Biochimica et Biophysica Acta, 491 (1977) 581-590
© Elsevier/North-HollandBiomedicalPress BBA 37632 THE SECONDARY STRUCTURE OF MYELIN BASIC PROTEIN EXTRACTED BY DEOXYCHOLATE
ROSS SMITH Department of Physical Chemistry, University of Sydney, Sydney, N.S.W. (Australia)
(Received October 1lth, 1976)
SUMMARY Because of the implication of myelin basic protein in some neurological diseases its in vivo structure is of particular interest. The protein is usually isolated using organic solvents and acid solutions and has previously been shown to contain little a-helical or fl-structure; but it is not known how the extraction methods influence the structure. Following recent observations that deoxycholate generally causes minimal structural perturbation when used to dissolve membrane proteins, this detergent has been used to extract the basic protein from bovine myelin. The protein contained in deoxycholate washes of myelin has been purified by gel chromatography and its secondary structure examined by circular dichroism spectroscopy. This protein and conventionally prepared bovine and human basic protein to which 1 ~o deoxycholate has been added appear to have the same structure: they contain 8-14 ~ more helical structure than the chloroform/methanol-extracted protein in pH 4.8 acetate buffer or in pH 9.15 Tris buffer. This conformational change is unaffected by addition of 0.25 M NaC1. The helical content will approach the upper limit if, as is expected, these ordered segments are short. It is suggested that basic protein may adopt this more ordered structure in myelin and possess activity not apparent in its water-soluble unordered conformation. Retention of its encephalitogenic activity following severe treatment may result from an ability to rapidly refold to the original conformation rather than from this activity being inherent in the unordered form.
INTRODUCTION Myelin basic protein is a major constituent of central nervous system myelin. Particular attention has focussed on this component because of its ability to induce allergic encephalomyelitis on injection into animals, and because of its possible role in multiple sclerosis in humans. The amino acid sequences of basic protein from several species have been elucidated [1, 2]: they are marked by a large content of basic residues and an overall content of about 47 ~ polar residues (calculated following Vanderkooi and Capaldi [3]), a figure typical of most water-soluble proteins and a few membrane proteins. The apolar residues are not concentrated in one polypeptide
582 domain as observed, for example, in the bimodal membrane protein cytochrome
b5 [4]. Basic protein is generally prepared by acid extraction of the precipitate formed by adding chloroform/methanol to white matter or to whole brain tissue. This product is water-soluble and monomeric below pH 7 [5] and has very little helical or fl-structure as evidenced by its ORD spectrum, which yields a Moffitt-Yang parameter (b0) close to zero [6-8]. Analysis of its CD spectra at a variety of pH values appears to confirm this conclusion [5, 9]. Hydrodynamic studies of its tertiary structure suggested that the protein was behaving either as a prolate ellipsoid or as a flexible coil [10]. For example the observed intrinsic viscosity in dilute buffer (9.3 [11] to 16.9 [37] ml/g) is far above the 3--4 ml/g characteristic of globular proteins indicating asymmetry (or unusually high solvation): it is somewhat lower than the value obtained in 6 M guanidinium.HCl, expansion of a flexible coil in this (moderately good) solvent is however expected. Recently Krigbaum and Hsu [12] have obtained detailed low-angle X-ray scattering results which cannot be satisfactorily fitted by compact scattering models, but which are consistent with a flexible coil model. Because of the implication of this protein in neurological diseases its in vivo structure is of particular importance. The water-soluble protein may not display the same structure as obtains in vivo. Indeed the use of organic solvents and acid in the preparation is likely to induce conformational transitions: these solvents are known to denature other proteins [13]. If denatured, the protein might refold to its original conformation on reassociation with lipids, but there are no a priori grounds for making such an assumption. Our efforts have consequentlycentred on the extraction of basic protein from myelin by procedures expected to cause minimal structural perturbation, and on the determination of the secondary structure of this protein in association with an amphiphile, deoxycholate. METHODS Myelin was prepared from bovine brain white matter following the method of Autilio et al. [14] to the stage described as yielding crude myelin. For experimental convenience centrifugation was performed successively in 0.32 and 0.66 M sucrose rather than by layering one solution on top of the other. Thin sections of embedded myelin examined by electron microscopy were found to be essentially free of other subcellular structures. Included in the myelin preparation are three washings with water to remove sucrose. Additional washings with water and with 0.1 M NaCI were used to determine the lability of the basic protein. In these experiments a few grams of packed myelin were stirred at 4 °C with 20 ml of the appropriate solvent: after an hour the supernatant was obtained by centrifugation (20 000 × g for 30 min) and its protein content established by the method of Lowry et al. [15] and by absorption at 280 nm. For extraction with deoxycholate 2-3 g of lyophilized myelin were suspended in 20-40 ml of a solution containing 1 ~ sodium deoxycholate (Merck, Darmstadt, for microbiology) and 0.1 M Tris/Chloride (Sigma, St. Louis, analytical grade), titrated to pH 9.20 with dilute HC1 at 4 °C. Following 0.5-1 h stirring the suspension
583 was centrifuged (90 000 x g, 30 min) and the residue re-extracted twice. The supernatants were pooled and concentrated by ultrafiltration before dialysis against pH 9.2 buffer containing 1 ~ deoxycholate, recentrifugation (20 000 x g, 20 min), and then application to a Sephadex G-75 column equilibrated with the same buffer. Additional purification was effected by repeating the last step once or twice. The purity of the protein was established by electrophoresis in dodecyl sulphate [16] and in acid solution [171. The molecular weight and purity of the basic protein were verified by equilibrium ultracentrifugation using the meniscus depletion method [18]. For sedimentation studies the protein in deoxycholate was dialysed against 0.01 M, pH 9.30, Tris buffer then against water to remove the detergent: sodium acetate was then added to 0.1 M and the pH adjusted to 5 with dilute HC1. Interference optics were used to establish the equilibrium protein distribution: the initial concentration was 0.4 mg/ml. The partial specific volume (0.72 ml/g) of the protein was calculated from the amino acid composition. The efficacy of gel permeation chromatography in removing dissolved lipid was ascertained by testing the protein for residual phospholipid, by measuring the phosphorus content [19]. Basic protein from human and bovine brain was also prepared following Oshiro and Eylar [20]. The CD spectra were obtained on Cary 61 and Jobin Yvon Mark III spectrophotometers. The spectrometer calibration was verified with a standard solution of 5-a-cholestan-7-one (kindly supplied by Dr. Ruth Gall, Organic Chemistry Department, Sydney University). Spectra were recorded using 0.1 or 0.01 cm pathlength cells with protein concentrations in the range 0.2-3.4 mg/ml. Baseline corrections were made by running solvent under identical machine conditions. Mean residue rotations and ellipticities were calculated assuming an average residue molecular weight of 108.5 [10]. No corrections were applied for the solvent refractive index. The secondary structure of basic protein was deduced from CD measurements using a least-squares method to fit the observed spectra with a combination of the spectra for pure a-helices, fl-structure and random segments in proteins [21]. The observed ellipticities at 2.5-nm intervals (from 240 nm) were applied to simultaneous equations expressing this ellipicity, [0]z, as a linear combination of the ellipticities ([0]~, [O]°a,and [0]~) of the separate structures at the same wavelengths:
[Oh = fa [0]] + fa [01~ + % [01~
(1)
After an initial calculation with data from three widely separated wavelengths, all of the data were used to obtain the best values of fa, fm and fr, the fractions of the three structures in the protein, using a function minimization program. The residual was obtained by a simplex minimization of the sum of the values obtained by squaring the difference between the calculated and observed ellipticities after division by the observed ellipticity at the same wavelength. The author is indebted to Mr. E. Tysoe, Physical Chemistry Department, Sydney University, for writing the computer program, and for carrying out many of the computations.
584 RESULTS
Purification of basic protein Washing of myelin with water or 0.1 M NaC1 released no protein, but the latter solvent did yield a suspension which required higher speed centrifugation to produce a clear supernatant essentially devoid of protein. In contrast deoxycholate gave supernatants which were initially cloudy but after concentration and recentrifugation were clear and contained considerable amounts of protein. Typically 75 ~ of the myelin proteins were extracted in three washings. Only a few percent of the lipid dissolved. On gel permeation chromatography the extracts yielded two main peaks, their relative heights being dependent on the method of protein analysis (Fig. 1). Dodecyl sulphate gel electrophoresis (Fig. 2) revealed that the second peak was largely a single polypeptide of molecular weight 19 500 ± 10 ~ corresponding to the myelin basic protein. The ionic strength of the phosphate buffer used for electrophoresis was 0.12: it has recently been reported [22] that under these conditions apparent molecular weight of the basic protein is anomalously high by about 6-8 ~. The first chromatographic peak showed at least two components on dodecyl sulphate electrophoresis (Fig. 2); the majority of the protein remained at the.gel origin but some basic protein was present and frequently a small amount of protein of apparent molecular weight near 26 000 was visible. It appears that despite the precautions [23] taken in preparing samples for electrophoresis the majority of the proteolipid protein was aggregated and did not penetrate 5 or 10 ~ acrylamide gels. The behaviour of this 1.0 3.0
2.0
0.5 1.0 ¸ E c
E
~o
o~
(b)
0.2-
t--\
01
0.1-
0
0
IPO
20
30 Tube
4'0
0
number
Fig. 1. Chromatography of deoxycholate extracts of myelin on Sephadex G-75. The 2.5 cm internal diameter by 70 cm column was eluted with 1 ~ sodium deoxycholate in 0.1 M Tris buffer, pH 9.30. The flow rate was 20 ml/h, and 20-min fractions were collected at 4 °C. (a) First run of extract from myelin. (b) Rechromatography of the pooled protein indicated by the horizontal bar in a. Protein in the effluent was detected colorimetrically (dashed line), using 10pl (in a) or 50/~1 (in b)samples from each tube, and by absorbance measurements at 280 nm (solid line). The fraction indicated by the bar in b was again rechromatographed before use for circular dichroism measurements.
585
Fig. 2. (a) Polyacrylamide gel electrophoresis in dodecyl sulphate. Gel 1, the first peak from the chromatographic column (Fig. la) ; 2, second peak from the column; Gel 3, bovine chloroform-methanolextracted protein; 4, a-chymotrypsinogen (top) and cytochrome c. Electrophoresis was from top to bottom. (b) Gel electrophoresis at pH 4.3. Gel 5, basic protein extracted with deoxycholate (from the second peak of Fig. la); Gel 6, human basic protein extracted using chloroform/methanol. The protein from the first chromatographic peak (Figs. 1a and I b) was largely insoluble in the absence of detergent.
protein on electrophoresis, its abundance relative to the basic protein (Fig. 1), its elution position from the gel permeation column, and its amino acid composition (Gallagher, G.A. and Smith, R., unpublished), leave little doubt that it is the major proteolipid protein of myelin. The lipid applied to the column eluted in the tail of the basic protein peak. Phosphate analyses failed to detect any phospholipids in the basic protein under conditions where the binding of less than one molecule of a typical phospholipid per protein molecule would have been measurable: this indicates effective separation from the dissolved lipids [24]. Martenson et al. [38] have reported a phosphorus content of 0.10 mol per mol of bovine basic protein, which is beyond the lower limit of the present assay. Basic protein extracted with chloroform/methanol was subjected to similar procedures and also found to be phospholipid-free and homogeneous. Equilibrium ultracentrifugation verified both the homogeneity and molecular weight of the basic protein extracted with deoxycholate, a single weight-average molecular weight of 18 000 i 1000 being consistent with the protein distribution throughout the centrifuge cell. Circular Dichroism Studies Basic protein has previously been shown (e.g. refs. 6-9) to contain little a- or fl-structure in water or buffers. The present CD spectra (Fig. 3) confirm this conclusion and indicate that there is little structural alteration when the solvent is changed to 0.1 M Tris, pH 9.15: the solubility of the protein is however low in alkaline solutions and slight turbidity in the solution used for CD precluded measurement of the spectrum below 205 nm. This apparent conformational stability toward pH change is
586
J a3
-6 E ~ -5" "o
% o
d~ ~ -10
o ×
260
21o A (nm)
Fig. 3. Circular dichroism spectra of basic protein in 0.1 M sodium acetate buffer, pH 4.8. The vertical bars indicate the standard deviations in eight spectra run on three different samples of bovine and human basic protein. There were no significant differences in the spectra of the protein from the two species. Protein concentrations were derived from the absorbance at 2~0 nm assuming an extinction co-efficient (deduced from ref. 5) E]c°/m ° -- 5.35.
consistent with earlier CD studies and with the recent observation from proton N M R studies that the spin-spin relaxation times vary only slightly in traversing the pH range from 5.6 to 9.2 [5]. The spectrum obtained for basic protein in acetate buffer (Fig. 3) is very similar to several previously published [6, 25] but differs quantitatively from that of Liebes et al. [5]. The origin of this difference is not apparent: Fig. 3 incorporates data from five different preparations, the spectra of which were obtained on two different CD spectrometers, with no systematic deviations between spectra. The instruments provided satisfactory quantitative data for both 5-a-cholestan-7-one and for a standard solution of lysozyme. Basic protein extracted with, and examined in, deoxycholate solution exhibited a considerably different spectrum (Fig. 4) from that of the protein in acetate buffer. No diminution in this difference was observed when 0.25 M NaC1 was added to the deoxycholate solution; higher salt concentrations caused precipitation of the deoxycholate. Comparison of Figs. 3 and 4 reveals several changes. The peak near 198 nm in acetate buffer is shifted to 203 nm and its negative ellipticity is diminished in the presence of deoxycholate; a pronounced shoulder is also introduced near 220 nm. These changes in the spectrum are well accounted for by the introduction of a moderate amount of helical structure, which is characterized by a large positive ellipticity at 192 nm and two smaller negative peaks at 209 and 222 nm [21]. Spectra from several sources [21, 26-28] for protein a-helices, fl-structure and unordered regions (the basis spectra) failed to yield satisfactory fits to the basic protein spectra, even if in Eqn. l [0]z was replaced by C[O]zwhere C is a wavelength-independent parameter adjusted to attain the best correspondence between the calculated and experimental spectra. The difficulty is most simply seen in fitting the spectrum of the chloroform/methanolextracted protein in water: none of these sets of basis spectra could reproduce this curve despite the fact that is now well-established (ref. 6 and the present work). Failure to fit this curve may stem from inadequacies in the basis spectra. There is
587
.~ o-
~
? o
-5
x
g -10 I
I
200
210
~
I
220 ?~ (nm)230
I
240
Fig. 4. Circular dichroism spectrum of basic protein extracted from myelin by deoxycholate and examined in 1% sodium deoxycholate (in 0.1 M Tris buffer, pH 9.30), and of organically extracted human and bovine basic protein in the same buffer. The vertical bars indicate the standard deviations from five spectra from 240 to 220 nm and of three spectra from 217.5 to 195 nm. The solid line is drawn through the average ellipticity at each wavelength. The spectrum computed using basis spectra B (Table I) is indicated by (x). Use of basis spectra C resulted in a similar spectrum. Protein concentrations were derived from the absorbance at 280 nm relative to the buffer. reasonable agreement on the standard spectra for infinite helices and for fl-structures but there is marked divergence in,the spectra accepted for unordered protein segments. As a consequence, the fitting of spectra of proteins containing appreciable ordered structure may be carried out with some confidence, but it is to be expected that the fit will be far less satisfactory for proteins possessing little ordered structure, as indeed proved to be the case with the basic protein in acetate buffer. As an alternative approach we have started from the assumption that basic protein in water contains negligible a- or fl-structure'. As noted above, there are reasonable experimental grounds for this assumption in that earlier O R D studies have shown that the Moffitt-Yang parameter is close to zero, indicating the absence of ordered structures [29]. And although the analysis of the CD spectrum is beset by the difficulties mentioned above it does not suggest the presence of e- or E-structure. In particular, the ellipticity at 222 nm [27] is consistent with the absence of e-helix. ~, To elucidate the structure of basic protein in deoxycholate we have used the spectrum of the protein in acetate buffer with published e- and/~-structure spectra [21] as the basis spectra for curve fitting. We have additionally fitted the spectra for protein in acetate buffer and in deoxycholate using all three basis spectra derived from Chen et al. [21]. The results are presented in Table I. Separate analyses of the spectra of deoxycholate-extracted protein and of basic protein in pH 9.15 Tris buffer were not carried out as the former essentially mimicked the chloroform/methanol-extracted protein in deoxycholate solution and the latter did not differ significantly from the spectra in water or acetate buffer. The folding of the protein appears to be rapid as the spectrum run directly after addition of deo" This is not in conflict with the observation that when dissolved in guanidinium. HCI solution the protein exhibits a slightly different spectrum [10]: we are assuming the absence of a- and flstructure, not a unique spectrum for the disordered states. Protein intramolecular hydrophobic interactions in water may well alter the optical activity from that expected for the random coil in guanidinium- HCI solution.
588 TABLEI SECONDARY STRUCTURE OF BASIC PROTEIN COMPUTED FROM CD SPECTRA The averaged spectra of Figs. 3 and 4 were used in the calculations. Basis spectra: A, from Chen et al. [21] interpolated from their Table II (i.e. 11-residue a-helix spectrum). B, a-helix and fl-structure spectra as in A, but with the spectrum of basic protein in acetate buffer replacing the spectrum from ref. 21 for unordered protein segments. C, as in B except 5-residue a-helix spectrum from ref. 21 substituted.
Basic protein in acetate buffer Basic protein in deoxycholate
Basis spectra
a-helix (~)
fl-structure (~)
Unordered (~)
A
l0
16
74
A B C
22 9 14
13 0 3
65 91 82
xycholate differed little from the spectrum of the protein extracted with this surfactant. Although not analysed in detail the O R D spectra obtained also revealed a higher helical content in basic protein in the presence of deoxycholate. DISCUSSION Intense interest in the conformation of basic protein has been generated by its unique encephalitogenic activity. The seeming lack of helical or fl-structure in water has been used to explain the retention of this activity following severe heating and chemical treatment. All of these conformational studies have however been conducted with protein extracted under conditions likely to cause structural alteration. In contrast, deoxycholate has been widely used for the isolation of other membrane proteins and has been shown to effect minimal structural perturbation [24]: particularly noteworthy are studies [30] showing no significant changes in the CD spectra of lymphocyte, heine-, and other membrane proteins on isolation with deoxycholate. Independent of the method of analysis of the C D spectra, there appears to be about 8-14 ~ difference in helix content of basic protein in water and in 1 ~ deoxycholate. This difference has been calculated assuming an average length of either 5 or 11 amino acid residues in each helical segment: if in fact the helical segments in basic protein are short the proportion of a-helix will be close to 1 4 ~ . Application of the empirical structure-prediction algorithm of Chou and Fasman [31] reveals several regions which are potentially helix forming, the average length of these is less than 11 : the efficacy of empirical prediction algorithms has however been questioned [23]. Difficulties inherent in structural analysis by C D [33] necessitate some restraint in interpretation of the quantitative differences in structure. The exact nature of the conformational change is obscured a little by the unusual spectrum of the protein in acetate buffer (see above), which has the form expected for a randomly coiled polypeptide but appears flattened in a way not attributable to the introduction of either ahelical or fl-structure. Basic protein is also monomolecularly dispersed in the acetate buffer, hence the flattening cannot be accounted for by light scattering. Basic protein extracted with organic solvents is able to refold quickly on addition of deoxycholate; a similar folding has been observed on the addition of dodecyl
589 sulphate, oleate, lysolecithin [6] and trifluoroethanol [5]. Successive additions of trifluoroethanol produced step-like changes in the CD spectrum suggestive of three domains in the polypeptide separately undergoing co-operative coil-to-helix transitions. In view of these results it is unlikely that the change in ellipticity in deoxycholate results from an enhancement of the optical activity of the detergent per se upon binding. The apparent lack of effect of severe treatment on the encephalitogenic properties of the protein may well result from its ability to refold quickly from the denatured state (for example, when added to Freund's adjuvant), rather than the activity being intrinsic in the disordered protein. Basic protein is not freely water-soluble at near-neutral pH, despite the considerable net positive charge on the protein. Aggregation has been demonstrated by light scattering and N M R spectroscopy [5]. This aggregation appears to be unaccompanied by significant changes in secondary structure, the CD spectra at neutral and alkaline pH being superimposable (see above). Moscarello et al. [9] did observe minor changes in the alkaline spectrum as the protein concentration was varied: such changes may in part be attributable to scattering and absorption flattening effects [34]. The protein appears to be soluble and apparently monomeric at high pH in the presence of deoxycholate, a surfactant similar in structure to cholesterol, a major myelin lipid. This enhanced solubility is presumably due to substitution of intermolecular proteinprotein hydrophobic interactions with detergent-protein contacts. Basic protein has been demonstrated to interact hydrophobically with lipid monolayers, the incorporation showing a positive correlation with lipid alkyl chain length [35]. It also alters the temperature and enthalpy change of the phase transition in vesicles of dipalmitoyl phosphatidylcholine and dipalmitoyl phosphatidylglycerol [36]. Preliminary experiments (Smith, R., unpublished) show also appreciable binding of deoxycholate to basic protein at alkaline pH in 0.25 M NaC1 demonstrating more directly its possession of hydrophobic binding sites. These studies and the solubility characteristics of the protein point to some hydrophobic character consistent with partial penetration of the bilayer in myelin. As a consequence of this basic protein may adopt a more ordered structure in myelin and may possess activities not apparent in its water-soluble conformation. ACKNOWLEDGEMENTS The co-operation of Dr. C. J. Hawkins, Inorganic Chemistry Department, University of Queensland, and Dr. J. N. Douglas, Solid State Physics Department, Australian National University, in providing access to their CD spectrometers is gratefully acknowledged. The author has benefited from numerous discussions with Professor Walter J. Moore and Mr. E. Tysoe of the Physical Chemistry Department, Sydney University. This work was partially supported by the Australian Research Grants Committee. REFERENCES 1 Carnegie, P. R. (1971) Nature 229, 25-28 2 Eylar, E. H., Brostoff, S., Hashim, G., Caccam, J. and Burnett, P. (1971) J. Biol. Chem. 246, 5770-5784
590 3 Vanderkooi, G. and Capaldi, R. A. (1972) Ann. N.Y. Acad. Sci. 195, 135-138 4 Spatz, L. and Strittmatter, P. (1971) Proc. Natl. Acad .Sci. U.S. 68, 1042-1046 5 Liebes, L. F., Zand, R. and Phillips, W. D. (1975) Biochim. Biophys. Acta 405, 27-39 6 Anthony, J. S. and Moscarello, M. A. (1971) Biochim. Biophys. Acta 243, 429-433. 7 Palmer, F. B. and Dawson, R. M. C. (1969) Biochem. J. 111, 629-636 80shiro, Y. and Eylar, E. H. (1970) Arch. Biochem. Biophys. 138, 606-613 9 Moscarello, M. A., Katona, E., Neumann, A. W. and Epand, R. M. (1974) Biophys. Chem. 2, 290-295 l0 Epand, R. M., Moscarello, M. A., Zierenberg, B. and Vail, W. J. (1974) Biochemistry 13, 12641267 11 Eylar, E. H. and Thompson, M. (1969) Arch. Biochem. Biophys. 129, 468-479 12 Krigbaum, W. R. and Hsu, T. S. (1975) Biochemistry 14, 2542-2546 13 Tanford, C. (1968) in Advances in Protein Chemistry (Anfinsen, C. B., Anson, M. L., Edsall, J. T. and Richards, F. M., eds.), Vol. 23, pp. 121-282, Academic Press, New York 14 Autilio, L. A., Norton, W. T. and Terry, R. D. (1964) J. Neurochem. 11, 17-27 15 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 16 Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412 17 Reisfeld, R. A., Lewis, U. J. and Williams, D. E. (1962) Nature 195, 281-283 18 Yphantis, D. A. (1964) Biochemistry 3, 297-317 19 Bartlett, G. R. (1959) J. Biol. Chem. 234, 466-468 20 Oshiro, Y. and Eylar, E. H. (1970) Arch. Biochem. Biophys. 138, 392-396 21 Chen, Y-H., Yang, J. T. and Chou, K. H. (1974) Biochemistry 13, 3350-3359 22 Campagnoni, A. T. and Magpo, C. J. (1974) J. Neurochem. 23, 887-890 23 Morell, P., Wiggins, R. C. and Gray, M. J. 0975) Anal. Biochem. 68, 148-154 24 Helenius, A. and Simons, K. (1975) Biochim. Biophys. Acta 415, 29-79 25 Block, R. E., Brady, A. H. and Joffe, S. (1974) Biochem. Biophys. Res. Commun. 54, 1595-1602 26 Saxena, V. P. and Wetlaufer, D. B. (1971) Pro¢. Natl. Acad. Sci. U.S. 68, 969-972 27 Chen, Y-H. and Yang, J. T. (1971) Biochem. Biophys. Res. Commun. 44, 1285-1291 28 Greenfield, N. and Fasman, G. D. (1969) Biochemistry 8, 4108-4116 29 Urnes, P. and Doty, P. (1961) Adv. Protein Chem. 16, 401-544 30 Bayley, P. M. (1973) Prog. Biophys. Mol. Biol. 27, 3-76 31 Chou, P. Y. and Fasman, G. D. 0974) Biochemistry 13, 222-245 32 Burgess, A. W. and Scheraga, H. A. (1975) Proc. Natl. Acad. Sci. U.S. 72, 1221-1225 33 Sears, D. W. and Beychok, S. (1973) in Physical Principles and Techniques of Protein Chemistry (Leach, S. J., ed.), Part C, pp. 445-593, Academic Press, New York 34 Gordon, D. J. and Holzwarth, G. (1971) Proc. Natl. Acad Sci. U.S. 68, 2365-2369 35 Demel, R. A., London, Y., van Kessel, W. S. M. G., Vossenberg, F. G. A. and van Deenen, L. L. M. (1973) Biochim. Biophys. Acta 311, 507-519 36 Papahadjopoulas, D., Moscarello, M., Eylar, E. H. and Isac, T. (1975) Biochim. Biophys. Acta 401,317-335 37 Chao, L.-P. and Einstein, E. R. (1970) J. Neurochem. 17, 1121-1132 38 Martenson, R. E., Kramer, A. J. and Deibler, G. E. (1976) J. Neurochem. 26, 733-736
© Elsevier/North-HollandBiomedicalPress BBA 37632 THE SECONDARY STRUCTURE OF MYELIN BASIC PROTEIN EXTRACTED BY DEOXYCHOLATE
ROSS SMITH Department of Physical Chemistry, University of Sydney, Sydney, N.S.W. (Australia)
(Received October 1lth, 1976)
SUMMARY Because of the implication of myelin basic protein in some neurological diseases its in vivo structure is of particular interest. The protein is usually isolated using organic solvents and acid solutions and has previously been shown to contain little a-helical or fl-structure; but it is not known how the extraction methods influence the structure. Following recent observations that deoxycholate generally causes minimal structural perturbation when used to dissolve membrane proteins, this detergent has been used to extract the basic protein from bovine myelin. The protein contained in deoxycholate washes of myelin has been purified by gel chromatography and its secondary structure examined by circular dichroism spectroscopy. This protein and conventionally prepared bovine and human basic protein to which 1 ~o deoxycholate has been added appear to have the same structure: they contain 8-14 ~ more helical structure than the chloroform/methanol-extracted protein in pH 4.8 acetate buffer or in pH 9.15 Tris buffer. This conformational change is unaffected by addition of 0.25 M NaC1. The helical content will approach the upper limit if, as is expected, these ordered segments are short. It is suggested that basic protein may adopt this more ordered structure in myelin and possess activity not apparent in its water-soluble unordered conformation. Retention of its encephalitogenic activity following severe treatment may result from an ability to rapidly refold to the original conformation rather than from this activity being inherent in the unordered form.
INTRODUCTION Myelin basic protein is a major constituent of central nervous system myelin. Particular attention has focussed on this component because of its ability to induce allergic encephalomyelitis on injection into animals, and because of its possible role in multiple sclerosis in humans. The amino acid sequences of basic protein from several species have been elucidated [1, 2]: they are marked by a large content of basic residues and an overall content of about 47 ~ polar residues (calculated following Vanderkooi and Capaldi [3]), a figure typical of most water-soluble proteins and a few membrane proteins. The apolar residues are not concentrated in one polypeptide
582 domain as observed, for example, in the bimodal membrane protein cytochrome
b5 [4]. Basic protein is generally prepared by acid extraction of the precipitate formed by adding chloroform/methanol to white matter or to whole brain tissue. This product is water-soluble and monomeric below pH 7 [5] and has very little helical or fl-structure as evidenced by its ORD spectrum, which yields a Moffitt-Yang parameter (b0) close to zero [6-8]. Analysis of its CD spectra at a variety of pH values appears to confirm this conclusion [5, 9]. Hydrodynamic studies of its tertiary structure suggested that the protein was behaving either as a prolate ellipsoid or as a flexible coil [10]. For example the observed intrinsic viscosity in dilute buffer (9.3 [11] to 16.9 [37] ml/g) is far above the 3--4 ml/g characteristic of globular proteins indicating asymmetry (or unusually high solvation): it is somewhat lower than the value obtained in 6 M guanidinium.HCl, expansion of a flexible coil in this (moderately good) solvent is however expected. Recently Krigbaum and Hsu [12] have obtained detailed low-angle X-ray scattering results which cannot be satisfactorily fitted by compact scattering models, but which are consistent with a flexible coil model. Because of the implication of this protein in neurological diseases its in vivo structure is of particular importance. The water-soluble protein may not display the same structure as obtains in vivo. Indeed the use of organic solvents and acid in the preparation is likely to induce conformational transitions: these solvents are known to denature other proteins [13]. If denatured, the protein might refold to its original conformation on reassociation with lipids, but there are no a priori grounds for making such an assumption. Our efforts have consequentlycentred on the extraction of basic protein from myelin by procedures expected to cause minimal structural perturbation, and on the determination of the secondary structure of this protein in association with an amphiphile, deoxycholate. METHODS Myelin was prepared from bovine brain white matter following the method of Autilio et al. [14] to the stage described as yielding crude myelin. For experimental convenience centrifugation was performed successively in 0.32 and 0.66 M sucrose rather than by layering one solution on top of the other. Thin sections of embedded myelin examined by electron microscopy were found to be essentially free of other subcellular structures. Included in the myelin preparation are three washings with water to remove sucrose. Additional washings with water and with 0.1 M NaCI were used to determine the lability of the basic protein. In these experiments a few grams of packed myelin were stirred at 4 °C with 20 ml of the appropriate solvent: after an hour the supernatant was obtained by centrifugation (20 000 × g for 30 min) and its protein content established by the method of Lowry et al. [15] and by absorption at 280 nm. For extraction with deoxycholate 2-3 g of lyophilized myelin were suspended in 20-40 ml of a solution containing 1 ~ sodium deoxycholate (Merck, Darmstadt, for microbiology) and 0.1 M Tris/Chloride (Sigma, St. Louis, analytical grade), titrated to pH 9.20 with dilute HC1 at 4 °C. Following 0.5-1 h stirring the suspension
583 was centrifuged (90 000 x g, 30 min) and the residue re-extracted twice. The supernatants were pooled and concentrated by ultrafiltration before dialysis against pH 9.2 buffer containing 1 ~ deoxycholate, recentrifugation (20 000 x g, 20 min), and then application to a Sephadex G-75 column equilibrated with the same buffer. Additional purification was effected by repeating the last step once or twice. The purity of the protein was established by electrophoresis in dodecyl sulphate [16] and in acid solution [171. The molecular weight and purity of the basic protein were verified by equilibrium ultracentrifugation using the meniscus depletion method [18]. For sedimentation studies the protein in deoxycholate was dialysed against 0.01 M, pH 9.30, Tris buffer then against water to remove the detergent: sodium acetate was then added to 0.1 M and the pH adjusted to 5 with dilute HC1. Interference optics were used to establish the equilibrium protein distribution: the initial concentration was 0.4 mg/ml. The partial specific volume (0.72 ml/g) of the protein was calculated from the amino acid composition. The efficacy of gel permeation chromatography in removing dissolved lipid was ascertained by testing the protein for residual phospholipid, by measuring the phosphorus content [19]. Basic protein from human and bovine brain was also prepared following Oshiro and Eylar [20]. The CD spectra were obtained on Cary 61 and Jobin Yvon Mark III spectrophotometers. The spectrometer calibration was verified with a standard solution of 5-a-cholestan-7-one (kindly supplied by Dr. Ruth Gall, Organic Chemistry Department, Sydney University). Spectra were recorded using 0.1 or 0.01 cm pathlength cells with protein concentrations in the range 0.2-3.4 mg/ml. Baseline corrections were made by running solvent under identical machine conditions. Mean residue rotations and ellipticities were calculated assuming an average residue molecular weight of 108.5 [10]. No corrections were applied for the solvent refractive index. The secondary structure of basic protein was deduced from CD measurements using a least-squares method to fit the observed spectra with a combination of the spectra for pure a-helices, fl-structure and random segments in proteins [21]. The observed ellipticities at 2.5-nm intervals (from 240 nm) were applied to simultaneous equations expressing this ellipicity, [0]z, as a linear combination of the ellipticities ([0]~, [O]°a,and [0]~) of the separate structures at the same wavelengths:
[Oh = fa [0]] + fa [01~ + % [01~
(1)
After an initial calculation with data from three widely separated wavelengths, all of the data were used to obtain the best values of fa, fm and fr, the fractions of the three structures in the protein, using a function minimization program. The residual was obtained by a simplex minimization of the sum of the values obtained by squaring the difference between the calculated and observed ellipticities after division by the observed ellipticity at the same wavelength. The author is indebted to Mr. E. Tysoe, Physical Chemistry Department, Sydney University, for writing the computer program, and for carrying out many of the computations.
584 RESULTS
Purification of basic protein Washing of myelin with water or 0.1 M NaC1 released no protein, but the latter solvent did yield a suspension which required higher speed centrifugation to produce a clear supernatant essentially devoid of protein. In contrast deoxycholate gave supernatants which were initially cloudy but after concentration and recentrifugation were clear and contained considerable amounts of protein. Typically 75 ~ of the myelin proteins were extracted in three washings. Only a few percent of the lipid dissolved. On gel permeation chromatography the extracts yielded two main peaks, their relative heights being dependent on the method of protein analysis (Fig. 1). Dodecyl sulphate gel electrophoresis (Fig. 2) revealed that the second peak was largely a single polypeptide of molecular weight 19 500 ± 10 ~ corresponding to the myelin basic protein. The ionic strength of the phosphate buffer used for electrophoresis was 0.12: it has recently been reported [22] that under these conditions apparent molecular weight of the basic protein is anomalously high by about 6-8 ~. The first chromatographic peak showed at least two components on dodecyl sulphate electrophoresis (Fig. 2); the majority of the protein remained at the.gel origin but some basic protein was present and frequently a small amount of protein of apparent molecular weight near 26 000 was visible. It appears that despite the precautions [23] taken in preparing samples for electrophoresis the majority of the proteolipid protein was aggregated and did not penetrate 5 or 10 ~ acrylamide gels. The behaviour of this 1.0 3.0
2.0
0.5 1.0 ¸ E c
E
~o
o~
(b)
0.2-
t--\
01
0.1-
0
0
IPO
20
30 Tube
4'0
0
number
Fig. 1. Chromatography of deoxycholate extracts of myelin on Sephadex G-75. The 2.5 cm internal diameter by 70 cm column was eluted with 1 ~ sodium deoxycholate in 0.1 M Tris buffer, pH 9.30. The flow rate was 20 ml/h, and 20-min fractions were collected at 4 °C. (a) First run of extract from myelin. (b) Rechromatography of the pooled protein indicated by the horizontal bar in a. Protein in the effluent was detected colorimetrically (dashed line), using 10pl (in a) or 50/~1 (in b)samples from each tube, and by absorbance measurements at 280 nm (solid line). The fraction indicated by the bar in b was again rechromatographed before use for circular dichroism measurements.
585
Fig. 2. (a) Polyacrylamide gel electrophoresis in dodecyl sulphate. Gel 1, the first peak from the chromatographic column (Fig. la) ; 2, second peak from the column; Gel 3, bovine chloroform-methanolextracted protein; 4, a-chymotrypsinogen (top) and cytochrome c. Electrophoresis was from top to bottom. (b) Gel electrophoresis at pH 4.3. Gel 5, basic protein extracted with deoxycholate (from the second peak of Fig. la); Gel 6, human basic protein extracted using chloroform/methanol. The protein from the first chromatographic peak (Figs. 1a and I b) was largely insoluble in the absence of detergent.
protein on electrophoresis, its abundance relative to the basic protein (Fig. 1), its elution position from the gel permeation column, and its amino acid composition (Gallagher, G.A. and Smith, R., unpublished), leave little doubt that it is the major proteolipid protein of myelin. The lipid applied to the column eluted in the tail of the basic protein peak. Phosphate analyses failed to detect any phospholipids in the basic protein under conditions where the binding of less than one molecule of a typical phospholipid per protein molecule would have been measurable: this indicates effective separation from the dissolved lipids [24]. Martenson et al. [38] have reported a phosphorus content of 0.10 mol per mol of bovine basic protein, which is beyond the lower limit of the present assay. Basic protein extracted with chloroform/methanol was subjected to similar procedures and also found to be phospholipid-free and homogeneous. Equilibrium ultracentrifugation verified both the homogeneity and molecular weight of the basic protein extracted with deoxycholate, a single weight-average molecular weight of 18 000 i 1000 being consistent with the protein distribution throughout the centrifuge cell. Circular Dichroism Studies Basic protein has previously been shown (e.g. refs. 6-9) to contain little a- or fl-structure in water or buffers. The present CD spectra (Fig. 3) confirm this conclusion and indicate that there is little structural alteration when the solvent is changed to 0.1 M Tris, pH 9.15: the solubility of the protein is however low in alkaline solutions and slight turbidity in the solution used for CD precluded measurement of the spectrum below 205 nm. This apparent conformational stability toward pH change is
586
J a3
-6 E ~ -5" "o
% o
d~ ~ -10
o ×
260
21o A (nm)
Fig. 3. Circular dichroism spectra of basic protein in 0.1 M sodium acetate buffer, pH 4.8. The vertical bars indicate the standard deviations in eight spectra run on three different samples of bovine and human basic protein. There were no significant differences in the spectra of the protein from the two species. Protein concentrations were derived from the absorbance at 2~0 nm assuming an extinction co-efficient (deduced from ref. 5) E]c°/m ° -- 5.35.
consistent with earlier CD studies and with the recent observation from proton N M R studies that the spin-spin relaxation times vary only slightly in traversing the pH range from 5.6 to 9.2 [5]. The spectrum obtained for basic protein in acetate buffer (Fig. 3) is very similar to several previously published [6, 25] but differs quantitatively from that of Liebes et al. [5]. The origin of this difference is not apparent: Fig. 3 incorporates data from five different preparations, the spectra of which were obtained on two different CD spectrometers, with no systematic deviations between spectra. The instruments provided satisfactory quantitative data for both 5-a-cholestan-7-one and for a standard solution of lysozyme. Basic protein extracted with, and examined in, deoxycholate solution exhibited a considerably different spectrum (Fig. 4) from that of the protein in acetate buffer. No diminution in this difference was observed when 0.25 M NaC1 was added to the deoxycholate solution; higher salt concentrations caused precipitation of the deoxycholate. Comparison of Figs. 3 and 4 reveals several changes. The peak near 198 nm in acetate buffer is shifted to 203 nm and its negative ellipticity is diminished in the presence of deoxycholate; a pronounced shoulder is also introduced near 220 nm. These changes in the spectrum are well accounted for by the introduction of a moderate amount of helical structure, which is characterized by a large positive ellipticity at 192 nm and two smaller negative peaks at 209 and 222 nm [21]. Spectra from several sources [21, 26-28] for protein a-helices, fl-structure and unordered regions (the basis spectra) failed to yield satisfactory fits to the basic protein spectra, even if in Eqn. l [0]z was replaced by C[O]zwhere C is a wavelength-independent parameter adjusted to attain the best correspondence between the calculated and experimental spectra. The difficulty is most simply seen in fitting the spectrum of the chloroform/methanolextracted protein in water: none of these sets of basis spectra could reproduce this curve despite the fact that is now well-established (ref. 6 and the present work). Failure to fit this curve may stem from inadequacies in the basis spectra. There is
587
.~ o-
~
? o
-5
x
g -10 I
I
200
210
~
I
220 ?~ (nm)230
I
240
Fig. 4. Circular dichroism spectrum of basic protein extracted from myelin by deoxycholate and examined in 1% sodium deoxycholate (in 0.1 M Tris buffer, pH 9.30), and of organically extracted human and bovine basic protein in the same buffer. The vertical bars indicate the standard deviations from five spectra from 240 to 220 nm and of three spectra from 217.5 to 195 nm. The solid line is drawn through the average ellipticity at each wavelength. The spectrum computed using basis spectra B (Table I) is indicated by (x). Use of basis spectra C resulted in a similar spectrum. Protein concentrations were derived from the absorbance at 280 nm relative to the buffer. reasonable agreement on the standard spectra for infinite helices and for fl-structures but there is marked divergence in,the spectra accepted for unordered protein segments. As a consequence, the fitting of spectra of proteins containing appreciable ordered structure may be carried out with some confidence, but it is to be expected that the fit will be far less satisfactory for proteins possessing little ordered structure, as indeed proved to be the case with the basic protein in acetate buffer. As an alternative approach we have started from the assumption that basic protein in water contains negligible a- or fl-structure'. As noted above, there are reasonable experimental grounds for this assumption in that earlier O R D studies have shown that the Moffitt-Yang parameter is close to zero, indicating the absence of ordered structures [29]. And although the analysis of the CD spectrum is beset by the difficulties mentioned above it does not suggest the presence of e- or E-structure. In particular, the ellipticity at 222 nm [27] is consistent with the absence of e-helix. ~, To elucidate the structure of basic protein in deoxycholate we have used the spectrum of the protein in acetate buffer with published e- and/~-structure spectra [21] as the basis spectra for curve fitting. We have additionally fitted the spectra for protein in acetate buffer and in deoxycholate using all three basis spectra derived from Chen et al. [21]. The results are presented in Table I. Separate analyses of the spectra of deoxycholate-extracted protein and of basic protein in pH 9.15 Tris buffer were not carried out as the former essentially mimicked the chloroform/methanol-extracted protein in deoxycholate solution and the latter did not differ significantly from the spectra in water or acetate buffer. The folding of the protein appears to be rapid as the spectrum run directly after addition of deo" This is not in conflict with the observation that when dissolved in guanidinium. HCI solution the protein exhibits a slightly different spectrum [10]: we are assuming the absence of a- and flstructure, not a unique spectrum for the disordered states. Protein intramolecular hydrophobic interactions in water may well alter the optical activity from that expected for the random coil in guanidinium- HCI solution.
588 TABLEI SECONDARY STRUCTURE OF BASIC PROTEIN COMPUTED FROM CD SPECTRA The averaged spectra of Figs. 3 and 4 were used in the calculations. Basis spectra: A, from Chen et al. [21] interpolated from their Table II (i.e. 11-residue a-helix spectrum). B, a-helix and fl-structure spectra as in A, but with the spectrum of basic protein in acetate buffer replacing the spectrum from ref. 21 for unordered protein segments. C, as in B except 5-residue a-helix spectrum from ref. 21 substituted.
Basic protein in acetate buffer Basic protein in deoxycholate
Basis spectra
a-helix (~)
fl-structure (~)
Unordered (~)
A
l0
16
74
A B C
22 9 14
13 0 3
65 91 82
xycholate differed little from the spectrum of the protein extracted with this surfactant. Although not analysed in detail the O R D spectra obtained also revealed a higher helical content in basic protein in the presence of deoxycholate. DISCUSSION Intense interest in the conformation of basic protein has been generated by its unique encephalitogenic activity. The seeming lack of helical or fl-structure in water has been used to explain the retention of this activity following severe heating and chemical treatment. All of these conformational studies have however been conducted with protein extracted under conditions likely to cause structural alteration. In contrast, deoxycholate has been widely used for the isolation of other membrane proteins and has been shown to effect minimal structural perturbation [24]: particularly noteworthy are studies [30] showing no significant changes in the CD spectra of lymphocyte, heine-, and other membrane proteins on isolation with deoxycholate. Independent of the method of analysis of the C D spectra, there appears to be about 8-14 ~ difference in helix content of basic protein in water and in 1 ~ deoxycholate. This difference has been calculated assuming an average length of either 5 or 11 amino acid residues in each helical segment: if in fact the helical segments in basic protein are short the proportion of a-helix will be close to 1 4 ~ . Application of the empirical structure-prediction algorithm of Chou and Fasman [31] reveals several regions which are potentially helix forming, the average length of these is less than 11 : the efficacy of empirical prediction algorithms has however been questioned [23]. Difficulties inherent in structural analysis by C D [33] necessitate some restraint in interpretation of the quantitative differences in structure. The exact nature of the conformational change is obscured a little by the unusual spectrum of the protein in acetate buffer (see above), which has the form expected for a randomly coiled polypeptide but appears flattened in a way not attributable to the introduction of either ahelical or fl-structure. Basic protein is also monomolecularly dispersed in the acetate buffer, hence the flattening cannot be accounted for by light scattering. Basic protein extracted with organic solvents is able to refold quickly on addition of deoxycholate; a similar folding has been observed on the addition of dodecyl
589 sulphate, oleate, lysolecithin [6] and trifluoroethanol [5]. Successive additions of trifluoroethanol produced step-like changes in the CD spectrum suggestive of three domains in the polypeptide separately undergoing co-operative coil-to-helix transitions. In view of these results it is unlikely that the change in ellipticity in deoxycholate results from an enhancement of the optical activity of the detergent per se upon binding. The apparent lack of effect of severe treatment on the encephalitogenic properties of the protein may well result from its ability to refold quickly from the denatured state (for example, when added to Freund's adjuvant), rather than the activity being intrinsic in the disordered protein. Basic protein is not freely water-soluble at near-neutral pH, despite the considerable net positive charge on the protein. Aggregation has been demonstrated by light scattering and N M R spectroscopy [5]. This aggregation appears to be unaccompanied by significant changes in secondary structure, the CD spectra at neutral and alkaline pH being superimposable (see above). Moscarello et al. [9] did observe minor changes in the alkaline spectrum as the protein concentration was varied: such changes may in part be attributable to scattering and absorption flattening effects [34]. The protein appears to be soluble and apparently monomeric at high pH in the presence of deoxycholate, a surfactant similar in structure to cholesterol, a major myelin lipid. This enhanced solubility is presumably due to substitution of intermolecular proteinprotein hydrophobic interactions with detergent-protein contacts. Basic protein has been demonstrated to interact hydrophobically with lipid monolayers, the incorporation showing a positive correlation with lipid alkyl chain length [35]. It also alters the temperature and enthalpy change of the phase transition in vesicles of dipalmitoyl phosphatidylcholine and dipalmitoyl phosphatidylglycerol [36]. Preliminary experiments (Smith, R., unpublished) show also appreciable binding of deoxycholate to basic protein at alkaline pH in 0.25 M NaC1 demonstrating more directly its possession of hydrophobic binding sites. These studies and the solubility characteristics of the protein point to some hydrophobic character consistent with partial penetration of the bilayer in myelin. As a consequence of this basic protein may adopt a more ordered structure in myelin and may possess activities not apparent in its water-soluble conformation. ACKNOWLEDGEMENTS The co-operation of Dr. C. J. Hawkins, Inorganic Chemistry Department, University of Queensland, and Dr. J. N. Douglas, Solid State Physics Department, Australian National University, in providing access to their CD spectrometers is gratefully acknowledged. The author has benefited from numerous discussions with Professor Walter J. Moore and Mr. E. Tysoe of the Physical Chemistry Department, Sydney University. This work was partially supported by the Australian Research Grants Committee. REFERENCES 1 Carnegie, P. R. (1971) Nature 229, 25-28 2 Eylar, E. H., Brostoff, S., Hashim, G., Caccam, J. and Burnett, P. (1971) J. Biol. Chem. 246, 5770-5784
590 3 Vanderkooi, G. and Capaldi, R. A. (1972) Ann. N.Y. Acad. Sci. 195, 135-138 4 Spatz, L. and Strittmatter, P. (1971) Proc. Natl. Acad .Sci. U.S. 68, 1042-1046 5 Liebes, L. F., Zand, R. and Phillips, W. D. (1975) Biochim. Biophys. Acta 405, 27-39 6 Anthony, J. S. and Moscarello, M. A. (1971) Biochim. Biophys. Acta 243, 429-433. 7 Palmer, F. B. and Dawson, R. M. C. (1969) Biochem. J. 111, 629-636 80shiro, Y. and Eylar, E. H. (1970) Arch. Biochem. Biophys. 138, 606-613 9 Moscarello, M. A., Katona, E., Neumann, A. W. and Epand, R. M. (1974) Biophys. Chem. 2, 290-295 l0 Epand, R. M., Moscarello, M. A., Zierenberg, B. and Vail, W. J. (1974) Biochemistry 13, 12641267 11 Eylar, E. H. and Thompson, M. (1969) Arch. Biochem. Biophys. 129, 468-479 12 Krigbaum, W. R. and Hsu, T. S. (1975) Biochemistry 14, 2542-2546 13 Tanford, C. (1968) in Advances in Protein Chemistry (Anfinsen, C. B., Anson, M. L., Edsall, J. T. and Richards, F. M., eds.), Vol. 23, pp. 121-282, Academic Press, New York 14 Autilio, L. A., Norton, W. T. and Terry, R. D. (1964) J. Neurochem. 11, 17-27 15 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 16 Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412 17 Reisfeld, R. A., Lewis, U. J. and Williams, D. E. (1962) Nature 195, 281-283 18 Yphantis, D. A. (1964) Biochemistry 3, 297-317 19 Bartlett, G. R. (1959) J. Biol. Chem. 234, 466-468 20 Oshiro, Y. and Eylar, E. H. (1970) Arch. Biochem. Biophys. 138, 392-396 21 Chen, Y-H., Yang, J. T. and Chou, K. H. (1974) Biochemistry 13, 3350-3359 22 Campagnoni, A. T. and Magpo, C. J. (1974) J. Neurochem. 23, 887-890 23 Morell, P., Wiggins, R. C. and Gray, M. J. 0975) Anal. Biochem. 68, 148-154 24 Helenius, A. and Simons, K. (1975) Biochim. Biophys. Acta 415, 29-79 25 Block, R. E., Brady, A. H. and Joffe, S. (1974) Biochem. Biophys. Res. Commun. 54, 1595-1602 26 Saxena, V. P. and Wetlaufer, D. B. (1971) Pro¢. Natl. Acad. Sci. U.S. 68, 969-972 27 Chen, Y-H. and Yang, J. T. (1971) Biochem. Biophys. Res. Commun. 44, 1285-1291 28 Greenfield, N. and Fasman, G. D. (1969) Biochemistry 8, 4108-4116 29 Urnes, P. and Doty, P. (1961) Adv. Protein Chem. 16, 401-544 30 Bayley, P. M. (1973) Prog. Biophys. Mol. Biol. 27, 3-76 31 Chou, P. Y. and Fasman, G. D. 0974) Biochemistry 13, 222-245 32 Burgess, A. W. and Scheraga, H. A. (1975) Proc. Natl. Acad. Sci. U.S. 72, 1221-1225 33 Sears, D. W. and Beychok, S. (1973) in Physical Principles and Techniques of Protein Chemistry (Leach, S. J., ed.), Part C, pp. 445-593, Academic Press, New York 34 Gordon, D. J. and Holzwarth, G. (1971) Proc. Natl. Acad Sci. U.S. 68, 2365-2369 35 Demel, R. A., London, Y., van Kessel, W. S. M. G., Vossenberg, F. G. A. and van Deenen, L. L. M. (1973) Biochim. Biophys. Acta 311, 507-519 36 Papahadjopoulas, D., Moscarello, M., Eylar, E. H. and Isac, T. (1975) Biochim. Biophys. Acta 401,317-335 37 Chao, L.-P. and Einstein, E. R. (1970) J. Neurochem. 17, 1121-1132 38 Martenson, R. E., Kramer, A. J. and Deibler, G. E. (1976) J. Neurochem. 26, 733-736
