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Complete separation of the triplet components of neurofilament by DE-52 column chromatography depends upon urea concentration
ANALYTICAL
BIOCHEMISTRY
l&203-207
(1984)
Complete Separation of the Triplet Components of Neurofilament by DE-52 Column Chromatography Depends upon Urea Concentration SATOSHI TOKUTAKE Psychiatric Research Institute of Tokyo, 2-l-8 Kamikitazawa,
Setagaya-ku, 156 Tokyo, Japan
Received December 27, 1983 In the separation of the triplet components of neurotilament (P 200, P 160, and P 68) by DE-52 column chromatography in the presence of urea, it was revealed that the efficiency of separation depended upon urea concentration. When chromatography was performed in the presence of 8 M urea and a linearly increasing sodium phosphate concentration from 10 to 400 mM at pH 6.8, P 160 and P 68 were eluted in the same peak, although P 200 was eluted faster. P 160 and P 68 were partiahy separated with 6 M urea, and completely separated with 4 M urea. But, under these conditions, P 200 was eluted at the same position as contaminated glial fibrillary acidic protein (GFA). From these results, two methods were recommended for the complete separation of the triplet components of neurohlament and GFA by DE-52 column chromatography. In one method, chromatography was performed in the presence of 8 M urea at hrst, and then P 160 and P 68 were separated in the presence of 4 M urea. In the other method, chromatography was performed with a linearly decreasing urea concentration from 8 to 0 M. KEY Worux: neurotilaments; triplet components; intermediate hlaments; glia tilament; glial fibrillary acidic protein; column chromatography.
DE-52 column chromatography in the presence of urea is used for the separation of the triplet components of neurolilament ( I ,2). In the method of Liem and Hutchinson (2), the P 200 component was eluted with 8 M urea containing 100 mM sodium phosphate, and the P 160 and P 68 components were eluted with urea containing 300 mM sodium phosphate. On the other hand, in the method of Geisler and Weber (l), a column was eluted with 6 M urea solution increasing sodium chloride concentration to 250 mM linearly. When the chromatography was performed under conditions similar to those of Geisler and Weber, P 160 and P 68 were not separated completely. Better separation may be obtained with longer columns or larger volumes of buffer. On the other hand, it is suspected that the concentration of urea in the buffers may have some influence on separation, and it was revealed that complete separation of P 160 and P 68 was obtained with 4 M urea buffer.
MATERIALS
AND METHODS
Isolation of neurojlaments and partial puriJication of neurofilament proteins with hydroxyapatite gel by a batchwise method. Crude neurolilaments were prepared from bovine brain white matter and spinal cord by axone floatation and subsequent isotonic lysis following the methods of Liem et al. (3-5). A 400-g quantity of bovine brain white matter and spinal cord was dissected and homogenized mildly in a Waring blender in 2.5 vol of buffer consisting of 10 mM NaHzP04, 1 mM EDTA, 0.1 M NaCI, and 0.85 M sucrose at pH 6.8 (Buffer A). Axones were floated by centrifugation, and were washed by suspending in a volume of Buffer A. Then, the washed axones were lysed in a volume of Buffer A plus 1% Triton X-100 overnight. The lysate was homogenized vigorously, and crude neurofilaments were collected by centrifugation. The crude neurolilaments were dissolved in 203
0003-2697184 63.00 Copyright 0 1984 by Academic Press, Inc. AU rights of reproduction in any form rserved.
204
SATOSHI
10 mM NaI-12P04, 1 mM EDTA, and 1% Bmercapmethanol (&ME),’ pH 6.8, and insoluble materials were removed by centrifugation. The neurohlament proteins were purified with hydroxyapatite gel by the batchwise extraction method (45). A 50-ml volume of the neurohlament protein solution was mixed with 10 g of swollen hydroxyapatite gel (BioGel HTP). The gels were suspended well and collected by centrihtgation. Unadsorbed proteins in the supematant (I) were few. Then, the gels were extracted twice with 8 M urea containing 140 mM NaH#04, pH 6.8. The main component in the supematant (II) was glial fibrillary acidic protein (GFA). Finally, the gels were extracted twice with 50 ml of 8 M urea containing 300 mM NaHZP04, pH 6.8. The triplet components of neurofilament were contained in this supematant (III), and a little GFA was contaminated (Fig. 1). Amino acid analysis. Several milligrams of lyophilized samples of the triplet components and GFA were hydrolyzed in 1 ml 6 N HCl acid at 110°C for 24 or 3 N ptoluenesulfonic h. The hydrolysate with 6 N HCl was lyophilized, and then dissolved in 5 ml of distilled water. A l-ml sample was used for ordinary amino acid analysis (6). The balance of the hydrolysate was used for the determination of cysteine content, by converting cysteine to sulfocysteine with sodium tetrathionate (7). To the hydrolysate with ptoluenesulfonic acid, 2 ml of 1 N NaOH and distilled water were added and used for the determination of tryptophan content (8). Amino acid analysis was performed with an Hitachi KLA-5 amino acid analyzer.
TOKUTAKE
200 ml of 8 M urea containing
123456 P 200 P 160
Fm. 1. SDS-gel electmphoretic pattern of neurohlament proteins in the process of purihcation by batchwise extraction of hydroxyapatite gel (Bio-Gel HTP). SDS-gel electrophoresis was performed following the method of Laemmli (9) using 7.5% gel. (1) Crude neurolilaments obtained by axone floatation and isotonic lysis in 1% Triton X-100 solution and dissolved in 8 M urea; (2) the supernatant (I), unadsorbed Ii-action to B&Gel HTP, (3) the supematant (II), fraction extracted from B&Gel HTP with 8 M urea containing 140 mM NaI-I$O, and 1 mM EDTA, pH 6.8; (4) the supematant (II), second extmction; (5) the supematant (III), fraction extracted with 8 M urea containing 300 mM NaI-I$O, and 1 mhi EDTA, pH 6.8; (6) the supematant (III), second extraction.
consisting of 10 mM NaH#Q , 1 mM EDTA, pH 6.8 (Buffer B) to remove urea and excess sodium phosphate. The dialysate was concentrated to 10 ml with dialysis tubing under reduced pressure. To the concentrated solution, urea and @-ME were added to 6 M and 1% respectively, and applied to a DE-52 column (1.5 X 50 cm) equilibrated with the starting buffer consisting of 6 M urea, 10 mM NaH2P04, 1 mM EDTA, 0.1% @-ME, pH 6.8. The column was eluted with 500 ml of 6 M urea solution with a linearly increasing sodium phosphate concentration from 10 to 400 mM. As shown in Fig. 2, five peaks (O-IV) were RESULTS AND DISCUSSION observed, and the protein components of the Separation of the triplet components of neu- peaks were examined by SDS-gel electrophorofdament and GFA. A lOO-ml volume of the resis (9). It is shown that Peak I is GFA, Peak supematant (III), which contained about 30 II is P 200, Peak III is P 160, and Peak IV is mg of protein, was dialyzed against buffer P 68. The separation of Peaks III and IV was not complete, and was not improved even if the final salt concentration was reduced to ’ Abbreviations used: @-ME, j+mercaptoethanol; GFA,
glial fibrillary acidic protein; SDS, sodium dodecyl sulfate.
300 rnM.
CHROMATOGRAPHIC
SEPARATION
OF NEUROFILAMENT
205
FIG. 2. Separation of the triplet components of neurofilament and GFA by DE-52 column chromatography (A) and examination of the protein components of each peak by SDS-gel electrophoresii (B). (A) DE-52 column (1.5 X 50 cm) was equilibrated with the starting buffer consisting of 6 M urea, 10 mM NaH$‘O,, 1 mM EDTA and 0.1% &ME, pH 6.8, and eluted with 500 ml of the buffer and a linearly increasing sodium phosphate concentration to 400 mM. Each 5 ml of the eluate was fractionated at the flow rate of 10 ml/h. Protein content was determined by dye-binding method of Bradford ( 13) using a Bio-Rad protein assaykit.
Then, chromatography was performed with the buffer containing 8 M urea. The elution profile and the electrophoretic pattern were shown in Figs. 3A and B. P 160 and P 68 were eluted in the same peak (III). From this result, it is suspected that lower concentrations of urea will be favorable for the separation of P 160 and P 68. Rechromatography of the
A
10
20 30 Fraction
40
50 60 Number
70
B
Peak III fraction was attempted with buffer containing 4 M urea, though it is not usual to use 4 M urea in the preparation of proteins. The results in Figs. 3C and D show that P 160 and P 68 were separated completely. By this chromatography, about 5 mg of each component of neurofdament was obtained. However, when the mixture of the triplet
C 50 60 70 60 90
D
FIG. 3. DE-52 column chromatography with the buffer containing 8 M urea (A), and separation of P 160 and P 68 by rechromatography of Peak III with 4 M urea buffer (C).
206
SATOSHI
components and GFA were chromatographed under these conditions, GFA and P 200 were eluted in the same peak. From these results, it was shown that GFA and P 200 were well separated with the urea solution above 6 M, and P 160 and P 68 were well separated with 4 M urea SOlUtiOn. Finally, the separation of the triplet components and GFA by one chromatographic process was attempted, using a linearly decreasing urea concentration from 8 to 0 M. The result is shown in Fig. 4. It appears that the recovery of P 68 was not good. The elution position of P 200 was almost constant at any urea concentration, but the positions of other components were changed by different urea concentrations. This must be due to conformational changes of proteins in urea solution, but changes in uv spectra were not observed at any urea concentration. Amino acid composition. The amino acid compositions of the triplet components of neurofilament and GFA are shown in Table 1. The results are almost comparable with the reported results ( IO,1 1). Significant amounts of tryptophan were not detected, even by hydrolysis with ptoluenesulfonic acid, but it is certain that at least one tryptophan is con-
A whm
TOKUTAKB TABLE AMINO
ASP
Thr Ser Glu pro
GUY Ala cys
Val Met Ileu LeU
TF Phe LYS His 4 Trp
1
ACID COMPOSITIONS OF THE TRIPLET COMPONENTS AND GFA P200
P 160
P 68
GFA
4.6 3.3 9.0 22.0 6.7 5.3 12.9 0.64 5.5 1.0 2.0 5.7 0.88 1.5 14.2 1.2 3.6 -
5.5 4.9 8.9 25.1 6.1 5.0 9.5 0.68 5.5 0.82 3.2 6.0 1.3 1.0 11.6 1.5 3.4 -
7.3 4.5 9.8 24.6 2.5 4.2 12.0 3.2 2.1 1.5 2.9 8.2 3.2 1.9 6.4 0.67 5.0 -
8.9% 4.5 8.4 19.2 2.9 3.3 10.1
5.6 1.9 2.9 12.1 2.6 2.1 4.5 1.5 9.5 -
Note Samples were hydrolyzed with 6 N HCI or 3 N ptoluenesulfonic acid at 110°C for 24 h. No siificant differences were observed between two values except for cysteine and tryptophan. No corrections for the decay of threonine and serine during hydrolysis and for the incomplete hydrolysis of valine and leucine were performed. Cysteine content was determined by converting cysteine to sulfocysteine with sodium tetrathionate (7). Determination oftryptophan content was attempted by hydrolysis with 3 N ptoluenesulfonic acid (S), but a significant amount was not detected.
tamed in P 68, from the sequence study (12). Both the triplet components and GFA have high levels of glutamic acid and alanine. In GFA, a high level of leucine seems characteristic. Among the basic amino acids, lysine has the highest level in the triplet components, and arginine in GFA. ACKNOWLEDGMENTS 10
20
30 40 Fraction
50
60 70 Number
80
SO
100
PIG. 4. DE-52 column chromatography with linearly increasing sodium phosphate concentration from 10 to 400 mM and decreasing urea concentration from 8 to 0 ht.
A part of this experiment was performed in the Department of Pharmacology, New York University Medical Center. Thanks am due Dr. Ronald K Liem and Professor Michael L. Shelanski for their useful suggestions and dis cussions. The author also thanks Ms. Michiko Hishida for her technical assistance.
CHROMATGGRAPHIC
SEPARATION
REFERENCES 1. Geisler, N., and Weber, K. (1981) J. Mol. Biol. 151, 565-571. 2. Liem, R., and Hutchinson, S. B. (1982) Biochemistry 21,3221-3226. 3. Liem, R., Yen, S. H., Salmon, G. D., and Shelanski, M. L. (1978) J. Cett. Biol. 76, 637-645. 4. Liem, R. (1982) J. Neurochem. 38, 142-l 50. 5. Tokutake, S., Hutchinson, S. B., Patcher, J., and Liem, R. (1983) Anal. B&hem. 135, 102-105. 6. Spackman, D. H. (1967) in Methods in Enzymology (Hi, C. H. W., ed.), Vol. 11, pp. 3-l 5, Academic Press, New York. 7. Lie, T-Y., and Inglis, A. (1972) in Methods in En-
8. 9. 10. 11. 12. 13.
OF NEUROFILAMENT
207
zymology (Him, C. H. W., and Timasheff, S. N., eds.), Vol. 25, pp. 55-60, Academic Press, New York. Lie, T-Y. (1972) in Methods in Enzymology (Hits, C. H. W., and Timasheff, S. N., eds.), Vol. 25, pp. 44-55, Academic Press, New York. Laemmli, U. K. (1970) Nature (Londonj 227, 680685. Hogue-Angelett, R., Wu, H.-L., and Schlaepfer, W. (1982) J. Neurochem. 38, 116-120. Rueger, D., Gardner, E., Simonian, H., Dahl, D., and Bignami, A. (1981) J. Biol. Chem. 256, 1060610612. Geisler, N., Plessmann, U., and Weber, K. (1982) Nature (London) 296, 448-450. Bradford, M. M. (1976) Anal. B&hem. 72,248-254.
BIOCHEMISTRY
l&203-207
(1984)
Complete Separation of the Triplet Components of Neurofilament by DE-52 Column Chromatography Depends upon Urea Concentration SATOSHI TOKUTAKE Psychiatric Research Institute of Tokyo, 2-l-8 Kamikitazawa,
Setagaya-ku, 156 Tokyo, Japan
Received December 27, 1983 In the separation of the triplet components of neurotilament (P 200, P 160, and P 68) by DE-52 column chromatography in the presence of urea, it was revealed that the efficiency of separation depended upon urea concentration. When chromatography was performed in the presence of 8 M urea and a linearly increasing sodium phosphate concentration from 10 to 400 mM at pH 6.8, P 160 and P 68 were eluted in the same peak, although P 200 was eluted faster. P 160 and P 68 were partiahy separated with 6 M urea, and completely separated with 4 M urea. But, under these conditions, P 200 was eluted at the same position as contaminated glial fibrillary acidic protein (GFA). From these results, two methods were recommended for the complete separation of the triplet components of neurohlament and GFA by DE-52 column chromatography. In one method, chromatography was performed in the presence of 8 M urea at hrst, and then P 160 and P 68 were separated in the presence of 4 M urea. In the other method, chromatography was performed with a linearly decreasing urea concentration from 8 to 0 M. KEY Worux: neurotilaments; triplet components; intermediate hlaments; glia tilament; glial fibrillary acidic protein; column chromatography.
DE-52 column chromatography in the presence of urea is used for the separation of the triplet components of neurolilament ( I ,2). In the method of Liem and Hutchinson (2), the P 200 component was eluted with 8 M urea containing 100 mM sodium phosphate, and the P 160 and P 68 components were eluted with urea containing 300 mM sodium phosphate. On the other hand, in the method of Geisler and Weber (l), a column was eluted with 6 M urea solution increasing sodium chloride concentration to 250 mM linearly. When the chromatography was performed under conditions similar to those of Geisler and Weber, P 160 and P 68 were not separated completely. Better separation may be obtained with longer columns or larger volumes of buffer. On the other hand, it is suspected that the concentration of urea in the buffers may have some influence on separation, and it was revealed that complete separation of P 160 and P 68 was obtained with 4 M urea buffer.
MATERIALS
AND METHODS
Isolation of neurojlaments and partial puriJication of neurofilament proteins with hydroxyapatite gel by a batchwise method. Crude neurolilaments were prepared from bovine brain white matter and spinal cord by axone floatation and subsequent isotonic lysis following the methods of Liem et al. (3-5). A 400-g quantity of bovine brain white matter and spinal cord was dissected and homogenized mildly in a Waring blender in 2.5 vol of buffer consisting of 10 mM NaHzP04, 1 mM EDTA, 0.1 M NaCI, and 0.85 M sucrose at pH 6.8 (Buffer A). Axones were floated by centrifugation, and were washed by suspending in a volume of Buffer A. Then, the washed axones were lysed in a volume of Buffer A plus 1% Triton X-100 overnight. The lysate was homogenized vigorously, and crude neurofilaments were collected by centrifugation. The crude neurolilaments were dissolved in 203
0003-2697184 63.00 Copyright 0 1984 by Academic Press, Inc. AU rights of reproduction in any form rserved.
204
SATOSHI
10 mM NaI-12P04, 1 mM EDTA, and 1% Bmercapmethanol (&ME),’ pH 6.8, and insoluble materials were removed by centrifugation. The neurohlament proteins were purified with hydroxyapatite gel by the batchwise extraction method (45). A 50-ml volume of the neurohlament protein solution was mixed with 10 g of swollen hydroxyapatite gel (BioGel HTP). The gels were suspended well and collected by centrihtgation. Unadsorbed proteins in the supematant (I) were few. Then, the gels were extracted twice with 8 M urea containing 140 mM NaH#04, pH 6.8. The main component in the supematant (II) was glial fibrillary acidic protein (GFA). Finally, the gels were extracted twice with 50 ml of 8 M urea containing 300 mM NaHZP04, pH 6.8. The triplet components of neurofilament were contained in this supematant (III), and a little GFA was contaminated (Fig. 1). Amino acid analysis. Several milligrams of lyophilized samples of the triplet components and GFA were hydrolyzed in 1 ml 6 N HCl acid at 110°C for 24 or 3 N ptoluenesulfonic h. The hydrolysate with 6 N HCl was lyophilized, and then dissolved in 5 ml of distilled water. A l-ml sample was used for ordinary amino acid analysis (6). The balance of the hydrolysate was used for the determination of cysteine content, by converting cysteine to sulfocysteine with sodium tetrathionate (7). To the hydrolysate with ptoluenesulfonic acid, 2 ml of 1 N NaOH and distilled water were added and used for the determination of tryptophan content (8). Amino acid analysis was performed with an Hitachi KLA-5 amino acid analyzer.
TOKUTAKE
200 ml of 8 M urea containing
123456 P 200 P 160
Fm. 1. SDS-gel electmphoretic pattern of neurohlament proteins in the process of purihcation by batchwise extraction of hydroxyapatite gel (Bio-Gel HTP). SDS-gel electrophoresis was performed following the method of Laemmli (9) using 7.5% gel. (1) Crude neurolilaments obtained by axone floatation and isotonic lysis in 1% Triton X-100 solution and dissolved in 8 M urea; (2) the supernatant (I), unadsorbed Ii-action to B&Gel HTP, (3) the supematant (II), fraction extracted from B&Gel HTP with 8 M urea containing 140 mM NaI-I$O, and 1 mM EDTA, pH 6.8; (4) the supematant (II), second extmction; (5) the supematant (III), fraction extracted with 8 M urea containing 300 mM NaI-I$O, and 1 mhi EDTA, pH 6.8; (6) the supematant (III), second extraction.
consisting of 10 mM NaH#Q , 1 mM EDTA, pH 6.8 (Buffer B) to remove urea and excess sodium phosphate. The dialysate was concentrated to 10 ml with dialysis tubing under reduced pressure. To the concentrated solution, urea and @-ME were added to 6 M and 1% respectively, and applied to a DE-52 column (1.5 X 50 cm) equilibrated with the starting buffer consisting of 6 M urea, 10 mM NaH2P04, 1 mM EDTA, 0.1% @-ME, pH 6.8. The column was eluted with 500 ml of 6 M urea solution with a linearly increasing sodium phosphate concentration from 10 to 400 mM. As shown in Fig. 2, five peaks (O-IV) were RESULTS AND DISCUSSION observed, and the protein components of the Separation of the triplet components of neu- peaks were examined by SDS-gel electrophorofdament and GFA. A lOO-ml volume of the resis (9). It is shown that Peak I is GFA, Peak supematant (III), which contained about 30 II is P 200, Peak III is P 160, and Peak IV is mg of protein, was dialyzed against buffer P 68. The separation of Peaks III and IV was not complete, and was not improved even if the final salt concentration was reduced to ’ Abbreviations used: @-ME, j+mercaptoethanol; GFA,
glial fibrillary acidic protein; SDS, sodium dodecyl sulfate.
300 rnM.
CHROMATOGRAPHIC
SEPARATION
OF NEUROFILAMENT
205
FIG. 2. Separation of the triplet components of neurofilament and GFA by DE-52 column chromatography (A) and examination of the protein components of each peak by SDS-gel electrophoresii (B). (A) DE-52 column (1.5 X 50 cm) was equilibrated with the starting buffer consisting of 6 M urea, 10 mM NaH$‘O,, 1 mM EDTA and 0.1% &ME, pH 6.8, and eluted with 500 ml of the buffer and a linearly increasing sodium phosphate concentration to 400 mM. Each 5 ml of the eluate was fractionated at the flow rate of 10 ml/h. Protein content was determined by dye-binding method of Bradford ( 13) using a Bio-Rad protein assaykit.
Then, chromatography was performed with the buffer containing 8 M urea. The elution profile and the electrophoretic pattern were shown in Figs. 3A and B. P 160 and P 68 were eluted in the same peak (III). From this result, it is suspected that lower concentrations of urea will be favorable for the separation of P 160 and P 68. Rechromatography of the
A
10
20 30 Fraction
40
50 60 Number
70
B
Peak III fraction was attempted with buffer containing 4 M urea, though it is not usual to use 4 M urea in the preparation of proteins. The results in Figs. 3C and D show that P 160 and P 68 were separated completely. By this chromatography, about 5 mg of each component of neurofdament was obtained. However, when the mixture of the triplet
C 50 60 70 60 90
D
FIG. 3. DE-52 column chromatography with the buffer containing 8 M urea (A), and separation of P 160 and P 68 by rechromatography of Peak III with 4 M urea buffer (C).
206
SATOSHI
components and GFA were chromatographed under these conditions, GFA and P 200 were eluted in the same peak. From these results, it was shown that GFA and P 200 were well separated with the urea solution above 6 M, and P 160 and P 68 were well separated with 4 M urea SOlUtiOn. Finally, the separation of the triplet components and GFA by one chromatographic process was attempted, using a linearly decreasing urea concentration from 8 to 0 M. The result is shown in Fig. 4. It appears that the recovery of P 68 was not good. The elution position of P 200 was almost constant at any urea concentration, but the positions of other components were changed by different urea concentrations. This must be due to conformational changes of proteins in urea solution, but changes in uv spectra were not observed at any urea concentration. Amino acid composition. The amino acid compositions of the triplet components of neurofilament and GFA are shown in Table 1. The results are almost comparable with the reported results ( IO,1 1). Significant amounts of tryptophan were not detected, even by hydrolysis with ptoluenesulfonic acid, but it is certain that at least one tryptophan is con-
A whm
TOKUTAKB TABLE AMINO
ASP
Thr Ser Glu pro
GUY Ala cys
Val Met Ileu LeU
TF Phe LYS His 4 Trp
1
ACID COMPOSITIONS OF THE TRIPLET COMPONENTS AND GFA P200
P 160
P 68
GFA
4.6 3.3 9.0 22.0 6.7 5.3 12.9 0.64 5.5 1.0 2.0 5.7 0.88 1.5 14.2 1.2 3.6 -
5.5 4.9 8.9 25.1 6.1 5.0 9.5 0.68 5.5 0.82 3.2 6.0 1.3 1.0 11.6 1.5 3.4 -
7.3 4.5 9.8 24.6 2.5 4.2 12.0 3.2 2.1 1.5 2.9 8.2 3.2 1.9 6.4 0.67 5.0 -
8.9% 4.5 8.4 19.2 2.9 3.3 10.1
5.6 1.9 2.9 12.1 2.6 2.1 4.5 1.5 9.5 -
Note Samples were hydrolyzed with 6 N HCI or 3 N ptoluenesulfonic acid at 110°C for 24 h. No siificant differences were observed between two values except for cysteine and tryptophan. No corrections for the decay of threonine and serine during hydrolysis and for the incomplete hydrolysis of valine and leucine were performed. Cysteine content was determined by converting cysteine to sulfocysteine with sodium tetrathionate (7). Determination oftryptophan content was attempted by hydrolysis with 3 N ptoluenesulfonic acid (S), but a significant amount was not detected.
tamed in P 68, from the sequence study (12). Both the triplet components and GFA have high levels of glutamic acid and alanine. In GFA, a high level of leucine seems characteristic. Among the basic amino acids, lysine has the highest level in the triplet components, and arginine in GFA. ACKNOWLEDGMENTS 10
20
30 40 Fraction
50
60 70 Number
80
SO
100
PIG. 4. DE-52 column chromatography with linearly increasing sodium phosphate concentration from 10 to 400 mM and decreasing urea concentration from 8 to 0 ht.
A part of this experiment was performed in the Department of Pharmacology, New York University Medical Center. Thanks am due Dr. Ronald K Liem and Professor Michael L. Shelanski for their useful suggestions and dis cussions. The author also thanks Ms. Michiko Hishida for her technical assistance.
CHROMATGGRAPHIC
SEPARATION
REFERENCES 1. Geisler, N., and Weber, K. (1981) J. Mol. Biol. 151, 565-571. 2. Liem, R., and Hutchinson, S. B. (1982) Biochemistry 21,3221-3226. 3. Liem, R., Yen, S. H., Salmon, G. D., and Shelanski, M. L. (1978) J. Cett. Biol. 76, 637-645. 4. Liem, R. (1982) J. Neurochem. 38, 142-l 50. 5. Tokutake, S., Hutchinson, S. B., Patcher, J., and Liem, R. (1983) Anal. B&hem. 135, 102-105. 6. Spackman, D. H. (1967) in Methods in Enzymology (Hi, C. H. W., ed.), Vol. 11, pp. 3-l 5, Academic Press, New York. 7. Lie, T-Y., and Inglis, A. (1972) in Methods in En-
8. 9. 10. 11. 12. 13.
OF NEUROFILAMENT
207
zymology (Him, C. H. W., and Timasheff, S. N., eds.), Vol. 25, pp. 55-60, Academic Press, New York. Lie, T-Y. (1972) in Methods in Enzymology (Hits, C. H. W., and Timasheff, S. N., eds.), Vol. 25, pp. 44-55, Academic Press, New York. Laemmli, U. K. (1970) Nature (Londonj 227, 680685. Hogue-Angelett, R., Wu, H.-L., and Schlaepfer, W. (1982) J. Neurochem. 38, 116-120. Rueger, D., Gardner, E., Simonian, H., Dahl, D., and Bignami, A. (1981) J. Biol. Chem. 256, 1060610612. Geisler, N., Plessmann, U., and Weber, K. (1982) Nature (London) 296, 448-450. Bradford, M. M. (1976) Anal. B&hem. 72,248-254.