THE JOURNAL. OF BKX.OG~CAL CHEMISTRY Vol. 254, No. 13, Issue of July 10, pp. 6107-6111, 1979 Printed in U.S.A.

Characterization of Brain Microtubule Proteins Prepared by Selective Removal of Mitochondrial and Synaptosomal Components*

(Received for publication, August 21, 1978, and in revised form, January 12, 1979)

Timothy L. Karr, Hillary D. White, and Daniel L. PurichS

From the Department of Chemistry, University of Cnlifornia, Snnta Barbara, California 93106

Tubulin purified by the method of Shelanski, M. L., Gaskin, F., and Cantor, R. C. ((1973) Proc. N&Z. Acud. Sci. U. S. A. 70, 765-768) contains significant levels of glutamate dehydrogenase resulting from hypotonic shock of mitochondria. Presumably, the anionic char- acter of tubulin leads to ionic associations with com- partmentalized proteins not normally associated with tubulin in Go. A new sucrose extraction method for tubulin purification, which maintains cellular organelle integrity during extraction while producing high yields, is described. Briefly, this involves extraction of cerebral cortical tissue in a sucrose medium and minor modification of the Shelanski protocol. Assays of two mitochondrial enzymes, cytochrome c oxidase and glu- tamate dehydrogenase, indicated that the sucrose ex- traction method contains lo- to 20-fold less enzyme activity than the former hypotonic method. Sodium dodecyl sulfate-polyacryalmide gel electrophoresis in- dicated distinctly less protein contamination of the mi- crotubules obtained by the sucrose extraction proce- dure at all stages of purification. High molecular weight microtubule-associated proteins had altered electro- phoretic behavior, and the slowest migrating band was consistently larger in the new protocol. The microtu- bule protein from the sucrose extraction method dem- onstrated normal assembly kinetics as well as Ca”+-, drug-, and cold-induced depolymerization. The signifi- cance of the lower critical concentration for assembly (0.09 mg/ml uersus 0.16 mg/ml) and the altered micro- tubule-associated proteins composition are currently under investigation.

Microtubules are composed of a principal protein compo- nent known as tubulin and several less abundant cationic high molecular weight proteins (l-3). These components remain in roughly constant stoichiometry through a number of warm- induced polymerization and cold-induced depolymerization cycles. In addition, microtubules apparently interact with other minor proteins of unknown stoichiometry and function (4). Depending upon the source of the microtubules, these minor proteins may number greater than 30 (5), and their association with tubulin represents a major obstacle for those interested in identifying proteins which regulate microtubule function. During the course of our experiments on tubulin, it became apparent that glutamate dehydrogenase activity was

* This research was supported in part by Research Grant SB-2 from the University of California Cancer Research Coordinating Committee. The costs of publication of this article were defrayed in part by the payment of page charges, This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Recipient of a United States Public Health Service Research Career Development Award and an Alfred P. Sloan Fellowship.

present in highly purified tubulin, and we recognized the presence of this marker enzyme as an indicator of mitochon- drial disruption during the early steps of tissue extraction. This suggested that various osmotically fragile organelles may be disrupted within the release of additional proteins free to associate with microtubules. Indeed, the current methods of brain microtubule protein purification generally use hypotonic extracting buffers, whole brain, and frequently, mechanical blenders (6-8). All of these contribute to the destruction of brain mitochondria and synaptosomes, particularly the emul- sification caused by the lipid-rich myelinated white matter present in whole brains (9).

We have developed a new protocol which minimizes the disruption of mitochondria and synaptosomes. The protein purification is improved considerably, and a single polymeri- zation-depolymerization cycle suffices for two such cycles in earlier methods. The details of this method and the charac- terization of the purified microtubule proteins form the basis for this report. Several significant differences between the purified protein components are discussed.

EXPERIMENTAL PROCEDURES

Methods

Purification of Microtubular Proteins-The initial stage of puri- fication was that of Basford (9). To ensure the structural integrity of mitochondria and synaptosomes, the extraction of cytosolic proteins was performed with a buffer having a tonicity of about 600 mosm. Bovine brains were stripped of meninges and 150 g of gray matter removed with scissors. (It is apparently unnecessary to free the gray matter completely of the white matter.) To this tissue was added 1 ml of extraction medium (0.52 M sucrose, 1 mM EGTA, 1 mM ATP, pH 7.0 with KOH)/g of wet tissue. The tissue was homogenized in a 50-ml glass-Teflon homogenizer with at least five passes of the pestle, and the homogenate was centrifuged at 75,000 X g for 60 min at 4°C. The crude extract (CE fraction) was diluted 10% with concentrated buffer (10 X MEM buffer, see below) containing 1 mM GTP to give a final concentration of 0.1 M 2-(N-morpholino)ethanesulfonic acid, 1 rnM EGTA, 1 mM MgC12, pH 6.8 (MEM buffer) with 0.1 mM GTP. Microtubules were further purified using a modification of the She- lanski method (6). First, glycerol was added to 3.5 M, and polymeri- zation was initiated by warming to 37°C. After a 45.min incubation period, microtubules were collected by centrifugation at 75,000 x g for 90 min at 37°C. The pellets were then resuspended in MEM buffer containing 1 mM GTP at 4”C, homogenized gently with one pass in a glass-Teflon homogenizer (HIP fraction), depolymerized on ice for 30 min, and centrifuged for 60 min at 75,ooO x g and 4°C. The superna- tant (CIS fraction) was brought to 3.5 M glycerol and was then polymerized for 45 min by raising the temperature to 37°C. Micro- tubules were pelleted by centrifugation (60 min, 75,000 X g, giving the HIP fraction), and this protein was subjected to another cycle of polymerization/depolymerization as described above (giving the CB and H ,P fractions). Protein was stored at -80°C in pellet form after rapid freezing in liquid nitrogen. At each step of the purification procedure, aliquots were withdrawn for protein determination and electrophoresis (see below).

Microtubular protein (from 150 g of whole brain) was purified in a parallel fashion without sucrose. This method employs a Waring

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6108 Brain Microtubule Proteins

blendor for the initial homogenization of brain tissue under noniso- tonic conditions with MEM buffer containing 1 mM ATP. The crude extract was made up to 0.1 mrvr GTP prior to the fust polymerization, and subsequent details of the purification are as stated above.

Later preparations were scaled up for 600-g quantities of gray matter. The brains were placed on ice in plastic bags with a 0.32 M sucrose, 1 mM EGTA (pH 7.0 with Tris buffer) solution during transit from the slaughterhouse. A large, 500-ml glass-Teflon homogenizer with a 0.005-inch radial clearance was designed to accommodate the larger homogenate volumes. The brain homogenate and polymerized crude extract were spun at 45,000 X g for 60 min and 3 h, respectively. The protein was stored in liquid nitrogen at the C&l stage with 3.5 M glycerol. Otherwise the procedure was the same as that given above.

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis- Sodium dodecyl sulfate-electrophoresis on 7% or 6% polyacrylamide gels was performed by the method of Laemmli (10). Protein samples (35 to 80 pg) were applied and run at 3 mA/tube for 6 h. Gels were fixed and stained in 5% methanol and stained in 7.5% acetic acid containing 0.1% Coomassie blue for 60 min. Gels were destained in 7.5% acetic acid overnight and scanned in a Gelman DCD-16 densi- tometer at 600 nm. The proteins used as molecular weight markers were; myosin (220,000), /3-galactosidase (130,009), phosphorylase a (94,006), hemocyanin (70,000), and bovine serum albumin (68,009).

Turbidity Assays-Microtubule polymerization was monitored by an increase in turbidity at 350 nm (11) with a Cary 118 recording spectrophotometer equipped with a thermostated sample chamber. Assembly was initiated in MEM buffer containing 1.5 mM GTP. The reaction was initiated by raising the temperature to 37’ and the presence of microtubules was confnmed by electron microscopy.

Enzyme Assays-Cytochrome c oxidase was assayed by the method of Wharton and Tzagoloff (12). The decrease in the a-spectral band was followed at 550 nm on a Cary model 118 recording spectro- photometer.

Glutamate dehydrogenase activity was monitored by the increase in absorbance at 340 nm. The reactions were initiated by addition of NADP’ (90 pM) to a mixture containing 0.1 mg of cell extract plus 1.5 mM glutamate in 100 mM KP, buffer, pH 8.1.

Electron Microscopy-Microtubules were negatively stained by standard procedures using cytochrome c and uranyl acetate, and inspected with a Siemens Elmiskop I.

RESULTS

To understand the regulation of microtubule assembly fully, it is necessary to determine the protein components and enzymatic activities associated with microtubules. This is a formidable task since the anionic character of tubulin may cause additional artificial associations with other cell compo- nents during cell lysis and organelle disruption. To simplify the task a new extraction protocol (see “Methods”) was de- veloped to minimize damage to mitochondrial and synapto- somal fractions, and the microtubular protein was character- ized.

Comparison of Sucrose Extraction Protocol and Typical Microtubule Purification Methods-The general method for microtubule protein purification relies upon whole brain tissue extraction without regard to the preservation of organelle integrity. The new protocol described in this report depends upon sucrose to maintain mitochondrial and synaptosomal proteins within their respective organelles. We used a loose- fitting (0.005-inch radical clearance) Teflon-glass homogenizer on cerebral cortical tissue to minimize organelle breakage by mechanical disruption or the detergent action of the lipid-rich white matter (9). Subsequent to homogenization and centrif- ugation, the samples were treated in an essentially identical manner, and the protein content of each fraction is indicated in Table I. These data clearly indicate that cell extracts at approximately 400 mosm have considerably less protein. Nonetheless, the overall yield of microtubules after two cycles of assembly-disassembly is appreciably greater than that ob- tained by the standard protocol used in many laboratories. It was also observed that the amount of protein pelleted in the cold depolymerized fractions was consistently less in the su- crose extraction method.

To determine the extent of mitochondrial and synaptosomal breakage in both extraction procedures, we measured the relative activities of two mitochondrial marker enzymes: glu- tamate dehydrogenase and cytochrome c oxidase. The activ- ities of each were greatly diminished in the cell extracts obtained by the sucrose extraction protocol. The former was reduced by at least a factor of 20, and the latter by a factor of 7. Together, these findings suggest that the mitochondria remain essentially intact during the initial steps of the new isolation method.

Electrophoretic Examination of the Microtubule Proteins Obtained by the Two Purification Methods-It was possible to gain a better understanding of the differences between the sucrose extraction method and the hypotonic method by the use of sodium dodecyl sulfate-gel electrophoresis. Represent-

TABLE I

Comparison of microtubule protein purification methods

These values are based upon 100 g of whole brain tissue (standard isolation method) or cerebral cortex (sucrose method). For additional details see “Experimental Procedures.”

Total protein Overall yield

Cell-free extract First warm pel-

let First cold super-

natant Second warm

pellet Second cold su-

pernatant

w 5% 93 CE 1368 765 100 100 HIP 210 148 15.3 19.4

CS 88 85 6.4 11.1

H2P 64 67 4.7 8.8

CS 41 56 3.0 7.3

C,S H2P C2S H3P

HMW

Tbn -

Dye -

HS HS HS HS FIG. 1. Gel electrophoretic comparison of microtubule protein pre-

pared by the sucrose extraction procedure and the standard Shelanski (hypotonic) protocol. Aliquots of protein were taken at various stages of the purification (indicated at the top of the figure) and prepared for electrophoretic analysis as described under “Experimental Pro- cedures.” The gels were loaded with 80 pg of protein from each stage of purification of both the hypotonic (H) and sucrose (S) extraction procedures. (*) presents differences seen in staining patterns of the two purification protocols. Tbn, tubulin; HMW, high molecular weight.

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Brain Microtubule Proteins 6109

ative protein profiles for both methods are shown in Fig. 1. Even in the impure fractions (A and B), it is noteworthy that the sucrose extraction method results in the conspicuous absence or substantial decrease of at least six protein bands. These are indicated by the asterisks. Significantly, the top asterisk points to the presence of a major change in the so- called high molecular weight protein fraction. The sucrose protocol greatly enhances one of the high M, bands, which is present only in relatively small amounts in the standard protocol. These changes may be observed more clearly in the gel densitometry tracings seen in Fig. 2 (A, B). Here, one may compare the C$ and CZS fractions for the sucrose extraction method and the standard protocol, respectively. Cursory ex- amination of these results indicates that the sucrose extraction protocol is essentially a one-polymerization method giving tubulin and associated proteins that are at least as pure as that obtained with two polymerization cycles using the other method. Closer inspection of the densitometry tracings reveals that the two high M, peaks are of comparable size with the sucrose-extracted preparation; in contrast, the standard method yields less of the more slowly migrating band. This trend is evident throughout the isolation, and the H3P sample from a third reassembly step is shown in Fig. 2B. These tracings demonstrate that the sucrose extraction method is superior in overall purity, and as shown in Table I, the yield is higher as well.

Properties of Three-Cycle Purified Microtubule Protein

A’ ’ ’ ’ ’ ’ ’ I I1 I I, 1 I1 I,

5 n

DISTANCE MIGRATED (ARE. UNITS)

H,PSucrole Rotocd

DISTANCE MIGRATED cARB.UNITS)

FIG. 2. A, densitometry tracings of disc gels from twice cycled “hypotonic” tubulin and once cycled “sucrose” tubulin. Equal amounts of protein (80 pg) were loaded onto the gels and electropho- resis was performed as discussed under “Experimental Procedures.” B, densitometry tracings of polyacrylamide gels loaded with 3 times cycled tubuiin. Approximately 80 pg of protein was loaded onto the gels and electrophoresis was performed as discussed under “Experi- mental Procedures.” The tracings were obtained by measuring ab- sorbance at 600 nm.

01

01

6

8 a

0.c

5-

O-

6-

1 0

r I “I ;TP-&ported

Assembly 0 old 4 1

I

1 I I “I

JTP -Supported C Assembly

e-

a*-

4 I

1 Time (minutes)

FIG. 3. Characterization of nucleotide supported microtubule as- sembly. Assembly (0.89 mg/ml) was initiated after the addition of either 0.5 mM GTP or UTP by warming to 37°C. A, GTP-supported assembly in the presence and absence of 2.5 x IO-” M podophyllotoxin. After approximately 40 min, microtubules were cold depolymerized (dashed line). B, UTP-supported assembly under identical conditions and subsequent depolymerization by addition of 2 mM CaCl2 (dashed Line).

FIG. 4. Electron micrograph of sucrose-prepared microtubules, showing typical dimensions and protofilament substructure. Details are given under “Experimental Procedures.”

Isolated by the Sucrose Extraction Method-As shown in Fig. 3, GTP supports the assembly of microtubules in a manner typical of that seen in other tubulin preparations. Prior addition of 25 pM podophyllotoxin prevents assembly, and this finding agrees with earlier reports (13). Assembled microtubules are sensitive to cold (4’C) and 2 InM calcium, both of which effect depolymerization. Electron micrographs of the microtubules are shown in Fig. 4. The tubules have a regular morphology with typical dimensions. It is noteworthy that UTP will replace the GTP requirement for assembly (Fig. 3). There is a lag of 3 to 5 min in the UTP-supported

process, which is consistent with the presence of the nucleo- side-5’-diphosphate kinase activity observed by other inves- tigators (14). This suggests that the enzymatic activity prob- ably does not result from leakage of mitochondria which are known to contain a high level of membrane-bound nucleoside- 5’-diphosphate kinase (15). Nonetheless, a clear demonstra- tion of the in viuo association of microtubules with the cyto- plasmic enzyme will require additional work.

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6110 Brain Microtubule Proteins

The differences in high M, protein profiles noted in Figs. 1 and 2 suggest that microtubules isolated from the two methods may have different polymerization characteristics. One fre- quently studied parameter of microtubule assembly is the critical concentration for polymerization, which is a measure of the minimal concentration of protein required for assembly. In Fig. 5, we present evidence that the critical concentration is 44% lower with the protein isolated by the sucrose extraction protocol; values of about 0.2 mg/ml have been typically re- ported for protein purified by the standard procedure. To ensure that this 44% reduction represented a true difference of the two protocols, the experiment was performed as follows: both determinations were performed on the same day at the same time. Five samples were monitored intermittently in a Cary 118’spectrophotometer equipped with a rotating carou- sel. To minimize hysteretic effects due to possible decay of the protein, three of the five samples contained hypotonic protein and the other two contained the sucrose-extracted material. After the assembly plateau level had been reached, the experiment was repeated again but in reverse order to obtain all five points for both protein samples. This protocol was repeated again to obtain duplicate point determinations. The measured critical tubulin concentration for the sucrose- extracted material was 0.075 and 0.185 mg/ml for the hypo- tonic protein. The experimental points were fit to a linear regression program which gave a correlation coefficient of 0.99. These results were repeated three times on different days using different protein preparations, yielding values between 44 to 60% decrease for all experiments. The significance of the different critical concentrations in terms of changes in the microtubule-associated protein fraction must await further study. A second criterion related to microtubule stability is the rate of drug-induced depolymerization. Margolis and Wil- son (16, 17) have utilized colchicine or podophyllotoxin to inhibit the addition of dimers to the assembly end of tubules. The rate of turbidity loss has been closely correlated with the rate of dimer release from the disassembly end of tubules, and the value obtained is approximately 7% microtubular protein released/h at 30°C. In our experiments (see Fig. 6), rates of depolymerization in the presence of 20 ,uM podophyllotoxin are approximately 8.5% h-’ for both the sucrose extraction and the standard preparations at 37°C. Control samples with-

/ 0 1 I I I

0 a5 10 15 20 PUOTElN (MG/ML)

FIG. 5. Determination of critical tubuhn concentration. Samples of tubulin from both hypotonic and sucrose extraction preparations were assembled to plateau as measured by turbidity with 1.5 mM GTP. Ten times the final absorbance reading at 350 nm is plotted t~ersus the protein concentration.

AOLi=Ol

-1

1 1 1 1 I I I I 1

40 60 120 160 2

Time (mmutes)

FIG. 6. Drug-induced depolymerization of microtubules. Microtu- bule protein (2.0 mg/ml) prepared by the Shelanski protocol (hypo- tonic) and the sucrose extraction technique was assembled to appar- ent equilibrium and then depolymerized by addition of 2.0 x lo-” M podophyllotoxin (arrows).

out drug showed no depolymerization by electron microscopy and turbidity.

DISCUSSION

The results presented in this report suggest that the sucrose extraction protocol may be valuable in delineating the action of important modulators of the assembly-disassembly process, such as the microtubule “treadmilling” observed by Margolis and Wilson (17) and microtubule associations with other cell processes. Certainly, the identification of specific regulatory molecules and their mode of action promises to be an impor- tant long term goal in understanding microtubule-related biochemistry of nerve cells. To date, the only proteins whose interaction with tubulin is rather firmly established are the cationic high molecular weight proteins and tau, but even their roles remain unclear. Microtubules contaminated by even trace levels of enzymes might have profound effects, especially if they interact with the protein or nucleotide components essential for assembly. The work of Vallee and Borisy (18), for example, illustrates the morphological effects of trypsin which clips the proteins projecting from microtu- bules. In this respect, the goal of establishing associations of proteins and enzymes with microtubules is obviously quite ambitious, and the problem becomes even more complex when there exists the possibility of numerous protein contaminants arising from osmotic shock of fragile cellular organelles. The tonicity of the MEM buffer used in this work (similar to buffers used by other investigators) was approximately 165 mosm as measured in a Fiske osmometer. This is substantially below the osmotic pressure necessary to maintain isotonicity. We have used a sucrose buffer in this work with a tonicity of about 600 mosm, tonicities generally used for the isolation of mitochondria (9) and synaptosomes (19). Berkowitz et al. (20) have identified 100 A neurofiiaments contaminating typical microtubule preparations. Further work by Schlaepfer (21) and Shelanski (22) have described the neurofiaments as being comprised of three proteins having molecular weights of 68,000, 160,000, and 210,000. Fig. 7 shows the molecular weight determinations of the six protein bands noted with asterisks in Fig. 1, and two of these bands (at approximately 68,000 and 220,000) could represent neurofiiament proteins. We see no

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Brain Microtubule Proteins 6111

4.4L ’ s . , , ’ . , . 1 0 01 02 0.3 0.4 05 0.6 0.7 0.8 0.9 1.0

Ralative Mii,(+)

FIG. 7. Molecular weight determination of the proteins asterisked in Fig. 1 using sodium dodecyl sulfate electrophoresis with 6% poly- acrylamide as described under “Experimental Procedures.” The mi- crotubule protein used was twice cycled by the Shelanski (hypotonic) protocol and 35 or 75 pg was loaded.

significant staining at the 160,000-dalton position on our gels of the hypotonic material. The fact that these proteins are absent in the C,S fraction (see Fig. 1) of the sucrose extraction suggests that they are not extracted under slightly hypertonic conditions. The sucrose extraction procedure has already per- mitted us to conclude that certain mitochondrial contami- nants may be avoided by preventing orgallellar destruction. Activities of the mitochondrial marker enzymes, glutamate dehydrogenase and cytochrome c oxidase, are markedly di- minished when care is taken to preserve the mitochondria. In addition, at least six protein bands are absent in the sodium dodecyl sulfate-gel electrophoretograms of the material de- rived by the new method. Since we found no differences in the polymerization competence of the tubulin, one may al- ready infer that none of these particular proteins was a major modulator of assembly.

Another interesting property of the new sucrose procedure is the decreased amount of protein that may be sedimented after cold-induced depolymerization. With microtubular pro- tein prepared by hypotonic extraction and processed further in either the absence or presence of glycerol, we have observed that this sedimented fraction can comprise as much as 25% of the microtubular protein even after three polymerization-de- polymerization cycles. On the other hand, this fraction has never constituted more than 5 to 10% with the material derived from the sucrose extraction method. It will be of interest to learn if the protein which sediments after cold depolymerization is selectively removed in the initial centrif- ugation step or if it is prevented from sedimenting in subse- quent cold centrifugations by the conditions of the new ex- traction method. The former notion may account for the success of the method described by Kirkpatrick et al. (23) for purifying intact microtubules from brain tissue in the presence of 1 M hexylene glycol at 1°C.

The method has been scaled up to use 600 g of cerebral cortex scrapings, and one may expect about 250 to 350 mg of protein after the second polymerization. The care taken to preserve organellar integrity does require an additional ex- penditure of 1 to 2 h, but the sucrose apparently stabilizes the homogenate against the loss of the pol.ymerization compe- tency of microtubular protein. Undoubtedly, there will be additional improvements to reduce the time required for the two-cycle method, but the complete preparation (without the use of the large capacity glass-Teflon homogenizer) now takes only 10 to 12 h.

Certainly the most provocative differences between the sucrose extraction and hypotonic methods regard the micro- tubule-associated proteins fraction and the lowered critical

concentration for microtubule assembly. As shown earlier, there are no apparent changes in the rate of drug-induced depolymerization. The work of Margolis and Wilson (16, 17) demonstrated that colchicine and podophyllotoxin complex with tubulin to block dimer addition to the assembly end of tubules but not dimer dissociation from the disassembly end. Thus, the drug-induced drop in turbidity is a measure of dimer release, and we see no evidence of any altered behavior with the new preparation. Interestingly, we also observed indistinguishable rates of assembly at protein concentrations above the respective critical concentrations for the material from either preparation. Thus far, the ease of nucleation at low microtubule concentrations represents the major differ- ence between the two methods of purification in the assembly- disassembly process. Clearly, this matter will require addi- tional experimentation before more answers can be given.

In conclusion, the approach presented in this report deals with the need to preserve many of the physiologic properties of microtubules during cell disruption and extraction. This is a problem that must be resolved before in vitro studies may begin to provide a faithful indication of in viva events.

Acknowledgments-We are especially grateful to Mr. Mel Alder- man for providing the bovine brain tissue. We thank Brian J. Terry for his help in several experiments.

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T L Karr, H D White and D L Purichof mitochondrial and synaptosomal components.

Characterization of brain microtubule proteins prepared by selective removal

1979, 254:6107-6111.J. Biol. Chem. 

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