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Characterization of a myelin-related fraction (SN 4) isolated from rat forebrain at two developmental stages
Brain Research, 138 (1977) 2943 © Elsevier/North-Holland Biomedical Press
29
C H A R A C T E R I Z A T I O N OF A M Y E L I N - R E L A T E D F R A C T I O N (SN 4) ISOLATED F R O M RAT F O R E B R A I N AT TWO D E V E L O P M E N T A L STAGES
THOMAS V. WAEHNELDT, JEAN-MARIE MATTHIEU* and VOLKER NEUHOFF Forschungsstelle Neurochemie, Max-Planek-lnstitut fiir experimentelle Medizin, 3400 G6ttingen (G.F.R.)
(Accepted March 23rd, 1977)
SUMMARY A myelin-related fraction (SN 4) was isolated from forebrain of 17- and 40-dayold rats. Fraction SN 4 was obtained as a supernatant in a slow speed differential centrifugation of a myelin fraction. In contrast to multilamellar myelin fraction, SN 4 consisted of small vesicular profiles of a mixture of single membranes and some triplelayered structures. All typical myelin components were found in the SN 4 fraction from adult rat brain but their relative proportion was different from that of myelin: Wolfgram protein, myelin glycoproteins and 2',3'-cyclic nucleotide 3'-phosphohydrolase were increased, while basic proteins and proteolipid protein were decreased significantly. In contrast, the lipid composition appeared very similar to the one found in myelin. SN 4 from 17-day-old rat brains was essentially similar to that from adults, except that the major myelin glycoprotein was not enriched in comparison to myelin. Developmental changes found in myelin were also present in the SN 4 fraction. The specific radioactivity of the fucose-labeled major myelin glycoprotein was similar in SN 4 and in myelin. The particular composition of fraction SN 4 suggests that this material is not significantly contaminated by non-myelin-related membranes but rather supports the hypothesis that it could be enriched in a membrane representing a zone of transition during the formation of myelin and which is subjected to a remodelling of its protein components.
INTRODUCTION Myelin isolated by density gradient centrifugation is probably a mixture of membranes related to the process of myelin formation, consisting mostly of myelin * Service de P6diatrie, C.H.U.V., 1011 Lausanne, Switzerland.
30 lamellae besides oligodendroglial plasma membranes and their regions of transition to myelin, paranodal glial membranes, newly formed myelin and possibly some axolemma still attached to myelin. In an attempt to separate these different myelin particles a number of investigators have subfractionated myelin and got biochemically and morphologically distinct subfractions. Recent reports suggest that immature myelin or premyelin can fractionate into particular myelin subfractions: myelin-like fractionZ,a, 27, lower layer 20, membrane fraction s, heavy myelinS,6, 37, or SN 4 fraction32,34. This investigation was undertaken to isolate myelin from immature and young adult rat forebrain and this fraction was subfractionated after hypo-osmotic treatment. Purified myelin and a myelin-related fraction (SN 4) were characterized and compared to each other by electron microscopy and enzyme markers for membranes. Proteins and glycoproteins were separated by polyacrylamide gel electrophoresis and quantitated by densitometric scanning of protein bands. The incorporation of radioactive fucose into the myelin glycoproteins was studied in these fractions and their electrophoretic mobility on polyacrylamide gels was analyzed by using the radioactive double labeling technique. A preliminary report of some of these results was published elsewhere 34. MATERIALS AND METHODS
Fractionation of forebrain Rats of either sex (Wistar strain TNO/W70), aged 17 days and 40 days, were obtained from F. Winkelmann, 4791 Borchen, G.F.R. The animals were slightly anaesthetized with ether and each animal was intracerebrally injected with either 20 #Ci of L-[1,5,6-ZH]fucose or with 8 /~Ci of L-[1-14C]fucose (New England Nuclear, Boston, Mass.) in 10 #1 of 0 . 8 5 ~ NaCI. After 16-18 h, the rats were killed by decapitation and the forebrains removed and chilled. In a typical experiment, 30 brains of 17-day-old animals (15.41 g) or 15 brains of 40-day-old animals (28.75 g) RAT F O R E B R A I N '~"1 Total Homogenate in 0.32 M Sucrose
11,000 g 20 rain) 11,000 g 20 rain,)
T
11,000 g 20 rain) 100,0001~ 30 rain PELLET Homog. 0.88 M Sucrose 0.32 M Sucrose Overlay
I
T
/,000g 20 rain
I
50,000g 60rain ) 0.32/0.88 Layer Water Shock 25"C l
[]
Fig. 1. Flow sheet for the preparation of myelin and SN 4 fractions from forebrains of 17- and 40day-old rats. Thick arrows indicate iso- or hyper-osmotic media, thin lines stand for hypo-osmotic conditions. Further details are given in Materials and Methods.
31 were homogenized in 0.32 M sucrose (300 ml at 17 days, 250 ml at 40 days) with 3 4 strokes in a loosely fitting Teflon-glass homogenizer (Thomas, Philadelphia) at 750 rev./min (Fig. 1). The total homogenate was then centrifuged for 20 min at 11,000 gay in a fixed angle rotor. The supernatant (SN 1) was carefully removed with a syringe, avoiding the residual portion above the pellet, and the pellet was gently homogenized by hand leaving the tightly packed red portion in the tube. Centrifugation, removal of the supernatant, and rehomogenization was repeated twice, giving supernatants SN 2 and SN 3. The washed pellet was densely packed by high speed centrifugation at 100,000 gay for 30 min (fixed angle rotor). The resulting pellet was homogenized with 8 strokes at 750 rev./min in either 125 ml (17 days) or 70 ml (40 days) 0.88 M sucrose, placed into two tubes (17 days) or three tubes (40 days), overlayered with 0.32 M sucrose, and centrifuged at 40,000 gay for 75 min (swing-out rotor) or at 50,000 gay for 60 min (swing-out rotor). The tightly packed 0.32/0.88 M sucrose layer was removed with a syringe, hypo-osmotically shocked in 180 ml water of 25 °C, and homogenized with 6-8 strokes in a tightly fitting Teflon-glass homogenizer (Braun, 3508 Melsungen) at 2600 rev./min. After cooling in an ice-water bath the homogenate was centrifuged at 4,000 gay for 20 min (with the break-off). The slightly turbid supernatant (SN 4) was carefully removed with a syringe, avoiding the portion above the myelin (MY) pellet. The supernatant SN 4 was then centrifuged for 30 min at 60,000 gay, to give a faintly brownish pellet which is less opaque than the myelin pellet. Both the myelin pellet and fraction SN 4 were rehomogenized in water and recentrifuged 3 times. The final homogenates (about 1 mg protein/ml) were stored at --70 °C. Similarly, the particulates of fractions SN 1, SN 2, SN 3, and of the pellet formed in 0.88 M sucrose (Fig. 1, P) were centrifuged, washed with water and stored at --70 °C. Electron microscopy Suspensions of myelin and SN 4 preparations were fixed in 1.5 ~ glutaraldehyde0.5 ~ formaldehyde buffered with phosphate (pH 7.4). After a 5-min centrifugation at 25,000 gay the pellets were post-fixed with 1 ~ osmic acid, thereafter subjected to stepwise dehydration with ethanol ranging from 35 ~ to 100 ~. Ethanol was replaced by propylene oxide and final embedding was done in Epon. Thin sections were stained with saturated uranyl acetate and 3 ~ lead citrate and were examined in a Zeiss EM 9 or a Jeol 100 B electron microscope. Protein determination Samples were assayed for protein content using the method of Lowry et al. 14, with crystalline bovine serum albumin as a standard. Enzyme assays The activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP, EC 3.1.4.16) was determined by a photometric method 24. The concentration of CNP was doubled and care was taken to sufficiently dilute the samples to maintain saturating conditions. Acetylcholinesterase (ACHE, EC 3.1.1.7) was assayed with acetylthiocholine as a substrate 9.
32
Determination of individual proteins Two different techniques z5 were used to analyse proteins from myelin and SN 4 fractions by polyacrylamide gel electrophoresis on 5 ~ or 15 ~ gels in the presence of SDS. Gels were stained with 1 ~o Fast Green and scanned on a spectrophotometer at 580 nm according to Morell et al. 2°. The amounts of protein in the different bands were calculated from the densitometric scans and the results expressed as percentage of the total dye-binding capacity 1~.
PAS staining and radioactive counting of the glycoprotein Lyophilized myelin or SN 4 was delipidated with chloroform-methanol (2:1, v/v), solubilized in SDS and electrophoresed on 5 ~ polyacrylamide gels 25. Glycoprotein quantitation was performed by staining with periodic acid-Schiffreagent and scanning at 520 nm 15. Fetuin was used as a standard, and one unit of the myelin glycoprotein was defined as the amount which stained with the same intensity as 1 #g of fetuin. Radioactive-labeled glycoproteins were determined by cutting 5 ~o gels into I mm slices and counting according to method B of Quarles et al. 25. The data were corrected for quenching and double labeling and graphically displayed using a computer program. The results are plotted as the percentage of isotope in each fraction of the gel or in some experiments of a segment of the gel containing the major myelin glycoprotein.
Lipid separation Lipids from lyophilized fractions were extracted with chloroform-methanol (2:1, v/v), partitioned 11,2s, and separated by TLC. The lower phase lipids were separated on silica gel G plates developed in chloroform-methanol-ammonia (80:20:0.4 by vol.) 22 and charred with sulfuric acid. The positions of the lipids were related to lipid standards run on the same plate which was scanned on a photometer fitted with a 480 nm filter. Gangliosides extracted in the upper phase were separated by TLC 30 and stained with the resorcinol reagent 29. The spots were compared to ganglioside standards run on the same plate. RESULTS
Morphology Electron microscopy of myelin and fraction SN 4 (Figs. 2 and 3) showed that at both ages the individual fragments of myelin contained more layers than the average of the profiles seen in SN 4. This difference was more pronounced in 40-day-old forebrain as compared to 17-day-old forebrain. Multilayered myelin was rarely seen in the SN 4 fraction at both ages, however, small vesicular profiles with only a few layers were observed, many of which were triple-layered (Fig. 2B and 3B, inserts). Structures other than myelin, e.g. mitochondria or axonal contents, were virtually absent in the preparations.
Protein and enzyme determination In Table I, the protein yield and the activities of CNP and of AChE are shown
Fig. 2. Electron micrographs, myelin fraction (A) and SN 4 fraction (B) from 40-day-old rat forebrains. Large circular profiles of multilayered myelin contrast with smaller and thinner vesicular structures in myelin-related fraction SN 4. Very few small vesicular profiles are present in compact myelin, x 17,000, inserts x 215,000.
Fig. 3. Electron micrographs, myelin fraction (A) and SN 4 fraction (B) from 17-day-old rat forebrains. As in 40-day-old brains substantial compaction of rather large circular structures is evident in myelin whereas very few layers are seen in the SN 4 fraction with many small vesicles besides some singleor double-layered larger circular profiles, x 17,000, inserts x 215,000.
35 TABLE I Developmental changes in protein yield and in activities of two marker enzymes in total homogenate and in myelin subfractions from rat ]brebrain
The figures represent the means of several determinations (numbers in brackets) -4- S.E.M. Postnatal Fraction age of rats (days)
40
17
YieM of protein (mg/g fresh tissue)
CNP specific activity*
AChE specific activity*
CNP/A ChE
Total homogenate Myelin SN 4 SN 4/Myelin
94.54 ± 4.67 (4) 286 -4- 10 (14) 3.52 -4- 0.36 (8) 1470 -4- 113 (14) 0.52 4- 0.02 (8) 3458 -- 260 (14) 0.15 2.35
7.52 ± 0.99 (8) 0.77 4- 0.03 (8) 3.85 ± 0.48 (8) 5.00
38 1909 898
Total homogenate Myelin SN 4 SN 4/Myelin
80.71 4- 7.22 (4) 106 -4- 16 (14) 0.81 -4- 0.07 (8) 1627 4- 164 (14) 0.21 -4- 0.25 (8) 3124 -4- 410 (14) 0.26 1.92
5.77 4- 0.45 (8) 1.39 -4- 0.13 (8) 4.29 4- 0.91 (8) 3.09
18 1171 728
* /~moles hydrolysed/mg protein/h. for the total homogenate, myelin, and for SN 4. The protein yield in the total homogenate increased only slightly with age, in agreement with an increase from about 6 ~ in 5-day-old forebrain to about 10 ~ in adult tissue 3a. By contrast, SN 4 and particularly myelin displayed a substantial increase of respectively 2.5 and 7 times the original values. Although an increase in C N P activity was evident in the total homogenate from day 17 to day 40, the values for myelin and for SN 4 were much higher and were similar at either age. In both cases SN 4 displayed about twice the activity of C N P than myelin, which is characteristic of fraction SN 4. The specific activity of ACHE, despite an increase in the total homogenate with age, showed higher levels in myelin and in SN 4 in 17-day-old animals. Proteins
The protein profile of myelin and SN 4 fractions from 40-day-old rat brain is shown in Fig. 4. Both fractions contained the typical myelin proteins: Wolfgram protein, proteolipid protein, DM-20 or intermediate protein, large and small components of the myelin basic protein. An additional band in the high molecular weight region of the gel called X was not specific to myelin (Matthieu and Waehneldt, unpublished). The SN 4 fraction showed low levels of basic proteins, proteolipid protein and DM-20. The high molecular weight proteins were increased in SN 4 and this was particularly pronounced for the Wolfgram protein and the X band. The different protein values expressed in per cent of the dye-binding capacity are presented in Table II. In myelin from 17-day-old animals there was relatively more of the high molecular weight proteins (Fig. 5). This contrasted with SN 4 from immature brains where the percentage of high molecular weight proteins was similar to adult values.
36
o
Fig. 4. Densitometric scans of Fast Green-stained proteins in myelin and SN 4 fraction of 40-day-old rats after separation on 15 ~ polyacrylamide gels in the presence of SDS. Each gel contained 150/~g of protein. X, high molecular weight component, probably of non-myelin origin; W, Wolfgram protein; PLP, proteolipid protein; DM-20 or intermediate protein; LBP, SBP, large and small components of myelin basic protein; D, degradation product.
TABLE II
Densitome!ric quantitation of individual proteins in fractions isolated from 40-day-old rats Per cent of the dye-binding capacit~ (15% gels stained with Fast Green). Mean ± S.E.M. of 4 separate experiments compared with the Student's test. n.s., not significant.
High molecular weight proteins (--W) Wolfgram protein (W) Proteolipid protein (PLP) Intermediate protein or DM-20 Large basic protein (LBP) Small basic protein (SBP) Rest SBP/LBP
Myelin
SN 4
P
S N 4~Myelin
21.4 ± 0.3
45.7 ~ 1.4
< 0.001
2.1
7.9 ± 23.3 ± 5.7 ± 14.3 ~20.2 ± 7.2 ± 1.41
14.6 ± 9.1 ± 3.2 ± 7.5 ± 10.1 ± 10.0 ± 1.35
< 0.001 < 0.001 < 0.001 < 0.001 < 0.001 n.s.
1.9 0.4 0.6 0.5 0.5 1.4
0.4 1.1 0.2 0.4 0.5 1.6
0.8 0.5 0.3 0.4 0.8 0.7
37
Fig. 5. Densitometric scans of proteins in myelin and SN 4 of 17-day-old rats. Details as for Fig. 4.
Myelin basic proteins, DM-20 and proteolipid protein were decreased in the SN 4 fraction when compared to myelin, while the Wolfgram protein was practically unchanged (Table III). In contrast to the values found in adults, the difference of the individual proteins between myelin and SN 4 was smaller in immature animals. Nevertheless, when the non-Wolfgram high molecular weight proteins were omitted from the calculation, the Wolfgram protein value in SN 4 was 1.65-fold that of myelin TABLE III
Densitometric quantitation of individuat proteins in fractions isolated from 17-day-old rats Per cent of the dye-binding capacity (15 ~ gels stained with Fast Green). Mean ± S.E.M. of 4 separate experiments compared with the Student's test. n.s., not significant.
Myelin High molecular weight proteins (--W)28.6 Wolfgram protein (W) 9.0 Proteolipid protein (PLP) 16.4 Intermediate protein or DM-20 6.1 Large basic protein (LBP) Small basic protein (SBP) Rest SBP/LBP
q- 1.4 -I- 1.3 ± 0.1 ± 0.3
16.5 ± 0.1 15.7 d_ 0.1 7.9 ± 0.5 0.95
SN 4 46.0 10.6 7.8 4.0
:k :k :k ±
0.7 0.6 0.7 0.1
12.2 ± 0.1 10.8 q- 0.1 8.7 d- 0.3 0.89
P
S N 4~Myelin
< 0.001 n.s. < 0.001 0.001 < P < 0.01 < 0.001 < 0.001 n.s.
1.6 1.2 0.5 0.7 0.7 0.7 1.1
38 TABLE IV Quantitat&n o] the PAS-stained major myelin glycoprotein
Glycoprotein unit/rag protein (chloroform-methanol insoluble proteins). Mean ± S.E.M. of 4 separate experiments compared with the Student's test. n.s., not significant.
40-day-old rats 17-day-old rats
Myelin
SN 4
P
S N 4~Myelin
42.8 ± 5.6 40.7 :L 7.9
84.2 4:_ 7.5 39.0 ~: 7.1
0.001 < P < 0.01 1.97 n.s. 0.96
and this was close to the two-fold increase observed in the adults. In myelin and SN 4 from adult animals, the small component of the myelin basic proteins was present in higher proportion than the large component (Fig. 4 and Table II). In contrast, these two components were present in similar proportions in myelin and SN 4 from immature animals (Fig. 5 and Table III). Glycoproteins
In adult rats, PAS-stained gels from chloroform-methanol-delipidated SN 4 fraction showed the presence of a major band which exhibited a similar electrophoretic mobility as the one from myelin. There was twice as much of this major glycoprotein in the SN 4 fraction than in myelin (Table IV). In contrast, in SN 4 from immature animals, the amount of PAS-stained major glycoprotein was similar to that found in myelin (Table IV). After 16 h of incorporation, the specific radioactivity of the in vivo fucoselabeled major glycoprotein (dpm/unit of glycoprotein) was similar in myelin and in SN 4, but there was a 5-fold increase in 17-day-old fractions when compared to 40-day-old fractions. The amount of radioactivity in the major glycoprotein was 40 ~ of the total radioactivity bound to chloroform-methanol-insoluble proteins of myelin and SN 4. The patterns of the fucose-labeled glycoproteins from SN 4 and myelin coincided (Fig. 6) at both ages considered. The newly synthesized glycoprotein in immature rat myelin showed a slightly higher apparent molecular weight than that in if- 16.
H [ 1 4 C ] fucose, myelin °--'°[3HI fucose, SN-4
o o
b [. 10 FRACTION NUMBER
20
i0
Fig. 6. Comparison of the fucose labeled glycoproteins in myelin (O 0) and SN 4 (©----()) fractions from 40-day-old rats which had been injected with [14C]fucoseand [3H]fucose, respectively. The gel was divided into 3-mm fractions and contained 800/~g of protein, 5573 dpm 14C and 12,158 dpm 3H.
39 SN4
MYELIN
~'PI o..or.14C]fucose,lTd.
;i0 (_~ 0 o
: 4
0
: [3H]fucose, 17d. d -o [14C] . / f ucose, %
mr
/
,~ 0
z u ~d
o. I
I
I
10 A
/A
gs
7 UJ U
nO
'~._.r3.l,ooo,~,~o~.
FRACTION
NUMBER
I 20
0
I
I
I
I0 FRACTION NUMBER
I 20 B
Fig. 7. Fucose-labeled glycoproteins in myelin and SN 4 fractions from 17- and 40-day-old rat forebrains. High resolution was obtained by using 1-mm fractions. Figure shows the portion of the gel 2-4 cm from the top. A: comparison of the major labeled glycoprotein in myelin of 17-day-old rats injected with [SH]fucose (9965 dpm, • • ) with that of 40-day-old rats injected with [14C]fucose (10,736 dpm, O - - - O). B : comparison of the major labeled glycoprotein in SN 4 of 17-day-old rats injected with [taC]fucose (9557 dpm O ---O) with that of 40-day-old rats injected with [3H]fucose (3940 dpm, • •). The gel contained 870 Mg of protein. mature myelin (Fig. 7A) as previously reported 18,~6. This developmental difference in electrophoretic mobility was also found in the SN 4 fraction from 17- and 40-day-old animals (Fig. 7B).
L~/ds The SN 4 fraction contained slightly less lipids, on a dry weight basis, than did myelin: 75.3 mg/100 mg dry weight and 78.4 mg/100 mg dry weight, respectively. Cholesterol, cerebrosides and phosphatidyl ethanolamine were the most prominent lipids. Sulfatides were also present but in smaller amounts. The ratios of cerebrosides to sulfatides in myelin and in SN 4 from 40-day-old animals were 3.1 and 3.2, respectively. G m ganglioside was present in similar amounts in myelin and in SN 4. Traces of higher polysialogangliosides were present in the SN 4 fraction but these gangliosides were barely detectable in myelin. DISCUSSION It has been observed in several laboratories 1,8,1°,19,81 that myelin isolated from adult CNS tissue has a limited number of fairly low molecular weight protein components, ranging from 15,000 to 25,000 daltons and commonly known as 'major myelin proteins'. Among these the basic protein(s) and the proteolipid protein are the most prominent lz. Electrophoresis of myelin proteins in the presence of detergent reveals, however, an additional large number of minor protein components with molecular weights higher than 25,000 daltons of which the Wolfgram protein and the major myelin-associated glycoprotein have been characterized in some detail 18,25. Since it is possible to prepare myelin fractions with changing proportions of high molecular weight proteins 32, it has to be considered that some of these high molecular weight proteins might not be intrinsic myelin components. Rather they may be
40 involved in the process of myelin formation in single membranes leading to compact myelin, such as oligodendroglial processes, loose lamellae, external loops, and paranodal membranes. Moreover, the possibility of contamination with alien membranes has to be seriously taken into account. In this study the isolation of a myelin-related membrane fraction was based on two crucial preparative steps: (1) elimination of microsomal material and (2) hypo-osmotic shock, coupled with slow speed differential centrifugation. Elimination of microsomal fragments was achieved by three-fold washing of the total particulate fraction in iso-osmotic medium, thus concentrating large-sized compact myelin. Compact myelin was separated from other subcellular particles such as nuclei, large mitochondria and large synaptosomes in a 'floating up' density gradient centrifugation. It was only at this degree of purity that myelin was subjected to hypo-osmotic conditions, in contrast to other methods~, zl. By subjecting the shocked material to a second slow speed differential centrifugation multilayered myelin was found as a pellet and small fragments of membranes and vesicular structures remained in the supernatant (SN 4), as shown by electron microscopy. Thus, up to the stage of the hypoosmotic shock, the multilayered structure of myelin helped to protect cytoplasmcontaining portions, such as paranodal loops and possibly external and internal tongues. One of the characteristics of the myelin-related fraction SN 4 was its high specific activity of CNP. As shown in Table I, fraction SN 4 had specific activities twice those of myelin, both at 40 days and at 17 days, although the activities of the total homogenate showed an age-dependent increase reflecting the process of myelination. The high activity of CNP in fraction SN 4 indicates that this enzyme is not only a marker for myelin but could already be present in loosely wrapped myelin and in oligodendroglial membranes 23. The levels of AChE specific activity, an assumed neuronal and endoplasmic reticular marker 13, are more difficult to assess. Compared with the total homogenate, lower levels of AChE specific activity were found in myelin and in SN 4 at both ages examined (Table I). This points to substantial elimination of non-myelin material. A higher degree of purity was reached in myelin and SN 4 from 40-day-old rats which can be explanied by the greater extent of in situ compaction and by the larger amount of myelin in older animals facilitating the isolation of myelin 21. The ratio CNP/AChE (Table !) clearly indicates that a substantial purification of myelin has been achieved at both ages. The SN 4 fraction is closely related to myelin because all typical myelin components are present: myelin basic proteins, proteolipid protein, Wolfgram protein, cerebrosides, CNP and the major myelin glycoprotein. Furthermore, it also exhibits the developmental and metabolic features specific to myelin : (1) the developmental change in the proportion of the two myelin basic proteins previously reported 7,16,20,36 was als~ observed in SN 4 and was similar to that measured in myelin; (2) the developmental change in the apparent molecular weight of the major myelin glycoprotein "~8,26 was also found in SN 4; (3) the specific radioactivity of fucose incorporated into the major myelin glycoprotein was the same in both SN 4 and myelin fractions but was much higher in young and actively myelinating rats than in
41 adult animals; (4) the proportion of the different glycoproteins separated by electrophoresis and measured by PAS staining or radioactive counting was identical in myelin and SN 4 and this strongly suggests that SN 4 did not contain significantly more contaminants than myelin. Nevertheless, these typical myelin components were present in SN 4 in different proportions. Three of them were enriched in SN 4: the Wolfgram protein which is a minor component in myelin became the prominent protein in SN 4. The CNP activity and the myelin major glycoprotein were increased by a two-fold factor in SN 4 from adult animals. Two specific myelin proteins were significantly decreased: the myelin basic proteins and the proteolipid protein. In contrast, the lipid composition appeared very similar in myelin and SN 4. Another difference between SN 4 and myelin is the fact that in young myelinating animals the protein pattern of SN 4 was very similar to the adult SN 4 fraction, and this contrasts with immature myelin, which contained a higher proportion of high molecular weight proteins than adult myelin. The SN 4 fraction presents some similitude with the heavy myelin fraction isolated by Matthieu et al. 16 in the adult rat and by Zimmermann et al. 37 during development. Density distributions on zonal gradients (Matthieu and Waehneldt, in preparation) demonstrated that SN 4 is a heavier fraction (maximum at 0.73 M sucrose) than the corresponding myelin fraction (maximum at 0.67 M sucrose). This is consistent with a slightly lower lipid content of SN 4 when compared to myelin, as shown in this communication. Also CNP, ACHE, high molecular weight proteins and myelin glycoproteins were enriched in both adult SN 4 and heavy myelin, while basic proteins and proteolipid protein were decreased. But one main difference when SN 4 is compared to the heavy myelin was the specific enrichment in Wolfgram protein and in CNP in fraction SN 4 and its similar protein composition in immature and adult animals, while the heavy myelin fraction from immature rats appeared heavily contaminated37. Therefore we think that SN 4 contains membranes which are also present in heavy myelin subfractions but in a higher yield and less contaminated. The structural origin of membranes in the SN 4 fraction, like the heavy myelin subfraction, is not yet established. These myelin-related membranes could be transitional between the plasma membrane of the oligodendrocyte and multilamellar myelin16. Another possible location would be the paranodal region or the inner and outer loops of the myelin sheath. In conclusion, in view of the similar lipid composition, the very different distribution of the myelin protein components observed between myelin and SN 4 suggests a rearrangement of the membrane proteins within the lipid bilayer rather than a dilution of the SN 4 fraction by non-myelin-related membranes. These results suggest that SN 4 represents a highly purified fraction derived from, or closely related to, myelin, although its exact location and role during the process of myelination is still a matter of conjecture.
42 ACKNOWLEDGEMENTS We w o u l d like to t h a n k Dr. R. S a m m e c k , Mrs. M. M e y e r m a n n an d Dr. J.-C. Krieg for the electron micrographs. This w o r k was s u p p o r t e d in part by the Deutsche F o r s c h u n g s g e m e i n s c h a f t (SFB 33) and by the Swiss N a t i o n a l Science F o u n d a t i o n , G r a n t 3.225.74.
REFERENCES 1 Adams, D. H. and Fox, M. E., The homogeneity and protein composition of rat brain myelin, Brain Research, 14 (1969) 647-661. 2 Agrawal, H. C., Banik, N. L., Bone, A., Davison, A. N., Mitchell, R. F. and Spohn, M., The identity of myelin-like fraction isolated from developing brain, Biochem. J., 120 (1970) 635-642. 3 Agrawal, H. C., Trotter, J. L., Burton, R. M. and Mitchell, R., Metabolic studies on myelin: evidence for a precursor role of a myelin subfraction, Biochem. J., 140 (1974) 99 109. 4 Banik, N. L. and Davison, A. N., Enzyme activity and composition of myelin and subcellular fractions in the developing rat brain, Biochem. J., 115 (1969) 1051-1062. 5 Benjamins, J. A., Miller, S. L. and Morell, P., Metabolic relationships between myelin subfractions: entry of galactolipids and phospholipids, J. Neurochem., 27 (1976) 565-570. 6 Benjamins, J. A., Gray, M. and Morell, P., Metabolic relationship between myelin subfractions: entr~r of proteins, J. Neurochem., 27 (1976) 571-575. 7 Cammer, W. and Norton, W. T., Disc gel electrophoresis of myelin proteins: new observations on development of the intermediate proteins (DM-20), Brain Research, 109 (1976) 643-648. 8 Cotman, C. W. and Mahler, H. R., Resolution of insoluble proteins in rat brain subcellular fractions, Arch. Biochem., 120 (1967) 384-396. 9 Ellman, G. L., Courtney, K. D., Andres, V., Jr. and Featherstone, R. M., A new and rapid colorimetric determination of acetylcholinesterase, Biochem. Pharmacol., 7 (1961) 88-95. 10 Eng, L. F., Chao, F.-C., Gerstl, B., Pratt, D. and Tavaststjerna, M. G., The maturation of human white matter m3,elin. Fractionation of the myelin membrane proteins, Biochemistry, 7 (1968) 44554465. 11 Folch, J., Lees, M. and Sloane-Stanley, G. H., A simple method for the isolation and purification of total lipids from animal tissues, J. biol. Chem., 266 (1957) 497 509. 12 Guidotti, G., Membrane proteins, Ann. Rev. Bioehem., 41 (1972) 731-752. 13 Kokko, A., Mautner, H. G. and Barrnett, R. J., Fine structural localization of acetyl-betamethylthiocholine and acetyl selenocholine as substrates, J. Histochern. Cytochem., 17 (1969) 625-640. 14 Lowry, O. H., Rosebrough, N. A., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 15 Matthieu, J.-M. and Quarles, R. H., Quantitative scanning of glycoproteins on polyacrylamide gels stained with periodic acid-Schiff reagent (PAS), Analyt. Biochem., 55 (1973) 313-316. 16 Matthieu, J.-M., Quarles, R. H., Brady, R. O. and Webster, H. deF., Variation of proteins, enzyme markers and gangliosides in myelin subfractions, Bioehim. biophys. Acta (Amst.), 329 (1973) 305-317. 17 Matthieu, J.-M., Quarles, R. H., Poduslo, J. F. and Brady, R. O., [z6S]sulfate incorporation into myelin glycoproteins I. Central nervous system, Biochim. biophys. Acta (Amst.), 392 (1975) 159-166.
18 Matthieu, J.-M., Brady, R. O. and Quarles, R. H., Change in a myelin-associated glycoprotein in rat brain during development: metabolic aspects, Brain Research, 86 (1975) 55 65. 19 Mehl, E. and Halaris, A., Stoichiometric relation of protein components in cerebral m),elin from different species, J. Neurochem., 17 (1970) 659-668. 20 Morell, P., Greenfield, S., Costantino-Ceccarini, E. and Wigniewski, H., Changes in the protein composition of mouse brain myelin during development, J. Neurochem., 19 (1972) 2545-2554. 21 Norton, W. T. and Poduslo, S. E., Myelination in rat brain: method of myelin isolation, J. Neurochem., 21 (1973) 749-758.
43 22 O'Brien, J. S., Fillerup, D. L. and Mead, J. F., Brain lipids: I. Quantification and fatty acid composition of cerebroside sulfate in human cerebral gray and white matter, J. Lipid Res., 5 (1964) 109-116. 23 Poduslo, S. E., The isolation and characterization of plasma membrane and a myelin fraction derived from oligodendroglia of calf brain, J. Neurochem., 24 (1975) 647-654. 24 Prohaska, J. R., Clark, D. A. and Wells, W. W., Improved rapidity and precision in the determination of brain 2',3'-cyclic nucleotide 3'-phosphohydrolase, Analyt. Biochem., 56 (1973) 275-282. 25 Quarles, R. H., Everly, J. L. and Brady, R. O., Evidence for the close association of a glycoprotein with myelin in rat brain, J. Neurochem., 21 (1973) 1177-1191. 26 Quarles, R. H., Everly, J. L. and Brady, R. O., Myelin-associated glycoprotein: a developmental change, Brain Research, 58 (1973) 506-509. 27 Sabri, M. I., Tremblay, C., Banik, N. L., Scott, T., Gohl, K. and Davison, A. N., Biochemical and morphological changes in the subcellular fractions during myelination of rat brain, Biochem. Soc. Trans. (Land.), 554 (1975) 275-276. 28 Suzuki, K., The pattern of mammalian brain gangliosides: II. Evaluation of the extraction procedures, postmortem changes and the effect of formalin preservation, J. Neurochem., 12 (1965) 629-638. 29 Svennerholm, L., Quantitative estimation of sialic acids: II. A colorimetric resorcinol-hydrochloric acid method, Biochim. biophys. Acta (Amst.), 24 (1957) 604-611. 30 Van den Eijnden, D. H., Chromatographic separation of gangliosides on precoated silicagel thin-layer plates, Hoppe-Seyler's Z. Physiol. Chem., 352 (1971) 1601-1602. 31 Waehneldt, T. V. and Mandel, P., Proteins of rat brain myelin. Extraction with sodium dodecylsulphate and electrophoresis on analytical and preparative scale, FEBS Lett., 9 (1970) 209-212. 32 Waehneldt, T. V. and Mandel, P., Isolation of rat brain myelin, monitored by polyacrylamide gel electrophoresis of dodecylsulfate-extracted proteins, Brain Research, 40 (1972) 419~,36. 33 Waehneldt, T. V. and Neuhoff, V., Membrane proteins of rat brain: compositional changes during postnatal development, J. Neurochem., 23 (1974) 71-78. 34 Waehneldt, T. V., Ontogenetic study of a myelin derived fraction with 2',3'-cyclic nucleotide 3'-phosphohydrolase activity higher than that of myelin, Biochem. J., 151 (1975) 435-437. 35 Wolfgram, F., A new proteolipid fraction of the nervous system. I. Isolation and amino acid analysis, J. Neurochem., 13 (1966) 461~70. 36 Zgorzalewicz, B., Neuhoff, V. and Waehneldt, T. V., Rat myelin proteins. Compositional changes in various regions of the nervous system during ontogenetic development, Neurobiology, 4 (1974) 265-276. 37 Zimmermann, A. W., Quarles, R. H., Webster, H. deF., Matthieu, J.-M. and Brady, R. O., Characterization and protein analysis of myelin subfractions in rat brain: developmental and regional comparisons., J. Neurochem., 25 (1975) 749-757.
29
C H A R A C T E R I Z A T I O N OF A M Y E L I N - R E L A T E D F R A C T I O N (SN 4) ISOLATED F R O M RAT F O R E B R A I N AT TWO D E V E L O P M E N T A L STAGES
THOMAS V. WAEHNELDT, JEAN-MARIE MATTHIEU* and VOLKER NEUHOFF Forschungsstelle Neurochemie, Max-Planek-lnstitut fiir experimentelle Medizin, 3400 G6ttingen (G.F.R.)
(Accepted March 23rd, 1977)
SUMMARY A myelin-related fraction (SN 4) was isolated from forebrain of 17- and 40-dayold rats. Fraction SN 4 was obtained as a supernatant in a slow speed differential centrifugation of a myelin fraction. In contrast to multilamellar myelin fraction, SN 4 consisted of small vesicular profiles of a mixture of single membranes and some triplelayered structures. All typical myelin components were found in the SN 4 fraction from adult rat brain but their relative proportion was different from that of myelin: Wolfgram protein, myelin glycoproteins and 2',3'-cyclic nucleotide 3'-phosphohydrolase were increased, while basic proteins and proteolipid protein were decreased significantly. In contrast, the lipid composition appeared very similar to the one found in myelin. SN 4 from 17-day-old rat brains was essentially similar to that from adults, except that the major myelin glycoprotein was not enriched in comparison to myelin. Developmental changes found in myelin were also present in the SN 4 fraction. The specific radioactivity of the fucose-labeled major myelin glycoprotein was similar in SN 4 and in myelin. The particular composition of fraction SN 4 suggests that this material is not significantly contaminated by non-myelin-related membranes but rather supports the hypothesis that it could be enriched in a membrane representing a zone of transition during the formation of myelin and which is subjected to a remodelling of its protein components.
INTRODUCTION Myelin isolated by density gradient centrifugation is probably a mixture of membranes related to the process of myelin formation, consisting mostly of myelin * Service de P6diatrie, C.H.U.V., 1011 Lausanne, Switzerland.
30 lamellae besides oligodendroglial plasma membranes and their regions of transition to myelin, paranodal glial membranes, newly formed myelin and possibly some axolemma still attached to myelin. In an attempt to separate these different myelin particles a number of investigators have subfractionated myelin and got biochemically and morphologically distinct subfractions. Recent reports suggest that immature myelin or premyelin can fractionate into particular myelin subfractions: myelin-like fractionZ,a, 27, lower layer 20, membrane fraction s, heavy myelinS,6, 37, or SN 4 fraction32,34. This investigation was undertaken to isolate myelin from immature and young adult rat forebrain and this fraction was subfractionated after hypo-osmotic treatment. Purified myelin and a myelin-related fraction (SN 4) were characterized and compared to each other by electron microscopy and enzyme markers for membranes. Proteins and glycoproteins were separated by polyacrylamide gel electrophoresis and quantitated by densitometric scanning of protein bands. The incorporation of radioactive fucose into the myelin glycoproteins was studied in these fractions and their electrophoretic mobility on polyacrylamide gels was analyzed by using the radioactive double labeling technique. A preliminary report of some of these results was published elsewhere 34. MATERIALS AND METHODS
Fractionation of forebrain Rats of either sex (Wistar strain TNO/W70), aged 17 days and 40 days, were obtained from F. Winkelmann, 4791 Borchen, G.F.R. The animals were slightly anaesthetized with ether and each animal was intracerebrally injected with either 20 #Ci of L-[1,5,6-ZH]fucose or with 8 /~Ci of L-[1-14C]fucose (New England Nuclear, Boston, Mass.) in 10 #1 of 0 . 8 5 ~ NaCI. After 16-18 h, the rats were killed by decapitation and the forebrains removed and chilled. In a typical experiment, 30 brains of 17-day-old animals (15.41 g) or 15 brains of 40-day-old animals (28.75 g) RAT F O R E B R A I N '~"1 Total Homogenate in 0.32 M Sucrose
11,000 g 20 rain) 11,000 g 20 rain,)
T
11,000 g 20 rain) 100,0001~ 30 rain PELLET Homog. 0.88 M Sucrose 0.32 M Sucrose Overlay
I
T
/,000g 20 rain
I
50,000g 60rain ) 0.32/0.88 Layer Water Shock 25"C l
[]
Fig. 1. Flow sheet for the preparation of myelin and SN 4 fractions from forebrains of 17- and 40day-old rats. Thick arrows indicate iso- or hyper-osmotic media, thin lines stand for hypo-osmotic conditions. Further details are given in Materials and Methods.
31 were homogenized in 0.32 M sucrose (300 ml at 17 days, 250 ml at 40 days) with 3 4 strokes in a loosely fitting Teflon-glass homogenizer (Thomas, Philadelphia) at 750 rev./min (Fig. 1). The total homogenate was then centrifuged for 20 min at 11,000 gay in a fixed angle rotor. The supernatant (SN 1) was carefully removed with a syringe, avoiding the residual portion above the pellet, and the pellet was gently homogenized by hand leaving the tightly packed red portion in the tube. Centrifugation, removal of the supernatant, and rehomogenization was repeated twice, giving supernatants SN 2 and SN 3. The washed pellet was densely packed by high speed centrifugation at 100,000 gay for 30 min (fixed angle rotor). The resulting pellet was homogenized with 8 strokes at 750 rev./min in either 125 ml (17 days) or 70 ml (40 days) 0.88 M sucrose, placed into two tubes (17 days) or three tubes (40 days), overlayered with 0.32 M sucrose, and centrifuged at 40,000 gay for 75 min (swing-out rotor) or at 50,000 gay for 60 min (swing-out rotor). The tightly packed 0.32/0.88 M sucrose layer was removed with a syringe, hypo-osmotically shocked in 180 ml water of 25 °C, and homogenized with 6-8 strokes in a tightly fitting Teflon-glass homogenizer (Braun, 3508 Melsungen) at 2600 rev./min. After cooling in an ice-water bath the homogenate was centrifuged at 4,000 gay for 20 min (with the break-off). The slightly turbid supernatant (SN 4) was carefully removed with a syringe, avoiding the portion above the myelin (MY) pellet. The supernatant SN 4 was then centrifuged for 30 min at 60,000 gay, to give a faintly brownish pellet which is less opaque than the myelin pellet. Both the myelin pellet and fraction SN 4 were rehomogenized in water and recentrifuged 3 times. The final homogenates (about 1 mg protein/ml) were stored at --70 °C. Similarly, the particulates of fractions SN 1, SN 2, SN 3, and of the pellet formed in 0.88 M sucrose (Fig. 1, P) were centrifuged, washed with water and stored at --70 °C. Electron microscopy Suspensions of myelin and SN 4 preparations were fixed in 1.5 ~ glutaraldehyde0.5 ~ formaldehyde buffered with phosphate (pH 7.4). After a 5-min centrifugation at 25,000 gay the pellets were post-fixed with 1 ~ osmic acid, thereafter subjected to stepwise dehydration with ethanol ranging from 35 ~ to 100 ~. Ethanol was replaced by propylene oxide and final embedding was done in Epon. Thin sections were stained with saturated uranyl acetate and 3 ~ lead citrate and were examined in a Zeiss EM 9 or a Jeol 100 B electron microscope. Protein determination Samples were assayed for protein content using the method of Lowry et al. 14, with crystalline bovine serum albumin as a standard. Enzyme assays The activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP, EC 3.1.4.16) was determined by a photometric method 24. The concentration of CNP was doubled and care was taken to sufficiently dilute the samples to maintain saturating conditions. Acetylcholinesterase (ACHE, EC 3.1.1.7) was assayed with acetylthiocholine as a substrate 9.
32
Determination of individual proteins Two different techniques z5 were used to analyse proteins from myelin and SN 4 fractions by polyacrylamide gel electrophoresis on 5 ~ or 15 ~ gels in the presence of SDS. Gels were stained with 1 ~o Fast Green and scanned on a spectrophotometer at 580 nm according to Morell et al. 2°. The amounts of protein in the different bands were calculated from the densitometric scans and the results expressed as percentage of the total dye-binding capacity 1~.
PAS staining and radioactive counting of the glycoprotein Lyophilized myelin or SN 4 was delipidated with chloroform-methanol (2:1, v/v), solubilized in SDS and electrophoresed on 5 ~ polyacrylamide gels 25. Glycoprotein quantitation was performed by staining with periodic acid-Schiffreagent and scanning at 520 nm 15. Fetuin was used as a standard, and one unit of the myelin glycoprotein was defined as the amount which stained with the same intensity as 1 #g of fetuin. Radioactive-labeled glycoproteins were determined by cutting 5 ~o gels into I mm slices and counting according to method B of Quarles et al. 25. The data were corrected for quenching and double labeling and graphically displayed using a computer program. The results are plotted as the percentage of isotope in each fraction of the gel or in some experiments of a segment of the gel containing the major myelin glycoprotein.
Lipid separation Lipids from lyophilized fractions were extracted with chloroform-methanol (2:1, v/v), partitioned 11,2s, and separated by TLC. The lower phase lipids were separated on silica gel G plates developed in chloroform-methanol-ammonia (80:20:0.4 by vol.) 22 and charred with sulfuric acid. The positions of the lipids were related to lipid standards run on the same plate which was scanned on a photometer fitted with a 480 nm filter. Gangliosides extracted in the upper phase were separated by TLC 30 and stained with the resorcinol reagent 29. The spots were compared to ganglioside standards run on the same plate. RESULTS
Morphology Electron microscopy of myelin and fraction SN 4 (Figs. 2 and 3) showed that at both ages the individual fragments of myelin contained more layers than the average of the profiles seen in SN 4. This difference was more pronounced in 40-day-old forebrain as compared to 17-day-old forebrain. Multilayered myelin was rarely seen in the SN 4 fraction at both ages, however, small vesicular profiles with only a few layers were observed, many of which were triple-layered (Fig. 2B and 3B, inserts). Structures other than myelin, e.g. mitochondria or axonal contents, were virtually absent in the preparations.
Protein and enzyme determination In Table I, the protein yield and the activities of CNP and of AChE are shown
Fig. 2. Electron micrographs, myelin fraction (A) and SN 4 fraction (B) from 40-day-old rat forebrains. Large circular profiles of multilayered myelin contrast with smaller and thinner vesicular structures in myelin-related fraction SN 4. Very few small vesicular profiles are present in compact myelin, x 17,000, inserts x 215,000.
Fig. 3. Electron micrographs, myelin fraction (A) and SN 4 fraction (B) from 17-day-old rat forebrains. As in 40-day-old brains substantial compaction of rather large circular structures is evident in myelin whereas very few layers are seen in the SN 4 fraction with many small vesicles besides some singleor double-layered larger circular profiles, x 17,000, inserts x 215,000.
35 TABLE I Developmental changes in protein yield and in activities of two marker enzymes in total homogenate and in myelin subfractions from rat ]brebrain
The figures represent the means of several determinations (numbers in brackets) -4- S.E.M. Postnatal Fraction age of rats (days)
40
17
YieM of protein (mg/g fresh tissue)
CNP specific activity*
AChE specific activity*
CNP/A ChE
Total homogenate Myelin SN 4 SN 4/Myelin
94.54 ± 4.67 (4) 286 -4- 10 (14) 3.52 -4- 0.36 (8) 1470 -4- 113 (14) 0.52 4- 0.02 (8) 3458 -- 260 (14) 0.15 2.35
7.52 ± 0.99 (8) 0.77 4- 0.03 (8) 3.85 ± 0.48 (8) 5.00
38 1909 898
Total homogenate Myelin SN 4 SN 4/Myelin
80.71 4- 7.22 (4) 106 -4- 16 (14) 0.81 -4- 0.07 (8) 1627 4- 164 (14) 0.21 -4- 0.25 (8) 3124 -4- 410 (14) 0.26 1.92
5.77 4- 0.45 (8) 1.39 -4- 0.13 (8) 4.29 4- 0.91 (8) 3.09
18 1171 728
* /~moles hydrolysed/mg protein/h. for the total homogenate, myelin, and for SN 4. The protein yield in the total homogenate increased only slightly with age, in agreement with an increase from about 6 ~ in 5-day-old forebrain to about 10 ~ in adult tissue 3a. By contrast, SN 4 and particularly myelin displayed a substantial increase of respectively 2.5 and 7 times the original values. Although an increase in C N P activity was evident in the total homogenate from day 17 to day 40, the values for myelin and for SN 4 were much higher and were similar at either age. In both cases SN 4 displayed about twice the activity of C N P than myelin, which is characteristic of fraction SN 4. The specific activity of ACHE, despite an increase in the total homogenate with age, showed higher levels in myelin and in SN 4 in 17-day-old animals. Proteins
The protein profile of myelin and SN 4 fractions from 40-day-old rat brain is shown in Fig. 4. Both fractions contained the typical myelin proteins: Wolfgram protein, proteolipid protein, DM-20 or intermediate protein, large and small components of the myelin basic protein. An additional band in the high molecular weight region of the gel called X was not specific to myelin (Matthieu and Waehneldt, unpublished). The SN 4 fraction showed low levels of basic proteins, proteolipid protein and DM-20. The high molecular weight proteins were increased in SN 4 and this was particularly pronounced for the Wolfgram protein and the X band. The different protein values expressed in per cent of the dye-binding capacity are presented in Table II. In myelin from 17-day-old animals there was relatively more of the high molecular weight proteins (Fig. 5). This contrasted with SN 4 from immature brains where the percentage of high molecular weight proteins was similar to adult values.
36
o
Fig. 4. Densitometric scans of Fast Green-stained proteins in myelin and SN 4 fraction of 40-day-old rats after separation on 15 ~ polyacrylamide gels in the presence of SDS. Each gel contained 150/~g of protein. X, high molecular weight component, probably of non-myelin origin; W, Wolfgram protein; PLP, proteolipid protein; DM-20 or intermediate protein; LBP, SBP, large and small components of myelin basic protein; D, degradation product.
TABLE II
Densitome!ric quantitation of individual proteins in fractions isolated from 40-day-old rats Per cent of the dye-binding capacit~ (15% gels stained with Fast Green). Mean ± S.E.M. of 4 separate experiments compared with the Student's test. n.s., not significant.
High molecular weight proteins (--W) Wolfgram protein (W) Proteolipid protein (PLP) Intermediate protein or DM-20 Large basic protein (LBP) Small basic protein (SBP) Rest SBP/LBP
Myelin
SN 4
P
S N 4~Myelin
21.4 ± 0.3
45.7 ~ 1.4
< 0.001
2.1
7.9 ± 23.3 ± 5.7 ± 14.3 ~20.2 ± 7.2 ± 1.41
14.6 ± 9.1 ± 3.2 ± 7.5 ± 10.1 ± 10.0 ± 1.35
< 0.001 < 0.001 < 0.001 < 0.001 < 0.001 n.s.
1.9 0.4 0.6 0.5 0.5 1.4
0.4 1.1 0.2 0.4 0.5 1.6
0.8 0.5 0.3 0.4 0.8 0.7
37
Fig. 5. Densitometric scans of proteins in myelin and SN 4 of 17-day-old rats. Details as for Fig. 4.
Myelin basic proteins, DM-20 and proteolipid protein were decreased in the SN 4 fraction when compared to myelin, while the Wolfgram protein was practically unchanged (Table III). In contrast to the values found in adults, the difference of the individual proteins between myelin and SN 4 was smaller in immature animals. Nevertheless, when the non-Wolfgram high molecular weight proteins were omitted from the calculation, the Wolfgram protein value in SN 4 was 1.65-fold that of myelin TABLE III
Densitometric quantitation of individuat proteins in fractions isolated from 17-day-old rats Per cent of the dye-binding capacity (15 ~ gels stained with Fast Green). Mean ± S.E.M. of 4 separate experiments compared with the Student's test. n.s., not significant.
Myelin High molecular weight proteins (--W)28.6 Wolfgram protein (W) 9.0 Proteolipid protein (PLP) 16.4 Intermediate protein or DM-20 6.1 Large basic protein (LBP) Small basic protein (SBP) Rest SBP/LBP
q- 1.4 -I- 1.3 ± 0.1 ± 0.3
16.5 ± 0.1 15.7 d_ 0.1 7.9 ± 0.5 0.95
SN 4 46.0 10.6 7.8 4.0
:k :k :k ±
0.7 0.6 0.7 0.1
12.2 ± 0.1 10.8 q- 0.1 8.7 d- 0.3 0.89
P
S N 4~Myelin
< 0.001 n.s. < 0.001 0.001 < P < 0.01 < 0.001 < 0.001 n.s.
1.6 1.2 0.5 0.7 0.7 0.7 1.1
38 TABLE IV Quantitat&n o] the PAS-stained major myelin glycoprotein
Glycoprotein unit/rag protein (chloroform-methanol insoluble proteins). Mean ± S.E.M. of 4 separate experiments compared with the Student's test. n.s., not significant.
40-day-old rats 17-day-old rats
Myelin
SN 4
P
S N 4~Myelin
42.8 ± 5.6 40.7 :L 7.9
84.2 4:_ 7.5 39.0 ~: 7.1
0.001 < P < 0.01 1.97 n.s. 0.96
and this was close to the two-fold increase observed in the adults. In myelin and SN 4 from adult animals, the small component of the myelin basic proteins was present in higher proportion than the large component (Fig. 4 and Table II). In contrast, these two components were present in similar proportions in myelin and SN 4 from immature animals (Fig. 5 and Table III). Glycoproteins
In adult rats, PAS-stained gels from chloroform-methanol-delipidated SN 4 fraction showed the presence of a major band which exhibited a similar electrophoretic mobility as the one from myelin. There was twice as much of this major glycoprotein in the SN 4 fraction than in myelin (Table IV). In contrast, in SN 4 from immature animals, the amount of PAS-stained major glycoprotein was similar to that found in myelin (Table IV). After 16 h of incorporation, the specific radioactivity of the in vivo fucoselabeled major glycoprotein (dpm/unit of glycoprotein) was similar in myelin and in SN 4, but there was a 5-fold increase in 17-day-old fractions when compared to 40-day-old fractions. The amount of radioactivity in the major glycoprotein was 40 ~ of the total radioactivity bound to chloroform-methanol-insoluble proteins of myelin and SN 4. The patterns of the fucose-labeled glycoproteins from SN 4 and myelin coincided (Fig. 6) at both ages considered. The newly synthesized glycoprotein in immature rat myelin showed a slightly higher apparent molecular weight than that in if- 16.
H [ 1 4 C ] fucose, myelin °--'°[3HI fucose, SN-4
o o
b [. 10 FRACTION NUMBER
20
i0
Fig. 6. Comparison of the fucose labeled glycoproteins in myelin (O 0) and SN 4 (©----()) fractions from 40-day-old rats which had been injected with [14C]fucoseand [3H]fucose, respectively. The gel was divided into 3-mm fractions and contained 800/~g of protein, 5573 dpm 14C and 12,158 dpm 3H.
39 SN4
MYELIN
~'PI o..or.14C]fucose,lTd.
;i0 (_~ 0 o
: 4
0
: [3H]fucose, 17d. d -o [14C] . / f ucose, %
mr
/
,~ 0
z u ~d
o. I
I
I
10 A
/A
gs
7 UJ U
nO
'~._.r3.l,ooo,~,~o~.
FRACTION
NUMBER
I 20
0
I
I
I
I0 FRACTION NUMBER
I 20 B
Fig. 7. Fucose-labeled glycoproteins in myelin and SN 4 fractions from 17- and 40-day-old rat forebrains. High resolution was obtained by using 1-mm fractions. Figure shows the portion of the gel 2-4 cm from the top. A: comparison of the major labeled glycoprotein in myelin of 17-day-old rats injected with [SH]fucose (9965 dpm, • • ) with that of 40-day-old rats injected with [14C]fucose (10,736 dpm, O - - - O). B : comparison of the major labeled glycoprotein in SN 4 of 17-day-old rats injected with [taC]fucose (9557 dpm O ---O) with that of 40-day-old rats injected with [3H]fucose (3940 dpm, • •). The gel contained 870 Mg of protein. mature myelin (Fig. 7A) as previously reported 18,~6. This developmental difference in electrophoretic mobility was also found in the SN 4 fraction from 17- and 40-day-old animals (Fig. 7B).
L~/ds The SN 4 fraction contained slightly less lipids, on a dry weight basis, than did myelin: 75.3 mg/100 mg dry weight and 78.4 mg/100 mg dry weight, respectively. Cholesterol, cerebrosides and phosphatidyl ethanolamine were the most prominent lipids. Sulfatides were also present but in smaller amounts. The ratios of cerebrosides to sulfatides in myelin and in SN 4 from 40-day-old animals were 3.1 and 3.2, respectively. G m ganglioside was present in similar amounts in myelin and in SN 4. Traces of higher polysialogangliosides were present in the SN 4 fraction but these gangliosides were barely detectable in myelin. DISCUSSION It has been observed in several laboratories 1,8,1°,19,81 that myelin isolated from adult CNS tissue has a limited number of fairly low molecular weight protein components, ranging from 15,000 to 25,000 daltons and commonly known as 'major myelin proteins'. Among these the basic protein(s) and the proteolipid protein are the most prominent lz. Electrophoresis of myelin proteins in the presence of detergent reveals, however, an additional large number of minor protein components with molecular weights higher than 25,000 daltons of which the Wolfgram protein and the major myelin-associated glycoprotein have been characterized in some detail 18,25. Since it is possible to prepare myelin fractions with changing proportions of high molecular weight proteins 32, it has to be considered that some of these high molecular weight proteins might not be intrinsic myelin components. Rather they may be
40 involved in the process of myelin formation in single membranes leading to compact myelin, such as oligodendroglial processes, loose lamellae, external loops, and paranodal membranes. Moreover, the possibility of contamination with alien membranes has to be seriously taken into account. In this study the isolation of a myelin-related membrane fraction was based on two crucial preparative steps: (1) elimination of microsomal material and (2) hypo-osmotic shock, coupled with slow speed differential centrifugation. Elimination of microsomal fragments was achieved by three-fold washing of the total particulate fraction in iso-osmotic medium, thus concentrating large-sized compact myelin. Compact myelin was separated from other subcellular particles such as nuclei, large mitochondria and large synaptosomes in a 'floating up' density gradient centrifugation. It was only at this degree of purity that myelin was subjected to hypo-osmotic conditions, in contrast to other methods~, zl. By subjecting the shocked material to a second slow speed differential centrifugation multilayered myelin was found as a pellet and small fragments of membranes and vesicular structures remained in the supernatant (SN 4), as shown by electron microscopy. Thus, up to the stage of the hypoosmotic shock, the multilayered structure of myelin helped to protect cytoplasmcontaining portions, such as paranodal loops and possibly external and internal tongues. One of the characteristics of the myelin-related fraction SN 4 was its high specific activity of CNP. As shown in Table I, fraction SN 4 had specific activities twice those of myelin, both at 40 days and at 17 days, although the activities of the total homogenate showed an age-dependent increase reflecting the process of myelination. The high activity of CNP in fraction SN 4 indicates that this enzyme is not only a marker for myelin but could already be present in loosely wrapped myelin and in oligodendroglial membranes 23. The levels of AChE specific activity, an assumed neuronal and endoplasmic reticular marker 13, are more difficult to assess. Compared with the total homogenate, lower levels of AChE specific activity were found in myelin and in SN 4 at both ages examined (Table I). This points to substantial elimination of non-myelin material. A higher degree of purity was reached in myelin and SN 4 from 40-day-old rats which can be explanied by the greater extent of in situ compaction and by the larger amount of myelin in older animals facilitating the isolation of myelin 21. The ratio CNP/AChE (Table !) clearly indicates that a substantial purification of myelin has been achieved at both ages. The SN 4 fraction is closely related to myelin because all typical myelin components are present: myelin basic proteins, proteolipid protein, Wolfgram protein, cerebrosides, CNP and the major myelin glycoprotein. Furthermore, it also exhibits the developmental and metabolic features specific to myelin : (1) the developmental change in the proportion of the two myelin basic proteins previously reported 7,16,20,36 was als~ observed in SN 4 and was similar to that measured in myelin; (2) the developmental change in the apparent molecular weight of the major myelin glycoprotein "~8,26 was also found in SN 4; (3) the specific radioactivity of fucose incorporated into the major myelin glycoprotein was the same in both SN 4 and myelin fractions but was much higher in young and actively myelinating rats than in
41 adult animals; (4) the proportion of the different glycoproteins separated by electrophoresis and measured by PAS staining or radioactive counting was identical in myelin and SN 4 and this strongly suggests that SN 4 did not contain significantly more contaminants than myelin. Nevertheless, these typical myelin components were present in SN 4 in different proportions. Three of them were enriched in SN 4: the Wolfgram protein which is a minor component in myelin became the prominent protein in SN 4. The CNP activity and the myelin major glycoprotein were increased by a two-fold factor in SN 4 from adult animals. Two specific myelin proteins were significantly decreased: the myelin basic proteins and the proteolipid protein. In contrast, the lipid composition appeared very similar in myelin and SN 4. Another difference between SN 4 and myelin is the fact that in young myelinating animals the protein pattern of SN 4 was very similar to the adult SN 4 fraction, and this contrasts with immature myelin, which contained a higher proportion of high molecular weight proteins than adult myelin. The SN 4 fraction presents some similitude with the heavy myelin fraction isolated by Matthieu et al. 16 in the adult rat and by Zimmermann et al. 37 during development. Density distributions on zonal gradients (Matthieu and Waehneldt, in preparation) demonstrated that SN 4 is a heavier fraction (maximum at 0.73 M sucrose) than the corresponding myelin fraction (maximum at 0.67 M sucrose). This is consistent with a slightly lower lipid content of SN 4 when compared to myelin, as shown in this communication. Also CNP, ACHE, high molecular weight proteins and myelin glycoproteins were enriched in both adult SN 4 and heavy myelin, while basic proteins and proteolipid protein were decreased. But one main difference when SN 4 is compared to the heavy myelin was the specific enrichment in Wolfgram protein and in CNP in fraction SN 4 and its similar protein composition in immature and adult animals, while the heavy myelin fraction from immature rats appeared heavily contaminated37. Therefore we think that SN 4 contains membranes which are also present in heavy myelin subfractions but in a higher yield and less contaminated. The structural origin of membranes in the SN 4 fraction, like the heavy myelin subfraction, is not yet established. These myelin-related membranes could be transitional between the plasma membrane of the oligodendrocyte and multilamellar myelin16. Another possible location would be the paranodal region or the inner and outer loops of the myelin sheath. In conclusion, in view of the similar lipid composition, the very different distribution of the myelin protein components observed between myelin and SN 4 suggests a rearrangement of the membrane proteins within the lipid bilayer rather than a dilution of the SN 4 fraction by non-myelin-related membranes. These results suggest that SN 4 represents a highly purified fraction derived from, or closely related to, myelin, although its exact location and role during the process of myelination is still a matter of conjecture.
42 ACKNOWLEDGEMENTS We w o u l d like to t h a n k Dr. R. S a m m e c k , Mrs. M. M e y e r m a n n an d Dr. J.-C. Krieg for the electron micrographs. This w o r k was s u p p o r t e d in part by the Deutsche F o r s c h u n g s g e m e i n s c h a f t (SFB 33) and by the Swiss N a t i o n a l Science F o u n d a t i o n , G r a n t 3.225.74.
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