Proc. Natl. Acad. Sci. USA Vol. 77, No. 8, pp. 4425-4429, August 1980 Biochemistry Observation of an exothermic process associated with the in vitro polymerization of brain tubulin (microtubule assembly/exothermic reactions/multiple pathways/differential scanning calorimetry) S. A. BERKOWITZ*, G. VELICELEBIt, J. W. H. SUTHERLANDt, AND J. M. STURTEVANT§ Kline Chemistry Laboratory, Yale University, New Haven, Connecticut 06520 Contributed by Julian M. Sturtevant, April 7,1980

ABSTRACT The polymerization of tubulin has been studied MATERIALS AND METHODS with a high-sensitivity differential scannin microcalorimeter, with results which indicate that microtu ule assembly can Microtubule Preparation. The purification of microtubules proceed via one or possibly two exothermic reactions. The from calf brains was carried out as described by Berkowitz et amount of heat evolution has been found to be far in excess of al. (14). The cold high-speed supernatant after the first poly- GTP hydrolysis. The heat liberated has been observed to depend merization was stored in liquid nitrogen. Before experiments strongly upon the exact experimental conditions, varying from many hundreds of kilocalories per mole of tubulin dimer when were conducted, microtubule samples were rapidly thawed, dilute tubulin solutions are heated rapidly to a few kilocalories cycled (depolymerized and polymerized) an additional time, per mole of tubulin dimer when concentrated tubulin solutions and then placed into PM 2M buffer [2 M glycerol/100 mM are heated slowly. The results are tentatively interpreted in 1,4-piperazinediethanesulfonic acid (Pipes)/2 mM ethylene terms of the existence of at least two pathways or the ormation glycol bis(/3-aminoethyl ether)-NN,N',N'-tetraacetic acid of energetically distinct polymers. These findings indicate the importance of kinetic factors in studying tubulin polymeriza- (EGTA)/1 mM MgSO4/1 mM dithioerythritol, pH 6.9], by tion. exchanging bulk solvent on a Sephadex G-25 column. A mi- crotubule preparation from porcine brain, prepared in the Microtubules are a class of organelles that appear to be present complete absence of glycerol in a buffer containing 100 mM in all eukaryotic cells. These structures are thought to function Pipes, 1 mM EGTA, 0.5 mM MgSO4 at pH 6.9, was a gift from as a cytoskeleton (to maintain the shape and form of cells) or Robert B. Scheele and G. Borisy, Laboratory of Molecular in the transport of various macromolecules and supramolecular Biology, University of Wisconsin. Prior to its use in some ex- structures [for a recent review of these organelles see Dustin (1)]. periments this material was cycled an additional time. Because the temporal and spatial control of the assembly and Tubulin Concentrations. Unless otherwise stated, all tubulin disassembly of microtubules is believed to be a key factor in concentrations were determined by measuring the absorbance cellular function, an understanding of the mechanism or at 280 nm, using the extinction coefficient 1.21 liter g'l cm-1 mechanisms involved in this process is of great importance. (15). Measured values of A28o were corrected for tubulin purity Since the discovery of the conditions necessary for the in vitro (which averaged 77.5% wt/wt) by assuming the average ex- assembly of cytoplasmic microtubules (2), a considerable tinction coefficient of all nontubulin proteins to be the same as amount of biochemical and biophysical work has been directed that of tubulin. No correction was made for the small levels of toward unraveling the mechanism of the polymerization and light scattering present. The estimated uncertainty in the depolymerization of tubulin, the major protein subunit of mi- concentrations reported is 10%. crotubules, which has a molecular weight of 110,000 (3). Al- Electrophoresis. Sodium dodecyl sulfate/polyacrylamide though various models have been proposed [which are sum- electrophoresis was conducted as outlined by Berkowitz et al. marized in a recent review by Kirschner (4)], there is no general (14). Preparations used in this study were found to contain agreement as to their validity. In all these models it has been 75-80 (wt/wt) tubulin as determined by quantitative electro- assumed that the assembly of microtubules is reversible, and phoresis. that its temperature-dependent behavior shows it to be an en- DSC. DSC experiments were conducted in an instrument dothermic reaction. However, if GTP hydrolysis is indeed designed by Privalov et al. (16). Addition of various effectors coupled to this process, a question arises as to its thermodynamic such as GTP and CaCI2 to both sample and reference solutions reversibility. The recent results of Margolis and Wilson (5), were made volumetrically from concentrated stock solutions. which showed that the pathway for microtubule assembly is Maximum additions represented no more than 1.5% of the total not the pathway for microtubule disassembly, raise further volume. The heat evaluated for the first exothermic process was questions concerning reversibility. A point of additional con- calculated as described by Velicelebi and Sturtevant (17). fusion is the numerous and varied enthalpies which have been Baselines (or what we assumed to be baselines) used in these reported from studies using van't Hoff analysis (6-9) and direct calorimetry (10-12). Abbreviations: DSC, differential scanning calorimetry (or calorimeter); In this paper we report a study of the polymerization of EGTA, ethylene glycol bis(,3-aminoethyl ether)-NNN',N'-tetraacetic tubulin, using a high-sensitivity differential scanning calo- acid; HMW-MAPs, high molecular weight microtubule-associated rimeter (DSC). The data obtained have revealed the presence proteins; LMW-MAPs, low molecular weight microtubule-associated of a major, and perhaps also a minor, exothermic process asso- proteins; Pipes, 1,4-piperazinediethanesulfonic acid. ciated with tubulin polymerization. An initial report of this * Present address: Clinical Endocrinology Branch, National Institute of Arthritis, Metabolism and Digestive Diseases, National Institutes work has appeared elsewhere (13). of Health, Bethesda, MD 20205. t Present address: The Biological Laboratories, Harvard University, The publication costs of this article were defrayed in part by page 16 Divinity Avenue, Cambridge, MA 02138. charge payment. This article must therefore be hereby marked "ad- tPresent address: Eastman Kodak, Research Laboratories, Rochester, vertisement" in accordance with 18 U. S. C. §1734 solely to indicate NY 14627. this fact. § To whom correspondence should be addressed. 4425 Downloaded by guest on September 24, 2021 4426 Biochemistry: Berkowitz et al. Proc. Natl. Acad. Sci. USA 77 (1980) calculations were obtained by extrapolating data on both sides It is evident from the curves in Fig. 1 that no correlation exists of the negative peak to the temperature of half completion of between the apparent excess heat capacity and GTP hydrolysis. the process as determined by planimeter integration, as illus- Thus, the hydrolysis of GTP cannot account for the released trated for one DSC scan in Fig. 1. heat. This conclusion is further supported by the fact that the Turbidity Measurements. Turbidity measurements were heats calculated to result from GTP hydrolysis, shown in Table made in a Gilford-Beckman DU spectrophotometer at 350 nm, 1, are in some cases very much smaller than the observed exo- using 1-cm cuvettes, and are expressed as optical densities. The thermic heats. temperature of water-jacketed cuvettes was controlled by a Characterization of the Posttransitional Region of EXO Lauda bath, which was modified by the addition of an elec- I. An interesting feature of all DSC results showing the exo- tronic controller that enabled the bath temperature to be in- thermic transition EXO I is the decrease in the apparent specific creased linearly at rates up to approximately 2 K min1. heat that accompanies this process. Values for this decrease, Phosphorus Analysis. Inorganic phosphorus was measured estimated from the difference between the extrapolated base- according to the procedure of Taussky and Shorr (18). lines at half completion, are given in Table i. The apparent heat capacity in the posttransitional region has RESULTS displayed various types of temperature-dependent behavior, Dependence of Excess Heat Capacity, Turbidity, and GTP ranging from a positive monotonic function of temperature to Hydrolysis on Scan Rate. The scan rate dependence of the a strongly negative monotonic function of temperature, as excess specific heat, the turbidity, and the hydrolysis of GTP shown in curves A and B in Fig. 2. Under appropriate experi- during tubulin polymerization are shown in Fig. 1. An increase mental conditions (which include the concentrations of GTP in the scan rate results in shifts in the corresponding curves to and tubulin and the scan rate) we have even observed a second higher temperature. The DSC and turbidity traces show an negative peak in this region, as seen in curve C of Fig. 2. exothermic transition (labeled EXO I), which occurs during the Effect of GTP and Protein Concentration on the Excess early phase of net polymerization, as indicated by the increase Specific Heat. The- size, shape, and position on the temperature in turbidity, and reaches completion after approximately 50% axis of the DSC traces for tubulin polymerization are strongly or less of the total mass of polymer has been formed. The heats influenced by the concentration of GTP and tubulin, as shown for this exothermic process for the three different scan rates in Fig. 3 A and B. In fact, in the absence of GTP (Fig. 3A) or shown in Fig. 1 are given in Table 1, expressed in terms of the microtubule proteins, no exothermic process was observed. For total amount of tubulin dimer initially present. The amount of example, a solution of 31 tiM tubulin in PM 2M buffer was heat evolved can be seen to be strongly dependent on the scan heated to 100'C for 5 min, clarified by centrifugation, made rate. Although the accuracy of these numbers is not high, due 2 mM in GTP, and scanned in the DSC at 1 K min1; no trace primarily to baseline uncertainties, the order of magnitude of of the exotherm was seen. Similarly, no heat evolution was the values is very surprising. We have observed heat evolution observed when a 31 ALM solution of tubulin was polymerized ranging from 4 to 850 kcal (1 kcal = 4.184 kJ) per mol of tubulin at 370C in the presence of 2 mM GTP and centrifuged at 35,000 dimer depending on scan rate and tubulin concentration. At rpm in a Beckman T35 rotor at 250C for 60 min, and the su- present we can offer no explanation for either the unusually pernatant was scanned at 1 K min-. We have also observed the wide range of heat effects observed or the unusually large absence of the exothermic effect when the tubulin concentra- magnitudes encountered. We wish to emphasize that all our tion is below its critical value (0.2-0.6 mg ml-l for twice-cycled DSC results are satisfactorily reproducible if experimental material in PM 2M buffer at 20-40'C). It is surprising that the conditions are carefully controlled.

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20 25 30 I I Temperature, 10 15 20 25 30 35 40 Temperature, FIG. 1. Effect of scan rate on the excess specific heat, the increase in turbidity, and the hydrolysis of GTP during the polymerization FIG. 2. Variations in the excess specific heat after the exothemic 0.5 K of tubulin. Scan rates: -- -, 0.5 K min1; -, 1 K min'; - -, 1.8 K process EXO I. Curve A, 31 uM tubulin, 2 mM GTP, scan rate mink. All experiments were conducted in PM 2M buffer containing min1; curve B, 91 ,uM tubulin, 3.1 mM GTP, scan rate 1 K min'; 2 mM GTP at a tubulin concentration of 31 tgM. curve C, 125 uM tubulin, 1 mM GTP, scan rate 1 K mink. Downloaded by guest on September 24, 2021 Biochemistry: Berkowitz et al. Proc. Natl. Acad. Sci. USA 77 (1980) 4427

Table 1. Enthalpy and specific heat changes associated with the exothermic process EXO I when tubulin is polymerized in PM 2M buffer containing 2 mM GTP Change in Scan rate, Observed heat for AH for EXO I,* kcal Enthalpy due to GTP heat capacity,* Tubulin, uM K min-1 EXO I, mcal (mol tubulin)-1 hydrolysis, mcalt kcal K-1 molh 10 1.8 8.S -850 -64 1.0 2.7 -270 -46 0.5 0.9 -90 -35 31 1.8 7.4 -240 0.1 (23-310C) -15 1.0 2.1 -68 0.2 (21-270C) -11 0.5 0.2 -7 0.2 (19-220C) -3.2 89 1.8 3.8 -42 -5.2 1.0 1.0 -11 -1.5 0.9 0.4 -4 -0.2 * Estimated maximal uncertainty is +20%. t Heat generated from the hydrolysis of GTP was based on an assumed heat of hydrolysis of -5 kcal/mol (9). The numbers in parentheses are the temperature range used in determining the amount of inorganic phosphorus released. molar exothermic enthalpies increase sharply with decreasing As the concentration of calcium was increased, both the DSC tubulin concentration. The increase in the temperature required and turbidity traces were found to be shifted to higher tem- for the reaction to take place as the concentration is lowered is, peratures, a correlation that supports the conclusion that the on the other hand, not unexpected for a polymerization pro- DSC traces reflect the polymerization of tubulin. cess. The rather high concentration of calcium required to inhibit Effect of Calcium on the DSC Results. Additional evidence polymerization is due in part to the presence of glycerol at high for associating the exothermic process appearing in the DSC concentration (19, 20). The required concentration is also in- traces with tubulin polymerization was obtained by studying creased by an increase in tubulin concentration; calcium in the the effect of calcium ions on the polymerization process. The micromolar range suffices for tubulin near its critical concen- results of these experiments are illustrated in Fig. 4 A and B. tration. Dependence of DSC Results on Nontubulin Proteins. In order to eliminate the possibility that the observed DSC traces were due to the presence of the 100-A filaments, high molecular weight microtubule-associated proteins (HMW-MAPs) or low 5 molecular weight microtubule-associated proteins (LMW- I0 B T

Y ~~~~~~~~~~~~I0.0I~o1

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15 20 25 30 35 Temperature, 0C FIG. 3. (A) Effect of GTP concentration on the DSC traces ob- Temperature, 00 served in the polymerization of tubulin. The mM concentration of GTP is indicated for each curve. The tubulin concentration was 31 FIG. 4. Effect of Ca2+ concentration on the polymerization of ,uM and a scan rate of 1 K min-' was used. (B) Effect oftubulin con- tubulin observed calorimetrically (A) and turbidimetrically (B) in centration on the DSC traces. The ,M total concentration of tubulin PM 2M buffer containing 2 mM GTP. The tubulin concentration was is indicated for each curve. The GTP concentration was held constant 31 uM and a scan rate of 1 K min-' was used. The mM concentration at 2 mM and a scan rate of 1 K min-1 was used. The two scans marked of Ca2+ in excess of the EGTA present is indicated for each experi- by asterisks are identical. ment. Downloaded by guest on September 24, 2021 4428 Biochemistry: Berkowitz et al. Proc. Natl. Acad. Sci. USA 77 (1980) DISCUSSION Calorimetry is the most direct technique for evaluating the enthalpy and heat capacity changes associated with chemical and physical processes. In using this technique, however, one must keep in mind its lack of specificity (22), which in principle allows all chemical and physical transformations having non- 0.2 caIK I zero enthalpies to be detected and measured. Calorimetric data are thus the summation of the heats of all the reactions occur- ring in a given system, whether or not the reactions are perti- nent to the process under study. In considering this problem, it is our conclusion that the curves of excess heat capacity shown in this paper are indeed due to the polymerization of tubulin. pieces of evidence: 15 20 25 30 35 This conclusion is based on the following Temperature, 00 (i) the approximate correlation in temperature between the changes in the apparent excess heat capacity and the turbidity; FIG. 5. Calorimetric scan of a tubulin preparation freedofPMoA (ii) the consistency of this correlation on perturbing the mi- filaments and most of the HMW MAPs andLMW M) of calcium ions; (iii) the buffer containing 2 mM GTP. This material was obtained by gel fi2 crotubule assembly by the addition tration on a Bio-Gel A-15m column, concentrated by'ultrafiltration, requirement of tubulin and GTP for the exothermic reactions and equilibrated with PM 2M buffer on a Sephadex G-2 25 column. The to occur; (iv) the failure to eliminate the exothermic reactions tubulin concentration was 57,uM and a scan rate of 11 K min1 was after the prominent minor proteins had been removed; and (v) used. the observation of the exothermic reactions whether or not glycerol is present. The exothermic that we call EXO I is the most distinct MAPs), which are the major contaminants in our preparations and well-characterizedprocessfeature present in our DSC results. It (14), tubulin preparations were further purified byzgelfiltration is most clearly observed at high scan rate and low tubulin con- chromatography (Bio-Gel A-15m). A scan obtaijned withthis centration. Atlow scan rate andhightubulin concentration only material is shown in Fig. 5; it is qualitatively sinnilar to those a smallexothermiceffect isobserved, although polymerization shown in earlier figures-for example, the bottonniscan in Fig. nevertheless takes place. 3A-except that it occurs at a somewhat higher temperature An additional feature of EXO I is the large decrease in the than expected on the basis of the results obtained Nwith less pure apparent heat capacity of the product or products of this process preparations. in comparison to the reactants. This decrease can account for Role of Glycerol in the Calorimetric Results. Experiments a sizeable fraction of the changes in the enthalpy resulting from were also conducted to determine if the presence of glycerol variations in experimental parameters that cause changes in the could in some way account for the observed DSC results. polymerization temperature. This decrease is consistent with Tubulin samples prepared and polymerized in1 the complete the view that hydrophobic interactions play an important role absence of glycerol were run in the DSC in a buffer similar to in tubulin polymerization (23). However, this simplistic view PM 2M (100mM Pipes/i mM EGTA/0.5 mM MgrSO4, pH 6.9). is complicated in the present case by the extremely large neg- Results from these experiments, shown in Fig. 6, indicate that ative enthalpies, which have not been approached in any other an exothermic process is present, the behaviorc )f which as a reaction thought to be significantly influenced by hydrophobic function of protein concentration is qualitatively y the same as forces. Also, the complex temperature-dependent behavior of seen in the presence of glycerol. It is apparent, biowever, that the excess heat capacity after the completion of EXO I suggests glycerol markedly lowers the temperature at whicIhthis process the formation of more than one type of polymer having dif- occurs and increases its sharpness. ferent heat capacities. An alternative explanation for the complex behavior of the excess heat capacity is the presence of a second exothermic reaction (EXO II) that partially overlaps EXO I. In either case, experimental conditions control the mass 16 ratio and the interconversion of different polymers, or the ex- tent of EXO II. At present our data cannot distinguish which explanation is correct, or possibly whether both explanations are involved. Any exothermic process that can be observed on scanning the 3 temperature in the positive direction must be primarily kine- tically rather than thermodynamically limited. An estimate of T the activation energy for EXO I can be calculated from the DSC .g 5.0 cal K g trace (24) if it is assumed that the reaction goes to completion X I\ at all temperatures within the DSC peak. The sharpness of the I exothermic peaks corresponding to this reaction shows that the reaction has an extraordinarily large activation energy. For II trace 10 15 20 25 30 35 40 example, the downward slope in the range 25-260C in the Temperature, 0C at a scan rate of 2 K minI in Fig. 1 corresponds to an activation energy of 280 kcal molh. As a result of the large activation Calorimetric scans of tubulin polymeri FIG. 6. zOn energy associated with EXO I, it is understandable that this complete absence of glycerol in a buffer containing 1 mM Pipes process makes a small contribution to the DSC curve under 2 mM GTP, 1 mM EGTA, and 0.5 mM MgSO4 at p1 H 6.9. Tubulin only concentrations, determined by the method of Lowry et al. (21), using experimental conditions that permit polymerization to take bovine serum albumin as a standard, are given for eaci h trace in units place at temperatures only slightly below the range in which of AtM. The scan rate for all experiments was 1.7 K m in-1. EXO I is observed. Downloaded by guest on September 24, 2021 Biochemistry: Berkowitz et al. Proc. Natl. Acad. Sci. USA 77 (1980) 4429 The above facts and others reported in this paper lead us to changes in enthalpy and in apparent heat capacity observed suggest that there are at least two pathways for microtubule by Hinz et al. were much smaller than those that we have re- polymerization. The first route is initiated at a low temperature, ported here. These differences appear to be mainly the result has a relatively small activation energy, and does not involve of their not having used as low tubulin concentrations or as high or possibly requires little of EXO I. The second route has a large scan rates as we employed. Other differences between their activation energy, proceeds via EXO I or permits a large results and ours may be mentioned: (i) their exothermic peaks amount of EXO I to occur, and requires a higher temperature occur at higher temperatures than ours under equivalent con- than the first route before it can be initiated. If it is assumed that ditions; and (fi) they observed a more complete correlation material polymerized by either route can undergo no further between turbidity increases and excess heat capacity decreases reaction within the time course of the experiment, then it is than we did. These differences may stem at least in part from possible to obtain a qualitative understanding of various features differences in the methods of tubulin preparation or buffer of our DSC data. This includes the decrease in the mholar exo- conditions employed or both. thermic enthalpy as the scan rate is decreased, the tubulin The authors thank Drs. P. D. Ross and M. B. Jackson for their helpful concentration is increased, or the calcium concentration is de- comments on reading this paper, and Drs. J. Wolff and R. C. Williams, creased. These factors are all influences that lower the poly- Jr., for their support and encouragement. This work was supported in merization temperature range and thus favor the lower-tem- part by grants from the National Institutes of Health (GM 04725) and perature pathway over the higher-temperature pathway. the National Science Foundation (PCM 7681012). A major requirement in this proposal of two pathways for 1. Dustin, P. (1978) Microtubules (Springer, New York). polymerization is that the products of the two pathways cannot 2. Weisenberg, R. S. (1972) Science 177, 1104-1105. be identical because they differ drastically in enthalpy of for- 3. Lee, J. C., Frigon, R. P. & Timasheff, S. N. (1973) J. Biol. Chem. mation from the common starting material, unpolymerized 248,7253-7262. tubulin at low temperature. That nonidentical microtubules 4. Kirschner, M. W. (1978) Int. Rev. Cytol. 54, 1-65. can in fact be formed under differing conditions is supported 5. Margolis, R. L. & Wilson, L. (1978) Cell 13, 1-8. by the recently published work of Pierson et al. (25) showing 6. Gaskin, F., Cantor, C. R. & Shelanski, M. L. (1974) J. Mol. Biol. 89,686-689. that in vitro polymerization of tubulin at different temperatures 7. Engelborghs, Y., Heremans, K. A. H. & de Maeyer, L. C. M. leads to microtubules of different structures. (1976) Nature (London) 259,686-689. Several of our observations find no ready explanation in this 8. Lee, J. C. & Timasheff, S. N. (1977) Biochemistry 16, 1754- proposal. First and foremost of these is the extremely large heat 1764. evolution observed under certain conditions. At maximum this 9. Johnson, K. A. & Borisy, G. G. (1979) J. Mol. Biol. 133, 199- amounts to about 8 cal per g of tubulin, which is similar to the 216. heat absorption observed in the complete thermal unfolding 10. Sutherland, J. W. H. & Sturtevant, J. M. (1976) Proc. Natl. Acad. of globular proteins. Another unexplained observation is that Sci. USA 73,3084-3089. in cases continues to increase at temperatures 11. Hard, R. & Klump, H. (1977) J. Cell Biol. 75,286 (abst.). all the turbidity 12. Hinz, H.-J., Gorbunoff, M. H., Price, B. & Timasheff, S. N. (1979) well above the temperature of completion of EXO I. This could Biochemistry 18, 3084-3089. result, at least in part, from the fact that enthalpy and light 13. Berkowitz, S. A., Velicelebi, G. & Sturtevant, J. M. (1979) Biophys. scattering presumably give different measures of the extent of J. 25, 35 (abst.). polymerization, the former responding approximately equally 14. Berkowitz, S. A., Katagiri, J., Binder, H. K. & Williams, R. C., Jr. to the formation of intersubunit bonds regardless of whether (1977) Biochemistry 16, 5610-5617. they are formed in small or in large polymers, whereas the latter 15. Detrich, H. W., III & Williams, R. C., Jr. (1978) Biochemistry is disproportionately affected by material present in the form 17,3900-3907. of large polymers. 16. Privalov, P. L., Plotnikov, V. V. & Filimonov, V. V. (1975) J. Chem. Thermodyn. 7,41-47. The decreases in apparent specific heat that accompany EXO 17. Velicelebi, G. & Sturtevant, J. M. (1979) Biochemistry 18, I have values up to approximately the levels found for the in- 1180-1186. creases which accompany the thermal unfolding of globular 18. Taussky, H. & Shorr, E. (1953) J. Biol. Chem. 202,673-685. proteins. 19. Rebhun, L. I., Mellon, M., Jemiolo, D., Nath, J. & Ivy, N. (1974) The polymerization of tubulin has been the subject of two J. Supramol. Struct. 2,466-485. recent investigations (11, 12) using the same model DSC as we 20. Nishida, E. (1978) J. Biochem. 84,507-512. have used. We have been unable to duplicate in any significant 21. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. respect the results reported by Hard and Klump (11), and we (1951) J. Biol. Chem. 193,265-275. have no explanation to offer for our differences. Hinz et al. (12) 22. Sturtevant, J. M. (1972) Methods Enzymol. 26, 227-253. 23. Sturtevant, J. M. (1977) Proc. Natl. Acad. Sci. USA 74, 2236- observed an exothermic phase in the polymerization as have 2240. we, but they found this to be preceded by an endothermic 24. Borchardt, H. J. & Daniels, F. (1957) J. Am. Chem. Soc. 79, phase, which has not appeared in our work. They also observed, 41-46. as have we, a decrease in the apparent heat capacity of the 25. Pierson, G. B., Burton, P. R. & Himes, R. H. (1974) J. Cell Sci. protein resulting from the polymerization. However, the 39,89-99. Downloaded by guest on September 24, 2021