- Email: [email protected]
A comparison of microtubule assembly in brain extracts from young and old rats
100
mol,'c,lar Brain Research, 18 (1993) 100 106 ,~c~1993 Elsevier Science Publishers B.V. All rights reserved (1169-328x/93/$06.(10
BRESM 70587
A comparison of microtubule assembly in brain extracts from young and old rats A. Qian a, P.R. Burton a and R . H . H i m e s b " Department of Physiology and Cell Biology and I, Department o]' Biochemism', Unit'ersity (7]"Kansas, Lawrenee, KS 66045 (USA) (Accepted 24 November 1992)
Key words': Microtubule assembly; Aging; Tubulin; GTP; GTPase activity
Microtubule assembly was examined in the high-speed supernatant from homogenates of young (2-4 months old) and old (more than 24 months old) rat brains and significant age-related differences in microtubule assembly were found in the absence of exogenous GTP. In extracts from young brains, the increase in absorbance at 350 nm, which reflects the assembly reaction, was characterized by three phases (lag, elongation, and steady state) superimposed on a slow continuous increase due to non-specific aggregation. However, assembly in extracts from old brains was very sluggish, in some cases barely more rapid than the non-specific aggregation reaction. Two to three times as much protein was assembled into cold-labile microtubules in extracts from young brains than from old brains. W h e n 1 mM G T P was included in the assembly solutions the patterns of assembly in extracts from young and old brains became similar, with about the same amount of protein assembled into cold-labile microtubules. The assembly of tubulin purified from rat brains showed no differences between young and old. Results showed that extracts from old brains contained a higher GTPase activity than extracts from young brains. The sluggish assembly in extracts from old brains could be due to a deficiency in GTP or an inefficient regeneration of GTP.
INTRODUCTION
The structural integrity and function of neurons depend, in part, on the regulated assembly and disassembly of microtubules. Neuronal degeneration, which is a characteristic of brains of aged mammals, including loss of neurons and change in dendritic extent (for review see Coleman and Flood3), could be related to age-related changes in brain microtubles. Some studies on age-related changes of brain tubulin and microtubule-associated proteins (MAPs) have previously been reported. Yan et al. reported that soluble tubulin of human cerebral cortex decreased during aging ~1. Matus and Green examined the protein composition of microtubules prepared from 3 month and 27 month rat brains, and found that a cathepsin D-like protease activity which degrades high molecular weight MAPs increased with age 7. However, information on how microtubule assembly is affected during aging of the brain is lacking. In this study, we compared microtubule assembly in brain extracts from young rats ( 2 - 4 momhs) with old rats (more than 24 months). Our
results show that microtubule assembly in brain extracts from aged rats is very sluggish when compared with young rats. This difference is noted only when exogenous G T P is omitted from the assembly reaction mixture indicating that the aged brain extract has an endogenous G T P concentration which is not sufficient to support the in vitro formation of microtubules. MATERIALS
AND METHODS
Materials Pipes, EGTA, and DMSO were obtained from Sigma Chemical Co. GTP was from Boehringer Mannheim. and purified glutaraldehyde was from Ted Pella, Inc. The tannic acid used was manufactured by Merck and marketed by Baker Chemical Co.. Nocodazole and T B A were from Aldrich.
Animals Male Fischer rats (strain F344) were used. These age-designated rats were obtained from colonies maintained by the National Institute of Aging.
Brain extract preparation and microtubule assembly Rat brains were removed quickly and homogenized in 0.1 M Pipes containing 1 mM E G T A and 1 mM MgSO 4 (PEM buffer), pH
Correspondence: R.H. Himes, Department of Biochemistry, University of Kansas, Lawrence, KS 66045, USA. Fax: (1) (913) 864-5321.
101 6.90 at 4°C. The homogenate was kept on ice for 30 min and centrifuged at 100,000x g for 30 rain. The supernatant after this centrifugafion step is referred to as the extract. The total protein concentration of extracts used in all microtubule assembly studies was adjusted to 7 m g / m l . Microtubule assembly was detected by monitoring the increase in turbidity at 350 nm and 37°C in a Shimadzu multisample spectrophotometer (Model U V 2100U).
Cold disassembly of microtubules After 30 min of assembly, part of the sample was put on ice for 30 rain, after which the turbidity at 350 nm was measured. A n o t h e r part was loaded on a 5 ml 40% sucrose cushion in P E M buffer and centrifuged at 200,000x g and 37°C for 90 min. The resultant pellet, containing microtubules, was suspended in cold P E M buffer and subsequently centrifuged at 1 0 , 0 0 0 x g and 4°C for 5 rain. The supernatant contained proteins released from cold-labile microtubules initially assembled in the extracts.
Electron microscopy T E M was used to confirm that microtubules were assembled in extracts. After about 40 min of assembly, extracts were centrifuged through a sucrose cushion as described above to sediment microtubule pellets. The sedimented microtubules were then fixed in 3% glutaraldehyde and 4% tannic acid. Thin sections were cut and stained by uranyl acetate and lead citrate, and examined in a Philips 300 electron microscope.
Tubulin determination The amount of tubulin in extracts and supernatants of cold-depolymerized microtubules was determined by ELISA. Purified bovine brain tubulin was used as a standard. Monoclonal anti-/3 tubulin antibody was derived from ascites tumors in Balb-C mice that were injected with hybridoma cells 3B1.5.9., originally supplied by Bonnie Neighbors, University of Colorado, Boulder, Colorado, and was previously characterized s. The second antibody used in the ELISA, peroxidase conjugated goat anti-mouse IgG, was purchased from Jackson l m m u n o r e s e a r c h Laboratories. The color reagent ABTS (2,2-azino-di[3-ethylbenzthiazoline] sulfonic acid) was purchased from Zymed.
centrifuged at 200,000x g (Beckman TL-100 ultracentrifuge) for 4 min at 4°C and the resultant pellet was suspended in 100/zl of P E M buffer. An aliquot portion of this solution was added to 50 IzM G T P in P E M buffer and incubated for different periods of time 37°C. Nucleotides were analyzed by H P L C as descibed above.
Other procedures Tubulin from young and old rat brains was analyzed by 2-D gel electrophoresis as described by Supernant et alJ °. Protein concentration was determined by the Bradford assay 2. Bovine brain tubulin was purified as described by Algaier and Himes 1.
RESULTS Microtubule assembly in rat brain extracts in the absence of exogenous GTP Because of the low turbidity of rat brain extracts when compared to brain extracts from some other mammals, in vitro assembly of microtubules in rat brain high speed extract supernatants can be monitored using light scattering measurements 6. In addition, such an assembly reaction occurs in the absence of added stimulators such as glycerol and DMSO and in the absence of added G T P 6. Microtubule assembly in brain extracts from at least 25 rats from both young and old age groups was examined. A similar absorbance curve, representing the assembly reaction, was obtained in all young brain extracts (two representative curves are shown in Fig. 1). The reaction was characterized by a short lag (nucleation) phase, followed by a rapid elongation phase, and finally a steady-state phase (Fig. 1). The assembly curve was
Rat brain tubulin purification After 40 min of microtubule assembly in brain extracts with 1 m M GTP and 10% D M S O at 37°C, microtubules were centrifuged at 55,000 x g for l h. The pellet was suspended in cold PEM, and then centrifuged at 100,000x g and 4°C for 1 h. The supernatant was mixed with an equal volume of 0.8 M PIPES, pH 6.90-20% DMSO, and incubated with 1 m M G T P at 37°C for 30 min 4. Microtubules were pelleted at 100,000× g and 37°C for 1 h, and the pellet was suspended in cold PEM buffer. The second cycle of polymerization was repeated. Tubulin was freed from MAPs by phosphocellulose ( W h a t m a n P11)-Biogel P-10 chromatography as described by Algaier and Himes I.
Endogenous GTPase acti~'ity GTPase activity of brain extracts was measured by incubating extract at a protein concentration of 0.5 m g / m l with 50 p.M G T P for different periods of time at 37°C, stopping the reaction with 2.5% perchloric acid. The supernatant obtained after centrifugation was then neutralized with 4 M potassium acetate-10 M KOH, and the precipitate removed by centrifugation. The supernatant containing nucleotides was injected into an anion exchange H P L C column. Elution was performed with an isocratic buffer 0.4 M N a H 2 P O 4 - 0 . 2 M NaCI, pH 4.3, and followed by absorbance detection at 254 nm. The G T P concentration was determined by comparing the area of the peak with the same elution time as G T P to G T P standards. In some experiments, reversed phase chromatography was used, in which case elution was performed isocratically with 50 m M K 2 H P O 4, 5 m M TBA, and 10% acetonitrile, p H 6.90. GTPase activity of pellets obtained after centrifugation of extracts was measured as follows: 1 ml of brain extract at 7 m g / m l was
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Fig. 1. Microtubule assembly in brain extracts from young and old rats. T h e assembly reaction was performed at 37°C and a protein concentration of 7 m g / m l . G T P was not added.
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Fig. 3. Assembly in extracts eluted from Sephadex G-25. Gel-filtered extract at a protein concentration of 7 m g / m l was incubated at 37°C in the absence (A) and presence (B) of 1 m M GTP,
MINUTES Fig. 2. The effect of exogenous GTP on microtubule assembly in brain extracts. The assembly reaction was carried out at a protein concentration of 7 m g / m l at 37°C with 1 mM GTP present.
superimposed on a line representing a continuous absorbance increase. This linear absorbance increase was not reversed by cold-treatment and was probably caused by non-specific aggregation. When 20 ~,M nocodazole was included in the assembly reaction, only the slow linear absorbance increase was observed. The ab-
sorbance patterns of assembly in brain extracts from old brains were quite different. They were usually characterized by a slow and steady increase without a clear indication of lag and elongation phases (Fig. 1). The assembly in extracts from old brains also included cold-irreversible non-specific aggregation. When 20 ~ M nocodazole was included in the assembly solution, a slow absorbance increase, similar to that seen in extracts from young brains, was observed.
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Fig. 4. Microtubules assembled in extracts from 3 month (A) and 27 month (B) old rat brains were fixed in 3% glutaraldehyde and 4% tannic acid and examined by TEM. Bar = 100 nm.
103
Microtubule assembly in rat brain extracts with added exogenous GTP Microtubule assembly in the extracts was significantly accelerated in the presence of 1 mM GTP, with the assembly in the extracts from old rat brains affected to a greater extent. The presence of G T P caused the assembly patterns in extracts from young and old brains to become similar. Both were characterized by a rapid elongation phase followed by a steady state phase, and the nucleation phase was shortened (Fig. 2). W h e n brain extracts were passed through Sephadex G-25 before incubation at 37°C, the extracts showed only linear increases in absorbance due to non-specific aggregation (Fig. 3). But when 1 mM G T P was included in the gel-filtered extracts, they exhibited the same type of assembly as the original extracts in the presence of 1 mM G T P (Fig. 3). Examination o f microtubules assembled in extracts by transmission electron microscopy Microtubules assembled in extracts from young and old rats were s e d i m e n t e d by centrifugation, and microtubule pellets were fixed in 3% glutaraldehyde with 4% tannic acid. Thin sections of the fixed samples were examined by using TEM. Fig. 4A shows microtubules assembled in an extract from a 3 month old rat brain, and Fig. 4B shows microtubules assembled in an extract from a 27 month old brain. It was confirmed that no m a t t e r how different the assembly curves looked, microtubules were formed in both cases. Cold-depolymerization o f microtubules formed in brain extracts and the resultant cold-labile microtubule protein Two methods were used to measure the amount of cold-labile microtubules formed in the assembly reaction. In the first, the extent of absorbance decrease after a 30 min incubation at 0°C was measured. The decrease in absorbance, which was taken to reflect the amount of polymer assembled as cold-labile microtubules, was roughly equivalent to the increase in ab-
sorbance that occurred in the elongation phase. The results, summarized in Table I, indicate that there was a 2.7-fold higher amount of cold-labile microtubules assembled in brain extracts from young rats than from old rats. W h e n G T P was included in the assembly reaction, the amount of cold-labile microtubules assembled in extracts from old brains was significantly increased, reducing the difference between young and old. G T P had little effect on the assembly of cold-labile microtubules in brain extracts from young rats. In the second method, the samples were centrifuged through a sucrose cushion. The pellets were suspended in cold-buffer, centrifuged, and the supernatant analyzed for protein. The results are also summarized in Table I. The data indicated that approximately 2.3 times as much cold-labile microtubules were assembled in brain extracts from young rats than from old rats. The addition of exogenous G T P to extracts from old brains resulted in a significant increase in the amount of cold-labile microtubules, however, exogenous G T P had little effect on extracts from young brains. The results from the two approaches are in good agreement.
Tubulin determination To quantify the amount of tubulin in extracts and in s u p e r n a t a n t s of c o l d - d e p o l y m e r i z e d microtubules, E L I S A s were performed. Results from four rats in each age group showed that extracts from young brains contained 0.15 + 0.06 mg of tubulin per mg of total protein, and extracts from old brains contained 0.16 + 0.06 mg of tubulin p e r mg of protein. A b o u t 16.5% of the tubulin in extracts from young brains was assembled into cold-labile microtubules, while only about 6.8% of the tubulin in extracts from old brains was assembled into cold-labile microtubules. Does tubulin differ in young and old brains? In order to know whether the sluggish microtubule assembly in brain extracts from old rats was caused by
TABLE I
Comparison of the amount of cold-labile microtubules formed in brain extracts Age Young Old
Cold-induced decreasein absorbance a
Amount of coM-solublepolymer b (mg)
Without GTP 0.155 + 0.022 (n = 5) 0.056 + 0.036 (n = 4) * * P < 0.01
Without GTP 0.130 + 0.010 (n = 5) 0.057 _+0.012 (n = 5) * * P < 0.01
With GTP 0.169 + 0.024 (n = 4) 0.128 _+0.005 (n = 3) P > 0.05
With GTP 0.126 _+0.004 (n = 3) 0.118 + 0.012 (n = 4) P > 0.05
a Microtubules were assembled in extracts at 37°C in the absence and presence of 1 mM GTP, then incubated at 0°C for 30 min. b Microtubules were assembled in extracts at 37°C in the absence and presence of 1 mM GTP. The sample (2.1 mg protein)was centrifuged through sucrose, the pellets suspended in PEM at 0°C, centrifuged, and the protein content in the resultant supernatants determined, n = number of individual rats.
104
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MINUTES Fig. 5. Microtubule assembly from purified rat brain tubulin. Tubulin purified from young and old rat brains was polymerized at a concentration of 1.2 mg/ml in PEM buffer containing 1 mM GTP and 10% DMSO at 37°C.
tracts from young and old brains suggests a lower GTP concentration
and/or
a higher GTPase
tracts from old brains. The in t u b u l i n , b r a i n and
30
Fig. 6. Microtubule assembly in brain extract before and after high speed centrifugation. Extracts from 3 month old and 28 month old rat brains were assembled in the absence of GTP before (A) and after (B) centrifugation at 200,000 × g for 4 min.
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tubulin was purified examined
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a c t i v i t y in ex-
activity of brain
e x t r a c t s w a s e x a m i n e d a t a p r o t e i n c o n c e n t r a t i o n o f 0.5 mg/ml,
at which concentration
microtubule
assembly
t r o p h o r e s i s . In 2 - D gels, a a n d /3 s u b u n i t s o f t u b u l i n
would not occur. As summarized
from young brains overlapped with that from old brains,
a c t i v i t y in e x t r a c t s f r o m o l d b r a i n s w a s a p p r o x i m a t e l y
i n d i c a t i n g t h a t c h a n g e s in t h e p o l y p e p t i d e s t h a t w o u l d
t w i c e as h i g h as in y o u n g b r a i n e x t r a c t s .
affect isoelectric points and molecular weights had not
in T a b l e II, G T P a s e
U p o n c e n t r i f u g a t i o n o f t h e b r a i n e x t r a c t s at 2 0 0 , 0 0 0 × g f o r 4 m i n , it w a s f o u n d t h a t t h e a s s e m b l y r e a c t i o n
occurred. Microtubule
assembly from phosphocellulose-puri-
fled rat brain tubulin was also compared. GTP and 10% DMSO
With 1 mM
present, tubulin from young and
o c c u r r e d m o r e e f f i c i e n t l y in t h e r e s u l t a n t s u p e r n a t a n t than
in t h e o r i g i n a l b r a i n e x t r a c t (Fig. 6). A t y p i c a l
observation was that a distinct elongation phase could
o l d r a t b r a i n s a s s e m b l e d i n t o m i c r o t u b u l e s at t h e s a m e r a t e a n d t o a b o u t t h e s a m e e x t e n t (Fig. 5).
05 F !
E n d o g e n o u s G T P a s e a c t i v i t y in e x t r a c t s f r o m y o u n g a n d o l d rat b r a i n s
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TABLE II The initial rate of GTP hydrolysis in extracts from young and old rat brains n = 3 individual rats for each determination. Results are reported as nmol/mg/min, a GTPase activity of extracts was measured by incubating extract at a protein concentration of 0.5 mg/ml with 50 /LM GTP for different periods of time, stopping the reaction with perchloric acid, and measuring the decrease in GTP concentration by HPLC. b One ml of extract at 7 mg/ml was centrifuged at 200,000 :x: g for 4 min at 4°C, and the resultant pellet was suspended in 100 /xl PEM buffer. An aliquot portion of the solution was added to 50 txM GTP and incubated for different periods of time at 37°C, The decrease in GTP concentration was measured by HPLC. Age
Total extract
Young Old
5.2 _+0.7 11.2 _+2.6 * P < 0.05
"
Pellet from extract ~ 173 _+28 365 _+78 * P < 0.05
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46
MINUTES Fig. 7. The effect of the high speed pellet from a 3 month brain extract on bovine brain tubulin polymerization. Bovine brain tubulin (2.5 mg/ml) was polymerized in the presence of 0.25 mM GTP. An aliquot (containing 7.5 /*g protein) of the resuspended pellet from a rat brain extract after a 200,000 × g centrifugation was added at the time shown. After depolymerization GTP (final concentration, 0.25 mM) was added.
105 be observed in the assembly of such a supernatant from a 28 month old rat. When the pellet obtained after centrifugation was suspended in PEM buffer and was added to microtubules assembled from purified bovine brain tubulin, microtubules were rapidly depolymerized, but reassembly occurred when G T P was added back (Fig. 7). This result suggested that the pellet might contain G T P a s e activity. The initial rate of G T P hydrolysis ( n m o i / m g / m i n ) was about twice as high in the pellets from old brain as in young brain extracts (Table II). In addition, we consistently found about 20% more protein in pellets from old brain extracts than from young brain extracts (112 _+ 21 p,g vs 87 + 16/zg from 7 mg extract protein, n = 5 individual rats for each age group). DISCUSSION In this study we found significant differences in the pattern of microtubule assembly curves in brain extracts from old and young rats. The assembly curves obtained in extracts from young brains in the absence of added G T P was characteristic of that obtained with purified microtubule protein, which consists of three phases: nucleation, elongation, and steady state. However, there was also a slow steady increase in absorbance due to non-specific aggregation. In contrast, assembly in extracts from old brains was very sluggish, in some cases barely more rapid than the non-specific aggregation reaction. The differences in assembly curves between young and old was clearly due to differences in the amount of tubulin being assembled. Differences in the amount of tubulin assembled into microtubules was demonstrated by the large differences in the decrease in absorbance after incubation at 0°C, in the amount of polymer centrifuged through sucrose, and the amount of tubulin determined by E L I S A of the sample which was centrifuged through sucrose. These assays showed that 2 to 3 times as much assembly occurred in the extracts from young brains. The differences we observed apparently are not due to differences in tubulin which would affect the assembly reaction. E L I S A showed that the tubulin content in both types of extracts was essentially equivalent (this result does not appear to be consistent with that of Yan, et al.'l); 2-D electrophoresis detected no differences in the a and /3 subunits of tubulin purified from young and old rat brains; and no difference could be found in the assembly of purified tubulin in the presence of exogenous GTP. G T P is required for microtubule assembly in extracts from rat brains. This was demonstrated by the fact that essentially no assembly occurred after the
extract had been passed through Sephadex G-25, and normal assembly occurred when 1 mM G T P was added back. These facts, together with the finding that the addition of G T P to the extracts removed the differences in assembly characteristics in the extracts between young and old, points to a deficiency in G T P in the extracts of old brains. This deficiency probably results from the higher GTPase activity in old brain extracts. Schr6der et al. found a higher GTPase activity in microtubule protein preparations from old bovine brains when compared to young brains 9. Whether this activity is related to the G T P a s e activity we have measured is not clear. It was also found that centrifugation of the extract at a high g value improved the assembly reaction in the resultant supernatant. The pellet of such a centrifugation contained G T P a s e activity, and this fraction from old brain extract had a higher GTPase activity than the same fraction from young brain extracts. Koehn and Olsen also reported the presence of a particulate inhibitor of microtubule assembly in mouse brain but this inhibitor appeared to be composed of R N A 5. Our studies do not explain the differences in the G T P a s e activities which are relative and not absolute values. For example, differences in an intrinsic G T P regenerating system could result in an apparent difference in GTPase activities. Margolis and Rauch found that the endogenous G T P level in rat brain extract rose and fell in an oscillatory manner when incubated at 37°C 6. This oscillation could be caused by an intrinsic and active nucleoside triphosphate regenerating system 6. Conceivably this regenerating system is less active in old brain extracts, thus resulting in an insufficient G T P concentration to support microtubule assembly and to prevent microtubule depolymerization. We did not address the question of intrinsic G T P regenerating systems in this study. Other possible explanations of the apparent lower content of G T P in old brain extracts are a lower content of GTPase inhibitors or a higher content of other G T P binding proteins. Acknowledgments. This work was supported in part by grants from the Wesley Foundation, Wichita, Kansas, the University of Kansas General Research Fund, and the Marion Merrell Dow-Scientific Education Partnership. ABBREVIATIONS DMSO ELISA MAPs PEM buffer PIPES TBA TEM
dimethyl sulfoxide enzyme-linked immunosorbent assay microtubule-associated proteins 0.1 M PIPES, 1 mM EGTA, 1 mM MgSO4, pH 6.9 piperazine-N,N'-bis[2-ethane-sulfonicacid] tetrabutylammonium dihydrogen phosphate transmission electron microscopy
106 REFERENCES 7 1 Algaier, J. and Himes, R.H., The effects of dimethyl sulfoxide on the kinetics of tubulin assembly, Biochim. Biophys. Acta, 954 (1988) 235-243. 2 Bradford, M.M.., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72 (1976) 248-254. 3 Coleman, P.D. and Flood, D.G, Neuron numbers and dendritic extent in normal aging and Alzheimer's disease, Neurobiol. Aging, 8 (1987) 521-545. 4 Himes, R.H., Burton, P.R. and Gaito, J.M., Dimethyl sulfoxideinduced self-assembly of tubulin lacking associated proteins, J. Biol, Chem., 252 (1977) 6222-6228. 5 Koehn, J.A. and Olsen, R.W., Endogenous particulate inhibitor of microtubule assembly in developing mammalian brain, Arch. Biochem. Biophys., 201 (1980) 207-215. 6 Margolis, R.L. and Rauch, C.T., Characterization of rat brain crude extract microtubule assembly: correlation of cold stability
8
9
10
11
with the phosphorylation state of a microtubule-associated 64K protein, Biochemistry, 20 ( 1981 ) 4451-4458. Matus, A. and Green, G.D.J., Age-related increase in a cathepsin D like protease that degrades brain microtubule-associated proteins, Biochemistry, 26 (1987) 8(183-8086. Scholey, J.M., Neighbors, B., Mclntosh, J.R. and Sa[mon, E.D., Isolation of microtubules and a dynein-like MgATPase from unfertilized sea urchin eggs, J. Biol. Chem., 259 (1984) 6516-6525. Schr6der, H.C., Bernd, A., Zahn, R.K. and Miiller, W.E.G., Age-dependent alterations of microtubule-associated enzyme activities from bovine brain (protein kinase, adenosine triphosphatase, guanosine triphosphatase), Mech. Ageing Decel., 22 (1983) 35-50. Supernant, K.A., Hays, E., LeCluyse, E. and Dentter, W.L.. Multiple forms of tubulin in the cilia and cytoplasm of 7k,trahymena thermophila, Proc. Natl. Acad. Sci. USA, 82 (1985) 69086912. Yan, S.B., Hwang, S., Rustan, T.D. and Frey, W.H., Human brain tubulin purification: decrease in soluble tubulin with age, Neurochem. Res.. 10 (1985) 1-17.
mol,'c,lar Brain Research, 18 (1993) 100 106 ,~c~1993 Elsevier Science Publishers B.V. All rights reserved (1169-328x/93/$06.(10
BRESM 70587
A comparison of microtubule assembly in brain extracts from young and old rats A. Qian a, P.R. Burton a and R . H . H i m e s b " Department of Physiology and Cell Biology and I, Department o]' Biochemism', Unit'ersity (7]"Kansas, Lawrenee, KS 66045 (USA) (Accepted 24 November 1992)
Key words': Microtubule assembly; Aging; Tubulin; GTP; GTPase activity
Microtubule assembly was examined in the high-speed supernatant from homogenates of young (2-4 months old) and old (more than 24 months old) rat brains and significant age-related differences in microtubule assembly were found in the absence of exogenous GTP. In extracts from young brains, the increase in absorbance at 350 nm, which reflects the assembly reaction, was characterized by three phases (lag, elongation, and steady state) superimposed on a slow continuous increase due to non-specific aggregation. However, assembly in extracts from old brains was very sluggish, in some cases barely more rapid than the non-specific aggregation reaction. Two to three times as much protein was assembled into cold-labile microtubules in extracts from young brains than from old brains. W h e n 1 mM G T P was included in the assembly solutions the patterns of assembly in extracts from young and old brains became similar, with about the same amount of protein assembled into cold-labile microtubules. The assembly of tubulin purified from rat brains showed no differences between young and old. Results showed that extracts from old brains contained a higher GTPase activity than extracts from young brains. The sluggish assembly in extracts from old brains could be due to a deficiency in GTP or an inefficient regeneration of GTP.
INTRODUCTION
The structural integrity and function of neurons depend, in part, on the regulated assembly and disassembly of microtubules. Neuronal degeneration, which is a characteristic of brains of aged mammals, including loss of neurons and change in dendritic extent (for review see Coleman and Flood3), could be related to age-related changes in brain microtubles. Some studies on age-related changes of brain tubulin and microtubule-associated proteins (MAPs) have previously been reported. Yan et al. reported that soluble tubulin of human cerebral cortex decreased during aging ~1. Matus and Green examined the protein composition of microtubules prepared from 3 month and 27 month rat brains, and found that a cathepsin D-like protease activity which degrades high molecular weight MAPs increased with age 7. However, information on how microtubule assembly is affected during aging of the brain is lacking. In this study, we compared microtubule assembly in brain extracts from young rats ( 2 - 4 momhs) with old rats (more than 24 months). Our
results show that microtubule assembly in brain extracts from aged rats is very sluggish when compared with young rats. This difference is noted only when exogenous G T P is omitted from the assembly reaction mixture indicating that the aged brain extract has an endogenous G T P concentration which is not sufficient to support the in vitro formation of microtubules. MATERIALS
AND METHODS
Materials Pipes, EGTA, and DMSO were obtained from Sigma Chemical Co. GTP was from Boehringer Mannheim. and purified glutaraldehyde was from Ted Pella, Inc. The tannic acid used was manufactured by Merck and marketed by Baker Chemical Co.. Nocodazole and T B A were from Aldrich.
Animals Male Fischer rats (strain F344) were used. These age-designated rats were obtained from colonies maintained by the National Institute of Aging.
Brain extract preparation and microtubule assembly Rat brains were removed quickly and homogenized in 0.1 M Pipes containing 1 mM E G T A and 1 mM MgSO 4 (PEM buffer), pH
Correspondence: R.H. Himes, Department of Biochemistry, University of Kansas, Lawrence, KS 66045, USA. Fax: (1) (913) 864-5321.
101 6.90 at 4°C. The homogenate was kept on ice for 30 min and centrifuged at 100,000x g for 30 rain. The supernatant after this centrifugafion step is referred to as the extract. The total protein concentration of extracts used in all microtubule assembly studies was adjusted to 7 m g / m l . Microtubule assembly was detected by monitoring the increase in turbidity at 350 nm and 37°C in a Shimadzu multisample spectrophotometer (Model U V 2100U).
Cold disassembly of microtubules After 30 min of assembly, part of the sample was put on ice for 30 rain, after which the turbidity at 350 nm was measured. A n o t h e r part was loaded on a 5 ml 40% sucrose cushion in P E M buffer and centrifuged at 200,000x g and 37°C for 90 min. The resultant pellet, containing microtubules, was suspended in cold P E M buffer and subsequently centrifuged at 1 0 , 0 0 0 x g and 4°C for 5 rain. The supernatant contained proteins released from cold-labile microtubules initially assembled in the extracts.
Electron microscopy T E M was used to confirm that microtubules were assembled in extracts. After about 40 min of assembly, extracts were centrifuged through a sucrose cushion as described above to sediment microtubule pellets. The sedimented microtubules were then fixed in 3% glutaraldehyde and 4% tannic acid. Thin sections were cut and stained by uranyl acetate and lead citrate, and examined in a Philips 300 electron microscope.
Tubulin determination The amount of tubulin in extracts and supernatants of cold-depolymerized microtubules was determined by ELISA. Purified bovine brain tubulin was used as a standard. Monoclonal anti-/3 tubulin antibody was derived from ascites tumors in Balb-C mice that were injected with hybridoma cells 3B1.5.9., originally supplied by Bonnie Neighbors, University of Colorado, Boulder, Colorado, and was previously characterized s. The second antibody used in the ELISA, peroxidase conjugated goat anti-mouse IgG, was purchased from Jackson l m m u n o r e s e a r c h Laboratories. The color reagent ABTS (2,2-azino-di[3-ethylbenzthiazoline] sulfonic acid) was purchased from Zymed.
centrifuged at 200,000x g (Beckman TL-100 ultracentrifuge) for 4 min at 4°C and the resultant pellet was suspended in 100/zl of P E M buffer. An aliquot portion of this solution was added to 50 IzM G T P in P E M buffer and incubated for different periods of time 37°C. Nucleotides were analyzed by H P L C as descibed above.
Other procedures Tubulin from young and old rat brains was analyzed by 2-D gel electrophoresis as described by Supernant et alJ °. Protein concentration was determined by the Bradford assay 2. Bovine brain tubulin was purified as described by Algaier and Himes 1.
RESULTS Microtubule assembly in rat brain extracts in the absence of exogenous GTP Because of the low turbidity of rat brain extracts when compared to brain extracts from some other mammals, in vitro assembly of microtubules in rat brain high speed extract supernatants can be monitored using light scattering measurements 6. In addition, such an assembly reaction occurs in the absence of added stimulators such as glycerol and DMSO and in the absence of added G T P 6. Microtubule assembly in brain extracts from at least 25 rats from both young and old age groups was examined. A similar absorbance curve, representing the assembly reaction, was obtained in all young brain extracts (two representative curves are shown in Fig. 1). The reaction was characterized by a short lag (nucleation) phase, followed by a rapid elongation phase, and finally a steady-state phase (Fig. 1). The assembly curve was
Rat brain tubulin purification After 40 min of microtubule assembly in brain extracts with 1 m M GTP and 10% D M S O at 37°C, microtubules were centrifuged at 55,000 x g for l h. The pellet was suspended in cold PEM, and then centrifuged at 100,000x g and 4°C for 1 h. The supernatant was mixed with an equal volume of 0.8 M PIPES, pH 6.90-20% DMSO, and incubated with 1 m M G T P at 37°C for 30 min 4. Microtubules were pelleted at 100,000× g and 37°C for 1 h, and the pellet was suspended in cold PEM buffer. The second cycle of polymerization was repeated. Tubulin was freed from MAPs by phosphocellulose ( W h a t m a n P11)-Biogel P-10 chromatography as described by Algaier and Himes I.
Endogenous GTPase acti~'ity GTPase activity of brain extracts was measured by incubating extract at a protein concentration of 0.5 m g / m l with 50 p.M G T P for different periods of time at 37°C, stopping the reaction with 2.5% perchloric acid. The supernatant obtained after centrifugation was then neutralized with 4 M potassium acetate-10 M KOH, and the precipitate removed by centrifugation. The supernatant containing nucleotides was injected into an anion exchange H P L C column. Elution was performed with an isocratic buffer 0.4 M N a H 2 P O 4 - 0 . 2 M NaCI, pH 4.3, and followed by absorbance detection at 254 nm. The G T P concentration was determined by comparing the area of the peak with the same elution time as G T P to G T P standards. In some experiments, reversed phase chromatography was used, in which case elution was performed isocratically with 50 m M K 2 H P O 4, 5 m M TBA, and 10% acetonitrile, p H 6.90. GTPase activity of pellets obtained after centrifugation of extracts was measured as follows: 1 ml of brain extract at 7 m g / m l was
0.4
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28 mo
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Fig. 1. Microtubule assembly in brain extracts from young and old rats. T h e assembly reaction was performed at 37°C and a protein concentration of 7 m g / m l . G T P was not added.
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Fig. 3. Assembly in extracts eluted from Sephadex G-25. Gel-filtered extract at a protein concentration of 7 m g / m l was incubated at 37°C in the absence (A) and presence (B) of 1 m M GTP,
MINUTES Fig. 2. The effect of exogenous GTP on microtubule assembly in brain extracts. The assembly reaction was carried out at a protein concentration of 7 m g / m l at 37°C with 1 mM GTP present.
superimposed on a line representing a continuous absorbance increase. This linear absorbance increase was not reversed by cold-treatment and was probably caused by non-specific aggregation. When 20 ~,M nocodazole was included in the assembly reaction, only the slow linear absorbance increase was observed. The ab-
sorbance patterns of assembly in brain extracts from old brains were quite different. They were usually characterized by a slow and steady increase without a clear indication of lag and elongation phases (Fig. 1). The assembly in extracts from old brains also included cold-irreversible non-specific aggregation. When 20 ~ M nocodazole was included in the assembly solution, a slow absorbance increase, similar to that seen in extracts from young brains, was observed.
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Fig. 4. Microtubules assembled in extracts from 3 month (A) and 27 month (B) old rat brains were fixed in 3% glutaraldehyde and 4% tannic acid and examined by TEM. Bar = 100 nm.
103
Microtubule assembly in rat brain extracts with added exogenous GTP Microtubule assembly in the extracts was significantly accelerated in the presence of 1 mM GTP, with the assembly in the extracts from old rat brains affected to a greater extent. The presence of G T P caused the assembly patterns in extracts from young and old brains to become similar. Both were characterized by a rapid elongation phase followed by a steady state phase, and the nucleation phase was shortened (Fig. 2). W h e n brain extracts were passed through Sephadex G-25 before incubation at 37°C, the extracts showed only linear increases in absorbance due to non-specific aggregation (Fig. 3). But when 1 mM G T P was included in the gel-filtered extracts, they exhibited the same type of assembly as the original extracts in the presence of 1 mM G T P (Fig. 3). Examination o f microtubules assembled in extracts by transmission electron microscopy Microtubules assembled in extracts from young and old rats were s e d i m e n t e d by centrifugation, and microtubule pellets were fixed in 3% glutaraldehyde with 4% tannic acid. Thin sections of the fixed samples were examined by using TEM. Fig. 4A shows microtubules assembled in an extract from a 3 month old rat brain, and Fig. 4B shows microtubules assembled in an extract from a 27 month old brain. It was confirmed that no m a t t e r how different the assembly curves looked, microtubules were formed in both cases. Cold-depolymerization o f microtubules formed in brain extracts and the resultant cold-labile microtubule protein Two methods were used to measure the amount of cold-labile microtubules formed in the assembly reaction. In the first, the extent of absorbance decrease after a 30 min incubation at 0°C was measured. The decrease in absorbance, which was taken to reflect the amount of polymer assembled as cold-labile microtubules, was roughly equivalent to the increase in ab-
sorbance that occurred in the elongation phase. The results, summarized in Table I, indicate that there was a 2.7-fold higher amount of cold-labile microtubules assembled in brain extracts from young rats than from old rats. W h e n G T P was included in the assembly reaction, the amount of cold-labile microtubules assembled in extracts from old brains was significantly increased, reducing the difference between young and old. G T P had little effect on the assembly of cold-labile microtubules in brain extracts from young rats. In the second method, the samples were centrifuged through a sucrose cushion. The pellets were suspended in cold-buffer, centrifuged, and the supernatant analyzed for protein. The results are also summarized in Table I. The data indicated that approximately 2.3 times as much cold-labile microtubules were assembled in brain extracts from young rats than from old rats. The addition of exogenous G T P to extracts from old brains resulted in a significant increase in the amount of cold-labile microtubules, however, exogenous G T P had little effect on extracts from young brains. The results from the two approaches are in good agreement.
Tubulin determination To quantify the amount of tubulin in extracts and in s u p e r n a t a n t s of c o l d - d e p o l y m e r i z e d microtubules, E L I S A s were performed. Results from four rats in each age group showed that extracts from young brains contained 0.15 + 0.06 mg of tubulin per mg of total protein, and extracts from old brains contained 0.16 + 0.06 mg of tubulin p e r mg of protein. A b o u t 16.5% of the tubulin in extracts from young brains was assembled into cold-labile microtubules, while only about 6.8% of the tubulin in extracts from old brains was assembled into cold-labile microtubules. Does tubulin differ in young and old brains? In order to know whether the sluggish microtubule assembly in brain extracts from old rats was caused by
TABLE I
Comparison of the amount of cold-labile microtubules formed in brain extracts Age Young Old
Cold-induced decreasein absorbance a
Amount of coM-solublepolymer b (mg)
Without GTP 0.155 + 0.022 (n = 5) 0.056 + 0.036 (n = 4) * * P < 0.01
Without GTP 0.130 + 0.010 (n = 5) 0.057 _+0.012 (n = 5) * * P < 0.01
With GTP 0.169 + 0.024 (n = 4) 0.128 _+0.005 (n = 3) P > 0.05
With GTP 0.126 _+0.004 (n = 3) 0.118 + 0.012 (n = 4) P > 0.05
a Microtubules were assembled in extracts at 37°C in the absence and presence of 1 mM GTP, then incubated at 0°C for 30 min. b Microtubules were assembled in extracts at 37°C in the absence and presence of 1 mM GTP. The sample (2.1 mg protein)was centrifuged through sucrose, the pellets suspended in PEM at 0°C, centrifuged, and the protein content in the resultant supernatants determined, n = number of individual rats.
104
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MINUTES Fig. 5. Microtubule assembly from purified rat brain tubulin. Tubulin purified from young and old rat brains was polymerized at a concentration of 1.2 mg/ml in PEM buffer containing 1 mM GTP and 10% DMSO at 37°C.
tracts from young and old brains suggests a lower GTP concentration
and/or
a higher GTPase
tracts from old brains. The in t u b u l i n , b r a i n and
30
Fig. 6. Microtubule assembly in brain extract before and after high speed centrifugation. Extracts from 3 month old and 28 month old rat brains were assembled in the absence of GTP before (A) and after (B) centrifugation at 200,000 × g for 4 min.
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e x t r a c t s w a s e x a m i n e d a t a p r o t e i n c o n c e n t r a t i o n o f 0.5 mg/ml,
at which concentration
microtubule
assembly
t r o p h o r e s i s . In 2 - D gels, a a n d /3 s u b u n i t s o f t u b u l i n
would not occur. As summarized
from young brains overlapped with that from old brains,
a c t i v i t y in e x t r a c t s f r o m o l d b r a i n s w a s a p p r o x i m a t e l y
i n d i c a t i n g t h a t c h a n g e s in t h e p o l y p e p t i d e s t h a t w o u l d
t w i c e as h i g h as in y o u n g b r a i n e x t r a c t s .
affect isoelectric points and molecular weights had not
in T a b l e II, G T P a s e
U p o n c e n t r i f u g a t i o n o f t h e b r a i n e x t r a c t s at 2 0 0 , 0 0 0 × g f o r 4 m i n , it w a s f o u n d t h a t t h e a s s e m b l y r e a c t i o n
occurred. Microtubule
assembly from phosphocellulose-puri-
fled rat brain tubulin was also compared. GTP and 10% DMSO
With 1 mM
present, tubulin from young and
o c c u r r e d m o r e e f f i c i e n t l y in t h e r e s u l t a n t s u p e r n a t a n t than
in t h e o r i g i n a l b r a i n e x t r a c t (Fig. 6). A t y p i c a l
observation was that a distinct elongation phase could
o l d r a t b r a i n s a s s e m b l e d i n t o m i c r o t u b u l e s at t h e s a m e r a t e a n d t o a b o u t t h e s a m e e x t e n t (Fig. 5).
05 F !
E n d o g e n o u s G T P a s e a c t i v i t y in e x t r a c t s f r o m y o u n g a n d o l d rat b r a i n s
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TABLE II The initial rate of GTP hydrolysis in extracts from young and old rat brains n = 3 individual rats for each determination. Results are reported as nmol/mg/min, a GTPase activity of extracts was measured by incubating extract at a protein concentration of 0.5 mg/ml with 50 /LM GTP for different periods of time, stopping the reaction with perchloric acid, and measuring the decrease in GTP concentration by HPLC. b One ml of extract at 7 mg/ml was centrifuged at 200,000 :x: g for 4 min at 4°C, and the resultant pellet was suspended in 100 /xl PEM buffer. An aliquot portion of the solution was added to 50 txM GTP and incubated for different periods of time at 37°C, The decrease in GTP concentration was measured by HPLC. Age
Total extract
Young Old
5.2 _+0.7 11.2 _+2.6 * P < 0.05
"
Pellet from extract ~ 173 _+28 365 _+78 * P < 0.05
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46
MINUTES Fig. 7. The effect of the high speed pellet from a 3 month brain extract on bovine brain tubulin polymerization. Bovine brain tubulin (2.5 mg/ml) was polymerized in the presence of 0.25 mM GTP. An aliquot (containing 7.5 /*g protein) of the resuspended pellet from a rat brain extract after a 200,000 × g centrifugation was added at the time shown. After depolymerization GTP (final concentration, 0.25 mM) was added.
105 be observed in the assembly of such a supernatant from a 28 month old rat. When the pellet obtained after centrifugation was suspended in PEM buffer and was added to microtubules assembled from purified bovine brain tubulin, microtubules were rapidly depolymerized, but reassembly occurred when G T P was added back (Fig. 7). This result suggested that the pellet might contain G T P a s e activity. The initial rate of G T P hydrolysis ( n m o i / m g / m i n ) was about twice as high in the pellets from old brain as in young brain extracts (Table II). In addition, we consistently found about 20% more protein in pellets from old brain extracts than from young brain extracts (112 _+ 21 p,g vs 87 + 16/zg from 7 mg extract protein, n = 5 individual rats for each age group). DISCUSSION In this study we found significant differences in the pattern of microtubule assembly curves in brain extracts from old and young rats. The assembly curves obtained in extracts from young brains in the absence of added G T P was characteristic of that obtained with purified microtubule protein, which consists of three phases: nucleation, elongation, and steady state. However, there was also a slow steady increase in absorbance due to non-specific aggregation. In contrast, assembly in extracts from old brains was very sluggish, in some cases barely more rapid than the non-specific aggregation reaction. The differences in assembly curves between young and old was clearly due to differences in the amount of tubulin being assembled. Differences in the amount of tubulin assembled into microtubules was demonstrated by the large differences in the decrease in absorbance after incubation at 0°C, in the amount of polymer centrifuged through sucrose, and the amount of tubulin determined by E L I S A of the sample which was centrifuged through sucrose. These assays showed that 2 to 3 times as much assembly occurred in the extracts from young brains. The differences we observed apparently are not due to differences in tubulin which would affect the assembly reaction. E L I S A showed that the tubulin content in both types of extracts was essentially equivalent (this result does not appear to be consistent with that of Yan, et al.'l); 2-D electrophoresis detected no differences in the a and /3 subunits of tubulin purified from young and old rat brains; and no difference could be found in the assembly of purified tubulin in the presence of exogenous GTP. G T P is required for microtubule assembly in extracts from rat brains. This was demonstrated by the fact that essentially no assembly occurred after the
extract had been passed through Sephadex G-25, and normal assembly occurred when 1 mM G T P was added back. These facts, together with the finding that the addition of G T P to the extracts removed the differences in assembly characteristics in the extracts between young and old, points to a deficiency in G T P in the extracts of old brains. This deficiency probably results from the higher GTPase activity in old brain extracts. Schr6der et al. found a higher GTPase activity in microtubule protein preparations from old bovine brains when compared to young brains 9. Whether this activity is related to the G T P a s e activity we have measured is not clear. It was also found that centrifugation of the extract at a high g value improved the assembly reaction in the resultant supernatant. The pellet of such a centrifugation contained G T P a s e activity, and this fraction from old brain extract had a higher GTPase activity than the same fraction from young brain extracts. Koehn and Olsen also reported the presence of a particulate inhibitor of microtubule assembly in mouse brain but this inhibitor appeared to be composed of R N A 5. Our studies do not explain the differences in the G T P a s e activities which are relative and not absolute values. For example, differences in an intrinsic G T P regenerating system could result in an apparent difference in GTPase activities. Margolis and Rauch found that the endogenous G T P level in rat brain extract rose and fell in an oscillatory manner when incubated at 37°C 6. This oscillation could be caused by an intrinsic and active nucleoside triphosphate regenerating system 6. Conceivably this regenerating system is less active in old brain extracts, thus resulting in an insufficient G T P concentration to support microtubule assembly and to prevent microtubule depolymerization. We did not address the question of intrinsic G T P regenerating systems in this study. Other possible explanations of the apparent lower content of G T P in old brain extracts are a lower content of GTPase inhibitors or a higher content of other G T P binding proteins. Acknowledgments. This work was supported in part by grants from the Wesley Foundation, Wichita, Kansas, the University of Kansas General Research Fund, and the Marion Merrell Dow-Scientific Education Partnership. ABBREVIATIONS DMSO ELISA MAPs PEM buffer PIPES TBA TEM
dimethyl sulfoxide enzyme-linked immunosorbent assay microtubule-associated proteins 0.1 M PIPES, 1 mM EGTA, 1 mM MgSO4, pH 6.9 piperazine-N,N'-bis[2-ethane-sulfonicacid] tetrabutylammonium dihydrogen phosphate transmission electron microscopy
106 REFERENCES 7 1 Algaier, J. and Himes, R.H., The effects of dimethyl sulfoxide on the kinetics of tubulin assembly, Biochim. Biophys. Acta, 954 (1988) 235-243. 2 Bradford, M.M.., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72 (1976) 248-254. 3 Coleman, P.D. and Flood, D.G, Neuron numbers and dendritic extent in normal aging and Alzheimer's disease, Neurobiol. Aging, 8 (1987) 521-545. 4 Himes, R.H., Burton, P.R. and Gaito, J.M., Dimethyl sulfoxideinduced self-assembly of tubulin lacking associated proteins, J. Biol, Chem., 252 (1977) 6222-6228. 5 Koehn, J.A. and Olsen, R.W., Endogenous particulate inhibitor of microtubule assembly in developing mammalian brain, Arch. Biochem. Biophys., 201 (1980) 207-215. 6 Margolis, R.L. and Rauch, C.T., Characterization of rat brain crude extract microtubule assembly: correlation of cold stability
8
9
10
11
with the phosphorylation state of a microtubule-associated 64K protein, Biochemistry, 20 ( 1981 ) 4451-4458. Matus, A. and Green, G.D.J., Age-related increase in a cathepsin D like protease that degrades brain microtubule-associated proteins, Biochemistry, 26 (1987) 8(183-8086. Scholey, J.M., Neighbors, B., Mclntosh, J.R. and Sa[mon, E.D., Isolation of microtubules and a dynein-like MgATPase from unfertilized sea urchin eggs, J. Biol. Chem., 259 (1984) 6516-6525. Schr6der, H.C., Bernd, A., Zahn, R.K. and Miiller, W.E.G., Age-dependent alterations of microtubule-associated enzyme activities from bovine brain (protein kinase, adenosine triphosphatase, guanosine triphosphatase), Mech. Ageing Decel., 22 (1983) 35-50. Supernant, K.A., Hays, E., LeCluyse, E. and Dentter, W.L.. Multiple forms of tubulin in the cilia and cytoplasm of 7k,trahymena thermophila, Proc. Natl. Acad. Sci. USA, 82 (1985) 69086912. Yan, S.B., Hwang, S., Rustan, T.D. and Frey, W.H., Human brain tubulin purification: decrease in soluble tubulin with age, Neurochem. Res.. 10 (1985) 1-17.