Journal of Nruroohumtsrry Vol 30, pp 1543-1551 Pergamon Press Ltd. 1978. Printed in Great Britain @international Society kir Neurochemistry Ltd.
0022-3042 78'0601.1543 502.00,O
MYELIN BASIC PROTEIN MICROHETEROGENEITY IN SUBFRACTIONS OF RAT BRAIN MYELIN C. W. SHULTS,J. N. WHITAKER' and J. G. WOOD The Research Service, Memphis Veterans Hospital, and the Departments of Neurology and Atanomy, University of Tennessee Center for the Health Sciences, Memphis, TN 38104, U.S.A. (Receioed 15 September 1977. Accepted 23 November 1977) Abstract-Myelin subfractions were prepared from adult rat brain by discontinuous sucrose gradient ultracentrifugation. Gel electrophoretic studies at pH 10.6 in the presence of urea revealed differences in basic protein microheterogeneity among subfractions. With increasing myelin density there was a decrease in the most positively charged components of both large BP and small BP. Since these components are the least modified by deamidation and phosphorylation, it seems likely that the heavier myelin subfractions are enriched in the more modified components of the microheterogeneous population of BP. These observed differences may be related to the regulatory processes controlling biosynthesis, organization, and catabolism of BP in CNS myelin.
MYELINencephalitogenic or basic protein (BP). ac- modifications suggests that they might be related to the counts for 30%of the protein content of CNS myelin processes or regulatory mechanisms involved in the (ENG et al., 1968) where it is presumed to serve a metabolism of BP. The present study shows that there structural role. BP has a monomeric mol. wt of 18,500 are differences in the population of microheteroand contains 169 amino acidsZ(EYLAR, 1971; BROSTOFFgeneous components of BP in myelin subfractions from et al., 1974). Rats, mice and related species in the sub- rat brain. orders Sciuromorphu and Myomorpha have a second smaller but more abundant BP with a mol. wt of 14,300 MATERIALS AND METHODS 1970b; (MARTENSON et al., 1970a, b; MARTENSON, Materials MARTENSON et al., 1971). In the rat this smaller BP Sprague-Dawley rats of both sexes were obtained from is probably identical to the large BP except for an internal deletion of 40 amino acids in the carboxyl half Charles River (Wilmington, MA); N,N'-methylene-bis acrylamide and N,N,N',N'-tetramethylethylene-diamine from of the molecule (MARTENSON et a!., 1972; DUNKLEY & Eastman Kodak Co. (Rochester, NY); glycine from Pierce CARNEGIE, 1974). Chemical Co. (Rockford, IL); ultra-pure urea and Coomasie At alkaline pH, BP from all species studied shows blue from Schwarz-Mann (Orangeburg, NY); sodium microheterogeneity with resolution into 5 or more dodecyl sulfate(SDS)and ammonium persulfate from Fisher components3 (MARTENSON et al., 1971). In guinea-pig ScientificCo. (St. Louis, MO); Amido black IOB from Sigma BP there is no difference among the 5 components in Chemical Co. (St. Louis, MO); carboxymethylcellulose amino acid composition or encephalitogenicity for the (CM-cellulose) (CM52) from Whatman (Clifton, NJ); and guinea pig (DEIBLER & MARTENSON, 1973) and the loss Sephadex from Pharmacia (Piscataway, NJ). All other of carboxy-terminal arginines plays a role in the micro- chemicals were reagent grade. Slab polyacrylamide gel heterogeneity (DEIBLER et a/., 1975). The microhetero- electrophoresis was performed in an assembly (10 x 14cm) purchased from Hoefer Instruments (San Francisco, CA). geneity in bovine BP is related to specific alterations in the BP molecule consisting of phosphorylation at Methods threonine 97 and serine 164 and deamidation of glutaIsolation of myelin and myelin subfractions. Myelin was mine at residues 102 and 146 (CHOU et al., 1976). isolated from fresh brains of 60-120 day-old Spraguee Although the functional relevance of BP microheteroDawley rats according to the method of NORTON& geneity has not been delineated, the specificity of the PODUSLO (1973) with the initial homogenization done in a To whom reprint requests should be sent, at Memphis Veterans Hospital, 1030 Jefferson Avenue, Memphis, TN 38104, U.S.A. The numbering of amino acid residues is based on that et d., 1971)with the corrected deletion of bovine BP (EYLAR et a/., 1974). of serine at position number two (BROSTOFF Numbering of BP components is based on the termin& MARTENSON (1973) in which the most ology of DEIBLER positively charged component at alkaline pH is designed number one. Abbreuiations used: BP, myelin basic or encephalitogenic protein; CM-cellulose, carboxymethylcellulose; SDS, sodium dodecyl sulfate.
10% (w/v) solution of brain in 0.32~-sucrose.All tissue processing and centrifugation were performed at 4°C. Centrifugation steps at 25,000 rev./min were done in a SW27 head in an L5-65 Beckman Ultracentrifuge. Centrifugation on a discontinuous 0.32-0.85 M-sucrose gradient was & performed twice with an osmotic shock step (NORTON PODUSLO,1973) between the two sucrose gradients. This preparation was designated crude myelin. If crude myelin was the product sought, this preparation was washed with H,O (NORTON& PODUSLO,1973) and lyophilized. For myelin subfractionation, crude myelin recovered from the sucrose interface of the second 0.32-0.85 M-discontinuous gradient was left in the sucrose solution overnight at 4°C. Myelin subfractions were prepared by the method of
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C. W. SHULTS,J. N. WHITAKER and J. G. W m i i
MATTHIEU et a/. (1973). The crude myelin, which had been isolated on the first day, was mixed with 2251111 H,O and centrifuged at 15,000 rev./min for 15 min at 4°C. The resulting pellets were pooled and resuspended in 72ml of 0.32 M-sucrose. Twelve ml-aliquots were layered into 6 tubes which contained 12ml of 0.7 M-sucrose overlaid with 14ml of0.62 M-sucrose. After centrifugation at 25,000 rev./min for 30 min, the crude myelin separated into 3 subfractions. The myelin at the interface of 0.32-0.62 M-sucrose, at the interface of 0.62-0.70~-sucrose, and in the pellet was designated light, medium, and heavy myelin subfractions, respectively. Each subfraction was mixed with 75 ml of H,O and centrifuged at 25,000 rev./min for 15 min. The pellet of each subfraction was suspended in 24ml of 0.32~-sucroseand the subfractionation procedure, as described above, repeated. Each subfraction collected at the same position it had during the initial subfractionation with only small amounts found at the other two positions. Each myelin subfraction was washed twice in 75 ml of H,O and lyophilized.
Tissue extraction BP was extracted from crude myelin and myelin subfractions by the method of WOOD & KING (1971). Ten milligrams of lyophilized myelin were mixed with 1.5ml of acetone and agitated for 2 h at 25°C. The mixture was centrifuged, the supernatant discarded, the pellet mixed with 1ml of acetone, and the mixture stirred for 20 min. The residue was collected by centrifugation and washed with 5 ml of H,O. The residue was again collected by centrifugation and mixed with lOml of IO-’M-HCI. In some of the initial experiments extraction was carried out with lo-’ M and i0-3~-HC1.This solution was agitated for 18 h at 4°C and was then centrifuged and the supernatant lyophilized. The residue was washed twice with 5 ml of H,O and lyophilized. For slab gel electrophoretic studies (see below) crude myelin was delipidated with ether-ethanol (3:2, v/v) (GREENFIELD et al., 1971).
Preparation of rat BPs Frozen adult rat brain was delipidated with chloroformmethanol (2:1, v/v) and acetone, the residue was washed with H,O and the BP was extracted at pH 3 (DEIBLER et a/., 1972). The pH 3 extract was subjected to ion exchange chromatography at 25°C on CM-cellulose equilibrated with 0.05 M-glycine-NaOH buffer, pH 10.5, containing 6 M-urea et a/., 1975). After the unretarded material had (WHITAKER passed through the column, BP-containing fractions were eluted with 0.05 M-glycine-NaOH, pH 10.5, containing 2 ~ - u r e aand 0.2NaC1. This material was desalted on a column of Sephadex G-25 equilibrated with 0.5% acetic acid and lyophilized. The lyophilized material was dissolved in 0.03 M-HCI and filtered through a column (1.5 x 90cm) of Sephadex G-100 (MARTENSON ei al., 1970h). The large BP and small BP were isolated by recycling of selected fractions over the same column.
Polyacrylarnide gel electrophoresis Disc gel electrophoresis at pH 10.6 or pH 2.5 in the presence of urea was performed according to the method of DEIBLER er al. (1972). For the alkaline urea gels 150-250 pg of extract were applied to each gel, and the electrophoresis was conducted for 3h with a current of 3.75mA/gel. For acetic acid-urea gels 25 pg of extract were applied to each gel, and electrophoresis was conducted for 75 min with 2.5 mA/gel. Gels were stained for 1 h in 1% Amido black 10B in 7% acetic acid and destained in 3% acetic acid.
Discontinuous slab gel electrophoresis was performed with a modification of the method of G R E E N F I ~etL IaI / . (1971). Delipidated extracts were mixed, at a concentration of 2mg/ml, with a 1% solution of SDS (w/v) in H,O and placed in boiling HzO for 10min (MORELL cf d.,1976). After cooling, 50 $ of the sample solution were mixed with 25 pl ofsample buffer. This solution was applied to each lane of the gel which was comprised of a 3% stacking gel and a 12.5%running gel. Electrophoresis was conducted at lOOmA with a bromphenol blue tracking dye. The gels were stained with 0.25% Coomasie brilliant blue in methanol-H,Oacetic acid (5:5:1) for 5 h and destained in methanol-H,Oacetic acid (1.6: 12.7: 1).
Other methods Electron microscooy was performed on crude myelin or myelin subfractions fixed and examined as described by WOOD et al. (1974). Myelin was fixed in the sucrose in plastic conical tubes prior to the final H,O wash. In order to obtain optimal tissue sampling, blocks were oriented for sectioning so that sections were cut perpendicular to the pelleted tissue. For quantitation of BP microheterogeneous bands, gels were photographed, and positive clear prints prepared. These prints were scanned in an Ortec 4310 Densitometer (PORTNER, 1977).Amounts in each band were determined by weighing the chart paper by the appropriate peak. The amount in each band was calculated as a percentage.of the total density of bands 1-6. RESULTS
Extruction o f B P Initial studies of acetone-delipidated crude myelin indicated that the relative proportions of the different bands reflecting the microheterogeneity of B P were essentiallyidentical in the 10- 10- a n d 10- M-HCI extracts and very little B P remained in the residue. Since the pattern of microheterogeneity was constant over this range of HCI concentrations, a single concentration, ~ O - ’ M , was utilized in all subsequent studies. By this extraction procedure virtually all of the BPs, trace amounts of higher molecular weight proteins, et al., usually only one of the intermediate (GREENFIELD 1971) or DM-20 (AGRAWALet al., 1972) proteins, and one or two diffuse bands migrating ahead of small BP in the slab gel system were solubilized (Fig. 1). Thus, BP was the predominant protein species extracted with only the two faster migrating bands being present in moderate amounts. As indicated by slab gel electrophoresis, there were no identifiable differences in the extent of extraction of BP among the subfractions with lo-’ M-HCI (Fig. 1). The two fast migrating bands (Fig. 1) most likely represent degradation products formed during the HCI extraction of this residue with lo-’ M-HCI.Performing migrating bands were present in myelin delipidated with ether-ethanol; they markedly increased after extraction ofthis residue with 10- M-HCI. Performing all of thedefatting and extraction a t 4°C did not prevent the formation of these bands. Electrophoretic studies of the extracts of the myelin subfractions at pII 2.5 i n the presence of urea revealed
’,
’,
’
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FIG.I. Electrophoresisin 12.5%discontinuous polyacrylamide slabgel containing SDS. (A) Ether-ethanol dclipidated crudc myelin, (B) acid extract from crude myelin, (C) acid extract from light myelin, (D) residue remaining after acid extraction of light myelin, (E) acid extract of medium myelin, (F) residue remaining after acid extraction of medium myelin, (C) acid extract of heavy myelin, and (H) residue remaining after acid extraction of heavy myelin. Comparable amounts (100 pg) applied to each lane resulted in overloading of lanes A, B, C, E and C but demonstrate the extent of removal of B P from the residue. Major bands designated as follows: Wolfgram protein (W), proteolipid (PL), DM-20 or intermediate protein (I), large basic protein (BP-L), and small basic protein (BP-s).
FIG.2. Disc gel electrophoresis a t pH 2.5 in the presence of urea of acid extracts from delipidated crude myelin (A) and the light (B), medium (C), and heavy (D) myelin subfractions. Acid extraction performed . enriched in rat large B P (E) and small B P (F) also shown. 25 pg applied t o in 10- M - H C ~Fractions each gel. Cathode at bottom. Abbreviations same as used for Fig. 1.
’
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FIG.3a. Disc gel electrophoresis at pH 10.6 in the presence of urea of rat brain pH 3 extract (A) and acid extracts ofdelipidatcd crude myelin (B), light myelin (C), medium myelin (D),and heavy myelin (E and F). 1 SO pg of protein were applied on gels A-E, and 250 pg applied on gel F. Diminution in bands 1 and 2 in heavy myclin remains apparent even on overloaded gel (F). Cathode at bottom. FIG 3b. Disc gel electrophoresis at pH 10.6 in the presence of urea of acid extract of delipidated crude myelin (A) and fractions enriched in large B P (B) and small B P (C) isolated by gel filtration. Bands 1-4 were comprised of small B P components whereas large BP components were present in bands 5-7. 150 pg applied per gel. Cathode at bottom.
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FIG.5. Electron micrographs of crude myelin (A) and light (B), medium (C), and heavy (D) myelin subfractions from adult rat brain. All micrographs are at the same magnification (19,ooOx ).
Basic protein microheterogeneity in myelin subfractions
1549
no appreciable differences in BPs among crude myelin or myelin subfractions (Fig. 2). I n addition to the two BP bands, each of the acid extracts contained similar bands migrating more slowly and more rapidly than BP in this system. Thus, the procedure chosen for extraction of BP appeared to show no preferential effect on extraction of BP among crude myelin and myelin subfractions.
ofmyelin subfraction (Fig. 3a). Based on the assumption that the bands of rat BPs have similar dye-binding capacities for Amido black IOB, as guinea-pig BP components appear to have (DEIBLER& MARTENSON, 1973), the bands observed o n electrophoresis were scanned by densitometry (Fig. 4). Densitometric analysis confirmed the differences among bands noted by visual inspection (Fig. 3a). Results representing the average of three different electrophoretic analyses of Microheterogeneity of BP’s acid extracts from two preparations of myelin and In contrast to the SDS-slab and acetic acid urea disc myelin subfractions were consistent and showed a gel electrophoresis, electrophoresis at pH 10.6 in the tendency for there to be a relative increase in the presence of urea revealed readily apparent differences of density of bands 3, 4 and 6 with increasing myelin BP bands among myelin subfractions (Fig. 3a). In density. Because of the variability in band 7, no attempt Accordance with the convention of others (MARTENSON was made to quantitate this band which attained its & GAITONDE, 1969; DEIBLER & MARTENSON, 1973) the largest relative amount in the heavy subfraction (Fig. 4). bands were numbered from the cathodal front toward In order to determine which of the BPs was reprethe anodal end. Six bands could be detected with cer- sented in the electrophoretic bands seen in alkalinetainty, but a seventh band to the anodal side of band 6 urea gels, rat B P was separated into fractions enriched sometimes occurred, especially in the denser myelin in large BP and small BP. Electrophoresis of these subfractions. Similar results were obtained from crude, fractions at pH 2.5 in the presence of urea (Fig. 2) and in light, medium, and heavy myelin in three subfraction- SDS-slab gels (data not shown) indicated their purity. ations. The most obvious difference was a progressive Only small amounts of contamination of the other size diminution of bands 1 , 2 and 5 with increasing density BP occurred. O n alkaline-urea gel electrophoresis (Fig. 3b) it could be shown that bands 1-4 were com6 5 4 1 2 1 ponents of small BP and bands 5-7 were components of large BP. Band 5 additionally had some small BP present. This indicates that the bands which diminished with increasing density of myelin are the more positively charged components of rat large and small BP. Electron microscopy Electron microscopy revealed that crude myelin and each myelin subfraction consisted almost exclusively of myelin with very little, or no, contamination (Fig. 5). The light subfraction was comprised of large whorls of myelin with 3-4 lamellae. The heavy subfraction contained whorls of smaller diameter and 7-8 lamellae and some single-walled vesicles. The medium subfraction was intermediate between the light and heavy subfractions in diameter of the whorl and number of lamellae.
DISCUSSION
FIG.4. Tracings of densitometric scans of positive clear photographic prints of disc gels following electrophoresis at pH 10.6 in the presence of urea. Relative differences in bands 1-6 of BP from crude myelin (A), light myelin (B), medium myelin (C), and heavy myelin (D) agree with those seen on visual inspection (Fig. 34, but changes in band 5 are more obvious. 150 pg of acid extract of delipidated myelin applied per gel. Cathode to right.
Many investigations have demonstrated that CNS myelin is not homogeneous but exists in a spectrum of varying densities (AUTILIOet al., 1964; ADAMS& FOX, 1969; CUZNER & DAVISON,1968; KURIHARAet al., 1971;MCMILLAN et al., 1972;WAEHNELDT & MANDEL, 1972; BENJAMINS et al., 1973; MAITHIEUet al., 1973; AGRAWALet al., 1974; ZIMMERMAN et al., 1975; FUJIMOTO et al., 1976; BENJAMINS et al., 1976; SHEADS et al., 1977). Different techniques have been used for the preparation of a number of subfractions, and the distinctive chemical and morphological features noted among the subfractions have not always been identical. Because of the probability that myelin subfractions are in some manner related to the organization, biosynthesis, maintenance or turnover of myelin, studies have been undertaken to characterize and compare
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C. W. SHULTS.J . N. WHITAKER and J. G . WOOII
protein constituents of myelin subfractions. In adult rat myelin subfractions. The distribution of BP componbrain total myelin BP decreased with increasing myelin ents among myelin may be a reflection of the regulatory density (MATTHIEU et d.,1973; ZIMMERMAN et al., 1975; processes influencing BP microhctcrogeneity eithcr in concert with or as a major controlling mechanism for BENJAMINS et a[., 1976). Results of the present investigation indicate that the changes in the composition of the myelin membrane. The exact basis for the observed differences remains population of components of microheterogeneous BP differs among myelin subfractions. With increasing to be elucidated. Characterization of the nature and density of the myelin subfraction there is a decrease in mechanism of these changes in BP microheterogeneity relative amounts of the more positively charged may provide an additional means for assessing chemical components of small BP (bands 1 and 2) and events during myelination. large BP (band 5). Although to a lesser extent, Acknowledgmirn~s-This work was conducted under Veterless positively charged components of small BP ans Administration Research Project 9351-03 and was also (bands 3 and 4) and large BP (band 6) increase. As supported by NIH grant NS-12590 and the Alfred P. Sloan observed by others (MARTENSON et al., 19706), the Foundation(JGW).We thank Drs. L. KINGand A. PORTNER multiple bands for both rat large and small BP at for their assistance in the densitometric analysis alkaline pH suggests that both are modified by processes contribating to BP microheterogeneity. Based on REFERENCES studies on microheterogeneity of bovine BP (CHOUrt AVAMSD. H. & Fox M. E. (1969) Brain Res. 14, 647-661. d., 1976), the microheterogeneity demonstrated in rat AGRAWAL H . C., BURTONR. M., FISHMAN M. A., MITCHELL small BP is most likely due to phosphorylation at R. F. & PRENSKY A. L. (1972)J. N~rtrochent.19,2083-2089. threonine 97 and serine 164 or deamidation at gluta- AGRAWALH. C., TROTTER J. L., BURTON R . M. & MITCHELL R . F. (1974) Biochem. J . 140,99-109. mine 102 for in rat small BP the potential site for deamidation at glutamine 146 is deleted (MARTENSON AUTILIOL. A,, NOKTONW. T. & TERRYR . D. (1964) J. Neiirochem. 11, 17--27. et d., 1972; DUNKLEY & CARNEGIE, 1974). In bovine BENJAMINS J. A., MILLERK. & MCKHANNG . M. (1973) BP there is good evidence that in uiuo microheteroJ. Neurocheni. 20, 1589-1603. geneity results from phosphorylation and, possibly, BENJAMINS J . A., GRAYM . & MORELL P.(1976)J . Neirrochmt. deamidation (CHOUet a!., 1977). 21, 571 -575. A number of explanations may account for the BROSTOFFS. W.. REUTER W., HICHENSM. & EYLARE. H. differences observed in the present study. These (1974) J. h i d . Cheni. 249, 559-567. differences may represent not only the conditions CHOWF. C-H.. CFIOUC-H. J., SHAPIRA R. & KIBLER R . F. (1976) J. bid. Cheni. 251, 2671-2679. existing in uiuo but also be a result of differential CHOU F. C-H., CHOWC-H. J., S H A P I RRA. & KIIKERR. F. alterations that occurred during the isolation and (1977) J . Nriirochem. 28, 1051-1059. extraction procedures. A restriction in BP extraction in CUZNER M. L. & DAVIIISON A. N . (1968) Biochent. J . 106, different myelin subfractions would seem to be excluded 29-34 since the extent of BP extraction from the subfractions DEIBLER G . E.. MARTENSON R . E. & KIESM. W. (1972) appeared similar and nearly complete. It is possible that Prep. &ochem. 2, 139-165. during the isolation procedures the more positively DEIHLER G. E., MAKrI
0022-3042 78'0601.1543 502.00,O
MYELIN BASIC PROTEIN MICROHETEROGENEITY IN SUBFRACTIONS OF RAT BRAIN MYELIN C. W. SHULTS,J. N. WHITAKER' and J. G. WOOD The Research Service, Memphis Veterans Hospital, and the Departments of Neurology and Atanomy, University of Tennessee Center for the Health Sciences, Memphis, TN 38104, U.S.A. (Receioed 15 September 1977. Accepted 23 November 1977) Abstract-Myelin subfractions were prepared from adult rat brain by discontinuous sucrose gradient ultracentrifugation. Gel electrophoretic studies at pH 10.6 in the presence of urea revealed differences in basic protein microheterogeneity among subfractions. With increasing myelin density there was a decrease in the most positively charged components of both large BP and small BP. Since these components are the least modified by deamidation and phosphorylation, it seems likely that the heavier myelin subfractions are enriched in the more modified components of the microheterogeneous population of BP. These observed differences may be related to the regulatory processes controlling biosynthesis, organization, and catabolism of BP in CNS myelin.
MYELINencephalitogenic or basic protein (BP). ac- modifications suggests that they might be related to the counts for 30%of the protein content of CNS myelin processes or regulatory mechanisms involved in the (ENG et al., 1968) where it is presumed to serve a metabolism of BP. The present study shows that there structural role. BP has a monomeric mol. wt of 18,500 are differences in the population of microheteroand contains 169 amino acidsZ(EYLAR, 1971; BROSTOFFgeneous components of BP in myelin subfractions from et al., 1974). Rats, mice and related species in the sub- rat brain. orders Sciuromorphu and Myomorpha have a second smaller but more abundant BP with a mol. wt of 14,300 MATERIALS AND METHODS 1970b; (MARTENSON et al., 1970a, b; MARTENSON, Materials MARTENSON et al., 1971). In the rat this smaller BP Sprague-Dawley rats of both sexes were obtained from is probably identical to the large BP except for an internal deletion of 40 amino acids in the carboxyl half Charles River (Wilmington, MA); N,N'-methylene-bis acrylamide and N,N,N',N'-tetramethylethylene-diamine from of the molecule (MARTENSON et a!., 1972; DUNKLEY & Eastman Kodak Co. (Rochester, NY); glycine from Pierce CARNEGIE, 1974). Chemical Co. (Rockford, IL); ultra-pure urea and Coomasie At alkaline pH, BP from all species studied shows blue from Schwarz-Mann (Orangeburg, NY); sodium microheterogeneity with resolution into 5 or more dodecyl sulfate(SDS)and ammonium persulfate from Fisher components3 (MARTENSON et al., 1971). In guinea-pig ScientificCo. (St. Louis, MO); Amido black IOB from Sigma BP there is no difference among the 5 components in Chemical Co. (St. Louis, MO); carboxymethylcellulose amino acid composition or encephalitogenicity for the (CM-cellulose) (CM52) from Whatman (Clifton, NJ); and guinea pig (DEIBLER & MARTENSON, 1973) and the loss Sephadex from Pharmacia (Piscataway, NJ). All other of carboxy-terminal arginines plays a role in the micro- chemicals were reagent grade. Slab polyacrylamide gel heterogeneity (DEIBLER et a/., 1975). The microhetero- electrophoresis was performed in an assembly (10 x 14cm) purchased from Hoefer Instruments (San Francisco, CA). geneity in bovine BP is related to specific alterations in the BP molecule consisting of phosphorylation at Methods threonine 97 and serine 164 and deamidation of glutaIsolation of myelin and myelin subfractions. Myelin was mine at residues 102 and 146 (CHOU et al., 1976). isolated from fresh brains of 60-120 day-old Spraguee Although the functional relevance of BP microheteroDawley rats according to the method of NORTON& geneity has not been delineated, the specificity of the PODUSLO (1973) with the initial homogenization done in a To whom reprint requests should be sent, at Memphis Veterans Hospital, 1030 Jefferson Avenue, Memphis, TN 38104, U.S.A. The numbering of amino acid residues is based on that et d., 1971)with the corrected deletion of bovine BP (EYLAR et a/., 1974). of serine at position number two (BROSTOFF Numbering of BP components is based on the termin& MARTENSON (1973) in which the most ology of DEIBLER positively charged component at alkaline pH is designed number one. Abbreuiations used: BP, myelin basic or encephalitogenic protein; CM-cellulose, carboxymethylcellulose; SDS, sodium dodecyl sulfate.
10% (w/v) solution of brain in 0.32~-sucrose.All tissue processing and centrifugation were performed at 4°C. Centrifugation steps at 25,000 rev./min were done in a SW27 head in an L5-65 Beckman Ultracentrifuge. Centrifugation on a discontinuous 0.32-0.85 M-sucrose gradient was & performed twice with an osmotic shock step (NORTON PODUSLO,1973) between the two sucrose gradients. This preparation was designated crude myelin. If crude myelin was the product sought, this preparation was washed with H,O (NORTON& PODUSLO,1973) and lyophilized. For myelin subfractionation, crude myelin recovered from the sucrose interface of the second 0.32-0.85 M-discontinuous gradient was left in the sucrose solution overnight at 4°C. Myelin subfractions were prepared by the method of
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C. W. SHULTS,J. N. WHITAKER and J. G. W m i i
MATTHIEU et a/. (1973). The crude myelin, which had been isolated on the first day, was mixed with 2251111 H,O and centrifuged at 15,000 rev./min for 15 min at 4°C. The resulting pellets were pooled and resuspended in 72ml of 0.32 M-sucrose. Twelve ml-aliquots were layered into 6 tubes which contained 12ml of 0.7 M-sucrose overlaid with 14ml of0.62 M-sucrose. After centrifugation at 25,000 rev./min for 30 min, the crude myelin separated into 3 subfractions. The myelin at the interface of 0.32-0.62 M-sucrose, at the interface of 0.62-0.70~-sucrose, and in the pellet was designated light, medium, and heavy myelin subfractions, respectively. Each subfraction was mixed with 75 ml of H,O and centrifuged at 25,000 rev./min for 15 min. The pellet of each subfraction was suspended in 24ml of 0.32~-sucroseand the subfractionation procedure, as described above, repeated. Each subfraction collected at the same position it had during the initial subfractionation with only small amounts found at the other two positions. Each myelin subfraction was washed twice in 75 ml of H,O and lyophilized.
Tissue extraction BP was extracted from crude myelin and myelin subfractions by the method of WOOD & KING (1971). Ten milligrams of lyophilized myelin were mixed with 1.5ml of acetone and agitated for 2 h at 25°C. The mixture was centrifuged, the supernatant discarded, the pellet mixed with 1ml of acetone, and the mixture stirred for 20 min. The residue was collected by centrifugation and washed with 5 ml of H,O. The residue was again collected by centrifugation and mixed with lOml of IO-’M-HCI. In some of the initial experiments extraction was carried out with lo-’ M and i0-3~-HC1.This solution was agitated for 18 h at 4°C and was then centrifuged and the supernatant lyophilized. The residue was washed twice with 5 ml of H,O and lyophilized. For slab gel electrophoretic studies (see below) crude myelin was delipidated with ether-ethanol (3:2, v/v) (GREENFIELD et al., 1971).
Preparation of rat BPs Frozen adult rat brain was delipidated with chloroformmethanol (2:1, v/v) and acetone, the residue was washed with H,O and the BP was extracted at pH 3 (DEIBLER et a/., 1972). The pH 3 extract was subjected to ion exchange chromatography at 25°C on CM-cellulose equilibrated with 0.05 M-glycine-NaOH buffer, pH 10.5, containing 6 M-urea et a/., 1975). After the unretarded material had (WHITAKER passed through the column, BP-containing fractions were eluted with 0.05 M-glycine-NaOH, pH 10.5, containing 2 ~ - u r e aand 0.2NaC1. This material was desalted on a column of Sephadex G-25 equilibrated with 0.5% acetic acid and lyophilized. The lyophilized material was dissolved in 0.03 M-HCI and filtered through a column (1.5 x 90cm) of Sephadex G-100 (MARTENSON ei al., 1970h). The large BP and small BP were isolated by recycling of selected fractions over the same column.
Polyacrylarnide gel electrophoresis Disc gel electrophoresis at pH 10.6 or pH 2.5 in the presence of urea was performed according to the method of DEIBLER er al. (1972). For the alkaline urea gels 150-250 pg of extract were applied to each gel, and the electrophoresis was conducted for 3h with a current of 3.75mA/gel. For acetic acid-urea gels 25 pg of extract were applied to each gel, and electrophoresis was conducted for 75 min with 2.5 mA/gel. Gels were stained for 1 h in 1% Amido black 10B in 7% acetic acid and destained in 3% acetic acid.
Discontinuous slab gel electrophoresis was performed with a modification of the method of G R E E N F I ~etL IaI / . (1971). Delipidated extracts were mixed, at a concentration of 2mg/ml, with a 1% solution of SDS (w/v) in H,O and placed in boiling HzO for 10min (MORELL cf d.,1976). After cooling, 50 $ of the sample solution were mixed with 25 pl ofsample buffer. This solution was applied to each lane of the gel which was comprised of a 3% stacking gel and a 12.5%running gel. Electrophoresis was conducted at lOOmA with a bromphenol blue tracking dye. The gels were stained with 0.25% Coomasie brilliant blue in methanol-H,Oacetic acid (5:5:1) for 5 h and destained in methanol-H,Oacetic acid (1.6: 12.7: 1).
Other methods Electron microscooy was performed on crude myelin or myelin subfractions fixed and examined as described by WOOD et al. (1974). Myelin was fixed in the sucrose in plastic conical tubes prior to the final H,O wash. In order to obtain optimal tissue sampling, blocks were oriented for sectioning so that sections were cut perpendicular to the pelleted tissue. For quantitation of BP microheterogeneous bands, gels were photographed, and positive clear prints prepared. These prints were scanned in an Ortec 4310 Densitometer (PORTNER, 1977).Amounts in each band were determined by weighing the chart paper by the appropriate peak. The amount in each band was calculated as a percentage.of the total density of bands 1-6. RESULTS
Extruction o f B P Initial studies of acetone-delipidated crude myelin indicated that the relative proportions of the different bands reflecting the microheterogeneity of B P were essentiallyidentical in the 10- 10- a n d 10- M-HCI extracts and very little B P remained in the residue. Since the pattern of microheterogeneity was constant over this range of HCI concentrations, a single concentration, ~ O - ’ M , was utilized in all subsequent studies. By this extraction procedure virtually all of the BPs, trace amounts of higher molecular weight proteins, et al., usually only one of the intermediate (GREENFIELD 1971) or DM-20 (AGRAWALet al., 1972) proteins, and one or two diffuse bands migrating ahead of small BP in the slab gel system were solubilized (Fig. 1). Thus, BP was the predominant protein species extracted with only the two faster migrating bands being present in moderate amounts. As indicated by slab gel electrophoresis, there were no identifiable differences in the extent of extraction of BP among the subfractions with lo-’ M-HCI (Fig. 1). The two fast migrating bands (Fig. 1) most likely represent degradation products formed during the HCI extraction of this residue with lo-’ M-HCI.Performing migrating bands were present in myelin delipidated with ether-ethanol; they markedly increased after extraction ofthis residue with 10- M-HCI. Performing all of thedefatting and extraction a t 4°C did not prevent the formation of these bands. Electrophoretic studies of the extracts of the myelin subfractions at pII 2.5 i n the presence of urea revealed
’,
’,
’
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FIG.I. Electrophoresisin 12.5%discontinuous polyacrylamide slabgel containing SDS. (A) Ether-ethanol dclipidated crudc myelin, (B) acid extract from crude myelin, (C) acid extract from light myelin, (D) residue remaining after acid extraction of light myelin, (E) acid extract of medium myelin, (F) residue remaining after acid extraction of medium myelin, (C) acid extract of heavy myelin, and (H) residue remaining after acid extraction of heavy myelin. Comparable amounts (100 pg) applied to each lane resulted in overloading of lanes A, B, C, E and C but demonstrate the extent of removal of B P from the residue. Major bands designated as follows: Wolfgram protein (W), proteolipid (PL), DM-20 or intermediate protein (I), large basic protein (BP-L), and small basic protein (BP-s).
FIG.2. Disc gel electrophoresis a t pH 2.5 in the presence of urea of acid extracts from delipidated crude myelin (A) and the light (B), medium (C), and heavy (D) myelin subfractions. Acid extraction performed . enriched in rat large B P (E) and small B P (F) also shown. 25 pg applied t o in 10- M - H C ~Fractions each gel. Cathode at bottom. Abbreviations same as used for Fig. 1.
’
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FIG.3a. Disc gel electrophoresis at pH 10.6 in the presence of urea of rat brain pH 3 extract (A) and acid extracts ofdelipidatcd crude myelin (B), light myelin (C), medium myelin (D),and heavy myelin (E and F). 1 SO pg of protein were applied on gels A-E, and 250 pg applied on gel F. Diminution in bands 1 and 2 in heavy myclin remains apparent even on overloaded gel (F). Cathode at bottom. FIG 3b. Disc gel electrophoresis at pH 10.6 in the presence of urea of acid extract of delipidated crude myelin (A) and fractions enriched in large B P (B) and small B P (C) isolated by gel filtration. Bands 1-4 were comprised of small B P components whereas large BP components were present in bands 5-7. 150 pg applied per gel. Cathode at bottom.
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FIG.5. Electron micrographs of crude myelin (A) and light (B), medium (C), and heavy (D) myelin subfractions from adult rat brain. All micrographs are at the same magnification (19,ooOx ).
Basic protein microheterogeneity in myelin subfractions
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no appreciable differences in BPs among crude myelin or myelin subfractions (Fig. 2). I n addition to the two BP bands, each of the acid extracts contained similar bands migrating more slowly and more rapidly than BP in this system. Thus, the procedure chosen for extraction of BP appeared to show no preferential effect on extraction of BP among crude myelin and myelin subfractions.
ofmyelin subfraction (Fig. 3a). Based on the assumption that the bands of rat BPs have similar dye-binding capacities for Amido black IOB, as guinea-pig BP components appear to have (DEIBLER& MARTENSON, 1973), the bands observed o n electrophoresis were scanned by densitometry (Fig. 4). Densitometric analysis confirmed the differences among bands noted by visual inspection (Fig. 3a). Results representing the average of three different electrophoretic analyses of Microheterogeneity of BP’s acid extracts from two preparations of myelin and In contrast to the SDS-slab and acetic acid urea disc myelin subfractions were consistent and showed a gel electrophoresis, electrophoresis at pH 10.6 in the tendency for there to be a relative increase in the presence of urea revealed readily apparent differences of density of bands 3, 4 and 6 with increasing myelin BP bands among myelin subfractions (Fig. 3a). In density. Because of the variability in band 7, no attempt Accordance with the convention of others (MARTENSON was made to quantitate this band which attained its & GAITONDE, 1969; DEIBLER & MARTENSON, 1973) the largest relative amount in the heavy subfraction (Fig. 4). bands were numbered from the cathodal front toward In order to determine which of the BPs was reprethe anodal end. Six bands could be detected with cer- sented in the electrophoretic bands seen in alkalinetainty, but a seventh band to the anodal side of band 6 urea gels, rat B P was separated into fractions enriched sometimes occurred, especially in the denser myelin in large BP and small BP. Electrophoresis of these subfractions. Similar results were obtained from crude, fractions at pH 2.5 in the presence of urea (Fig. 2) and in light, medium, and heavy myelin in three subfraction- SDS-slab gels (data not shown) indicated their purity. ations. The most obvious difference was a progressive Only small amounts of contamination of the other size diminution of bands 1 , 2 and 5 with increasing density BP occurred. O n alkaline-urea gel electrophoresis (Fig. 3b) it could be shown that bands 1-4 were com6 5 4 1 2 1 ponents of small BP and bands 5-7 were components of large BP. Band 5 additionally had some small BP present. This indicates that the bands which diminished with increasing density of myelin are the more positively charged components of rat large and small BP. Electron microscopy Electron microscopy revealed that crude myelin and each myelin subfraction consisted almost exclusively of myelin with very little, or no, contamination (Fig. 5). The light subfraction was comprised of large whorls of myelin with 3-4 lamellae. The heavy subfraction contained whorls of smaller diameter and 7-8 lamellae and some single-walled vesicles. The medium subfraction was intermediate between the light and heavy subfractions in diameter of the whorl and number of lamellae.
DISCUSSION
FIG.4. Tracings of densitometric scans of positive clear photographic prints of disc gels following electrophoresis at pH 10.6 in the presence of urea. Relative differences in bands 1-6 of BP from crude myelin (A), light myelin (B), medium myelin (C), and heavy myelin (D) agree with those seen on visual inspection (Fig. 34, but changes in band 5 are more obvious. 150 pg of acid extract of delipidated myelin applied per gel. Cathode to right.
Many investigations have demonstrated that CNS myelin is not homogeneous but exists in a spectrum of varying densities (AUTILIOet al., 1964; ADAMS& FOX, 1969; CUZNER & DAVISON,1968; KURIHARAet al., 1971;MCMILLAN et al., 1972;WAEHNELDT & MANDEL, 1972; BENJAMINS et al., 1973; MAITHIEUet al., 1973; AGRAWALet al., 1974; ZIMMERMAN et al., 1975; FUJIMOTO et al., 1976; BENJAMINS et al., 1976; SHEADS et al., 1977). Different techniques have been used for the preparation of a number of subfractions, and the distinctive chemical and morphological features noted among the subfractions have not always been identical. Because of the probability that myelin subfractions are in some manner related to the organization, biosynthesis, maintenance or turnover of myelin, studies have been undertaken to characterize and compare
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C. W. SHULTS.J . N. WHITAKER and J. G . WOOII
protein constituents of myelin subfractions. In adult rat myelin subfractions. The distribution of BP componbrain total myelin BP decreased with increasing myelin ents among myelin may be a reflection of the regulatory density (MATTHIEU et d.,1973; ZIMMERMAN et al., 1975; processes influencing BP microhctcrogeneity eithcr in concert with or as a major controlling mechanism for BENJAMINS et a[., 1976). Results of the present investigation indicate that the changes in the composition of the myelin membrane. The exact basis for the observed differences remains population of components of microheterogeneous BP differs among myelin subfractions. With increasing to be elucidated. Characterization of the nature and density of the myelin subfraction there is a decrease in mechanism of these changes in BP microheterogeneity relative amounts of the more positively charged may provide an additional means for assessing chemical components of small BP (bands 1 and 2) and events during myelination. large BP (band 5). Although to a lesser extent, Acknowledgmirn~s-This work was conducted under Veterless positively charged components of small BP ans Administration Research Project 9351-03 and was also (bands 3 and 4) and large BP (band 6) increase. As supported by NIH grant NS-12590 and the Alfred P. Sloan observed by others (MARTENSON et al., 19706), the Foundation(JGW).We thank Drs. L. KINGand A. PORTNER multiple bands for both rat large and small BP at for their assistance in the densitometric analysis alkaline pH suggests that both are modified by processes contribating to BP microheterogeneity. Based on REFERENCES studies on microheterogeneity of bovine BP (CHOUrt AVAMSD. H. & Fox M. E. (1969) Brain Res. 14, 647-661. d., 1976), the microheterogeneity demonstrated in rat AGRAWAL H . C., BURTONR. M., FISHMAN M. A., MITCHELL small BP is most likely due to phosphorylation at R. F. & PRENSKY A. L. (1972)J. N~rtrochent.19,2083-2089. threonine 97 and serine 164 or deamidation at gluta- AGRAWALH. C., TROTTER J. L., BURTON R . M. & MITCHELL R . F. (1974) Biochem. J . 140,99-109. mine 102 for in rat small BP the potential site for deamidation at glutamine 146 is deleted (MARTENSON AUTILIOL. A,, NOKTONW. T. & TERRYR . D. (1964) J. Neiirochem. 11, 17--27. et d., 1972; DUNKLEY & CARNEGIE, 1974). In bovine BENJAMINS J. A., MILLERK. & MCKHANNG . M. (1973) BP there is good evidence that in uiuo microheteroJ. Neurocheni. 20, 1589-1603. geneity results from phosphorylation and, possibly, BENJAMINS J . A., GRAYM . & MORELL P.(1976)J . Neirrochmt. deamidation (CHOUet a!., 1977). 21, 571 -575. A number of explanations may account for the BROSTOFFS. W.. REUTER W., HICHENSM. & EYLARE. H. differences observed in the present study. These (1974) J. h i d . Cheni. 249, 559-567. differences may represent not only the conditions CHOWF. C-H.. CFIOUC-H. J., SHAPIRA R. & KIBLER R . F. (1976) J. bid. Cheni. 251, 2671-2679. existing in uiuo but also be a result of differential CHOU F. C-H., CHOWC-H. J., S H A P I RRA. & KIIKERR. F. alterations that occurred during the isolation and (1977) J . Nriirochem. 28, 1051-1059. extraction procedures. A restriction in BP extraction in CUZNER M. L. & DAVIIISON A. N . (1968) Biochent. J . 106, different myelin subfractions would seem to be excluded 29-34 since the extent of BP extraction from the subfractions DEIBLER G . E.. MARTENSON R . E. & KIESM. W. (1972) appeared similar and nearly complete. It is possible that Prep. &ochem. 2, 139-165. during the isolation procedures the more positively DEIHLER G. E., MAKrI
