of chemistry vol. no 10529-10537. 1981 in fractionation ... · pp. 10529-10537. 1981 prrnted in 11....

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 256, No 20, Issue of Octoher 25. pp. 10529-10537. 1981 Prrnted in 11. S.A

Fractionation and Properties of a Chondroitin Sulfate Proteoglycan and the Soluble Glycoproteins of Brain*

(Received for publication, April 6, 1981, and in revised form, July 6, 1981)

Wei-Lai Kiang, Richard U. Margolis, and Renee K. Margolis From the Department of Pharmacology, New York University School of Medicine, New York, New York 10016, and the Department of Pharmacology, State University of New York, Downstate Medical Center, Brooklyn, New York 11203

The soluble glycoproteins and proteoglycans present in a phosphate-buffered saline extract of rat brain were fractionated by ion exchange chromatography and gel filtration. A 6.5 S proteoglycan purified by this procedure accounts for essentially all of the soluble chondroitin sulfate in rat brain and is composed of 56% protein (w/w), 24% glycosaminoglycans (predomi- nantly chondroitin 4-sulfate), and 20% glycoprotein oligosaccharides. Glutamic acid, aspartic acid, serine, leucine, and glycine (in descending order of concentration) account for half of the total amino acids present. The proteoglycan migrated electrophoretically as a single diffuse band on composite agarose/acrylamide gels, and gel filtration on Sepharose CL-4B in the presence of 4 M guanidine HCl indicated the presence of a single polydisperse macromolecule in which the chondroitin sulfate polysaccharide chains and the glycoprotein oligosaccharides are both covalently linked to a common protein core. However, the N- and 0-glycosidically linked glycoprotein oligosaccharides appear to be non- uniformly distributed in the proteoglycan, insofar as the larger molecular size species have a concentration of glycoprotein glucosamine over 10 times that of the smaller proteoglycan molecules, whereas the concentration of glycoprotein galactosamine is highest in the proteoglycans of intermediate size. The brain proteoglycan appears to be capable of at least a limited degree of interaction with hyaluronic acid to produce larger size aggregates, as is also known to occur with those from cartilage and cultured human glial cells, but differs from these proteoglycans in its smaller monomer size and higher protein content.

Until quite recently almost all studies of proteoglycans have concerned material isolated from connective tissue (for re- views see Kennedy, 1979; Roden, 1980), but relatively little is known concerning their structure, localization, and functional roles in other tissues. In brain, 85-90% of the glycoproteins are tightly bound to membranes, whereas much of the total glycosaminoglycan content is either soluble or easily extract- able by mild washing of microsomal membranes (Margolis et aZ., 1975; Kiang et al., 1978; Margolis and Margolis, 1979). Because the soluble pool of these complex carbohydrates in brain is more accessible for isolation, fractionation, and char- acterization than that present in the particulate fraction, the

of Health (NS-09348 and NS-13876) and the National Institute of * This work was supported by grants from the National Institutes

Mental Health (RSDA MH-00129 to R.U.M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertise- ment" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

soluble glycoproteins and proteoglycans have obvious advan- tages for studies aimed at investigating these compounds as intact macromolecules, rather than as glycopeptides or glycosaminoglycans obtained by such procedures as protease treatment of a lipid-free tissue residue.

We have previously reported in preliminary form (Margolis et al., 1976, 1979b) on a soluble chondroitin sulfate proteoglycan isolated from rat brain and have described a series of novel mannitol-containing oligosaccharides obtained from it by mild alkaline borohydride treatment (Finne et aZ., 1979). The present report provides more detailed information on the isolation and properties of the chondroitin sulfate proteoglycan and on the ion exchange chromatographic fractionation of several groups of soluble glycoproteins obtained from brain and isolated neurons.

EXPERIMENTAL PROCEDURES

IsoEation and Fractionation Procedures-Brains from 30- to 60- day-old Sprague-Dawley rats were homogenized in 9 volumes of cold 5 mM sodium phosphate buffer (pH 7.2) containing 0.15 M NaCl, using a glass Dual1 tissue grinder operated at 400 rpm. The soluble fraction was then obtained by centrifugation for 2 h at 140,000 X g. For the preparation of labeled fractions, one group of 20 rats (30 days old) was injected intracerebrally with 10-15 pl/rat of a saline solution containing 135-20 pCi of [6-3H]glucosamine or carrier-free sodium ["S]sulfate 18 h prior to decapitation, and the soluble extracts were first dialyzed overnight against running tap water (cooled to 3-5 "C) to thoroughly remove free precursor radioactivity.

The soluble protein obtained from 180-200 g of brain was dialyzed against deionized water, lyophilized, and redissolved by stirring overnight in 310 ml of 50 m M Tris-HCI buffer, pH 8.25, at 4 "C, containing 15 m M NaCI. (The buffer as routinely prepared contained 5.72 g of Tris-HCI and 1.66 g of Tris base per liter of distilled water, giving a pH of 8.2-8.3.) After stirring overnight at 4 "C and centrifugation for 30 min at 44,000 X g, the supernatant solution contained greater than 90% of the protein and at least 95% of the glycosaminoglycans and incorporated glucosamine or sulfate radioactivity. The resulting sample, representing approximately 2.5 g of soluble brain protein, was applied at a rate of 50-60 ml/h to a column (2.5 X 60 cm) of DEAE- cellulose (Whatman DE-52) previously equilibrated (5 column volumes over 65 h) with pH 8.25 Tris buffer containing 15 m~ NaCl. This buffer was also used to elute nonadsorbed proteins. Elution was then carried out stepwise at a rate of 30-40 ml/h with 0.09, 0.24, and 0.5 M NaCl in 50 mM Tris buffer (pH 8.25). Ten-ml fractions were collected and monitored for radioactivity and/or absorbance at 280 nm. Each elution step required 18-22 h for completion, and the pooled fractions were dialyzed against three changes of deionized water before lyophilization and analysis or further purification.

Electrophoresis-Electrophoresis was performed in composite 0.6% agarose/l.2% acrylamide gels by the general method of McDevitt and Muir (1971) as modified by Dr. P. Roughley.' The gels were made with 40 m M Tris-HCI buffer, pH 7.5, and poured at 42 "C. They were then cooled for 20 min at 4 "C and stored for no longer than 2 days in the refrigerator. The gels were warmed to room temperature before use, and electrophoresis was performed at room temperature in IO

' P. Roughley, personal communication.

10529

10530 Proteoglycans and Glycoproteins of Brain

mM Tris-HC1 buffer. Sodium sulfate and EDTA were omitted from the gel and running buffers, respectively. Samples (2-5 mg/ml) were dissolved in distilled water and diluted with an equal volume of 50% sucrose containing 0.01% bromphenol blue. Up to 30 pl of diluted sample was applied to the gel (without pre-electrophoresis) at a current of 1 mA/tube, which was then increased to 4 mA/tube after the bromphenol blue tracking dye entered the gel (15-20 min). Elec- trophoresis was continued at this current for the duration of the run (approximately 90 min total), by which time the bromphenol blue had migrated 2.5-3 cm. Longer runs led only to a greater diffuseness of the bands without any increase in resolution. The gels were stained overnight with 0.02% toluidine blue in 0.1 N acetic acid and destained in several changes of 3% acetic acid followed by water. Radioactivity was measured in 1-mm gel slices which were placed in scintillation counting vials together with 10 ml of toluene containing 0.4% Omni- fluor and 3% Protosol (New England Nuclear), followed by heating overnight at 37 "C.

Electrophoresis was also performed using 3.5% and 5% polyacrylamide gels with stacking gels as described by Maurer (1971). The separating gel was prepared with 0.37 M Tris buffer, pH 8.7, a t 25 'C, and the spacer gel buffer consisted of 0.06 M Tris-HC1 at pH 6.7. The samples were electrophoresed in a Tris-glycine buffer at pH 8.3, and when SDS' was used it was added to all solutions at a concentration of 0.1%. For electrophoresis in the presence of SDS, the samples were incubated for 1 h at 37 "C with 1% SDS containing 0.01 M dithiothreitol. Electrophoresis was performed at a constant current of 1 mA/ gel, and gels were stained with Coomassie blue as described by Weber and Osborn (1969).

Analytical Ultracentrifugation-Sedimentation velocity studies were performed using a Spinco model E ultracentrifuge at a rotor temperature of 5 "C and a speed of 44,000 rpm. Measurements were made at sample concentrations of 1-3.5 mg/ml in 0.01 M 2-(N-mor- pho1ino)ethanesulfonic acid buffer, pH 7.0, containing 0.15 M NaCl. The calculated sedimentation coefficient was corrected to 20 "C.

Analysis of Glycosaminoglycans and Glycopeptides-Fractions obtained from ion exchange or gel filtration columns were dialyzed, lyophilized, and digested with pronase as described previously (Mar- golis et al., 1975). The pronase digest was desalted by gel filtration on Sephadex G-15, and glycosaminoglycans were precipitated with cetylpyridinium chloride. Excess cetylpyridinium chloride was removed from the supernatant solution, containing the glycopeptides derived from the glycoproteins, by extraction with n-amyl alcohol. Sulfated glycosaminoglycans were separated from hyaluronic acid by differential precipitation with cetylpyridinium chloride. The concentrations of glycosaminoglycans were calculated from the glucosamine content of the hyaluronate fraction and from the glucosamine and galactosamine contents of the sulfated glycosaminoglycans (for heparan sulfate and chondroitin sulfate, respectively).

Hexosamines were determined by using the amino acid analyzer, and sialic acid was measured by the periodate-resorcinol method of Jourdian et al. (1971). Mannose, galactose, and fucose were assayed by automated ion exchange chromatography of their borate com- plexes (Lee et al., 1969) using a gradient containing 100 ml each of 0.27 M sodium borate buffer (pH 7.7) and 0.4 M borate buffer (pH 10) with a column (0.3 X 70 cm) of Hamilton HAXG resin. Amino sugars in glycosaminoglycans were determined after hydrolysis for 3 h in 6 N HC1 at 100 "C and in glycopeptides after hydrolysis in 4 N HCl for 8 h a t 100 "C, while neutral sugars were determined after hydrolysis of glycopeptides for 3 h in 2 N trifluoroacetic acid at 100 "C. For amino acid analysis, samples were hydrolyzed under vacuum in 6 N HCI for 22 h at 110 "c .

The unsaturated disaccharides resulting from treatment of the suifated glycosaminoglycan fraction with chondroitinase AC and ABC were quantitated by high performance liquid chromatography on an Altex Ultrasil NH2 column using a mobile phase of methanol-0.5 M ammonium formate, pH 4.8 (35:65, v/v), as described by Lee and Tieckelmann (1979).

Protease Treatment of Proteoglycan-The proteoglycan, dissolved in 0.05 M sodium phosphate buffer (pH 7.5) at a concentration of 1-3 mg/ml, was digested with either diphenyl carbamyl chloride-treated trypsin (Sigma type XI) or with twice crystallized trypsin (Sigma type 111) together with a-chymotrypsin (Sigma type 11) for 4 h at 37 "c. In each case a concentration of 0.1 mg of enzyme per mg of proteoglycan was used.

Reduction and Alkylation of the Crude Proteoglycan Fraction- The material eluted from the DEAE-cellulose column with 0.5 M

' The abbreviation used is: SDS, sodium dodecyl sulfate.

NaCl was dialyzed, lyophilized, and redissolved in 20 ml of deoxygen- ated 50 mM Tris-HC1 buffer (pH 8.5 at 20 "C) containing 4 M guanidine HCl and 10 mM dithiothreitol. All operations were performed under a nitrogen barrier (Crestfield, 1956) and with magnetic stirring. The sample was then sealed and allowed to stand for 3 h at 37 "C, after which iodoacetamide was added to a concentration of 40 mM with stirring under nitrogen, and the sample was allowed to stand for a further 3 h in the dark at room temperature. All of the material remained soluble after dialysis into 0.2 M sodium acetate buffer (pH 5.6) for gel filtration on Sepharose CL-GB.

Other Methods-Rat brain neurons were isolated by a modification of the method of Farooq et al. (1977), and the soluble fraction was obtained as described previously (Margolis et al., 1979a). Interaction of the brain proteoglycan with hyaluronic acid was assayed by mixing the two components (in a ratio of 1W1 based on glycosaminoglycan hexosamine content) in 0.5 M sodium acetate buffer, pH 7.0, and allowing it to stand for 18 h at 4 "C followed by gel filtration on Sepharose CL-2B. Protein was determined by the method of Lowry et al. (1951) and uronic acid was determined by the method of Bitter and Muir (1962).

Materials-[6-3H]Glucosamine (10-20 Ci/mmol) and [I-3H]fucose (1 Ci/mmol) were obtained from Amersham/Searle (Arlington Heights, IL), and sodium [35S]sulfate was from New England Nuclear (Boston, MA). Chondroitinase AC and ABC were obtained from Miles Laboratories (Elkhart, IN), and hyaluronic acid was prepared from umbilical cord. A characterized sample of bovine nasal cartilage proteoglycan subunit was kindly provided by Dr. Lawrence Rosen- berg, Montefiore Hospital and Medical Center, Bronx, NY.

RESULTS

Fractionation of Soluble Glycoproteins and Proteogly- cans-The soluble protein fraction (19.3 mg/g fresh weight of brain) has a relatively low content of glycoprotein carbohydrate (0.8% by weight) and accounts for 8% of the total glycoprotein carbohydrate in brain, calculated on a molar basis. However, there is considerable variation in the percentages of the individual glycoprotein sugars which are found in the soluble glycoproteins, i.e. mannose, 6% of the total in brain glycoproteins; N-acetylglucosamine, 7.3%; fucose, 8%; sialic acid, 9.3%; galactose, 11%; and N-acetylgalactosamine, 13%. These data also reflect our previous finding that the particulate glycoproteins of brain (e.g. in microsomal or synaptic membranes) have a relatively higher proportion of mannose and fucose residues and a lower proportion of galactose and N-acetylgalactosamine than do the soluble glycoproteins (Krusius et al., 1978). (Gangliosides account for less than 2% of the total sialic acid present in the 140,000 X g supernatant fraction.)

The concentration of glycosaminoglycans in the soluble

TABLE I DEAE-cellulose fractionation of soluble proteins from rat brain Soluble proteins of whole rat brain or isolated neurons were frac-

tionated by chromatography on DEAE-cellulose as described under "Experimental Procedures." The table gives the distribution of protein and radioactivity in fractions eluted by the indicated NaCl concentrations.

Radioactivity"

NaCl concentra- Protein Sulfate Glucosamine'

Per Relative Relative

'ent activity

tion specific Per cent specific ac-

tivity

M %

0.015 13 2 1.0 8 (7) 1.0

0.24 31 12 2.5 22 (25) 1.4 (0.8) 0.50 2.5 62 161 20 (21) 10.7 (2.2)

0.09 14 4 1.9 18 (16) 2.3 (1.9)

Total recovery 60 80 67 (69)

tration of sodium [%]sulfate or ['H]glucosamine.

from isolated neurons rather than whole brain.

Labeling of individual fractions 18 h after intracerebral adminis-

Values In parentheses refer to labeling of soluble fraction obtained

Proteoglycuns and Glycoproteins of Bruin 10531

TABLE 11 Glycoprotein carbohydrate composition of soluble proteins from rut brain fractionated on DEAE-cellulose

Fractions eluted from DEAE-cellulose column

0.015 M NaCl 0.09 M NaCl 0.24 M NaCl 0.50 M NaCl

-

Total soluble proteins

N-acetylglucosamine 1.04" 1.00b 0.51 1.00 1.97 1.00 0.86 N-acetylgalactosamine 0.20 0.19 0.12 0.23 0.24 0.12 0.28 Mannose 1.01 0.97 0.51 1.01 1.27 0.65 0.65 Fucose 0.38 0.36 0.20 0.40 0.49 0.25 Trace Galactose 0.76 0.73 0.28 0.55 0.82 0.42 0.58 N-acetylneuraminic acid 0.58 0.56 0.15 0.29 0.87 0.44 0.57

Total 3.97 1.77

Micromoles per 100 mg of protein. Molar ratio.

proteins extracted by phosphate-buffered saline (1.2 pmol of hexosamine/100 mg of protein) was essentially the same as that previously found in a 0.3 M sucrose supernatant resulting from differential centrifugation of a brain homogenate and involving several homogenization steps (Margolis et al., 1975). However, in the present study the proportion of chondroitin sulfate (86% of the total soluble glycosaminoglycans) was relatively greater and that of hyaluronic acid (10%) correspondingly less than found previously, whereas the amount of heparan sulfate was only 4-5% using either sucrose or saline.

When the soluble proteins from rat brain were chromato- graphed on DEAE-cellulose using a linear gradient of NaCl in 50 rnM Tris-HC1 buffer (pH 8.25), it was found that four distinct peaks of both protein and incorporated sulfate or glucosamine radioactivity were eluted at NaCl concentrations of 0.015, 0.09, 0.24, and 0.50 M. Stepwise elution using these NaCl concentrations yielded four fractions, which on rechro- matography with a linear NaCl gradient each eluted as single peaks of protein and radioactivity. In later work the soluble proteins were, therefore, routinely fractionated by stepwise elution using the NaCl concentrations given above. The total recovery of protein was approximately 60% with the distribution among the four fractions as listed in Table I. Higher NaCl concentrations or the addition of 4 M urea and 1 M NaCl to the Tris buffer did not result in the elution of any significant amounts of additional protein.

Whereas the radioactivity present in ["'Slsulfate-labeled material was primarily eluted with 0.5 M NaCl in a fraction containing the chondroitin sulfate proteoglycan (see below) together with 2.5% of the total protein applied to the column, each of the last three fractions contained approximately equd amounts of incorporated glucosamine radioactivity (Table I). The total recovery and proportions of glucosamine-labeled soluble proteins obtained from isolated neurons and Eraction- ated by the same procedure were very similar to those of whole brain (Table I), indicating that in this respect the soluble fraction of whole brain is representative of the neuronal cytoplasm. The considerably lower relative specific activity of the soluble neuronal proteins eluted with 0.5 M NaCl is due to the fact that a much larger proportion (18%) of the recovered protein is eluted in this fraction.

From the carbohydrate composition of the fractions given in Table 11, it can be seen that the molar ratio of sialic acid to glucosamine increases progressively with increasing ionic strength of the eluting buffer. Although the sialic acid concentration of the fraction eluted with 0.24 M NaCl is only two- thirds that of the preceding fraction, the 0.24 M NaCl eluate contains the major fraction of soluble sulfated glycoproteins (see below). It can, therefore, be assumed that the presence of sulfate residues plays an important role in determining the elution behavior of this fraction, which perhaps significantly is almost devoid of fucose.

To evaluate the possible contribution of plasma glycopro-

5.66 2.94

1.00 0.33 0.76

0.68 0.67

l - I

t

7.69 1.00

-

1.78 0.23 11.78 1.53 3.98 0.52

13.41 1.74 6.14 0.80

44.78

- 08

6 - 0 6 g R N 0 -

0 4 3

I 1 - 0 2

"0 +)P. O-O?-o.o.o~.n.n ,

16 20 24 28 32 36 40 44 48 52

FRACTION NUMBER

FIG. 1. Gel filtration on Sepharose CL-GB (2.5 x 45 cm) in 0.2 M sodium acetate buffer, pH 5.6, of the material eluted from DEAE-cellulose between 0.24 and 0.5 M NaCI. Four-ml fractions were collected and monitored for radioactivity (0- - -0) and absorbance at 280 nm (t"..). The fractions indicated by the bur were pooled and dialyzed to obtain the purified proteoglycan.

teins in metabolic studies of brain, rat plasma proteins were labeled for 18 h after intraperitoneal administration of ['HI glucosamine or [3H]fucose and fractionated (after dialysis and centrifugation at 140,000 X g) in a manner identical with that used for the soluble proteins of brain.3 It was found that the specific activity of plasma glycoproteins labeled with either precursor was very much greater than that of the actual soluble glycoproteins of brain (calculated after correction for the contribution of blood to the radioactivity present in the soluble fraction obtained from a brain homogenate). These data indicate that contamination of the soluble fraction of brain with even small amounts of highly labeled plasma proteins can introduce a significant error when the metabolism of soluble brain glycoproteins is studied following intraperitoneal administration of labeled precursor. This situation may account for the component with a rapid turnover of fucose ( t l / z = 1 day) previously reported for the soluble glycoproteins of rat brain (Margolis and Gomez, 1973) and suggests that studies on the metabolism of soluble glycoproteins of brain are best performed using intracerebrally administered precursor or after saline perfusion of the brain to remove blood and labeled plasma glycoproteins.

' In comparison with brain glycoproteins (Table I), the percentages of plasma protein glucosamine and fucose radioactivity recovered in the fractions eluted from DEAE-cellulose with 0.015, 0.09, and 0.24 M NaCl were 9-12%, 55-5996, and 28-2195, respectively, whiIe the proportion of protein recovered in these three fractions was 16, 63, and 20%. Only 1% of the plasma protein (and glucosamine or fucose radioactivity) was eluted from the DEAE-column with 0.5 M NaCI.


Glycosaminoglycans-The concentration of glycoprotein carbohydrate in the fraction eluted with 0.5 M NaCl is over 11 times that of the total soluble proteins of brain, with the major sugars being galactose and mannose (Table 11). This fraction also contains 96% of the proteoglycans (quantitated in terms of glycosaminoglycan hexosamine), only 4% of which is present in the previous fraction eluted with 0.24 M NaC1. There were significant differences in the glycosaminoglycan composition of the 0.24 and 0.5 M NaCl fractions, in that the former contained only 2-4% of the chondroitin sulfate and heparan sulfate eluted from the column but 13% of the hyaluronic acid. These two fractions also differed in their relative proportions of glycosaminoglycans and glycoproteins. In the 0.24 M NaCl fraction 91% of the total hexosamine was present in the form of glycoproteins, whereas glycoproteins accounted for only 40% of the amino sugar in the fraction eluted with 0.5 M NaC1.

As mentioned above, the glycoproteins eluted with 0.24 M NaCl contain a significant amount of sulfate radioactivity (Table I), although only small amounts of glycosaminoglycans are present (primarily hyaluronic acid). After pronase digestion of this fraction and precipitation of the glycosaminoglycans with cetylpyridinium chloride, 82-85% (in two experi- ments) of the sulfate radioactivity remained in the supernatant solution containing glycopeptides derived from sulfated glycoproteins. Although the glycoprotein oligosaccharides in the proteoglycan fraction (eluted with 0.5 M NaC1) are also sulfated, these contained a much smaller proportion (18%) of the incorporated sulfate radioactivity, most of which was present in the glycosaminoglycans (chondroitin sulfate, 73%; heparan sulfate, 9%).4

Fractions containing the crude proteoglycan (65-80 mg of protein) eluted from the DEAE-cellulose column with 0.5 M NaCl were dialyzed against deionized water, lyophilized, and stirred overnight at 4 “C in 20 ml of 0.2 M sodium acetate buffer, pH 5.6. Under these conditions 98-99% of the incorporated glucosamine or sulfate radioactivity redissolved, whereas 25-30% of the protein remained insoluble. This un- dissolved material (which contains only traces of glycosaminoglycans and glycoproteins) was removed by centrifugation, and 10-ml aliquots of the supernatant solution were eluted from columns (2.5 X 45 cm) of Sepharose CL-GB with the same acetate buffer used to dissolve the sample. The purified proteoglycan appeared in the void volume, well separated from a large retarded peak of protein and nucleic acid (Fig. 1).

When the crude proteoglycan fraction was reduced and alkylated it remained completely soluble after dialysis into 0.2 M acetate buffer. However, the component thus solubilized forms a highly polydisperse complex with the proteoglycan in the absence of 4 M guanidine HC1, as indicated by gel filtration and analytical ultracentrifugation. In this respect the brain proteoglycan differs markedly from that present in cartilage, since in the latter case reduction and alkylation leads to an irreversible dissociation of large molecular size aggregates into proteoglycan subunits, whereas similar treatment of the brain proteoglycan promotes aggregate formation (of a presumably different type).

Composition a n d Properties of the Proteoglycan-The proteoglycan purified by gel filtration on Sepharose CL-GB contains 56% protein by weight, 24% glycosaminoglycans, and 20% glycoprotein carbohydrate. After fractionation of the

After labeling hexosamine and sialic acid residues in the proteoglycan by administration of [“H]glucosamine, the glycoprotein oligosaccharides contained 43% of the tritium radioactivity (of which 14% was in sialic acid residues), and the glycosaminoglycans accounted for the remaining 57% (46% in chondroitin sulfate, 8% in heparan sulfate, and 3% in hyaluronic acid).

TABLE I11 Composition of the chondroitin sulfate proteoglycan

Glycosaminoglycans pmol hexosamine/100 mg P.

protein Chondroitin sulfate 60.05 93.1 Hyaluronic acid 2.92 4.5 Heparan sulfate 1.57 2.4

Total 100.0 Glycoprotein oligosaccharides

pmol / lOO protein mg Molar ratio

N-acetylglucosamine 45.25 1.00 N-acetylgalactosamine 8.67 0.19 Mannose 29.59 0.65 Fucose 16.93 0.37 Galactose 37.26 0.82 N-acetylneuraminic acid 30.58 0.68

TABLE IV - Amino acid composition of the brain proteoglycan

Residues/100 residues

Lysine 4.5 Histidine 2.3 Arginine 3.9 Aspartic acid 11.8 Threonine 6.9 Serine 9.9 Glutamic acid 13.7 Proline 6.7 Glycine 7.5 Alanine 6.9 Valine 7.4 Methionine 1.9 Isoleucine 3.4 Leucine 9.0 Tyrosine 2.1 Phenylalanine 3.1

glycosaminoglycans with cetylpyridinium chloride and digestion of the sulfated fraction with chondroitinase AC and ABC, it was found that 97% of the resulting disaccharides are 4- sulfated and the remaining 3% are 6-sulfated. There was no difference in the yield of 4-sulfated disaccharides after treatment with chondroitinase AC or ABC, indicating that the proteoglycan does not contain dermatan sulfate. Small amounts (2-4%) of heparan sulfate and hyaluronic acid are also present (Table 111), but the proportion of hyaluronic acid is reduced by half after further gel filtration on Sepharose CL- 4B in the presence of 4 M guanidine HC1 (see below).

The 20% of glycoprotein-type oligosaccharides cannot be separated from the remainder of the proteoglycan on the basis of density (Margolis et al., 1976), size, or charge and appears to be covalently linked by both N - and 0-glycosidic bonds to the same protein core as are the chondroitin sulfate polysaccharide chains. The glycoprotein portion of the proteoglycan also includes several novel mannitol-containing oligosaccharides produced by mild alkaline borohydride treatment of the proteoglycan glycopeptides (Finne et al., 1979).

The major amino acids (in descending order of concentration) are glutamic acid, aspartic acid, serine, leucine, and glycine, which together account for half of the total amino acids present (Table IV).

The proteoglycan begins to elute with the void volume from Sepharose CL-GB in 0.2 M sodium acetate buffer and is progressively retarded on Sepharose CL-4B and CL-2B (Fig. 2). It sediments as a single sharp peak in the analytical ultracentrifuge and has a sedimentation coefficient of 6.5 S (Figs. 3 and 4). This value is lower than might be expected from its

Proteoglycans and Glycoproteins of Brain 10533 1 SEPHAROSE 28

L 3-

z I-

2 -

0

y I -

LL

" I2 40

2 -

0 z I-

y I -

LL

f I

16 22 2.8 34 40 FRACTION NUMBER

FIG. 2. Gel filtration of [S6S]sulfate-labeled proteoglycan on columns of Sepharose CG2B (1 X 50 cm), CL4B (0.9 x 45 cm), and CG6B (1.2 X 45 cm) eluted with 0.2 M sodium acetate buffer, pH 5.6. The VO was measured with blue dextran and the V, with ["C]glucose. Fractions of 1.3 ml were collected and the total volume used for measurement of radioactivity.

gel filtration behavior on Sepharose CLSB, where there was found to be considerable overlap between the elution profiles of the brain proteoglycan and a preparation of bovine nasal cartilage proteoglycan subunit, with a sedimentation coefi- cient of 21 S (Fig. 5A).

After mixing with hyaluronic acid (in a ratio of 1:lOO based on chondroitin sulfate galactosamine) it was found that 10% of the proteoglycan eluted as a distinct peak in the void volume, as compared to 1% of a control sample without added hyaluronic acid (Fig. 5). Addition of greater or smaller amounts of hyaluronic acid to the proteoglycan (i.e. ratios of 1:50 and 1:2OO) decreased somewhat the amount of aggregate formed. These results indicate that the brain proteoglycan is capable of interacting with hyaluronic acid to at least a limited extent, in which respect it resembles cartilage proteoglycans (Hardingham and Muir, 1972, 1974) and the material synthesized by cultured human glial cells (Norling et al., 1978).

Gel Filtration in 4 M Guanidine HCl-After gel filtration on Sepharose CL4B in 4 M guanidine HCl, variable amounts of a protein component containing relatively little carbohydrate or glucosamine-derived radioactivity (Fig. 6, fraction I) could be separated from the single large peak of proteoglycan. It would appear that the material in fraction I represents some type of aggregation product of proteoglycan molecules containing a low concentration of carbohydrate, since rechroma- tography of pooled fractions 11-IV under identical conditions (after dialysis and lyophilization) again results in the appear- ance of a small distinct peak eluting at the position of the original fraction I. Moreover, the fraction I material is highly cross-reactive with antibodies raised to proteoglycan eluting in fractions II-IV.5 Since fractions I and V contained only 3.3%

'D. Aquino, R. U. Margolis, and R. K. Margolis, unpublished results.

FIG. 3. Sedimentation velocity patterns of the brain proteoglycan at 3.48 mg/ml (top) and 2.91 mg/ml (bottom). The direction of sedimentation is from left to right. Centrifugation was performed at 5 "C and 40,000 rpm; picture was taken 152 min after reaching speed.

S

0 I 2 3

CONCENTRATION (mglml)

FIG. 4. Concentration dependence of sedimentation coefficient of brain proteoglycan. Sedimentation coefficients at 5 "C extrapolat.ed to zero concentration gave a value of 4.24 S for s$.

and 0.5%, respectively, of the total incorporated glucosamine radioactivity and insignificant amounts of amino sugar, they were not studied further.

The composition of the three intermediate fractions is sum- marized in Table V. Fractions I1 and I11 contain identical proportions of chondroitin sulfate in relation to protein, whereas the chondroitin sulfate concentration of fraction IV is only one-third that of the two preceding fractions. All fractions of the proteoglycan contain 95-96% chondroitin sulfate as their glycosaminoglycan component, together with 2- 4% heparan sulfate and 1-2% hyaluronic acid.

In contrast to the distribution of glycosaminoglycans, the concentration of glycoprotein oligosaccharides decreases progressively in fractions 11, 111, and IV, suggesting that differences in their molecular size (and extent of labeling with glucosamine) are largely determined by the density of substi- tution with such oligosaccharides on the protein moiety of the proteoglycan. There is a high concentration of glucosamine and a very small amount of galactosamine in the glycoprotein


40

24

16

I

I I

0 I

I I

? I I

I I

?

0.6 D W

0.5

W W

0.4 0 rn

0.3 w 0

0.2 5 - 4 0 I

0. A -0.1 I

0.

FRACTION NUMBER FIG. 5. Elution of the brain proteoglycan (M) labeled

with [3H]glucosamine on a column (0.9 X 65 cm) of Sepharose CL-2B before ( A ) and after (B) addition of hyaluronic acid as described under "Experimental Procedures." For comparison, a sample of bovine nasal cartilage proteoglycan subunit (0- - -0) was eluted under identical conditions and monitored by measurement of uronic acid in the effluent. Elutions were carried out using 0.5 M sodium acetate buffer, pH 7.0, and 1.3-ml fractions were collected.

12 16 20 24 28 32 36 4 0 4 4 4 8 52 S

F-i--Y+

12 16 20 24 28 32 36 4 0 4 4 4 8 52 S FRACTION NUMBER

FIG. 6. Gel filtration of proteoglycan labeled with [3H]glucosamine on a column (1.2 X 85 cm) of Sepharose CG4B eluted with 50 m~ sodium acetate buffer (pH 5.6) containing 4 M guanidine HCl. Fractions of 2.5 ml were collected and monitored for radioactivity (0- - -0) and absorbance at 280 nm (t".). The fractions indicated by the bars were pooled and dialyzed for analysis. The VO was measured with blue dextran, and the V, was measured with ['4C]glucose.

oligosaccharides of fraction 11, while their relative proportions change as one proceeds to proteoglycan fractions of smaller molecular size, with equal amounts of the two amino sugars being present in fraction IV. It would, therefore, appear that

TABLE V Composition of proteoglycan fractions eluted from Sepharose

CL-4B Fraction Fraction Fraction

11" I11 IV

Chondroitin sulfate con- 58.0 57.6 19.4 centration (pmol of GalNAc/100 mg of protein)

Glycosaminoglycan composition

Chondroitin sulfate 95% 96% 96% Heparan sulfate 4% 2% 3% Hyaluronic acid 1% 2% 1%

Concentration of glycoprotein hexosamineh (and molar ratio to chondroitin sulfate)

N-acetylglucosamine 56.5 (0.97)' 31.8 (0.55) 5.4 (0.28) N-acetylgalactosamine 4.2 (0.07) 9.6 (0.17) 5.4 (0.28)

" Fractions are as indicated in Fig. 6. Micromoles per 100 mg of protein. ' Expressed as micromoles of glycoprotein hexosamine per pmol of

Total 60.7 41.4 10.8

chondroitin sulfate galactosamine.

the larger proteoglycans contain mainly asparagine-linked oligosaccharides, whereas the smaller molecules have considerably higher concentrations of the mono- and disialyl deriv- atives of galactosyl(p1 "-* 3)h"acetylgalactosamine linked to serine and threonine, as identified previously (Margolis and Margolis, 1973; Finne, 1975; Krusius et al., 1978; Finne et al., 1979).

Gel Electrophoresis-The proteoglycan purified by gel filtration on Sepharose CL-4B in 4 M guanidine HC1 migrated in agarose/acrylamide composite gels as a single diffuse band extending over approximately 4 mm and had a mobility of 0.83 relative to bromphenol blue, with ["S]sulfate radioactivity corresponding to the toluidine blue staining. This migration was slightly lower than the average for the two resolved bands which are obtained with a preparation of adult bovine nasal cartilage proteoglycan subunit, and no staining or radioactivity was found in the region corresponding to single chondroitin sulfate chains (relative mobility of 1.3). Although the molecular size of the brain proteoglycan is considerably less than that of cartilage proteoglycan subunits, its similar rate of electrophoretic migration is probably a consequence of its much higher protein content and correspondingly lower net charge.

Electrophoresis in conventional polyacrylamide gels, with or without the presence of sodium dodecyl sulfate, was not informative insofar as the proteoglycan failed to enter gels containing as low as 3.5% acrylamide (i.e. the lowest concentration it is possible to use in such gels). In the case of ["SI sulfate-labeled proteoglycan purified through the stage of Sepharose CL-GB gel fitration, almost all of the radioactivity remained at the interface between the running gel and a stacking gel (which was used to retain the sample after re- moval of the gel from the electrophoresis buffer), while a small amount of label was found to migrate with the tracking dye. Coomassie blue staining of such gels corresponded closely with the distribution of sulfate radioactivity.

Effect of Trypsin on the Proteoglycan-The proteoglycan was treated with purified trypsin (in which any residual chymotrypsin was inactivated with diphenyl carbamyl chloride) in an attempt to obtain partial degradation products containing both chondroitin sulfate polysaccharide chains and glycoprotein oligosaccharides linked to a common peptide frag- ment. Such products would presumably be more amenable to

Proteoglycans and Glycoproteins of Brain 10535

studies on the distribution of these two types of carbohydrate units in the proteoglycan than would the much larger intact molecule.

When proteoglycan, in which the hexosamine and sialic acid residues were labeled in vivo by administration of [3H] glucosamine, was digested with diphenyl carbamyl chloride- treated trypsin and applied to a column of Sepharose CL-GB, two incompletely separated labeled peaks were obtained, as well as a large retarded peak of peptide absorbing a t 280 nm (Fig. 7). The fractions comprising peaks I and I1 were pooled, dialyzed (with complete recovery of radioactivity), and ana- lyzed for their glycoprotein and glycosaminoglycan composition (Table VI). I t can be seen that over 90% of the glycosaminoglycans were found in fraction I, whereas over 80% of the glycoprotein oligosaccharides were present in fraction 11.

These results indicate that the chondroitin sulfate chains and the glycoprotein oligosaccharides are spaced sufficiently f a r apart on the protein core such that trypsin-susceptible peptide bonds are present between most or all of the carbohydrate units. After pronase digestion of the proteoglycan or alkali treatment (Carlson, 1968) of the sulfated glycosaminoglycan fraction, it was seen that the individual [35S]sulfate- labeled chondroitin sulfate polysaccharide chains eluted at a position just 3 to 4 fractions more retarded than that of peak I, indicating that only single chondroitin sulfate chains still remained linked to the peptide portion after trypsin treatment. Treatment of the proteoglycan with trypsin together with a-chymotrypsin gave the same results as trypsin alone. This is in contrast to the cartilage proteoglycans, in which digestion with trypsin together with chymotrypsin yields pep- tides with an average of four chondroitin sulfate chains at- tached, while trypsin alone yields even larger products (Hei- negdrd and Hascall, 1974).

va

4

300

260

2 2 0 0

180 3

n

2 rn

140 f 0 -

100 - 60

20

FRACTION NUMBER

FIG. 7. Proteoglycan labeled with [3H]glucosamine was digested with diphenyl carbamyl chloride-treated trypsin (see under “Experimental Procedures”) and eluted from a column of Sepharose CGGB (2.5 X 45 cm) with 0.2 M sodium acetate buffer, pH 5.6. Four-ml fractions were collected and monitored for radioactivity (0- - -0) and absorbance at 280 nm (U). Fractions I and I1 indicated by the bars were pooled and dialyzed for analysis.

TABLE vr Fractionation of trypsin-treated proteoglycan on Sepharose CL-GB

Fraction I Fraction I1 Total glycosaminoglycans” 93% 7%

Chondroitin sulfate Heparan sulfate

92% 8% 97% 3%

Hyaluronic acid 95% 5% Glycoprotein glucosamine 17% 83% Glycoprotein galactosamine 21% 79%

Sum of figures in each line = 1008.

DISCUSSION

An ion exchange chromatographic procedure using DEAE- cellulose has been established for the fractionation of the soluble glycoproteins and proteoglycans of brain. The chromatographic conditions described here yield four distinct though heterogeneous fractions, which are useful either as a preliminary separation for metabolic or other studies, or as a starting point for further fractionation based on other properties of these complex carbohydrates. The general utility of this simple high capacity method as an initial step in the fractionation of soluble glycoproteins and proteoglycans from nervous tissue is suggested by our finding that very similar conditions yield purified dopamine ,&hydroxylase, chromo- granins, and two chondroitin sulfate proteoglycans from the soluble matrix of adrenal chromaffin granules.

The major objective of this investigation was, however, to more completely characterize the soluble chondroitin sulfate proteoglycan of brain. In our previous studies we demonstrated that the brain proteoglycan has an unusually high protein content, and that unlike the prototypical cartilage proteoglycans, it cannot be dissociated into subunits which sediment in a cesium chloride density gradient in the presence of 4 M guanidine HC1 (Margolis et al., 1976). Its quantitative adsorption on a concanavalin A-Sepharose affinity column (and elution by a-methylglucoside) also indicated the presence in the proteoglycan of glycoprotein-type oligosaccharides in addition to chondroitin sulfate (and much smaller amounts of heparan sulfate and hyaluronic acid), since none of the glycosaminoglycan polysaccharide chains were significantly bound by concanavalin A after release from the proteoglycan by pronase digestion or alkali treatment. It was later reported by Schwermann et al. (1978) that sulfated proteoglycans secreted by human skin fibroblasts are also specifically bound by concanavalin A-Sepharose.

Investigation of the glycoprotein portion of the brain proteoglycan revealed that after mild alkaline borohydride treatment, a series of novel oligosaccharides terminated with mannitol was produced in addition to the more conventional oligosaccharides linked 0-glycosidically via N-acetylgalactosamine and alkali-stable oligosaccharides containing N-ace- tylglucosaminylasparagine linkages. One-third of the mannitol released from the brain proteoglycan by mild alkaline borohydride treatment was in the form of neutral compounds comprising approximately equal amounts of free mannitol, GlcNAc(P1 -+ 3)Manol and Gal(P1 + 4)[Fuc(al + 3)] GlcNAc(P1 -+ 3)Manol. Approximately one-half of the mannitol was present in the acidic tetrasaccharide, AcNeu(a2 + 3)Gal(P1+ 4)GlcNAc(Pl+ 3)Manol, and the remaining 20% was present in another acidic oligosaccharide which appears to contain sulfate residues (Finne et al., 1979; Krusius et al., 1980). Although mannitol-containing oligosaccharides have not up to now been obtained from other mammalian glycoproteins or proteoglycans, since our original report on the brain proteoglycan, glycoprotein-type oligosaccharides have been found in proteoglycans secreted by skin fibroblasts (Schwermann et al., 1978) and those present in ovarian follic- ular fluid (Yanagishita et al., 1979), articular cartilage (Thonar and Sweet, 1979), chick chondrocyte cultures (DeLuca et al., 1980), and rat chondrosarcoma (Lohmander et al., 1980), indicating that they may be a general structural feature of proteoglycans from a wide variety of tissues.

Our data demonstrate that the chondroitin sulfate proteoglycan from brain differs from the well characterized connective tissue proteoglycans mainly in terms of its much smaller size and higher protein content. Although the purified proteoglycan contains small amounts of hyaluronic acid (14%) and heparan sulfate (2-4%), keratan sulfate is not present and the


role, if any, of hyaluronic acid and heparan sulfate in the macromolecular structure of the brain proteoglycan is not yet clear. However, it is possible that the heparan sulfate found in the brain proteoglycan is analogous to the minor keratan sulfate component of cartilage proteoglycans. Immunochemi- cal studies have also demonstrated various degrees of cross- reactivity between the rat brain proteoglycan and rat chondrosarcoma core protein, as well as bovine nasal septum and articular cartilage proteoglycans,6 indicating the presence of similar antigenic determinants in both nervous tissue and connective tissue proteoglycans.

Although tbe brain proteoglycan is capable of interacting with hyaluronic acid to a limited extent, it more closely resembles the minor fraction of nonaggregating cartilage proteoglycans (HeinegHrd and Hascall, 1979) than the aggregating proteoglycans isolated in the presence of 4 M guanidine HC1 (Hardingham and Muir, 1972, 1974). Addition of hyaluronic acid to a chondroitin sulfate proteoglycan secreted by cultured human glial cells also results in an increase of approximately 20% in the amount of labeled material eluting in the void volume of a Sepharose CL2B column, but much of the proteoglycan secreted by these cells is apparently present in aggregate form before addition of hyaluronic acid. Whether this type of proteoglycan occurs as a constituent of normal brain parenchyma remains to be determined. In other respects, the brain proteoglycan described by us resembles the low buoyant density proteoglycans synthesized by embryonic chick cartilage (Kimata et al., 1978), which in addition to their presumably higher protein content are also capable (after concentration) of forming aggregates in the presence of 4 M guanidine HC1.

The properties of the isolated brain proteoglycan would not appear to be attributable in any significant degree to partial proteolytic degradation. There was no difference in the molecular size or composition of the final product when the isolation steps were carried out in the presence or absence of protease inhibitors,’ and these were, therefore, not routinely included during the purification procedure. Moreover, the proteoglycan described here has an unusually high protein content, and its extraction from brain by brief homogenization in saline would tend to minimize the release of lysosomal proteases. While all of these considerations suggest that the isolated proteoglycan is representative of its composition in situ, in any study of this type one cannot, of course, completely exclude the possibility of some proteolytic degradation.

We are currently studying the localization of the chondroitin sulfate proteoglycan in brain using immunocytochemical techniques. Previous biochemical analyses (reviewed by Mar- golis and Margolis, 1979) demonstrated the presence of significant amounts of chondroitin sulfate in neuronal cell bodies and axons (isolated in bulk from brain) as well as in purified nuclei, whereas there is little or no chondroitin sulfate in other cells or subcellular fractions such as oligodendroglia, myelin, mitochondria, and nerve endings (synaptosomes). Approxi- mately half of the chondroitin sulfate is found in the soluble fraction after high speed centrifugation of a brain homogenate, and much of the remainder is loosely associated with various membrane fractions (Margolis et al., 1975; Kiang et al., 1978). Chondroitin sulfate is also present in astrocyte-enriched fractions prepared from rat and bovine brain (Margolis and Mar- golis, 1974), but the chondroitin sulfate proteoglycan described in the present report appears to differ in certain respects from that produced by cultured human glial cells (Norling et al.,

A. R. Poole, D. Aquino, R. U. Margolis, and R. K. Margolis,

’ Phenylmethylsulfonyl fluoride (0.5 mM), henzamidine HC1 (5 unpublished results.

mM), EDTA (50 mM), and 6-aminohexanoic acid (100 mM).

1978; Glimelius et al., 1978, 1979), insofar as the latter has a monomer size and other properties more similar to those of cartilage proteoglycans.

When neurons are isolated in bulk from rat brain and lysed by a change in tonicity or pH, 82% of the chondroitin sulfate is released together with over 90% of the lactate dehydrogen- ase but only 20-25% of the total cell protein and glycoprotein hexosamine (Margolis et al., 1979a). Since the chondroitin sulfate was not removed during the previous washing steps in the cell isolation procedure or by mild trypsinization of the purified neurons, we concluded that a portion of the proteoglycan is present as a cytoplasmic component of neurons, where it may be involved in such processes as maintaining cell turgor.

Immunocytochemical studies (by the peroxidase-antiperox- idase technique) employing antibodies to the chondroitin sulfate proteoglycan demonstrated a predominant staining of neuronal elements including cytoplasm and axons (but not myelin) (Aquino et al., 1981). The possibility that the proteoglycan identifed at the periphery of cells may be partly present as an extracellular ground substance in brain is currently being investigated using these techniques at the elec- tron microscopic level.

Acknowledgments-We thank Dm. L. Rosenherg and L.-H. Tang for the analytical ultracentrifugation data, Dr. P. Roughley for advice on electrophoretic procedures, and R. Wong, C. Crockett, and B. Berman for technical assistance.

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