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The stabilization of calf-brain microtubule protein by sucrose
ARCHIVES
OF
The
BIOCHEMISTRY
AND
Stabilization
587-589
of Calf-Brain RONALD
Graduate
163,
TiIOPHYSICS
Department
of
Microtubule
P. FRIGON
Biochemistry,
AND
Brandeis
Received A sucrose solution activity of microtubule not to interfere with the sucrose solution binding activity.
JAMES
University,
July
Protein
by Sucrose’
C. LEE Waltham, Masswhusetts
02154
5, 1972
has been used as a stabilizing medium for the colchicine-binding protein isolated from calf brain. The sucrose itself is shown the binding ability and the protein could be stored below 0°C in for at least 2 weeks without any significant loss of colchicine-
In the course of studies of the Mg2+ ionand vinblastine-induced self-associations of calf-brain microtubule protein (tubulin) under way in this laboratory, it became desirable to find a way of stabilizing this protein. Xcrotubule proteins isolated from various sources are known to undergo a rapid aging process (l-4). Thus, purified calf-brain tubulin losespart of its colchicinebinding ability when stored overnight at 4°C in P1LIG2 buffer. Lyophilization has been employed for storing this protein, but in most casesthis results in irreversible aggregation, as indicated by partial loss of solubility in buffers and the appearance of fastmoving components in velocity sedimentation experiments. Hence, a search for conditions under which this protein could be stabilized for at least 2 weeks was undertaken. This communication presents data which indicate that calf-brain tubulin can be stabilized by 1 JI sucrosein PMG buffer. In this medium it is possible to store the protein below 0°C for at least 2 weeks without any significant loss of its colchicinebinding activity. EXPERIMENTAL Calf-brain of Weisenberg
(1972)
tubulin was isolated by the (1, 2). The colchicine-binding
method ac-
1 Publication No. 865, from the Graduate Department of Biochemistry, Brandeis University. ? Abbreviations used: PMG: buffer containing lo-* M sodium phosphate, 10e4 M GTP, 5 X 1w3 M MgCl2 at pH 7.0. 587 Copyright 3 1972 by Academic Press, Inc. All rights of reproduction in any form reserved.
tivit,y was monitored by a column-adsorption method which was found to be faster and more reproducible than the DEAE-chromatographic filter paper technique (1). Approximately 0.5 mg of tubulin was incubated with 5 X lO+ M [aH]colchicine in 1.0 ml of PMG buffer at pH 7.0, 37°C for 1 hr. After incubat#ion, the assay mixture was passed through a 0.5 X l.O-cm column of DEAESephadex A-50, which had been equilibrated with PMG buffer. The colchicine-protein complex was adsorbed to the column and the free labeled colchicine was washed away with 2 ml of unlabeled colchicine solution (1 X lo+ M in PMG). The column was then eluted by 10 ml of Bray’s solution (5) for t,he determination of radioactivity. DISCUSSION
One mole of colchicine has been found to bind to 110,000 g of fresh, native tubulin, within an estimated error of 10 %. If 1 M sucrose is included in the assay mixture, no interference with binding is observed, indicating that any differences in binding observed under various conditions reflect alterations in the protein molecule which result in the loss of colchicine-binding activity. Optimum stabilization of the binding activity was achieved by storing the protein solution at 4°C or below 0°C. Quick freezing and immediate thawing of the solution in the presenceor absenceof 1 M sucroseresulted in a binding activity within 10% of the initial activity. Storing the solution in the frozen state in sucrose for 14 days resulted in no change in the binding ability, whereas, in the absence of any sucrose, more than 95 % of the activity was lost.
588
FRIGON
From Fig. 1, the stabilizing effect of sucrose on tubulin is expressed by the increase in the half-life of colchicine-binding ability with an increase in the amount of sucrose in the system. Without sucrose, the half-life is about 30 hr, while in 1 M sucrose, there was no observable decrease in binding activity over the duration of the experiment, 67 hr. At intermediate concentrations of sucrose, the half-lives observed were of intermediate values. The optimum temperature for colchicinebinding activity of porcine-brain microtubule protein is 37”C, the activity decreasing sharply on both sides of this temperature (6). At 5O”C, the relative activity is approximately 40% of that at 37°C. In Table I are presented the results of experiments on the thermal stability and reversibility of the thermal loss of binding activity of tubulin in various concentrations of sucrose. The thermal stability is expressed as the percentage of binding activity in various sucrose concentrations, relative to the binding at 37°C without sucrose. The solutions were preincubated at various temperatures for 10 110 100 90 SO 70 60 F 0 .E z .z .$ 2 s ,L $
50 0 40 30 \ 0
20
I
IO 0
I
I
I
I
I
IO 20 30 40 50 60 Time of Storage in Hours
I
70
FIG. 1. Loss of colchicine-binding activity. The binding of 1 mole of [3H]colchicine per 110,000 g of protein, measured at 37”C, was arbitrarily chosen as 1007,. The protein was stored at 4°C in PMG containing varying amounts of sucrose: 0, no sucrose; n , 0.2 M sucrose; A, 0.5 M sucrose; 0, 1.0 M sucrose.
AND
LEE TABLE
THERM.~L
STABILITY
OF
AMOUNTS Condition
Styye M
T;myr-
Colchicine bindine (%I
I TUBULIN
Condition Swase
Temperature (“Cl
0.5
55 GO 75 3i 45 50 55 60 75 37 45 50 55 60 75
(“0 0.0 0.1
0.3
0.5
37 50 37 45 50 60 75 37 45 50 60 75 37 45 50
100 43 104 65 57 2 0 100 61 53 7 1 104 101 95
IX
V.IRYING
OF SUCROSK
0.7
1.0
(1 The binding to a fresh preparation 37°C without sucrose was arbitrarily
Colchicine binding“ (%)
83 38 2 99 100 105 85 40 2 98 104 loo 80 55 3
observed at set, as 100%.
min, followed by incubation wit’h [3H]colchitine at 37°C for 1 hr. It is shown that, in 0.7 M sucrose or higher, tubulin is stable to at least 50°C; above this temperature, the protein becomes irreversibly thermally denatured, as reflected by the lossof colchicinebinding capability. At low concentrations of sucrose (0.3 M and lower), there does not seem to be any stabilizing effect. In the absence of sucrose, the relative activity at 50°C is 43 %, in good agreement with Ventilla et al. (6). These results indicate that 1 11 sucroseeither stabilizes tubulin in its optimal state for colchicine binding or that the sucrose changes the optimum temperature for binding. Since the addition of sucrose does not affect colchicine binding (Table I), it is more likely that the first alternative is true. In addition to having no effect on the process of colchicine binding, sucrose does not measurably affect the conformation of tubulin, as monitored by circular dichroism, since the spectra in the presence of various concentrations of sucrose are identical with those obtained in its absence.3Furthermore, the sedimentation pattern of tubulin, stored 3 Lee, J. C., Frigon, R. P., and Timasheff? manuscript in preparation.
S. N.,
SUCROSE
STABILIZATION
in 1 11 sucrose PMG buffer shows no large nonsprcific aggregates,” which are always observed aftrr lyophilization of the protein or storage in the absence of sucrose. While the mechanism of tubulin stabilization by sucrow is not understood at present, thermodynamic c>xperiments currently in progress indicate! that, in this medium, the protein is prefcrcntially hydrated.4 ACKNOWLEDGMENTS The authors for his great
t,hank interest
4 Lee, J. C., Hirsh, sheff, S. N., manuscript
Professor Serge N. Timasheff and encouragement. in this J.,
Thomas, J , and in preparat,ion.
Tima-
OF work. NIH NSF
589
TUBULIN
These studies were supported Grants GM 14603, NS 5241, and Grant GB 12619.
in part by GM 212 and
REFERENCES 1. WI’:ISENBF:ILG,
R.
C.,
BORISY,
G.
G.,
AND
E. W. (1968) Biochemistry 7,4466. 2. WXISENBEKG, R. C., AND TIMASHEFF, S. N. (1970) Biochenzistry 9, 4110. 3. BORISY, G. cf., AND TAYLOR, E. W. (1967) J. Cell Biol. 34, 525, 535. 4. WILSON, L. (1970) Biochemistry 9,4999. 5. BR.YY, G. A. (1960) An,aZ. Biocl em. 1,279. TAYLOR,
6.
VKNTILLA,
M.
(1972)
M., CANTOR, Biochejt&ry
C. R., AND 11, 1554.
SHELANSKI,
OF
The
BIOCHEMISTRY
AND
Stabilization
587-589
of Calf-Brain RONALD
Graduate
163,
TiIOPHYSICS
Department
of
Microtubule
P. FRIGON
Biochemistry,
AND
Brandeis
Received A sucrose solution activity of microtubule not to interfere with the sucrose solution binding activity.
JAMES
University,
July
Protein
by Sucrose’
C. LEE Waltham, Masswhusetts
02154
5, 1972
has been used as a stabilizing medium for the colchicine-binding protein isolated from calf brain. The sucrose itself is shown the binding ability and the protein could be stored below 0°C in for at least 2 weeks without any significant loss of colchicine-
In the course of studies of the Mg2+ ionand vinblastine-induced self-associations of calf-brain microtubule protein (tubulin) under way in this laboratory, it became desirable to find a way of stabilizing this protein. Xcrotubule proteins isolated from various sources are known to undergo a rapid aging process (l-4). Thus, purified calf-brain tubulin losespart of its colchicinebinding ability when stored overnight at 4°C in P1LIG2 buffer. Lyophilization has been employed for storing this protein, but in most casesthis results in irreversible aggregation, as indicated by partial loss of solubility in buffers and the appearance of fastmoving components in velocity sedimentation experiments. Hence, a search for conditions under which this protein could be stabilized for at least 2 weeks was undertaken. This communication presents data which indicate that calf-brain tubulin can be stabilized by 1 JI sucrosein PMG buffer. In this medium it is possible to store the protein below 0°C for at least 2 weeks without any significant loss of its colchicinebinding activity. EXPERIMENTAL Calf-brain of Weisenberg
(1972)
tubulin was isolated by the (1, 2). The colchicine-binding
method ac-
1 Publication No. 865, from the Graduate Department of Biochemistry, Brandeis University. ? Abbreviations used: PMG: buffer containing lo-* M sodium phosphate, 10e4 M GTP, 5 X 1w3 M MgCl2 at pH 7.0. 587 Copyright 3 1972 by Academic Press, Inc. All rights of reproduction in any form reserved.
tivit,y was monitored by a column-adsorption method which was found to be faster and more reproducible than the DEAE-chromatographic filter paper technique (1). Approximately 0.5 mg of tubulin was incubated with 5 X lO+ M [aH]colchicine in 1.0 ml of PMG buffer at pH 7.0, 37°C for 1 hr. After incubat#ion, the assay mixture was passed through a 0.5 X l.O-cm column of DEAESephadex A-50, which had been equilibrated with PMG buffer. The colchicine-protein complex was adsorbed to the column and the free labeled colchicine was washed away with 2 ml of unlabeled colchicine solution (1 X lo+ M in PMG). The column was then eluted by 10 ml of Bray’s solution (5) for t,he determination of radioactivity. DISCUSSION
One mole of colchicine has been found to bind to 110,000 g of fresh, native tubulin, within an estimated error of 10 %. If 1 M sucrose is included in the assay mixture, no interference with binding is observed, indicating that any differences in binding observed under various conditions reflect alterations in the protein molecule which result in the loss of colchicine-binding activity. Optimum stabilization of the binding activity was achieved by storing the protein solution at 4°C or below 0°C. Quick freezing and immediate thawing of the solution in the presenceor absenceof 1 M sucroseresulted in a binding activity within 10% of the initial activity. Storing the solution in the frozen state in sucrose for 14 days resulted in no change in the binding ability, whereas, in the absence of any sucrose, more than 95 % of the activity was lost.
588
FRIGON
From Fig. 1, the stabilizing effect of sucrose on tubulin is expressed by the increase in the half-life of colchicine-binding ability with an increase in the amount of sucrose in the system. Without sucrose, the half-life is about 30 hr, while in 1 M sucrose, there was no observable decrease in binding activity over the duration of the experiment, 67 hr. At intermediate concentrations of sucrose, the half-lives observed were of intermediate values. The optimum temperature for colchicinebinding activity of porcine-brain microtubule protein is 37”C, the activity decreasing sharply on both sides of this temperature (6). At 5O”C, the relative activity is approximately 40% of that at 37°C. In Table I are presented the results of experiments on the thermal stability and reversibility of the thermal loss of binding activity of tubulin in various concentrations of sucrose. The thermal stability is expressed as the percentage of binding activity in various sucrose concentrations, relative to the binding at 37°C without sucrose. The solutions were preincubated at various temperatures for 10 110 100 90 SO 70 60 F 0 .E z .z .$ 2 s ,L $
50 0 40 30 \ 0
20
I
IO 0
I
I
I
I
I
IO 20 30 40 50 60 Time of Storage in Hours
I
70
FIG. 1. Loss of colchicine-binding activity. The binding of 1 mole of [3H]colchicine per 110,000 g of protein, measured at 37”C, was arbitrarily chosen as 1007,. The protein was stored at 4°C in PMG containing varying amounts of sucrose: 0, no sucrose; n , 0.2 M sucrose; A, 0.5 M sucrose; 0, 1.0 M sucrose.
AND
LEE TABLE
THERM.~L
STABILITY
OF
AMOUNTS Condition
Styye M
T;myr-
Colchicine bindine (%I
I TUBULIN
Condition Swase
Temperature (“Cl
0.5
55 GO 75 3i 45 50 55 60 75 37 45 50 55 60 75
(“0 0.0 0.1
0.3
0.5
37 50 37 45 50 60 75 37 45 50 60 75 37 45 50
100 43 104 65 57 2 0 100 61 53 7 1 104 101 95
IX
V.IRYING
OF SUCROSK
0.7
1.0
(1 The binding to a fresh preparation 37°C without sucrose was arbitrarily
Colchicine binding“ (%)
83 38 2 99 100 105 85 40 2 98 104 loo 80 55 3
observed at set, as 100%.
min, followed by incubation wit’h [3H]colchitine at 37°C for 1 hr. It is shown that, in 0.7 M sucrose or higher, tubulin is stable to at least 50°C; above this temperature, the protein becomes irreversibly thermally denatured, as reflected by the lossof colchicinebinding capability. At low concentrations of sucrose (0.3 M and lower), there does not seem to be any stabilizing effect. In the absence of sucrose, the relative activity at 50°C is 43 %, in good agreement with Ventilla et al. (6). These results indicate that 1 11 sucroseeither stabilizes tubulin in its optimal state for colchicine binding or that the sucrose changes the optimum temperature for binding. Since the addition of sucrose does not affect colchicine binding (Table I), it is more likely that the first alternative is true. In addition to having no effect on the process of colchicine binding, sucrose does not measurably affect the conformation of tubulin, as monitored by circular dichroism, since the spectra in the presence of various concentrations of sucrose are identical with those obtained in its absence.3Furthermore, the sedimentation pattern of tubulin, stored 3 Lee, J. C., Frigon, R. P., and Timasheff? manuscript in preparation.
S. N.,
SUCROSE
STABILIZATION
in 1 11 sucrose PMG buffer shows no large nonsprcific aggregates,” which are always observed aftrr lyophilization of the protein or storage in the absence of sucrose. While the mechanism of tubulin stabilization by sucrow is not understood at present, thermodynamic c>xperiments currently in progress indicate! that, in this medium, the protein is prefcrcntially hydrated.4 ACKNOWLEDGMENTS The authors for his great
t,hank interest
4 Lee, J. C., Hirsh, sheff, S. N., manuscript
Professor Serge N. Timasheff and encouragement. in this J.,
Thomas, J , and in preparat,ion.
Tima-
OF work. NIH NSF
589
TUBULIN
These studies were supported Grants GM 14603, NS 5241, and Grant GB 12619.
in part by GM 212 and
REFERENCES 1. WI’:ISENBF:ILG,
R.
C.,
BORISY,
G.
G.,
AND
E. W. (1968) Biochemistry 7,4466. 2. WXISENBEKG, R. C., AND TIMASHEFF, S. N. (1970) Biochenzistry 9, 4110. 3. BORISY, G. cf., AND TAYLOR, E. W. (1967) J. Cell Biol. 34, 525, 535. 4. WILSON, L. (1970) Biochemistry 9,4999. 5. BR.YY, G. A. (1960) An,aZ. Biocl em. 1,279. TAYLOR,
6.
VKNTILLA,
M.
(1972)
M., CANTOR, Biochejt&ry
C. R., AND 11, 1554.
SHELANSKI,