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Comparative electrophoretic study of the wolfgram proteins in myelin from several mammalia
582
&'ai, Research, 189 (1980) 582 587 I i Elsevier/North-Holland Biomedical Press
Comparative electrophoretic study of the wolfgram proteins in myelin from several mammalia
T. V. W A E H N E L D T and J. MALOTKA
Max-Planck-lnstitut fiir experimentelle Medizin, Forschungsstelle Neurochemie, 3400 GOttingen (G.F.R.) (Accepted January 3rd, 1980)
Key words: Wolfgram proteins - - myelin - - species
For several years it was not clear whether the Wolfgram proteins were myelinintrinsic or were components of membranes which contaminated myelin preparations. However, the concerted appearance during ontogeny of the Wolfgram proteins with the typical myelin proteins 20, their virtual absence from Jimpy mouse brain 9,1°, and their ultrastructural localization to the oligodendroglial plasma membrane-myelin continuum is leave no doubt that the Wolfgram proteins can be labelled as myelinintrinsic (for further discussion, see ref. 14). There appears to exist some discrepancy as to the number of components in the Wolfgram protein 'region' on SDS polyacrylamide gels. In some studies only a single band has been observed13,19, occasionally with one or two minor adjacent components 1,21, labelled as Wolfgram protein 1, 2 and 3 (see ref. 7). The majority of the reports, however, showed a protein doublet for Wolfgram proteins in myelin prepared from several species4,1~, le, also in two different electrophoretic systems 5,16. The reported molecular weights ranged from 54,000 and 62,000 Daltons in rat and beef ~,2~ to 47,000, 49,000 and 51,000 Daltons in mouseL The present comparative study was undertaken: (1) to examine systematically which of the numerous high molecular weight membrane proteins in brain are enriched in myelin; and (2) if the occurrence of a Wolfgram protein doublet is a phenomenon common to mammalian brain myelin. The brains were frozen immediately after death (--70 °C); in the case of ox and man the time between death and freezing was about I and 12 h, respectively. Total brains of adult mouse, rat, hamster, guinea pig and rabbit, and equal amounts of grey and white matter from brains o f o x and man were homogenized in 0.32 M sucrose (5 !!/ w/v). One portion of the homogenate was centrifuged at high speed ( > 100,000 g~v for 60 rain), and the pellet was designated the 'PART' fraction. Another portion of the homogenate was placed on 1.2 M sucrose and was centrifuged at 75,000 gay for 90 min, the floating layer forming t h e ' 1.2' fraction. Finally, a third portion was subjected
583
Fig. 1. A: SDS gel electrophoretic profiles of the 0.85 (myelin) fraction from brains of several mammals. MO, mouse; RA, rat; HA, hamster; GP, guinea pig; RB, rabbit, OX, beef; MA, man. Gradient slabs ranging from 6 to 30 ~ acrylamide, Coomassie blue stain, the anode is at the bottom. SBP, small basic protein; preSBP, presmall basic protein; LBP, large basic protein3; DM-20, intermediate or DM-20 protein; PLP, proteolipid protein1; Wl and W2, fast- and slow-migrating Wolfgram protein componentsZS; ACT, protein band comigrating with actin; TUB, protein band comigrating with tubulin. The bands migrating ahead of SBP (prominent in rat, hamster, rabbit and man) may be breakdown products of myelin basic protein(s), possibly due to differential lability of myelin to freezing and thawing, and to limited proteolysis. B: comparison of rates of migration of authentic actin (ACT) and tubulin (TUB) with rat 0.85 (myelin) fraction. The 4 horizontal lines to the left of the first lane show the positions of TUB, W2, Wl and ACT, from top to bottom.
"T U B" . ~ . . ~ W9
RA
RB Fig. 2. Close-up photographs of the gel region comprising the Wolfgram proteins in the 0.85 (myelin) fraction. Wl of MA (man) co-migrates with ACT; occasionally the two components were separated, with ACT forming a shoulder on the low molecular weight side of W1 (not shown here). For further abbreviations, see Fig. 1.
584 tO 3-fold elimination of microsomal material at 11,000 gay for 20 rain, followed by density gradient centrifugation on 0.85 M sucrose at 75,000 gay for 90 rain 2~, to give the '0.85' (myelin) fraction. All fractions obtained (PART; 1.2; 0.85) were washed 3 times with water to remove sucrose. They were dissolved in 1"o SDS and 1",, mercaptoethanol and were subjected to SDS gel electrophoresis on 6-30 % acrylamide gradient slabs 8. Staining was done with 0.1 ~ Coomassie blue in 50'~o methanol and 7.5 jo,~ acetic acid, and destaining with 5/o°J' methanol, 5 °/,o glycerol and 7.50/,i acetic acid; the gels were scanned at 550 nm and the areas under selected peaks were expressed as per cent of the total dye binding capacity. In addition to the presence of the major myelin proteins in the 0.85 fraction (Fig. 1 ; SBP, preSBP, LBP, DM-20, and PLP), the Wolfgram proteins were represented by a closely spaced doublet W1 and W2 in all species examined (Fig. 2; nomenclature of Nussbaum et al.l~). The significance of varying ratios WI/W2 in different mammals is not clear; however, other observations in rat have shown that the ratio decreases with increasing sucrose molarities on which myelin subfractions were collected 23. Moreover, this trend was also documented in the gel scans of PART, 1.2 and 0.85 fractions from guinea pig (Fig. 4). The Wolfgram protein doublet ran between two additional bands which were present in all animals and which showed identical rates of migration (Fig. IA): one 'fuzzy' band slower than the Wolfgram protein doublet and comigrating with tubulin (Figs. 1B and 2, tentatively labelled "TUB'), the other band positioned slightly ahead of the doublet and co-migrating with actin (Figs. 1B and 2, tentatively labelled 'ACT'S. Based on the molecular weights oftubulin (56,000 Daltons) and of actin (42,000
Fig. 3. Close-up photographs of the Wolfgram protein gel region of the 0.85 (myelin) fraction, demonstrating that rat Wl and hamster W2 components co-migrate, while rat Wl and man W2 components form a partly resolved doublet upon mixing (MIX). For molecular weight comparisons, see Table I.
585 TABLE I
Apparent molecular weights of the Wolfgram protein doublet W1 and W2* Values for W l and W2 columns are in thousand Daltons and are means from 3 separate experiments performed in duplicates 4- S.E.M.
W1
Mouse Rat Hamster Guinea pig Rabbit Ox Man
45.6 46.8 44.1 46.8 44.3 44.3 42.1
W2
4- 1.0 4- 0.8 4- 0.2 -4- 0.6 4- 0.3 4- 0.3 4- 0.2
48.8 49.5 47.0 49.3 47.2 46.9 45.3
4444444-
0.8 0.9 0.2 0.5 0.5 0.2 0.2
WI + W2
W2-
47.2 48.2 45.6 48.1 45.8 45.6 43.7
3.2 2.7 2.9 2.5 2.9 2.6 3.2
W1
* Based on rates of migration in SDS-PAGE, using actin (42,000 Daltons) and tubulin (56,000 Daltons) as references 17.
Dakons) 17 and using the TUB and ACT bands as inherent electrophoretic markers migrating to equal positions in different species (Figs. 1 and 3, rat and hamster), the apparent molecular weights of the Wolfgram protein doublet W1 and W2 showed small but significant differences among the species examined (Table I). Their mean
A
BP
B
PLP
60
40
~
PLP,DM-20,BP
W2 Wl
"TUB" "ACT" \ / <
~,,.
0.SS
z° 20 Z
1.2
I.u
>o 6
2 -p
PART
Wl + W2
"ACT" "TUB" I I I PART 1.2 0.85
Fig. 4. A: scans of the gel electrophoretic profiles of the PART, 1.2 and 0.85 fractions from guinea pig. B: the percentage dye binding was estimated for a selected number of bands from the gel profiles of the 7 species. BP includes SBP, preSBP and LBP in the case of mouse, rat and hamster, whereas only LBP is estimated in the case of guinea pig, rabbit, beef and man (see Fig. 1 for abbreviations).
586 apparent molecular weights ranged from 43,700 Daltons in man to 48,200 Daltons in rat, with an average spacing of about 2900 Daltons between the bands of the doublet. Mixing of extracts from two different species led either to overlap of two bands or to z~ more complex pattern of two largely separated doublets (Fig. 3). This demonstrates that the Wolfgram protein components are electrophoretically independent entities. The other fractions from the different species, i.e. PART and 1.2, werc also included in the S D S - P A G E analysis (Fig. 4A, guinea pig serves as an example), and the dye binding capacities of selected protein bands were determined in the species (Fig. 4B). With enrichment of myelin (PART -+ 1.2 - , 0.85) the major myelin proteins (PLP, DM-20, BP) more than doubled, while the sum of the Wolfgram protein components, W1 i W2, reached a plateau. The latter may be explained by the repeated elimination of uncompacted oligodendroglial membranes which contain the myelin-intrinsic, albeit not myelin-specific, Wolfgram proteins Is. By contrast, the A C T and TUB bands decreased finally towards the 0.85 (myelin) fraction. Provided A C T and TUB are in fact actin and tubulin, their reduction in the 0.85 (myelin) fraction tends to support a localization other than myelin ; however, small amounts of tubulin 6 and ofactin '~ have been found to be associated with isolated rat brain myelin. Unequivocal answers will require immunocytochemical techniques applied in situ. In conclusion, the Wolfgram proteins were consistently found as a doublet with a mean molecular weight below 50,000 D, showing maximal variation close to 5000 Daltons in the species examined. This result, together with the spacing of about 3000 Daltons agrees with that of Kelly and Luttges 7 but is at variance with a mean molecular weight of 58,000 Daltons and a wider spacing of 8000 Daltons in rat and beef ls,gs. Yet, this discrepancy may be due to different electrophoretic methods employed. The significance of a closely spaced doublet is not clear: the molecular weight spacing of about 3000 Daltons could however be related to that fotmd in myelin basic proteins :~ and may as such be a common phenomenon in membraneassociated proteins'~'L We would like to thank Prof. V. Neuhoff for discussion and Dr. H. H. Althaus for samples of tubulin and actin. Supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 33).
1 Agrawal, H. C., Trotter, J. L., Mitchell, R. F. and Burton, R. M., Criteria for identifying a myelin-like fraction from developing brain, Biochem. J., 136 (1973) 1117-1119. 2 Althaus, H. H. and Zechel, K., Demonstration of actin associated with isolated rat brain myelin, in preparation. 3 Barbarese, E., Carson, J. H., and Braun, P. E., Accumulation of the four myelin basic proteins in mouse brain during development, J. Neurochem., 31 (1978) 779-782. 4 Elam, J. S., Association of proteins undergoing slow axonal transport with goldfish visual system myelin, Brain Research, 97 (1975) 305-315. 5 Everly, J. L., Quarles, R. H. and Brady, R. O., Proteins and glycoproteins in myelin purified from the developing bovine and human central nervous system, J. Neurochem., 28 (1977) 95-10t. 6 Gozes, I. and Richter-Landsberg, C., Identification of tubulin associated with rat brain myelin, FEBS Lett., 95 (1978) 169-172. 7 Kelly, P. T. and Luttges, M. W., Mouse brain protein composition during postnatal development :
537 an electrophoretic analysis, J. Neurochem., 27 (1976) 1163-1172. 8 Laemmli, U. K., Cleavage of structural proteins during the assembly of the head of bacterieophage T4, Nature (Lond.), 227 (1970) 680-685. 9 Matthieu, J.-M., Quarles, R. H., Webster H. de F., Hogan, E. L. and Brady, R. O., Characterization of the fraction obtained from the CNS of Jimpy mice by a procedure for myelin isolation, J. Neurochem., 23 (1974) 517-523. 10 Matthieu, J.-M. and Waehneldt, T. V., Protein and enzyme distribution in microsomal and myelin fractions from rat and Jimpy mouse brain, Brain Research, 150 (1978) 307-318. 11 Mclntyre, R. J., Quarles, R. H., Webster, H. deF. and Brady, R. O., Isolation and characterization of myelin-related membranes, J. Neurochem., 30 (1978) 991-1002. 12 Mehl, E., Separation and characterization of myelin proteins, Adv. exp. reed. Biol., 32 (1972) 157-170.
13 MoreU, P., Greenfield, S., Costantino-Ceccarini, E. and Wisniewski, H., Changes in the protein composition of mouse brain myelin during development, J. Neurochem., 19 (1972) 2545-2554. 14 Norton, W. T., Isolation and characterization of myelin. In P. Morell (Ed.), Myelin, Plenum Press, New York and London, 1977, pp. 161-199. 15 Nussbaum, J. L., Delaunoy, J. P. and Mandel, P., Some immunochemical characteristics of Wl and W2 Wolfgram proteins isolated from rat brain myelin, J. Neurochem., 28 (1977) 183-191. 16 Poduslo, J. F., Everly, J. L. and Quarles, R. H., A low molecular weight glycoprotein associated with isolated myelin: distinction from myelin proteolipid protein, J. Neurochem., 28 (1977) 977-986. 17 Richter-Landsberg, C. and Yavin, E., Protein profiles of rat embryo cerebral cells during differentiation in culture, J. Neurochem., 32 (1979) 133-143. 18 Roussel, G., Delaunoy, J. P., Mandel, P. and Nussbaum, J.-L., Ultrastructural localization study of two Wolfgram proteins in rat brain tissue, J. NeurocytoL, 7 (1978) 155-163. 19 Toews, A. D., Horrocks, L. A. and King, J. S., Simultaneous isolation of purified microsomal and myelin fractions from rat spinal cord, J. Neurochem., 27 (1976) 25-31. 20 Waehneldt, T. V. and Neuhoff, V., Membrane proteins of rat brain: compositional changes during postnatal development, J. Neurochem., 23 (1974) 71-77. 21 Waehneldt, T. V., Ontogenetic study of a myelin-derived fraction with 2':3'-cyclic nucleotide 3'-phosphohydrolase activity higher than that of myelin, Bioehem. J., 151 (1975) 435-437. 22 Waehneldt, T. V., Matthieu, J.-M. and Neuhoff, V., Characterization of a myelin-related fraction (SN 4) isolated from rat forebrain at two developmental stages, Brain Research, 138 (1978) 29-43. 23 Waehneldt, T. V., Density and protein profiles of myelin from two regions of young and adult rat CNS, Brain Res. Bull., 3 (1978) 37-44. 24 Wickner, W., The assembly of proteins into biological membranes: the membrane trigger hypothesis, Ann. Rev. Biochem., 48 (1979) 23-45. 25 Wiggins, R. C., Joffe, S., Davidson, D. and Del Valle, U., Characterization of Wolfgram proteolipid protein of bovine white matter and fractionation of molecular weight heterogeneity, J. Neurochem., 22 (1974) 171-175.
&'ai, Research, 189 (1980) 582 587 I i Elsevier/North-Holland Biomedical Press
Comparative electrophoretic study of the wolfgram proteins in myelin from several mammalia
T. V. W A E H N E L D T and J. MALOTKA
Max-Planck-lnstitut fiir experimentelle Medizin, Forschungsstelle Neurochemie, 3400 GOttingen (G.F.R.) (Accepted January 3rd, 1980)
Key words: Wolfgram proteins - - myelin - - species
For several years it was not clear whether the Wolfgram proteins were myelinintrinsic or were components of membranes which contaminated myelin preparations. However, the concerted appearance during ontogeny of the Wolfgram proteins with the typical myelin proteins 20, their virtual absence from Jimpy mouse brain 9,1°, and their ultrastructural localization to the oligodendroglial plasma membrane-myelin continuum is leave no doubt that the Wolfgram proteins can be labelled as myelinintrinsic (for further discussion, see ref. 14). There appears to exist some discrepancy as to the number of components in the Wolfgram protein 'region' on SDS polyacrylamide gels. In some studies only a single band has been observed13,19, occasionally with one or two minor adjacent components 1,21, labelled as Wolfgram protein 1, 2 and 3 (see ref. 7). The majority of the reports, however, showed a protein doublet for Wolfgram proteins in myelin prepared from several species4,1~, le, also in two different electrophoretic systems 5,16. The reported molecular weights ranged from 54,000 and 62,000 Daltons in rat and beef ~,2~ to 47,000, 49,000 and 51,000 Daltons in mouseL The present comparative study was undertaken: (1) to examine systematically which of the numerous high molecular weight membrane proteins in brain are enriched in myelin; and (2) if the occurrence of a Wolfgram protein doublet is a phenomenon common to mammalian brain myelin. The brains were frozen immediately after death (--70 °C); in the case of ox and man the time between death and freezing was about I and 12 h, respectively. Total brains of adult mouse, rat, hamster, guinea pig and rabbit, and equal amounts of grey and white matter from brains o f o x and man were homogenized in 0.32 M sucrose (5 !!/ w/v). One portion of the homogenate was centrifuged at high speed ( > 100,000 g~v for 60 rain), and the pellet was designated the 'PART' fraction. Another portion of the homogenate was placed on 1.2 M sucrose and was centrifuged at 75,000 gay for 90 min, the floating layer forming t h e ' 1.2' fraction. Finally, a third portion was subjected
583
Fig. 1. A: SDS gel electrophoretic profiles of the 0.85 (myelin) fraction from brains of several mammals. MO, mouse; RA, rat; HA, hamster; GP, guinea pig; RB, rabbit, OX, beef; MA, man. Gradient slabs ranging from 6 to 30 ~ acrylamide, Coomassie blue stain, the anode is at the bottom. SBP, small basic protein; preSBP, presmall basic protein; LBP, large basic protein3; DM-20, intermediate or DM-20 protein; PLP, proteolipid protein1; Wl and W2, fast- and slow-migrating Wolfgram protein componentsZS; ACT, protein band comigrating with actin; TUB, protein band comigrating with tubulin. The bands migrating ahead of SBP (prominent in rat, hamster, rabbit and man) may be breakdown products of myelin basic protein(s), possibly due to differential lability of myelin to freezing and thawing, and to limited proteolysis. B: comparison of rates of migration of authentic actin (ACT) and tubulin (TUB) with rat 0.85 (myelin) fraction. The 4 horizontal lines to the left of the first lane show the positions of TUB, W2, Wl and ACT, from top to bottom.
"T U B" . ~ . . ~ W9
RA
RB Fig. 2. Close-up photographs of the gel region comprising the Wolfgram proteins in the 0.85 (myelin) fraction. Wl of MA (man) co-migrates with ACT; occasionally the two components were separated, with ACT forming a shoulder on the low molecular weight side of W1 (not shown here). For further abbreviations, see Fig. 1.
584 tO 3-fold elimination of microsomal material at 11,000 gay for 20 rain, followed by density gradient centrifugation on 0.85 M sucrose at 75,000 gay for 90 rain 2~, to give the '0.85' (myelin) fraction. All fractions obtained (PART; 1.2; 0.85) were washed 3 times with water to remove sucrose. They were dissolved in 1"o SDS and 1",, mercaptoethanol and were subjected to SDS gel electrophoresis on 6-30 % acrylamide gradient slabs 8. Staining was done with 0.1 ~ Coomassie blue in 50'~o methanol and 7.5 jo,~ acetic acid, and destaining with 5/o°J' methanol, 5 °/,o glycerol and 7.50/,i acetic acid; the gels were scanned at 550 nm and the areas under selected peaks were expressed as per cent of the total dye binding capacity. In addition to the presence of the major myelin proteins in the 0.85 fraction (Fig. 1 ; SBP, preSBP, LBP, DM-20, and PLP), the Wolfgram proteins were represented by a closely spaced doublet W1 and W2 in all species examined (Fig. 2; nomenclature of Nussbaum et al.l~). The significance of varying ratios WI/W2 in different mammals is not clear; however, other observations in rat have shown that the ratio decreases with increasing sucrose molarities on which myelin subfractions were collected 23. Moreover, this trend was also documented in the gel scans of PART, 1.2 and 0.85 fractions from guinea pig (Fig. 4). The Wolfgram protein doublet ran between two additional bands which were present in all animals and which showed identical rates of migration (Fig. IA): one 'fuzzy' band slower than the Wolfgram protein doublet and comigrating with tubulin (Figs. 1B and 2, tentatively labelled "TUB'), the other band positioned slightly ahead of the doublet and co-migrating with actin (Figs. 1B and 2, tentatively labelled 'ACT'S. Based on the molecular weights oftubulin (56,000 Daltons) and of actin (42,000
Fig. 3. Close-up photographs of the Wolfgram protein gel region of the 0.85 (myelin) fraction, demonstrating that rat Wl and hamster W2 components co-migrate, while rat Wl and man W2 components form a partly resolved doublet upon mixing (MIX). For molecular weight comparisons, see Table I.
585 TABLE I
Apparent molecular weights of the Wolfgram protein doublet W1 and W2* Values for W l and W2 columns are in thousand Daltons and are means from 3 separate experiments performed in duplicates 4- S.E.M.
W1
Mouse Rat Hamster Guinea pig Rabbit Ox Man
45.6 46.8 44.1 46.8 44.3 44.3 42.1
W2
4- 1.0 4- 0.8 4- 0.2 -4- 0.6 4- 0.3 4- 0.3 4- 0.2
48.8 49.5 47.0 49.3 47.2 46.9 45.3
4444444-
0.8 0.9 0.2 0.5 0.5 0.2 0.2
WI + W2
W2-
47.2 48.2 45.6 48.1 45.8 45.6 43.7
3.2 2.7 2.9 2.5 2.9 2.6 3.2
W1
* Based on rates of migration in SDS-PAGE, using actin (42,000 Daltons) and tubulin (56,000 Daltons) as references 17.
Dakons) 17 and using the TUB and ACT bands as inherent electrophoretic markers migrating to equal positions in different species (Figs. 1 and 3, rat and hamster), the apparent molecular weights of the Wolfgram protein doublet W1 and W2 showed small but significant differences among the species examined (Table I). Their mean
A
BP
B
PLP
60
40
~
PLP,DM-20,BP
W2 Wl
"TUB" "ACT" \ / <
~,,.
0.SS
z° 20 Z
1.2
I.u
>o 6
2 -p
PART
Wl + W2
"ACT" "TUB" I I I PART 1.2 0.85
Fig. 4. A: scans of the gel electrophoretic profiles of the PART, 1.2 and 0.85 fractions from guinea pig. B: the percentage dye binding was estimated for a selected number of bands from the gel profiles of the 7 species. BP includes SBP, preSBP and LBP in the case of mouse, rat and hamster, whereas only LBP is estimated in the case of guinea pig, rabbit, beef and man (see Fig. 1 for abbreviations).
586 apparent molecular weights ranged from 43,700 Daltons in man to 48,200 Daltons in rat, with an average spacing of about 2900 Daltons between the bands of the doublet. Mixing of extracts from two different species led either to overlap of two bands or to z~ more complex pattern of two largely separated doublets (Fig. 3). This demonstrates that the Wolfgram protein components are electrophoretically independent entities. The other fractions from the different species, i.e. PART and 1.2, werc also included in the S D S - P A G E analysis (Fig. 4A, guinea pig serves as an example), and the dye binding capacities of selected protein bands were determined in the species (Fig. 4B). With enrichment of myelin (PART -+ 1.2 - , 0.85) the major myelin proteins (PLP, DM-20, BP) more than doubled, while the sum of the Wolfgram protein components, W1 i W2, reached a plateau. The latter may be explained by the repeated elimination of uncompacted oligodendroglial membranes which contain the myelin-intrinsic, albeit not myelin-specific, Wolfgram proteins Is. By contrast, the A C T and TUB bands decreased finally towards the 0.85 (myelin) fraction. Provided A C T and TUB are in fact actin and tubulin, their reduction in the 0.85 (myelin) fraction tends to support a localization other than myelin ; however, small amounts of tubulin 6 and ofactin '~ have been found to be associated with isolated rat brain myelin. Unequivocal answers will require immunocytochemical techniques applied in situ. In conclusion, the Wolfgram proteins were consistently found as a doublet with a mean molecular weight below 50,000 D, showing maximal variation close to 5000 Daltons in the species examined. This result, together with the spacing of about 3000 Daltons agrees with that of Kelly and Luttges 7 but is at variance with a mean molecular weight of 58,000 Daltons and a wider spacing of 8000 Daltons in rat and beef ls,gs. Yet, this discrepancy may be due to different electrophoretic methods employed. The significance of a closely spaced doublet is not clear: the molecular weight spacing of about 3000 Daltons could however be related to that fotmd in myelin basic proteins :~ and may as such be a common phenomenon in membraneassociated proteins'~'L We would like to thank Prof. V. Neuhoff for discussion and Dr. H. H. Althaus for samples of tubulin and actin. Supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 33).
1 Agrawal, H. C., Trotter, J. L., Mitchell, R. F. and Burton, R. M., Criteria for identifying a myelin-like fraction from developing brain, Biochem. J., 136 (1973) 1117-1119. 2 Althaus, H. H. and Zechel, K., Demonstration of actin associated with isolated rat brain myelin, in preparation. 3 Barbarese, E., Carson, J. H., and Braun, P. E., Accumulation of the four myelin basic proteins in mouse brain during development, J. Neurochem., 31 (1978) 779-782. 4 Elam, J. S., Association of proteins undergoing slow axonal transport with goldfish visual system myelin, Brain Research, 97 (1975) 305-315. 5 Everly, J. L., Quarles, R. H. and Brady, R. O., Proteins and glycoproteins in myelin purified from the developing bovine and human central nervous system, J. Neurochem., 28 (1977) 95-10t. 6 Gozes, I. and Richter-Landsberg, C., Identification of tubulin associated with rat brain myelin, FEBS Lett., 95 (1978) 169-172. 7 Kelly, P. T. and Luttges, M. W., Mouse brain protein composition during postnatal development :
537 an electrophoretic analysis, J. Neurochem., 27 (1976) 1163-1172. 8 Laemmli, U. K., Cleavage of structural proteins during the assembly of the head of bacterieophage T4, Nature (Lond.), 227 (1970) 680-685. 9 Matthieu, J.-M., Quarles, R. H., Webster H. de F., Hogan, E. L. and Brady, R. O., Characterization of the fraction obtained from the CNS of Jimpy mice by a procedure for myelin isolation, J. Neurochem., 23 (1974) 517-523. 10 Matthieu, J.-M. and Waehneldt, T. V., Protein and enzyme distribution in microsomal and myelin fractions from rat and Jimpy mouse brain, Brain Research, 150 (1978) 307-318. 11 Mclntyre, R. J., Quarles, R. H., Webster, H. deF. and Brady, R. O., Isolation and characterization of myelin-related membranes, J. Neurochem., 30 (1978) 991-1002. 12 Mehl, E., Separation and characterization of myelin proteins, Adv. exp. reed. Biol., 32 (1972) 157-170.
13 MoreU, P., Greenfield, S., Costantino-Ceccarini, E. and Wisniewski, H., Changes in the protein composition of mouse brain myelin during development, J. Neurochem., 19 (1972) 2545-2554. 14 Norton, W. T., Isolation and characterization of myelin. In P. Morell (Ed.), Myelin, Plenum Press, New York and London, 1977, pp. 161-199. 15 Nussbaum, J. L., Delaunoy, J. P. and Mandel, P., Some immunochemical characteristics of Wl and W2 Wolfgram proteins isolated from rat brain myelin, J. Neurochem., 28 (1977) 183-191. 16 Poduslo, J. F., Everly, J. L. and Quarles, R. H., A low molecular weight glycoprotein associated with isolated myelin: distinction from myelin proteolipid protein, J. Neurochem., 28 (1977) 977-986. 17 Richter-Landsberg, C. and Yavin, E., Protein profiles of rat embryo cerebral cells during differentiation in culture, J. Neurochem., 32 (1979) 133-143. 18 Roussel, G., Delaunoy, J. P., Mandel, P. and Nussbaum, J.-L., Ultrastructural localization study of two Wolfgram proteins in rat brain tissue, J. NeurocytoL, 7 (1978) 155-163. 19 Toews, A. D., Horrocks, L. A. and King, J. S., Simultaneous isolation of purified microsomal and myelin fractions from rat spinal cord, J. Neurochem., 27 (1976) 25-31. 20 Waehneldt, T. V. and Neuhoff, V., Membrane proteins of rat brain: compositional changes during postnatal development, J. Neurochem., 23 (1974) 71-77. 21 Waehneldt, T. V., Ontogenetic study of a myelin-derived fraction with 2':3'-cyclic nucleotide 3'-phosphohydrolase activity higher than that of myelin, Bioehem. J., 151 (1975) 435-437. 22 Waehneldt, T. V., Matthieu, J.-M. and Neuhoff, V., Characterization of a myelin-related fraction (SN 4) isolated from rat forebrain at two developmental stages, Brain Research, 138 (1978) 29-43. 23 Waehneldt, T. V., Density and protein profiles of myelin from two regions of young and adult rat CNS, Brain Res. Bull., 3 (1978) 37-44. 24 Wickner, W., The assembly of proteins into biological membranes: the membrane trigger hypothesis, Ann. Rev. Biochem., 48 (1979) 23-45. 25 Wiggins, R. C., Joffe, S., Davidson, D. and Del Valle, U., Characterization of Wolfgram proteolipid protein of bovine white matter and fractionation of molecular weight heterogeneity, J. Neurochem., 22 (1974) 171-175.