecule (with cx and /3 subunits) contains four free -SH groups that are exposed to the outer environment and not buried. This has also been proved by denaturing the tubulin with 6 M urea followed by DTNB-tubulin reaction as shown in Table IIB. Figure 1 also shows that
E c 0.300 zt
TIME(minute) FIG. 5. Inhibition of tubulin polymerization by N-ethylmaleimide. Tubulin solution (10 pM) in glycerol assembly buffer containing 2 mM GTP was quickly mixed with 20 pM (B), 40 pM (C), 80 pM (D), or 120 pM (E) N-ethylmaleimide and incubated at 0°C for 1 h followed by incubation at 37°C; turbidity at 400 nm was measured against time. Curve A represents assembly of M. put&a tubulin in the absence of NEM. In (B), one thiol group per tubulin molecule was blocked. It was determined by measuring the absorbance at 302 nm using E = 620 M-’ cm-‘.
358
ROYCHAUDHURI
AND
BISWAS
FIG. 6. Electron micrograph of negatively stained M. pudica microtubules assembled in vitro. For in vitro assembly 0.2 ml of tubulin solution (1 mgprotein/ml) containing 2 mM GTP was divided into two aliquots, one of which was incubated at 37°C for 1 h to permit microtubule assembly; the other was preincubated with an excess concentration of N-ethylmaleimide (20 X lo-’ M) at 0°C for 1 h followed by incubation at 37°C for 1 h and centrifugation at 100,OOOg in a Beckman airfuge for 1 h at 25°C. Both pellets were resuspended in isolation buffer containing glycerol and grids were prepared for electron microscopic study (1% uranyl acetate was used to stain). (A) Electron microscopic photograph of self-assembled microtubular structure in the presence of glycerol. (B) Electron microscopic photograph of -SH-modified microtubular structure in the presence of glycerol.
-SH
GROUPS
IN
Mimosa
Wave lengthtnm) FIG. 7.
Identification of the specific group of M. pudica tubulin that determines the high affinity of tubulin-colchicine binding. M. pzdica tubulin solution (10 pM) was reached with 20 X lo- M fluorescine-5maleimide at 0°C for 2 min and the reaction was stopped immediately by adding 1 mM dithiothreitol. The reaction mixture was then dialyzed extensively against 50 mM Pipes buffer, pH 6.9, and loaded on 10% SDS-PAGE. After the complete run, the gel containing labeled tubulin was cut out and eluted with 50 mM Pipes buffer, pH 7.0, containing 0.1% SDS. The protein concentrations of elutants (a- and /3-tubulin) were measured by the Bradford method; fluorescence of the elutants (protein concentration of each 01 and /3, 400 pg) was measured at 520 nm. ‘C’ is the control of 50 mM Pipes buffer, pH 7.0, containing 0.1% SDS.
one thiol group is more highly exposed to the environment than other thiols. The results of the selective modification of -SH groups of tubulin and the [3H]colchicine-tubulin binding assay (Fig. 2) show that of colchicine-tubulin binding is reduced approximately 50-55% after modification of one -SH group, compared with unmodified tubulin-colchicine binding activity. According to Fig. 3, all the -SH groups are distributed between (Yand fl subunits and the modification is unequal between a! and /l sulfhydryl groups as far as the intensity of the band is concerned, which has also been proved by the experiment using N[14C]ethylmaleimide. Sulfhydryl groups distribute between the (Y and /3 subunits apparently in the ratio of 1:3, respectively. The results of colchicine or colcemid binding site studies on the tubulin molecule (Fig. 4) show that both (Yand p subunits of M. pudica bind with colchicine or colcemid, whereas in animal tubulin mainly the fl subunit is responsible for colchicine binding (U-20). This uniqueness of M. pudica tubulin is due to scanty resemblance between CYand /3 subunits of brain and Mimosa tubulin as shown earlier (10).
pudica
359
TUBULIN
Inhibition of polymerization by the sulfhydryl modifying agent N-ethylmaleimide raised the possibility that though 4 mol of -SH groups could be associated with the binding sites of tubulin molecules for microtubule assembly, modification of only one sulfhydryl group markedly decreased microtubule assembly, which was also consistent with the colchicine binding reaction (Fig. 1). In case of animal brain tubulin, although there are seven -SH groups per subunit, mainly two -SH groups are responsible for microtubule assembly, but seven -SH groups are responsible for the colchicine binding reaction (2). It is evident from the electron micrographs that not only very few microtubule filaments are present in the N-ethylmaleimide-treated fraction, in comparison to the untreated one, but also the filaments are somehow crumpled and distorted showing abnormality in their appearance. Moreover, CY-and ,&tubulin of M. pudica have greater homology with the /3 subunit than with the a! subunit of brain tubulin. This is concluded from both peptide mapping and Western blotting results (10). Lastly, in identification of the high-affinity sulfhydryl group in (Yand ,6 subunits of M. pudica tubulin, it has been found from Fig. 7 that the ,6 subunit of M. pudica gives maximum fluorescence at 520 nm, which supports the conclusion that the high-affinity sulfhydryl group taking part in both colchicine-tubulin binding and polymerization is present in the p subunit of M. pudica tubulin. Further, when M. pudica tubulin is polymerized, the MT shows resistance to dissociation at O”C, unlike animal MTs. That this is perhaps due to properties of M. pudica tubulin has recently been elucidated in our laboratory (data not shown). Though the assembly of animal tubulin into MTs has been worked out in detail (21), the involvement of -SH groups in the polymerization of plant tubulin has not yet been worked out. The structural differences between brain tubulin and M. pudica tubulin can be resolved only by cloning and sequencing tubulin cDNA from M. pudica. This work is now in progress in our laboratory. ACKNOWLEDGMENTS We are much indebted to Dr. Sabita Mazumder and Mr. Sailen Das for their help in electron microscopic studies. We also thank CSIR for financial support. We are thankful to Professor B. B. Biswas and Professor B. Bhattacharyya, Biochemistry Department, for encouragement and help during the progress of this work.
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