Partial characterization of heparan and dermatan sulfate proteoglycans synthesized by normal rat glomeruli.

Rat glomerular heparan sulfate (HS) and dermatan sulfate (DS) proteoglycan synthesis was studied in vitro and in vivo. Incorporation of [35S]sulfate into macromolecules was linear over 16 h in vitro, and DS was the predominant glycosaminoglycan (GAG), while HS dominated in vivo incubations. Proteoglycans were found in the bottom 2/5 (high density) CsCl gradient fractions and eluted as two overlapping peaks from DEAE-Sephacel columns. The proportion of low density 35S-glycoproteins and 35S-proteoglycans increased with time. Two high buoyant density HS proteoglycans were extracted from glomeruli and eluted in DEAE peak I. The first, HS-tIA, had an Mr of 130 X 10(3) with Mr 12.5 X 10(3) GAG chains. This proteoglycan was released from the tissue by trypsin and was partially displaced by heparin treatment. In addition, it was rapidly released into the medium of label-chase experiments after which it migrated slightly more rapidly than HS-tIA in gels, with HS chains similar in length to its tissue counterpart. The second, HS-tIB, had an Mr of 8.6 X 10(3) with little or no attached protein. This proteoglycan was characterized as intracellular as it resisted release by trypsin treatment or heparin extraction in medium and was not detected in the medium of label-chase experiments. Two tissue DS proteoglycans were characterized. The first, DS-tIA, co-purified with HS-tIA and was the predominant proteoglycan synthesized during 4-h in vitro incubations. Like HS-tIA, it was rapidly released into medium and displaced from cell surfaces or tissue "receptors" by heparin or trypsin treatments. A second, Sepharose CL-6B-excluded DS proteoglycan from DEAE peak II, DS-tII, accumulated in tissue over 16 h in vitro. This proteoglycan was self-associating and contained clusters of iduronic acid residues along its Mr 26 X 10(3) DS chains. It resisted extraction from the tissue with heparin, trypsin, and detergent. No DS-tII was detected in the incubation medium. Instead, medium proteoglycans eluted as single Sepharose CL-6B-included peaks. DS chains from medium proteoglycans were shorter (Mr 18 X 10(3)) and had more regularly spaced iduronic acid residues than GAGs from DS-tII. The length and sulfation patterns of DS-mII GAG were similar to GAG from DS-tIA. Thus, glomeruli rapidly synthesized and released Sepharose CL-6B-included heparin-displaceable DS and HS proteoglycans while retaining a Sepharose CL-6B-excluded self-associating DS proteoglycan and an intracellular HS.

Rat glomerular heparan sulfate (HS) and dermatan sulfate (DS) proteoglycan synthesis was studied in vitro and in vivo. Incorporation of [3SS]sulfate into macromolecules was linear over 16 h in vitro, and DS was the predominant glycosaminoglycan (GAG), while HS dominated in vivo incubations. Proteoglycans were found in the bottom Y. (high density) CsCl gradient fractions and eluted as two overlapping peaks from DEAE-Sephacel columns. The proportion of low density 35S-glycoproteins and 36S-proteoglycans increased with time.
Two high buoyant density HS proteoglycans were extracted from glomeruli and eluted in DEAE peak I.
The first, HS-tIA, had an M, of 130 X lo3 with M, 12.5 X lo3 GAG chains. This proteoglycan was released from the tissue by trypsin and was partially displaced by heparin treatment. In addition, it was rapidly released into the medium of label-chase experiments after which it migrated slightly more rapidly than HS-tIA in gels, with HS chains similar in length to its tissue counterpart. The second, HS-tIB, had an M, of 8.6 X lo3 with little or no attached protein. This proteoglycan was characterized as intracellular as it resisted release by trypsin treatment or heparin extraction in medium and was not detected in the medium of label-chase experiments.
Two tissue DS proteoglycans were characterized. The first, DS-tIA, co-purified with HS-tIA and was the predominant proteoglycan synthesized during 4-h in vitro incubations. Like HS-tIA, it was rapidly released into medium and displaced from cell surfaces or tissue "receptors" by heparin or trypsin treatments. A second, Sepharose CL-GB-excluded DS proteoglycan from DEAE peak 11, DS-tII, accumulated in tissue over 16 h in vitro. This proteoglycan was self-associating and contained clusters of iduronic acid residues along its M. 26 X lo3 DS chains. It resisted extraction from the tissue with heparin, trypsin, and detergent. No DS-tII was detected in the incubation medium. Instead, medium proteoglycans eluted as single Sepharose CL-GBincluded peaks. DS chains from medium proteoglycans were shorter (M, 18 X lo3) and had more regularly spaced iduronic acid residues than GAGS from DS-tII. The length and sulfation patterns of DS-mII GAG were * This work was supported in part by Grants AM17697, AM32372, and AM01157 from the National Institutes of Health and by grants from the Human Growth Foundation, American Heart Association, the American Diabetes Association Minnesota Affiliate, and the Viking Children's Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ The research presented in this study was performed in partial fulfillment of a Doctor of Philosophy degree in Biochemistry at the University of Minnesota. similar to GAG from DS-tIA.
Thus, glomeruli rapidly synthesized and released Sepharose CL-GB-included heparin-displaceable DS and HS proteoglycans while retaining a Sepharose CL-GB-excluded self-associating DS proteoglycan and an intracellular HS.
Heparan sulfate (HS)' proteoglycan is an integral component of basement membranes. This proteoglycan has been extracted from glomerular basement membrane (GBM) where it appears in a lattice-like pattern along the lamina rara externa and interna (1, 2) and helps maintain the glomerular capillary barrier to protein leak. Evidence for this role includes observations that in situ heparitinase treatment results in leakage of '251-albumin and native ferritin into the urinary space (3, 4) and that there is a diminution in the number of HS-related charge sites along the lamina rara externa of GBM from humans with a condition associated with proteinuria (5). In addition to its role in filtration, HS proteoglycan may anchor cells to their substratum, as suggested by its presence as an integral membrane macromolecule (6,7) and as a component of fibroblast adhesion sites in other tissues (8)(9)(10)(11). This function may be partially fulfilled through interaction with specific peptide domains of fibronectin and laminin Despite their potentially great functional importance, GBM proteoglycans account for only a small proportion (3-5%) of total glomerular proteoglycan (17). Their study has been complicated by frequent contamination of GBM preparations with mesangial components (18). Previous studies have focused on biochemical characterization of the principal M , 13 X lo4 HS proteoglycan from these GBM preparations (2, 19).
However, DS is the dominant proteoglycan synthesized by intact isolated glomeruli after in vitro [35S]~ulfate labeling (20).* DS proteoglycan has been localized to the glomerular mesangium (21) where it may maintain tissue integrity as suggested by its ability to be cross-linked to fibronectin at fibroblast cell surfaces (22) and to mediate enhanced binding of fibronectin to collagen in other tissues (23). Since the HS proteoglycan content of isolated glomeruli is greater than that which can be accounted for by that found in GBM (20, 241, this proteoglycan must, like DS proteoglycan, be synthesized and incorporated into non-GBM glomerular components such as the mesangium and cell surfaces. This study describes the biochemical characteristics and metabolic processing of proteoglycans from normal rat glomeruli. Emphasis is placed on descriptions of DS proteoglycan synthesized by isolated glomeruli in vitro. A large high buoyant density self-associating DS proteoglycan accumulated in the tissue over 16 h without being released intact into the incubation medium. A lower molecular weight DS was the predominant proteoglycan synthesized during shorter labeling experiments. This proteoglycan and an HS proteoglycan with similar biochemical characteristics were rapidly released from the tissue. These proteoglycans were displaced from cell surfaces or tissue GAG receptors by treatment with heparin or trypsin, while the larger DS proteoglycan resisted these extractions. A second HS with little or no protein core resisted heparin extractions and was, therefore, thought to be intracellular. Glomeruli isolated after in vivo [35S]sulfate labeling contained different proportions of proteoglycans which had similar properties to those synthesized in vitro.  (25); Aquasol I1 and EN3HANCE, New England Nuclear; CsC1, Kawecki Berylco; ultrapure guanidine HCl, Schwarz/Mann; papain, Cooper Biomedical (Worthington). All other chemicals were of reagent grade or of the best available quality and were used without further purification.

Glomerular Isolation and P5S]Sulfate Labeling
Glomeruli were isolated from normal 250-g male Sprague-Dawley rat kidneys (Holtzman Co., Madison, WI) by serial sieving through wire meshes (26). Intact glomeruli were retained on 150 (100 pm2) and 200 (74 pm2) mesh screens and contained less than 5% tubular elements when examined by phase contrast microscopy. Glomerular cells excluded trypan blue both before and after in vitro incubations. Glomeruli from 10 rat kidneys were gently rocked (80 rpm on a junior orbit shaker (Lab-line Instruments, Inc.)) in 10 ml of RPMI 1640 medium containing 0.1 mM NazS04 and 250 pCi/ml Na235S04 at 37 "C in a humidified mixture of 95% air and 5% COZ. The incubation media were separated by gentle centrifugation (200 X g for 5 min), and, in the case of in vitro label-chase experiments, glomeruli resuspended in RPMI medium containing 0.5 mM NazS04 without NaZBSO4 after two washes in nonradioactive medium.
For in vivo experiments, groups of 10 rats were injected intraperitoneally either with 1 mCi NaZ35S04 per animal 4 h prior to sacrifice or with 0.5 mCi Na~%O~/animal every 4 h and were sacrificed 16 h after the initial injection.

Proteoglycan Extraction and Purification
4 M Guanidine HC1 Extractions-Tissue 35S-macromolecules were extracted by shaking glomeruli at 60 rpm in 8 volumes/volume wet glomeruli of an extraction buffer containing 4 M guanidine HC1,0.05 M sodium acetate, 1 mM iodoacetamide, 5 mM benzamidine HC1, 10 mM sodium EDTA, and 0.1 M 6-aminohexanoic acid, pH 5.8, for 24 h (27). When the residual tissue was re-extracted with the 4 M guanidine HC1-protease inhibitor extraction mixture without iodoacetamide but containing 10 mM dithiothreitol and the reaction terminated after 24 h with iodoacetamide (final concentration, 30 mM), an additional 2-5% of the total incorporated radioactivity was released. Thus, the majority of glomerular 35S-proteoglycans are not disulfide bonded to the guanidine HC1-insoluble glomerular matrix. %-GAGS in the tissue residue were released from the twice-extracted tissue by papain digestion (17). The incubation media were diluted 1:l with 8 M guanidine HCl extraction buffer containing 2 X protease inhibitors.
Heparin Extractions of Radiolabeled Glomer~li-~~S-Proteoglycans were displaced from cell surface or intercellular matrix "GAG binding sites" by two different heparin treatment protocols. In one experiment performed in duplicate, glomerular proteoglycans were released from the tissue fraction by a 30-min treatment with 2 mg/ml heparin in serum-free RPMI 1640 medium containing 0.1 mM Na2S04 at 37 "C (28). Separate samples of [35S]sulfate-labeled glomeruli were treated for 10 min with trypsin (50 pg/ml in serum-free RPMI 1640 medium) to release extracellular tissue 35S-proteoglycans (28). The latter reaction was stopped by washing the tissue twice with medium containing 50 mM phenylmethylsulfonyl fluoride and combining the washes with the trypsin-containing medium. 35S-Macromolecules remaining in the tissue after these treatments were extracted with 4 M guanidine HC1 as described above. Cell membranes were intact by phase contrast microscopy after heparin and trypsin treatments (28).
In the second extraction protocol, glomeruli were treated with 8 volumes of 0.05 M Tris, 0.1 M NaCl, and protease inhibitors (27) containing 100 pg/ml heparin, pH 8.0, for 1 h at 4 "C (6, 29), centrifuged (400 X g for 10 min), and washed twice in the heparin extraction buffer. The residual tissue 35S-macromolecules were extracted with 0.05 M Tris and 0.35 M NaCl (high salt buffer), followed by a third extraction in high salt buffer containing 0.2% CHAPS each at pH 8.0 and 4 "C for 1 h in the presence of protease inhibitors. Tissue 35S-macromolecules remaining after these three treatments were extracted with the 4 M guanidine HCl extraction buffer described above for 24 h at 4 "C.
CsCl Gradient Ultracentrifugation-4 M guanidine-extracted 35Sproteoglycans were separated by CsCl density gradient ultracentrifugation (0.44 g of CsCl/g of extract) at 40,000 rpm and 4 "C for 60 h using a Ti-70 rotor and a Beckman model L5-65 ultracentrifuge. The gradients were cut into bottom Y 5 (high buoyant density), middle %, and top Y 5 (low buoyant density) fractions using a Beckman tube slicer, and the fractions were dialyzed against at least five 6-liter changes of 0.5 M sodium acetate and 0.1 M sodium sulfate in the presence of protease inhibitors (27). In preparation for DEAE-Sephacel chromatography, the final dialysis solution contained 6 M urea, 0.05 M Tris, 0.1% CHAPS, 0.1 M NaCl, and protease inhibitors, pH 7.0 (DEAE buffer). Aliquots of each fraction were counted in a Beckman LS-7500 scintillation counter using Aquasol 2 as the fluorophore.
DEAE-Sephacel Chromatography-DEAE-Sephacel chromatographic columns (2-ml total bed volume) were equilibrated in DEAE buffer (see above) and preconditioned by loading with 2 mg each of chondroitin sulfate proteoglycan from the Swarm rat chondrosarcoma (30), heparin, and bovine serum albumin. The columns were then washed with 10 volumes of DEAE buffer and stripped with the same buffer containing 2 M NaC1. After re-equilibration in DEAE buffer, samples were loaded and the columns washed with 10 column volumes of starting buffer. A 100-ml linear gradient from 0.1 to 1.0 M NaCl in DEAE buffer was then applied and 2-ml fractions collected. Fractions from early (peak I) and late (peak 11) eluting peaks were separately pooled, dialyzed against two 4-liter changes of distilled water, and lyophilized. The molarity at which each peak eluted was estimated by comparing conductivities measured using a Radiometer conductivity meter with standards of 0.1-1.0 M NaCl in DEAE buffer. Recovery of radioactivity from DEAE-Sephacel columns was 85-95%.

Analytical Methods
Gel Filtration Chromatography-Gel filtration was carried out on 0.9 X 110-cm columns of Sepharose CL-4B and CL-GB equilibrated and eluted with 0.5 M sodium acetate containing 0.2% CHAPS, pH 7.0, at 3.0-4.0 ml/h with 1.0-1.2-ml fractions collected (associative conditions). Rat chondrosarcoma chondroitin-sulfate proteoglycan (30) in association with 4% hyaluronic acid and glucuronolactone were used to mark the column void ( Vo) and total (V,) volumes, respectively (31). To dissociate proteoglycan aggregates, samples were either eluted from Sepharose columns run in the presence of 4 M guanidine HC1 (dissociative conditions), or columns were run under associative conditions after reducing 35S-proteoglycan samples overnight with 10 mM dithiothreitol in 4 M guanidine HCl, pH 7.4, at 25 "C under nitrogen followed by alkylation with 40 mM iodoacetamide in the dark at 25 "C for 16 h (32). Recovery of radioactivity from Sepharose columns varied from 80 to 95%. Proteoglycan and GAG size were estimated using the data of Heinegird and Hascall (33) and that of Wasteson (34), respectively.
GAG Cleauage from Intact Prote~glycan-~~S-GAGs were released from the proteoglycan core protein by alkaline 8-elimination in 0.05 N NaOH and 1 M NaBH4 for 48-60 h at 45 "C. The reaction mixture was neutralized by the dropwise addition of acetic acid and desalted on SeDhadex G-50 columns (17, 27. 35). "S-GAGs were recovered from the column Vo.
Determination of HS and DS3-GAG Content and Chain si~e-'~S-GAGS were subject to nitrous acid deaminative cleavage by methods modified from Conrad et al. (17,36). Insensitive (Vo) 35S-GAGs were digested with chondroitinase ABC or AC. When present, 35S-macromolecules excluded from Sephadex G-50 columns after both nitrous acid and chondroitinase ABC treatments were further digested with highly purified heparitinase (25) to cleave N-acetylated regions of HS. Equivalent results were obtained starting with either alkalireleased 35S-GAG or intact 35S-proteoglycans and were independent of the sequence of digestion. HS proteoglycan content was determined by percent sensitivity to nitrous acid and heparitinase while DS proteoglycan was that portion susceptible to chondroitinase ABC degradation. Material excluded from or partially included in Sephadex G-50 columns (Kav < 0.1) after the entire degradative sequence including heparitinase treatment was designated "sulfated glycoprotein" (35). This material has been previously shown to be insensitive to keratinase (17).
35S-GAGs present in the Sephadex G-50 VO after nitrous acid treatment were used for determining DS chain size on Sepharose CL-6B columns, while HS size was characterized using the chondroitinase ABC-insensitive 3SS-GAG.
Agarose-Polyacrylamide Gel Electrophoresk-Electrophoresis in 0.6% agarose-1.8% polyacrylamide gels was performed according to a method modified from the work of McDevitt and Muir (27,37). Rat chondrosarcoma chondroitin sulfate proteoglycan ( M , 2.6 X lo6 (30)) and chondroitin 4-sulfate (Mr 20,000) were used as markers. Labeled proteoglycans were detected by fluorography after impregnating gels with EN3HANCE. "R," was the ratio of the distance from the origin to an autoradiographic band as compared to the distance migrated by the chondroitin sulfate proteoglycan marker. The distance migrated by a proteoglycan varied between gels, but its position relative to markers (R,) showed little variation.
Chondroitin 4% Sulfate Ratios-The sulfation pattern of DS GAG was determined by paper chromatography after chondroitinase ABC or AC digestion and expressed as chondroitin 4 to 6 sulfate ratios (38).
Periodate Oxidation and Alkaline Cleavage-L-Iduronic acid residues lacking 2-0-sulfate substitution within DS chains were selectively cleaved by oxidation in 20 mM sodium metaperiodate and 50 mM sodium citrate, pH 3.0, at 4°C in the dark for 24 h using 2 mg/ ml pig skin DS as carrier (39,40). Addition of 20 mM sodium perchlorate to the reaction mixture during the oxidation step did not change the Sephadex G-50 elution profile (results not shown). The reaction was terminated by addition of a 20-fold molar excess of ethylene glycol. After dialysis against 2 changes of 2 liters of distilled water, the oxidized chains were cleaved at room temperature by adjusting the pH to 12 with 1 N NaOH. The reaction was stopped after 30 min by neutralization with 1 M acetic acid. The reaction products were applied to a 0.9 X 110-cm Sephadex G-50 column, and 1-ml fractions were collected. Oligosaccharides obtained after testicular hyaluronidase or chondroitinase AC digestion of rat chondrosarcoma chondroitin sulfate were used to calibrate the column. Periodate oxidation-alkaline cleavage products were detected in the Sephadex G-50 column void volume by eluting these oligosaccharides from Sepharose CL-GB columns.

Total Glomerular [3sS]Sulfate Uptake in Vitro
Glomeruli isolated from normal rats were incubated in serum-free medium for either 4 or 16 h. Total incorporation of Na235S04 into glomerular and incubation medium macromolecules was linear over this time (Table I). 35S-Macromolecules appeared progressively in the incubation medium. This fraction contained 41 f 3.1% of the total [3sS]~ulfate incor-porated after 4 h and 58 f 7% after 16 h. Extraction with a 4 M guanidine HC1 buffer containing protease inhibitors released at least 95% of tissue 35S-macromolecules.
Proteoglycans were separated from the majority of glomerular proteins and glycoproteins by CsCl gradient ultracentrif-~g a t i o n .~ 35S-Glycoproteins were found predominantly in the top */5 CsCl gradient fractions (Table I). In contrast, 35Sproteoglycans were found mostly in high buoyant density CsCl gradient fractions. This gradient fraction contained 95% of total glomerular 35S-proteoglycans after 4 h and 84% after 16 h i n uitro. Characterization of these high density 35S-proteoglycans, especially a previously uncharacterized tissue DS proteoglycan, was emphasized in these studies. The biochemical characteristics of high density glomerular proteoglycans described in detail below are summarized in Table II (Table I). This gradient fraction contained a mixture of HS and DS, with only 6% of this gradient fraction being 35S-glycoprotein. Before DEAE-Sephacel chromatographic purification, these high density 35S-proteoglycans eluted from Sepharose CL-GB columns as a partially excluded and a diffuse included peak (Fig. 1A). In contrast, when Stow et al. (20) incubated isolated rabbit glomeruli for 16 h in serum-containing medium, no Sepharose CL-GB-excluded 35S-proteoglycans were detected. We, therefore, performed additional 16-h isolated glomerular incubations in medium containing either 10% fetal calf serum or 5 ng/ml insulin (41). Total [35S]sulfate incorporation was not altered under these conditions, and Sepharose CL-GB-excluded 35S-proteoglycans were still present in high density CsCl gradient fractions (data not shown).

A ) .
Two HS proteoglycans (defined by sensitivity to nitrous acid cleavage or heparitinase digestion (see "Methods")) were present in DEAE peak I. HS-tIA eluted from Sepharose CL-GB columns within the Kay 0.24 peak ( M r 13 X lo4), and HS-tIB eluted at K,," 0.68 ( M , 8.6 x lo3) (Fig. 3A). Proteoglycans with similar elution volumes (Kav 0.30 and 0.68, respectively) were observed when high density glomerular 35S-macromo1ecules (prior to DEAE purification) were chromatographed after chondroitinase ABC digestion (Fig. 1B). After enzyme digestion, pooled radioactivity from Sepharose CL-GB-included peaks contained exclusively HS, while DS oligosaccharides appeared in the column V,.
' Proteoglycan nomenclature was based on: (a) their GAG component, either HS or DS; (b) whether the 35S-proteoglycan was extracted from the glomerular tissue (t) or was released into the incubation medium (m); ( e ) their elution position from DEAE-Sephacel columns (with DEAE peak I or peak 11); and (d) their relative hydrodynamic size analyzed by Sepharose CL-GB chromatography (A or B).   Dermatan sulfate (DS) and heparan sulfate (HS) proteoglycans were synthesized in vitro by glomeruli isolated from normal rats. Bottom V 5 CsCl gradient fraction proteoglycans from glomerular extracts (9) and incubation media (m) were further purified by DEAE-Sephacel chromatography. 35S-Macromolecules eluting in early (I) and late (11) portions of the linear salt gradient were pooled separately. Intact proteoglycan (2) and GAGS (3) were characterized by elution from Sepharose CL-GB columns (tissue fraction DEAE peak I contained two peaks, A and B) and by electrophoresis in 0.6% agarose-1.8% polyacrylamide gels (4). Dermatan sulfate chains were analyzed for the ratio of 4% sulfate groups after chondroitinase ABC digestion (5) and for the spacing of iduronic groups by susceptibility to periodate cleavage (pH 3.0, 4 "C) and alkaline elimination (6).
' These proteoglycans eluted predominantly in the front half of the Sepharose CL-GB peak.
Average molecular weight estimated from Sepharose CL-4B profile.
Migration in 0.6% agarose-1.8% acrylamide gels relative to an M, 2.6 X lo6 chondroitin-SO4 proteoglycan. (results not shown), while HS-tIB appeared in gels as a after alkaline borohydride treatment (results not shown). The chondroitinase ABC-resistant band in a position similar to hydrodynamic size of glomerular HS-tIA proteoglycan was that of the chondroitin sulfate marker (Fig. 3A, inset, lunes c similar to previously described rat GBM or rabbit glomerular and d). HS-GAG chains from HS-tIA had an M , of 12.5 x lo3 HS proteoglycan (2, 20), while HS-tIB resembled it in its size (Sepharose CL-GB K,," 0.58, Fig. 4A). HS-tIB contained no and resistance to alkaline borohydride treatment intracellular detectable protein since the elution volume did not change heparan sulfate from rat granulosa cells (42). DS proteoglycan, DS-tIA, was also present in DEAE peak I as determined from the partial sensitivity (46%) of alkaline borohydride-released 35S-GAGs from this peak to chondroitinase ABC digestion (Fig. 4A). DS-tIA had properties similar to glomerular HS-tIA proteoglycan. These proteoglycans both eluted from Sepharose CL-GB columns at K., = 0.24 and migrated as a single band in agarose-polyacrylamide gels (Fig.  3A, inset, lunes a and 6). The 35S-proteoglycan in this Sepharose peak did not appear to be a HS-DS hybrid proteoglycan since HS proteoglycan did not change position on agarosepolyacrylamide gel electrophoresis when samples were pretreated with chondroitinase ABC (Fig. 3A, inset, lune b). This is consistent with DS-tIA being a separate molecule although similar in hydrodynamic size and overall charge to HS-tIA. Indeed, 35S-GAG chains from this proteoglycan were larger than 35S-HS from HS-tIA proteoglycan (KaV 0.5, M , 20 X lo3,  A large DS proteoglycan (DS-tII) was present in DEAE peak 11. DS-tII was partially excluded from Sepharose CL-GB columns (Fig. 3B) and migrated as a chondroitinase ABCsensitive band to an R,; of 1.7 in agarose-polyacrylamide gels (compare Fig. 3B, inset, lanes u and b). When applied to Sepharose CL-4B columns run under associative conditions (see "Experimental Procedures"), DS-tII had a larger apparent hydrodynamic size (Fig. 5A, K., 0.29) than when eluted from a similar column in 4 M guanidine HC1 (Fig. 5B, K,, 0.48). The latter conditions would dissociate proteoglycans aggregated on a hyaluronic acid backbone (32) or those associated through interaction of GAG chains (43). To distinguish between these possibilities, DStII from the dissociative Sepharose CL-4B K,,, 0.48 peak was pooled, exhaustively dialyzed against distilled water, and lyophilized. The retentate was reduced and alkylated in the presence of 4 M guanidine HC1, conditions which denatured the hyaluronic acid binding region of cartilage proteoglycan protein core (32). The reduced and alkylated products eluted from an associative Sepharose CL-4B column in a position similar to untreated DS-tII (  A (lanes a and b) and B (lanes c and d). Equivalent amounts of radioactivity were electrophoresed before (lanes a and e) and after (lanes b and d)  aggregates through GAG chain-chain interaction or through interactions of the protein core not dependent for structural integrity on disulfide bond formation.
DS chains were released from the DS-tII proteoglycan protein core by alkaline &elimination, eluting with the V, , of Sephadex G-50 columns after nitrous acid treatment (see "Methods"). These chains had an apparent molecular weight of 26 X lo3 on Sepharose CL-GB (Kav 0.43, Fig. 4B) and were sulfated primarily at the 4 position of galactosamine, with a C4 to 6 sulfate ratio of 1.95:l (Table 11). The presence of iduronic acid within DS-tII 35S-GAG was evident from an 11% difference in sensitivity to chondroitinase ABC and chondroitinase AC digestions. This small difference was similar to that previously reported for DS from isolated rat glomeruli (24).
The presence of iduronic acid residues within DS-tII GAG was confirmed by oxidation of iduronic acid residues lacking 2-0-sulfate groups with periodate at pH 3.0 and 4 "C followed by alkaline cleavage (39, 40). After this treatment, a significant proportion (33%) of the reaction products was included in Sephadex G-50 columns (Fig. 6A). DS chains remaining in the column void volume were thought to be derived from unmodified regions of the DS chains. When rechromatographed on Sepharose CL-GB, these periodate-treated and alkaline-cleaved DS chains were more retarded (Kav 0.64, Fig.  6C) by the column than the original 35S-GAGs (Kav 0.43, Fig.  4B). When these periodate-resistant 35S-GAGs were treated with chondroitinase AC to confirm their derivation from unmodified chondroitin sulfate-rich regions, 10-20% of the reaction products were included in Sepharose CL-GB columns, eluting at K., 0.73 (Fig. 6D). The presence of 2-0-sulfated iduronic acid residues in DS-tII may account for this partial resistance to chondroitinase AC digestion after periodate oxidation and alkaline cleavage. 35S-GAGs were released by alkali under reductive conditions (see "Methods"). HS-tIA %-GAG was chromatographed on Sepharose CL-GB after digestion of glomerular extract DEAE peak I, Sepharose CL-GB peak A (Fig. 3A) with chondroitinase ABC (A). Oligosaccharides in the column V, after this treatment demonstrate that DEAE peak I contained a mixture of HS and DS proteoglycans. In B, %-GAGS from DS-tII (Fig. 3B, peak A) were chromatographed on Sepharose CL-GB, while C shows the elution profile of DS-tIA =S-GAG. The latter =S-GAG was isolated by treatment of DEAE peak I, Sepharose CL-GB peak A (=S-proteoglycans (Fig. 3A, peak A) with nitrous acid, and desalting on Sephadex G-50 (see "Methods"). DS-mII GAGS were purified from 35S-proteoglycans in Fig. 30, peak A, in a similar manner, and their elution profile is illustrated in D.
DS-tII GAG was further characterized by Sephadex G-50 chromatography after chondroitinase AC digestion. Disaccharides accounted for 59% of the chondroitinase AC digestion products of DS-tII GAG chains (Fig. 6B). Iduronic acid-rich sequences resistant to chondroitinase AC digestion eluted predominantly as tetrasaccharides (27%). Tetrasaccharides may result from digestion of regions along DS chains containing regularly spaced iduronic acid residues (occurring approximately every third repeat disaccharide unit). Longer chondroitinase AC-insensitive products, which eluted prior to the tetrasaccharide peak from Sephadex G-50 columns and constituted 14% of the total column radioactivity, would originate from regions containing more closely spaced iduronic acid moieties.
Partial Localization of Glomerular Proteoglycans by Heparin and Trypsin Treatment-35S-Proteoglycans noncovalently bound to tissue GAG binding sites were released using two different previously described heparin treatment protocols (6,28). Glomeruli were treated either with 2 mg/ml heparin in RPMI 1640 medium for lh h at 37 "C or with 100 pg/ml heparin in 0.05 M Tris buffer, pH 8.0, at 4 "C followed by detergent extraction. Residual glomerular 35S-macromolecules were released with 4 M guanidine HCl. 35S-Proteoglycans in each of these extracts were compared to those extracted with 4 M guanidine HCl directly (total tissue extracts).
Each heparin extraction protocol released similar proportions of total'tissue-associated radioactivity (33% with heparin in medium and 36% with heparin in Tris buffer, Table  111). This proportion was greater than that released upon exposure to medium alone (21%). A greater proportion of total glomerular h e~a r a n -~~S O~ proteoglycan was released by heparin in medium (47%) than by medium without heparin (31%). HS-tIA was the only HS proteoglycan released by heparin (Fig. 7A). No HS-tIB was detected in these extracts. Instead, the latter was detected when the heparin-treated tissues were extracted with 4 M guanidine HCl (Fig. 7C).
These observations point to the possibility that HS-tIA is a cell surface/matrix GAG receptor-bound HS proteoglycan while HS-tIB is intracellular. However, the second protocol which was carried out using heparin in Tris buffer, pH 8.0, at 4 "C with a subsequent detergent treatment released tissue 3sS-proteoglycans different from extractions with heparin in medium. Using this protocol, both HS-tIA and HS-tIB proteoglycans were released from the tissue. These 35S-proteoglycans eluted from Sepharose CL-GB columns at K,, values of 0.3 and 0.7 (not shown) and migrated to R, values of 2.3 and 3.9 in agarose-polyacrylamide gels (Fig. 8, lanes a-d). This is consistent with the heparin in Tris buffer protocol disrupting glomerular cell integrity and releasing both cell surface and intracellular glomerular proteoglycans in contrast to the reported specificity of this treatment in characterization of rat liver heparan sulfate (6).
DS proteoglycan was also released from the tissue by heparin in medium (Fig. 7B). As was true for HS proteoglycan, a greater proportion of total tissue DS proteoglycan was released by heparin in medium (28%) than was released by treatment with medium alone (19%). The hydrodynamic size of the major heparin-releasable DS proteoglycan as well as its 3sS-GAG chains (Kav 0.2 and 0.5, respectively) were similar to DS-MA. DS-tII proteoglycan, on the other hand, was only extracted with 4 M guanidine HC1 after heparin and detergent treatments (Fig. 70). Like the molecule released in the total guanidine HC1 extract, this proteoglycan was partially excluded from Sepharose CL-GB columns and migrated as a diffuse R, 1.8 chondroitinase ABC-sensitive autoradiographic band in agarose-polyacrylamide gels (Fig. 8, lunes g and h). Since most DS-tII proteoglycan resisted heparin and detergent treatments, it may be more tightly bound within the glomerular matrix. Thus, heparin did not preferentially release either HS or DS proteoglycan but displaced a specific subtype of each proteoglycan, HS-tIA and DS-tIA proteoglycans, from glomerular cell surfaces or intercellular matrix GAG binding sites.
Trypsin treatment without prior heparin extraction released 46% of total tissue-associated radioactivity (Table 111). These 35S-proteoglycans eluted from DEAE-Sephacel columns as two separate peaks at 0.34 and 0.47 M NaCl in DEAE buffer. Most (71%) tissue-associated h e~a r a n -~~S O~ proteoglycan was released by trypsin, accounting for all extracellular HS proteoglycan. Trypsin-released HS proteoglycan in DEAE peak I eluted later than its heparin-extracted counterpart from Sepharose CL-GB columns (ICav 0.4, Fig. 7E). As expected, HS-GAG from this peak was similar in length to that released by heparin (Kay 0.58, not shown). Thus, there are trypsin-sensitive sites within the protein core of extracellular HS proteoglycan. HS-tIB was not released from the tissue by trypsin treatment but was detected in the guanidine HClextracted residual tissue (KaV 0.7, Fig. 7G). Tissue DS proteoglycan fragments released by trypsin eluted from DEAE-Sephacel in peak I1 and from subsequent Sepharose CL-GB columns at K,, values of 0.2 and 0.4 (Fig. 7F), with both 35Sproteoglycans having GAGS similar in length to DS-tIA (Kay 0.5, not shown). On the other hand, DS-GAG from DEAE peak I1 3sS-proteoglycans remaining in the tissue after trypsin treatment (Fig. 7H)   (Sepharose CL-GB K., 0.43, not shown). Therefore, as occurred with heparin treatment, DS-tIA and HS-tIA were accessible to trypsin, while intracellular HS-tIB and tissue DS-tII resisted these treatments.
Partial Characterization of Low Buoyant Density 35S-Proteoglycan-Twenty-four percent of total tissue fraction 35Smacromolecules and 16% of total tissue 3sS-proteoglycans were in the top 2/5 CsCl gradient fraction after 16-h in uitro incubations (Table I). 35S-Glycoproteins in this fraction did not bind to DEAE-Sephacel columns (Fig. 9A), while 35Sproteoglycans eluted from the columns at 0.18 (peak I) and 0.34 M NaCl (peak 11). Peak I 35S-proteoglycans migrated in agarose-polyacrylamide gels as a doublet with R , values of 2.3 and 2.6 which was insensitive to chondroitinase ABC digestion (Fig. 9A, inset, lanes a and b). Peak I1 35S-proteoglycans migrated to an R, of 1.3 in gels (Fig. 9A, inset, lane c) and were partially sensitive to chondroitinase ABC digestion (Fig. 9A, inset, lane d). Similar low density 3sS-proteoglycans were released into the medium (Fig. 9B). Insufficient radioactivity was present in top gradient fractions for further analysis.

Incubation Medium 35S-Macromolecules
The medium fraction was examined for the presence of proteoglycan species different from those found in tissue fractions. The majority (72 f 2%) of 35S-macromolecules synthesized by glomeruli over 16 h in uitro and released into the incubation medium were recovered from the bottom 2/5 CsCl gradient fractions (Table I). This fraction contained both HS and DS proteoglycans which eluted from DEAE-Sephacel columns as two partially resolved peaks. These peaks eluted at 0.44 M (29%, peak I) and at 0.55 M NaCl(71%, peak 11, Fig. 2B) with no 35S-glycoprotein detected in the column wash. DEAE peak I 3sS-proteoglycans eluted from Sepharose CL-GB columns as a single peak (Kav 0.22, Fig. 3C) which contained both HS (40%) and DS (60%) proteoglycans, while DEAE peak I1 35S-proteoglycans eluted earlier from these columns (K,, 0.14, Fig. 3 0 ) and contained mainly (86%) DS proteoglycan.
Two HS proteoglycans were present in medium fractions.
These DEAE peak I proteoglycans migrated faster in agarosepolyacrylamide gels (R, values of 2.8 and 3.8. Fig. 3C, inset, lanes a and c ) than those from tissue (Fig. 3A, inset, lane a) and were partially resolved from each other by pooling the front or back halves of the K., 0.22 Sepharose CL-GB peak prior to gel electrophoresis (Fig. 3C). There was also a small amount of HS proteoglycan in DEAE peak I1 which formed a shoulder following the major K,, 0.14 DS proteoglycan peak (see below). HS chains from medium DEAE peak I proteoglycans had an M, of 12.5 X lo3 (Sepharose CL-GB K,, 0.58, not shown). This GAG chain length was similar to that of HS from extracellular tissue fraction proteoglycan.
Medium DS proteoglycan from DEAE peak I1 (DS-mII), after purification by Sepharose CL-GB chromatography, migrated in gels as a single band at R, 2.8 (Fig. 30), a position similar to that of medium HS proteoglycan. No small single chain DS was detected. DS-mII GAG chains had an M , of 18 X IO3 (Sepharose C1-6B Kay 0.53, Fig. 40) and were sulfated primarily at the 4 position of galactosamine, with a C4 to 6 sulfate ratio of 2.32:l (Table 11). These properties were similar to those described above for DS-tIA (see "Tissue Extraction with 4 M Guanidine HCl and Protease Inhibitors"). Like tissue fraction DS-tII, these GAGS were 11% less sensitive to chondroitinase AC than to chondroitinase ABC digestion, suggesting the presence of iduronic acid residues. DS-mII GAG was less sensitive (26%) to periodate oxidation/alkaline cleavage than DS-tII (Fig. 6F) and had a lower disaccharide to tetrasaccharide product ratio (0.9:l) than DS-tII GAG (21) after chondroitinase AC digestion (Fig. 6E), suggesting that DS-mII had more regularly spaced iduronic acid residues than DS-tII GAG.
cleavage eluted with the Sephadex G-50 void volume shown by the bar in A. These 35S-GAGs, which eluted at K., 0.43 before alkaline periodate treatment (Fig. 4B), were rechromatographed on Sepharose CL-GB both before (C) and after (D) chondroitinase AC digestion. Di-and tetrasaccharides eluted from Sephadex (2- 50 columns (A, B, E,  and F) 16 h in uitro were extracted with medium containing 2 mg/ml heparin for 30 min or with 50 Gg/ml trypsin for 10 min each at 37 "C (see "Methods"). The residual tissues were extracted with 4 M guanidine HCI in the presence of protease inhibitors. 35S-Proteoglycans released by each treatment were purified by DEAE-Sephacel chromatography, eluting as two partially separated peaks (I and 11, see Table 111). Sepharose CL-GB chromatographic profiles of 35S-proteoglycans released by heparin in medium are shown in A (DEAE peak I) and B (DEAE peak 11), while 35S-proteoglycans released by guanidine HCI extraction of the heparin-treated tissues are illustrated in C (DEAE peak I) and D (DEAE peak 11). Trypsin treatment released 35S-proteoglycans with Sepharose CL-GB elution profiles illustrated in E (DEAE peak I) and F (DEAE peak 11). 35S-Proteoglycans remaining in the tissue after this treatment eluted from Sepharose CL-GB as shown in G (DEAE peak I) and H (DEAE peak 11).

Glomerular Proteoglycans Synthesized during 4-h in Vitro Incubations
Since proteoglycans may be synthesized and degraded at different rates, the effect which time of incubation had on the type and percent of proteoglycan species present was next determined. After 4-h in vitro incubations, 95% of [35S]su1fate-labeled glomerular macromolecules were found in the bottom 2/5 CsCl gradient fractions. These proteoglycans eluted as two peaks from Sepharose CL-GB columns with K., values of 0.21 which contained both HS and DS proteoglycans (85% of the total eluted radioactivity) and 0.63 (15%) without prior DEAE-Sephacel purification (Fig. 1OA). HS proteoglycan accounted for 30% of high density tissue proteoglycan after 4 h in vitro (the remainder was DS proteoglycan) and eluted mainly with the second half of the K., 0.21 Sepharose CL-GB peak (38% of the pooled contents of this portion of the peak were sensitive to nitrous acid, while only 12% of earlier eluting proteoglycans were HS). Like HS-tIA, this proteoglycan eluted in DEAE peak I (0.3 M NaCl, Fig. llA), migrated to an R, of 2.3 in agarose-polyacrylamide gels (Fig. llA, inset, lune a), and had GAG chains with an M , of 12.5 x lo3 (Sepharose CL-GB K., 0.59).
No Sepharose CL-GB-excluded tissue DS proteoglycan similar to DS-tII from 16-h in vitro incubations was detectable after 4 h in vitro. DS proteoglycan eluted from DEAE-Sephacel columns with peak I1 (Fig. 1lA) and in the front half of the K., 0.21 Sepharose CL-GB peak. It migrated as two chondroitinase ABC-sensitive bands with R, values of 2.1 and 2.8 in agarose-polyacrylamide gels (Fig. 1L4, inset, lane  6). DS chains from 4-h tissue fraction proteoglycans had an average molecular weight of 18 X lo3 (Sepharose CL-GB K., 0.52) and a C4 to C6 sulfate ratio of 2.3:l (Table 11). After chondroitinase ABC digestion, a minor disaccharide component (15%) migrated more slowly than chondroitin 6-sulfate standards on paper chromatography, possibly representing chondroitin 4,g-sulfate or DS sulfated at the 2 position of iduronic acid (44). Tissue DS proteoglycan from 4-h in vitro incubations contained iduronic acid-rich regions as demonstrated by an 11% difference in sensitivity to chondroitinase ABC and AC digestions. It, therefore, resembled DS-tIA and DS-mII proteoglycans in hydrodynamic size, GAG chain length, iduronic acid content, as well as in C4 to C6 sulfate ratio. DS-tIA may be rapidly synthesized and released into medium, while DS-tII accumulates slowly in tissue, becoming detectable if the rapidly synthesized molecules are chased from the tissue. This hypothesis was tested in label-chase experiments described below.

Label-Chase Experiments
Glomerular macromolecules were [35S]sulfate labeled over 4 h in vitro and then chased for 2 h into medium containing unlabeled 0.5 mM Na2S0,. In three separate chase experiments (each run in duplicate), 31 f 1.6% of radioactivity originally incorporated into glomerular tissue was released into the medium. The Sepharose CL-GB elution profiles of proteoglycans from the pulse-labeled glomeruli and from the chase medium showed similar K., 0.21 chromatographic peaks (compare Fig. 10, A and C ) . HS-tIB, the K,, 0.63 intracellular proteoglycan, was not detected in the medium. 35S-Proteoglycans remaining in glomeruli after the chase eluted earlier (Knv of 0.12, Fig. 10B) than those from the pulse-labeled tissue fraction. Thus, lower molecular weight proteoglycans were rapidly cleared from the glomerulus, allowing the detection of a population of higher molecular weight tissue proteoglycans.

I n Vivo 35S-Proteoglycan Synthesis
In order to determine whether artifacts were introduced by in vitro incubation, in vivo [35S]sulfate-labeled proteoglycans were isolated. Glomeruli synthesized similar 35S-proteoglycans in vivo and in vitro. Maximal in vivo [35S]~ulfate incorporation into proteoglycan occurs 4 h after single intraperitoneal Na:5S04 injections (17). Glomeruli isolated after this time period contained 35S-macromolecules whose CsCl gradient distribution was similar to that seen after 4-h in vitro incubations (Table I) (Table I).
When rats received multiple intraperitoneal injections of Na235S04 over 16 h (see "Methods"), a greater proportion (62 f 0.5%) of 35S-macromolecules were present in the top 2/5 (less buoyantly dense) CsCl gradient fractions than 4 h after a single injection (17% , Table I), with 35S-glycoproteins accounting for 11% of the latter gradient fraction (Fig. 12B,  excluded peak). This change to less buoyantly dense CsCl gradient fractions after longer exposures to [35S]sulfate was similar to that seen after corresponding in vitro labeling periods (compare 4-and 16-h CsCl gradient distributions in Table I).
By far the major high density proteoglycan was HS-tIA,  (Table 111). Sepharose CL-GB chromatographic profiles of 35S-proteoglycans from each pooled DEAE peak were similar to their counterparts illustrated in Fig. 3. Equivalent quantities of radioactivity from each pooled Sepharose CL-GB chromatographic peak were subject to electrophoresis in 0.6% agarose-1.8% polyacrylamide gels both before (lanes a, c, e, and g) and after (lanes b, d, f, and h) chondroitinase ABC digestion. Heparin-extracted HS-tIA proteoglycan was electrophoresed in lunes a and b. HS-tIB (lanes c and d ) was also extracted by heparin under these conditions. Additional HSdIA proteoglycan was released from the tissue by detergent treatment (lanes e and fl, while DS-tII (lanes g and h) was released only after the heparin-and detergent-treated tissue was extracted with guanidine HCl. which eluted from DEAE-Sephacel columns at 0.4 M NaCl (Fig. 12A, DEAE peak I) and migrated as a chondroitinase ABC-resistant band to an R, of 2.3 in subsequent agarosepolyacrylamide gels (Fig. 12A, inset, lunes u and b). DEAE-Sephacel-purified high density proteoglycans synthesized over 16 h in vivo eluted as a single K,, 0.26 peak from Sepharose CL-GB columns which was composed largely (70%) of HS proteoglycan (Fig. 13A). HS-tIA (Fig. 13A, inset, lunes u and  b) as well as HS-tIB (Fig. 13A, inset, lunes c and d ) proteoglycans were detected in this peak. A minor slower migrating HS proteoglycan (R, 1.1) not detected in vitro was also present in this gradient fraction (Fig. 12A, inset, lanes u and b).
Several large HS proteoglycans were present in the top 2/5 CsCl gradient fractions. These proteoglycans eluted from DEAE-Sephacel columns as two included peaks at 0.11 and 0.35 M NaCl (Fig. 12B, peak I). After DEAE purification, these proteoglycans eluted from Sepharose CL-GB columns with the void volume and as diffuse included peaks (Fig. 13,   B and C). 35S-Proteoglycans eluting at 0.11 M NaCl from DEAE columns were partially excluded from Sepharose CL-6B (Fig. 13B) and migrated as a chondroitinase ABC-resistant band slightly slower than rat chondroitin sulfate proteoglycan standard in agarose-polyacrylamide gels (Fig. 13B, inset, lunes  u and b). 3sS-Proteoglycans eluting at 0.35 M NaCl migrated as two chondroitinase ABC-resistant bands in gels run both before (R, 2.2 and 2.8, Fig. 12B, inset, lunes u and b) and after Sepharose CL-GB chromatography (Fig. 13C, inset). Further characterization of HS proteoglycans was limited by low uptake of radioactivity in vivo.
The DS proteoglycan DS-tII was detected in the bottom 2/5 CsCl gradient fraction, eluting as a minor 0.47 M NaCl peak from a DEAE column (Fig. 12A, peak 11). This 35S-proteoglycan co-migrated with DS-tII from 16-h in uitro incubations in gels (compare Fig. 12A, inset, lune c with lune e) and was sensitive to prior chondroitinase ABC digestion (Fig. 124,  inset, lune f). DS chains cleaved from this proteoglycan had an average molecular weight similar to those synthesized in uitro (Ka, 0.42, M , 26,000). Low density DS proteoglycan synthesized in vivo also eluted from a DEAE column at 0.47 M NaCl (Fig. 12B, DEAE peak 11) and migrated slightly slower than high density DS-tII proteoglycan in agarosepolyacrylamide gels (compare Fig. 12B, inset, lanes c and e ) . labeled in vivo than was extracted from glomeruli isolated after in uitro labeling, but they had similar biochemical properties.

DISCUSSION
Rat glomeruli synthesized a mixture of HS and DS proteoglycans. A high density M, 13 x lo4 HS proteoglycan (HS-tIA) with M, 12.5 x lo3 GAG chains was extracted from glomeruli [35S]sulfate labeled both in uiuo and in uitro. This proteoglycan was similar in hydrodynamic size to that previously reported in extracts of rat and rabbit glomeruli as well as isolated rat, rabbit, and bovine GBM (3,20,24,45). However, GAG chain length estimates varied in these studies with those from in uitro [35S]sulfate-labeled isolated rabbit glomeruli and GBM (20) as well as from bovine GBM (19,45) having lengths by Sepharose CL-GB similar to those described in our studies. However, Kobayashi et al. (24) isolated HS chains from intact rat glomeruli which had an estimated Glomerular HS proteoglycan has been localized to the GBM by immunofluorescence microscopy and JY electron microscopic autoradiography (21,25,46). Antibody to intact rat glomerular HS proteoglycan failed to stain the mesangium or cell surfaces. This anti-HS proteoglycan core protein antibody also stained rough endoplasmic reticulum and Golgi apparatus within glomerular epithelial cells, suggesting that this cell was a source of GBM HS proteoglycan. However, antibody to bovine aorta HS proteoglycan core protein stained both the epithelial and endothelial aspects of GBM (25), and one high density HS proteoglycan synthesized by aortic endothelial cells in culture had an M, (13 X lo4) similar to that from isolated glomeruli (47). Therefore, endothelial cells cannot be eliminated as a source for GBM HS proteoglycan. A mesangial location for HS proteoglycan was suggested by studies which showed that total GBM HS could not account for the total glomerular HS content (17). In addition, anti-aorta HS proteoglycan antibody-stained mesangium (25) and heparitinase but not chondroitinase ABC treatment of tissue prior to electron microscopic autoradiography resulted in a partial loss of mesangial grains (21). Studies reported here describe the release of HS-tIA proteoglycan from tissue by heparin in medium, with an M , 8,600 HS (HS-tIB) having little or no protein core extracted from the remaining tissue by guanidine HCl. HS-tIB had properties similar to the intracellular HS described by Yanagishita and Hascall (42) in granulosa cells and may represent a product of proteoglycan endocytosis and degradation. However, HS-tIB was released by heparin extractions carried out in Tris buffer, indicating that this treatment, which releases liver cell surface heparan sulfate proteoglycan (6), may disrupt glomerular cell integrity. A possible cell surface location for heparinreleased proteoglycans was suggested by studies describing staining of glomerular epithelial and endothelial cell surfaces with anti-hepatocyte cell membrane HS proteoglycan antibody (48). Heparin-displaceable HS proteoglycan may bind to cell surfaces and/or tissue "GAG receptors" through interaction with specific domains of other extracellular matrix macromolecules such as fibronectin, type IV collagen, or laminin. Mild trypsin treatment released all extracellular HS proteoglycan, with only HS-tIB remaining in the tissue. Trypsin-released HS proteoglycan fragments were smaller (Sepharose CL-GB Ka., 0.42, M , 46,000) than HS-tIA but had similarly sized GAGs. Since more HS proteoglycan was released by trypsin than heparin, it is possible that a portion of glomerular HS proteoglycans is intercalated in cell membranes or bound to tissue receptors inaccessible to heparin.
Although HS was the predominant proteoglycan extracted from glomeruli [3sS]sulfate labeled in vivo and from GBM (17), DS accounted for 60% of high density glomerular macromolecules synthesized over 4 or 16 h in vitro (Table I). The proportion of glomerular 35S-macromo1ecules which were DS proteoglycan has varied between studies depending on incubation conditions. Glomeruli perfused ex situ with Na,3%04 synthesized a greater proportion of HS (85%) (3), while DS was the predominant proteoglycan extracted from in vitro "S04-labeled isolated glomeruli (75% (20) and 38% (24)).
Newly synthesized DS proteoglycans from in vivo and ex situ studies may be preferentially lost during the glomerular isolation process, while proteoglycans ["S]sulfate labeled in vitro were not subject to the serial sieving process prior to extraction. However, autoradiographic studies identified few, mostly mesangial, grains attributable to DS proteoglycan (21). It is, therefore, possible that glomeruli incubated in vitro may alter their proteoglycan phenotype as a reaction to incubation conditions or based on the preferential survival of cell populations responsible for DS proteoglycan synthesis. Two major DS proteoglycans were synthesized by isolated glomeruli in vitro. A small DS proteoglycan (DS-tIA) copurified with HS-tIA by CsCl density gradient ultracentrifugation, DEAE-Sephacel and Sepharose CL-GB chromatography, as well as by agarose-polyacrylamide gel electrophoresis (DS-tIA was detected at each purification step by partial sensitivity to chondroitinase ABC digestion). However, DS-tIA may be slightly larger than HS-tIA since it eluted predominantly with the front half of the K., 0.21 Sepharose CL-GB peak after 4-h in vitro incubations (Fig. lOA), and, after chondroitinase ABC treatment, HS-tIA eluted slightly later from Sepharose CL-GB than the proteoglycan mixture (Fig.  2B). DS-tIA contained longer GAG chains ( M , 18 x lo3) than HS-tIA. These GAGs had a chain length and chondroitin 4:6 sulfate ratio similar to medium DS proteoglycans (DS-mII) from 16-h in vitro incubations (Table 11). The possibility that HS-tIA and DS-tIA GAGs were attached to the same protein core could not be completely eliminated since protein core size and composition were not studied. Sepharose CL-GB-included HS and DS proteoglycans were rapidly released from the tissue, with 31% of these proteoglycans found in the medium after a 2-h chase of 4-h in vitro "S04-labeled glomeruli. The proportion of glomerular proteoglycans released into medium was comparable to the half-life reported for granulosa cell surface proteoglycans (4 h) and HS proteoglycan from adhesion sites (42, 49). Whereas heparin treatment of liver cell membranes released only HS proteoglycan (6), this treatment as well as limited tryptic digestion released both HS and DS proteoglycans from isolated glomeruli and from granulosa cell surfaces (28). Thus, DS-tIA as well as HS-tIA proteoglycans may be rapidly released from cell surface or extracellular matrix "GAG receptors" and appear in medium.
A second DS proteoglycan (DS-tII) accumulated in tissue fractions over time, without being found intact in the incubation medium. This proteoglycan eluted later than DS-tIA 2nd HS proteoglycans from DEAE-Sephacel columns. Intact DS-t!I proteoglycan (partially excluded from Sepharose CL- Glomeruli isolated from rats each given 0.5 nXi of intraperitoqeal Na,""SC), four times over 16 h were extracted with 4 M guanidine HCI in the prmence of protease inhibitors (see "Methods"). A, hcttom % CsCl gradient fraction "S-proteoglycans puriflled by DEAE-Sephacel chromatography were eluted from Sepharose CL-GB columns and pooled as two separate fractions indicated by bars -4 and B. Equivzlent quantities 6B) as well as its GAG chains (Mr 26 X lo3) were larger than other glomerular proteoglycans. That this proteoglycan may be "self-associating'' was evident from its differential elution from CL-4B columns in the presence and absence of 4 M guanidine HC1 without a change in elution volume when samples were reduced and alkylated? The latter procedure dissociatedproteoglycan-hyaluronate aggregates (32). DS proteoglycan with similar migration by gel electrophoresis and with similarly sized GAGS was detected in uiuo as a minor late-eluting DEAE-Sephacel peak in both high and low density CsCl gradient fractions. Thus, DS-tII was confirmed as a normal component of glomeruli.
DS-tII GAG chains contained iduronic acid residues as determined by a difference in sensitivity to chondroitinase ABC and AC digestions as well as by the appearance of short Sephadex G-50-included oligosaccharides after periodate oxidation and alkaline cleavage. A greater proportion of oligosaccharides (33%) was included in these columns than previously reported for the "self-associating" bovine scleral DS (50). In addition to a region of closely spaced iduronic acid residues, the presence of a large unmodified span within DS-tII GAG was suggested by Sepharose CL-GB chromatography of Sephadex G-50-excluded periodate-treated GAGS. Although reduced in size when compared to intact DS-tII GAG (Mr 26,000), periodate treatment and alkaline cleavage left a chain which had an M, of approximately 9,000 (& 0.64). The presence of long glucuronic acid-containing regions was confirmed by the generation of a large proportion (%) of disaccharides by chondroitinase AC digestion of DS-tII GAG. These studies, therefore, suggest that iduronic acid residues are clustered along DS-tII GAG.
Whether DS-tIA and DS-tII are distinct proteoglycans or part of a continuum of DS proteoglycans with varying numbers of GAG chains of polydisperse length/proteoglycan protein core was not resolved by these experiments. That these were distinct proteoglycans was suggested by their partial separation by DEAE-Sephacel and by Sepharose CL-GB chromatography. In addition, DS-tIA was preferentially released from tissue by heparin, detergent, or trypsin treatments, while the majority of DS-tII proteoglycan was detected in 4 M guanidine HC1 extracts of the residual tissue. 35S-Proteoglycans remaining in tissue fractions after a 2-h chase of 4-h pulse-labeled glomeruli eluted earlier from Sepharose CL-GB columns than those found in pulse-labeled glomeruli suggesting that larger proteoglycan accumulated in tissue over time, while the smaller ones were released into the medium. From the above results one might speculate that DS-tII represents a small but real component of the glomerulus which has a slow turnover rate, while HS-tIA and DS-tIA, the major glomerular proteoglycans, are rapidly synthesized and transported to cell surfaces or tissue "GAG receptors'' from which they are rapidly released into medium.
In addition, the Sepharose CL-2B elution volume (Kav 0.62) of glomerular proteoglycans failed to change after incubation with 2% hyaluronic acid (unpublished observation by authors).
of each sample were electrophoresed in 0.6% agarose-1.8% polyacrylamide gels before (inset, lanes a and c) and after (inset, lanes b and   d ) chondroitinase ABC digestion. B and C, top 2/5 CsCl gradient fraction DSAE-Sep'racel peak I "5S-nacromolec~~les eluted as two partially separable peaks (Fig. 32, peak I), aud ewh was chromatcg;aph.ed cn Sepharose C1-GB. Autoradiographs of gels run with samples pocled from Sepharose CL-GB peaks as indicated by burs (A,   after (lanes b and d ) chondroitinase ABC digestion. a and b; B, inset, lanes c and d ) before (lanes u and c) and

inset, lanes
These studies have characterized a previously described extracellular glomerular HS proteoglycan as being displaced from glomerular cell surfaces and/or tissue binding sites by heparin. A previously uncharacterized smaller intracellular HS proteoglycan with little or no protein core was also described. A heparin-displaceable DS proteoglycan was rapidly released from the tissue, leaving behind a large self-associating DS proteoglycan which contained GAGS with clusters of iduronic acid residues.