Are alpha-gliadins glycosylated?

Alpha-gliadins isolated by carboxymethylcellulose chromatography contain noncovalently bound glucose probably due to contaminating proteoglycans and to material shed from the column. Traces of carbohydrate remain strongly bound to alpha-gliadins even after harsh denaturation, but our results indicate alpha-gliadins are not glycoproteins. Suggestions that gliadins are glycoproteins are probably due to contamination with this glucose and the presence of these proteoglycans.     

Occurrence of the HNK-1 epitope (3-sulfoglucuronic acid) in PC12 pheochromocytoma cells, chromaffin granule membranes, and chondroitin sulfate proteoglycans

After biosynthetic labeling of sulfated glycoproteins in rat and goldfish brain and PC12 pheochromocytoma cells with sodium [35S]sulfate, it was observed that all of the bands reactive with the HNK-1 antibody on immunoblots of sodium dodecyl sulfate-polyacrylamide gels corresponded with sulfate-labeled proteins detected by fluorography. These results support data from other studies, which indicate that the HNK-1 epitope is a 3-sulfo-glucuronic acid residue. In addition to its presence in a wide range of nervous tissue glycoproteins, the HNK-1 epitope was also detected in chromaffin granule membranes, chondroitinase ABC, and in chondroitin sulfate proteoglycans of brain, cartilage, and chondrosarcoma. However, it is not present in the heparan sulfate proteoglycan of brain, or in either of two chondroitin sulfate/dermatan sulfate proteoglycans in the chromaffin granule matrix.     

Functional characterization of chondroitin sulfate proteoglycans of brain: interactions with neurons and neural cell adhesion molecules

Ng-CAM and N-CAM are cell adhesion molecules (CAMs), and each CAM can bind homophilically as demonstrated by the ability of CAM-coated beads (Covaspheres) to self-aggregate. We have found that the extent of aggregation of Covaspheres coated with either Ng-CAM or N-CAM was strongly inhibited by the intact 1D1 and 3F8 chondroitin sulfate proteoglycans of rat brain, and by the core glycoproteins resulting from chondroitinase treatment of the proteoglycans. Much higher concentrations of rat chondrosarcoma chondroitin sulfate proteoglycan (aggrecan) core proteins had no significant effect in these assays. The 1D1 and 3F8 proteoglycans also inhibited binding of neurons to Ng-CAM when mixtures of these proteins were adsorbed to polystyrene dishes. Direct binding of neurons to the proteoglycan core glycoproteins from brain but not from chondrosarcoma was demonstrated using an assay in which cell-substrate contact was initiated by centrifugation, and neuronal binding to the 1D1 proteoglycans was specifically inhibited by the 1D1 monoclonal antibody. Different forms of the 1D1 proteoglycan have been identified in developing and adult brain. The early postnatal form (neurocan) was found to bind neurons more effectively than the adult proteoglycan, which represents the C-terminal half of the larger neurocan core protein. Our results therefore indicate that certain brain proteoglycans can bind to neurons, and that Ng-CAM and N-CAM may be heterophilic ligands for neurocan and the 3F8 proteoglycan. The ability of these brain proteoglycans to inhibit adhesion of cells to CAMs may be one mechanism to modulate cell adhesion and migration in the nervous system.     

Biochemical characterization of mucous glycoproteins synthesized and secreted by hamster tracheal epithelial cells in primary culture

Hamster tracheal epithelial cells growing on type I collagen gel synthesize and secrete high molecular weight glycoconjugates which elute in the void volume upon Sepharose CL-4B column chromatography. The presence of any proteoglycans in this void volume material was ruled out based on both enzymatic analysis and behavior on DEAE-ion exchange chromatography. Based on the incorporation of radioactive precursors, followed by strong acid hydrolysis or neuraminidase digestion, the material was shown to contain sialic acid, fucose, galactose, N-acetylglucosamine, N-acetylgalactosamine, and sulfate. Complete susceptibility to papain digestion and reductive beta-elimination suggests that the material consists of O-linked glycoproteins. The identification of N-acetylgalactosaminitol in the beta-eliminated oligosaccharides confirms this notion. The molecular weight of the oligosaccharides following beta-elimination ranges from 4,000 to 15,000. We conclude that the high molecular weight glyconjugates produced by hamster tracheal epithelial cells in primary culture are mucous glycoproteins based on size, sensitivity to alkaline borohydride treatment, and monosaccharide composition. Further characterization of these mucous glycoproteins showed both size and charge microheterogeneity among molecules. Detailed structural analysis of oligosaccharides of these mucous glycoproteins is currently under way.     

The biochemistry and function of mucosubstances

Synopsis The mucosubstances embrace two types of molecule, the proteoglycans and the glycoproteins. These polymers have in common the existence of a single polypeptide chain to which is attached one or more polysaccharide structures. It is, however, possible to distinguish the two groups by means of several important structural characteristics of their carbohydrate components.The presence of large amounts of carbohydrate in the molecules of many mucosubstances puts special difficulties in the way of their fixation for microscopical examination. The staining reactions used for the proteoglycans and glycoproteins depend entirely on the chemical properties of their carbohydrate components; in the past, these reactions have lacked specificity, but new, improved methods are becoming available.In their biosynthesis, the mucosubstances seem to follow an intracellular pathway peculiar to those proteins destined for extracellular secretion, and distinct from that of the cytoplasmic proteins. The membrane systems of the endoplasmic reticulum and Golgi apparatus are of particular importance in the process.Little is known of the biological role of many of the mucosubstances, and of their carbohydrate components in particular. A theory is put forward, proposing an essential role for the proteoglycans of cartilage in the maintenance of the mechanical function of the tissue. Work now in progress is giving strong indications that a lysosomal proteinase, cathepsin D, plays an important part in the pathological degeneration of cartilage, through its action on the proteoglycans. Lysosomes seem well equipped in their complement of hydrolytic enzymes to mediate the catabolism of mucosubstances in general.     

Glycoproteins and glycosaminoglycans synthesized by human keratinocytes in culture. Their role in cell-substratum adhesion.

Glycoproteins and proteoglycans synthesized by human keratinocytes in medium containing D-[1-14C]glucosamine were extracted and analysed by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. Extraction of the labelled keratinocytes with 0.5% Triton X-100 removed most of the glycoconjugates and left the cytoskeleton and nuclear residue adherent to the substratum. In addition to the cytoskeletal proteins, there was a relatively simple profile of glycoproteins and glycosaminoglycans associated with this adherent cytoskeleton. These consisted of eight glycoproteins in the mol.wt. range 99000-232000, five proteins in the keratin region (mol.wt. 42000-61000), hyaluronic acid and a sulphated glycosaminoglycan. Surface labelling of the keratinocytes with galactose oxidase (with or without neuraminidase)/KB3H4 revealed that many of the glycoproteins were exposed on the cell surface. The importance of the glycoproteins and proteoglycans in attaching the keratinocytes to the substratum was examined by studying their expression after incubation in medium containing tunicamycin and their degradation after digestion with trypsin and hyaluronidase. These studies, together with an examination of the glycoconjugates released by sequential extraction with 0.5% Triton X-100 followed by 0.2% sodium dodecyl sulphate, revealed that the glycoprotein of mol.wt. 232000 has an important role in mediating the attachment of keratinocytes to the substratum.     

Immunohistochemistry and histochemistry of the inner ear gelatinous membranes and statoconia of the chick (Gallus domesticus)

This study was undertaken to investigate whether the gelatinous membranes (GMs) of the chick (Gallus domesticus) inner ear contain glycoproteins and proteoglycans, as is the case in mammals, and whether or not the relative concentrations of glycoproteins and proteoglycans change during development. We used electron-microscopic histochemistry with tannic acid, Alcian blue and ruthenium red and immunoperoxidase with monoclonal antibodies to fibronectin and keratan sulfate. Both techniques were applied to material from newly hatched chicks in various developmental stages. In hatchlings, tannic acid, which precipitates mainly proteoglycans, stained the mineralizing GMs of the utricle, saccule and lagenar macula darker than it did the nonmineralizing GMs of the cristae and tectorial membrane of the basilar papilla, while Alcian blue and ruthenium red stained all GMs in the vestibule and basilar papilla almost to the same degree. Antikeratan sulfate (a proteoglycan marker) stained the GMs intensively, while antifibronectin (glycoprotein monoclonal marker) stained them less intensively. In the mineralizing GMs of the embryos, the immunoreactivity (density) of keratan sulfate almost doubles between days 7 (stages 31) and 21 (stage 46), while the density of fibronectin does not seem to change as much. Different concentrations of glycoproteins and proteoglycans in each GM may impart to it the capacity to remain unmineralized, as is the case for cupulae of the vestibule and tectorial membrane of the basilar papilla, or mineralized, as is the case for the statoconial membrane of the maculae.     

An immunohistochemical study of the rabbit suprapatella, a sesamoid fibrocartilage in the quadriceps tendon containing aggrecan.

The rabbit suprapatella is a sesamoid fibrocartilage in the deep surface of the tendon of vastus intermedius and an integral part of the knee joint. We report the presence of a variety of proteoglycans (aggrecan and versican), glycosaminoglycans (chondroitin 4 and 6 sulfate, dermatan sulfate, keratan sulfate) and glycoproteins (tenascin) in its extracellular matrix and the intermediate filament vimentin in the fibrocartilage cells. The most significant finding is the presence of aggrecan in the extracellular matrix, along with its associated link protein and several of its integral glycosaminoglycans. Aggrecan probably enables the suprapatella to withstand compression. Although it can be assumed that aggrecan metabolites detected in synovial fluid from some human joints are predominantly associated with articular hyaline cartilage, the presence of aggrecan in the rabbit suprapatella means that this cannot be assumed for all animal knee joints. We conclude that it is important for orthopedic researchers who use animal models for arthritis research to check for the presence of a suprapatella when joint fluid analyses are interpreted.     

Evidence for an interaction between canine synovial cell proteoglycans and link proteins

Link proteins are glycoproteins which stabilize aggregates of proteoglycans and hyaluronic acid in cartilage. We recently identified link proteins in canine synovial cell cultures. We now find that link proteins and proteoglycans extracted from these cells under dissociative conditions sediment in the high-buoyant-density fractions of an associative cesium chloride density gradient, suggesting that link proteins interact with high-bouyant-density proteoglycans. In gradients containing [³⁵S]sulfate-labeled synovial cell extracts, 76% of the labeled sulfate and 54% of the uronic acid is found in the high-buoyant-density fractions. Under associative conditions, Sepharose 2B elution profiles of the crude synovial cell extract, synovial cell high-buoyant-density fractions, and culture medium indicate that synovial cell proteoglycans are present in monomeric form, rather than in aggregates. Synovial cell link proteins co-elute with the [³⁵S]sulfate-labeled material under the same conditions. These proteoglycans do not interact in vitro with exogenous hyaluronic acid. Dermatan sulfate, chondroitin sulfate and heparan sulfate are the major cell-associated sulfated glycosaminoglycans synthesized by cultured canine synovial cells, while hyaluronic acid is found in the culture medium. Although the proteoglycans synthesized by cultured synovial cells interact with link proteins, these data indicate that they do not interact with hyaluronic acid to form aggregates.     

The localization of sulfated glycoconjugates synthesized by the corneal epithelium of chick embryos

The localization of sulfated glycoconjugates in the corneal epithelium of 19-d-old chick embryo was investigated biochemically using epithelia labeled in vitro with [35S]sulfate, which exhibited autoradiographically a similar distribution of silver grains to that labeled in ovo. The radiolabeled tissues were dissociated into single cells by incubation in 0.25% trypsin containing 0.02% EDTA at 37 degrees C for 40 min. The proteoglycans and sulfated glycoproteins which were associated with the cells and those released into the dissociation medium were separated by DEAE-Sepharose CL-6B and analyzed on Sepharose CL-6B and SDS-PAGE. About 86% of the proteoglycans was released into the dissociation medium and more than 50% of the cell-associated ones was affected by trypsin. This indicates that the proteoglycans are mostly localized in an extracellular compartment. On the other hand, the extent of release of sulfated glycoproteins into the medium on dissociation of tissues was distinctly different depending upon their molecular weight (Mr): almost all of the sulfated glycoproteins of the family with Mr 48,000-70,000 (32% of the total sulfated glycoproteins) were recovered as intact molecules with the cells, whereas approximately 50% of those with Mr 70,000-150,000 (36%) and about 70% of those with Mr over 150,000 (28%) were released into the dissociation medium. These results indicate that the family with Mr 48,000-70,000 is localized intracellularly and that with Mr 70,000-150,000 in a compartment poorly affected by trypsin; in contrast to those, that with Mr more than 150,000 is localized in an extracellular compartment like the proteoglycans.     

Analysis of the proteoglycans synthesized by corneal explants from embryonic chicken. I. Characterization of the culture system with emphasis on stromal proteoglycan biosynthesis

Corneal explants with scleral rims were freshly prepared from day 18 chicken embryos and incubated in vitro for 3 h in the presence of various radioactive precursors. Radiolabeled proteoglycans were isolated from the stromal tissue and culture medium for analysis. Two predominant proteoglycans were identified in corneal stroma. One contains dermatan sulfate and the other contains keratan sulfate; a structural analysis of each is reported in the accompanying paper (Midura, R.J., and Hascall, V.C. (1989) J. Biol. Chem. 264, 1423-1430). A minor keratan sulfate proteoglycan distinct from the major form, a small amount of heparan sulfate proteoglycan, and some sulfated glycoproteins were also detected in stromal extracts. The biosynthesis of the dermatan sulfate proteoglycan was stable in vitro and in ovo, whereas that of the major keratan sulfate proteoglycan was stable only in ovo. Various treatments were tried to maintain a high rate of keratan sulfate synthesis with time in culture. Cooling the corneal explants to 5 degrees C was the only treatment that reduced this decline in keratan sulfate synthesis in vitro to any significant extent. Three major proteoglycans were observed in the culture medium. Two were dermatan sulfate proteoglycan and appeared to be mainly derived from the scleral tissue surrounding the corneal explant. The third proteoglycan contained keratan sulfate. It was smaller in size and lower in charge density compared to the keratan sulfate proteoglycan found in the stroma, but both appeared to have similar core protein sizes. It seems likely that this proteoglycan was synthesized in the stroma and secreted into the medium. A small amount of heparan sulfate proteoglycan and some sulfated glycoproteins were also detected in the medium.     

Further studies on the sub-units of α-crystallin

1. A new procedure is described for the purification of alpha-crystallin, including: preparative zone electrophoresis, density-gradient centrifugation and gel filtration. The total amino acid composition of highly purified samples prepared according to this procedure has been determined. 2. Evidence is presented for the presence of intermediates in the urea-induced splitting of alpha-crystallin into sub-units. A possible mechanism for this splitting is proposed. 3. The recombination of sub-units has been studied by polyacrylamide-gel electrophoresis and ultracentrifugal analysis. As judged from these criteria, only a partial recovery of starting material was obtained. 4. The origin of the minor bands in the electrophoretic pattern of alpha-crystallin on 7m-urea-polyacrylamide gel has been investigated. No evidence was found that their presence is due to carbamoylation or sulphide-disulphide interchange. They probably arise from isomerization. 5. The mean molecular weight of the sub-units was calculated to be 24000 (Archibald's method). Determination of the sedimentation-diffusion equilibrium revealed a value of 21000 at the meniscus. Assuming that all sub-units contain one cysteine residue/molecule, 23000 can be derived for the mean molecular weight.     

Short review. Inverse relationship between hyaluronan and collagens in development and angiogenesis

Abstract. The extracellular matrix plays a vital role in regulating normal tissue development and function - largely via the specific arrangement of macromolecules such as collagens, proteoglycans, glycosaminoglycans and glycoproteins. Previous reports have concentrated on associations between combinations of collagens/proteoglycans, collagens/glycoproteins and proteoglycans/glycosaminoglycans whilst little information is available on associations between collagens and free glycosaminoglycans.
In this review, we discuss possible associations between collagens and the glycosaminoglycan hyaluronan; macromolecules which are known to exhibit changes in amount and composition during development and under pathological conditions. We demonstrate two types of collagen/hyaluronan association in vivo: the first, during the formation of extracellular matrix structures where neither collagens nor hyaluronan are degraded, resulting in the regulation of collagen fibrillogenesis, and the second, involving an inverse correlation between collagen synthesis and hyaluronan degradation and vice versa. We suggest that associations between collagens and hyaluronan play an important role in the initiation and maintenance of angiogenesis and put forward a model of cartilage vascularisation which relies on these associations.     

Inverse relationship between hyaluronan and collagens in development and angiogenesis

Abstract. The extracellular matrix plays a vital role in regulating normal tissue development and function - largely via the specific arrangement of macromolecules such as collagens, proteoglycans, glycosaminoglycans and glycoproteins. Previous reports have concentrated on associations between combinations of collagens/proteoglycans, collagens/glycoproteins and proteoglycans/glycosaminoglycans whilst little information is available on associations between collagens and free glycosaminoglycans.
In this review, we discuss possible associations between collagens and the glycosaminoglycan hyaluronan; macromolecules which are known to exhibit changes in amount and composition during development and under pathological conditions. We demonstrate two types of collagen/hyaluronan association in vivo: the first, during the formation of extracellular matrix structures where neither collagens nor hyaluronan are degraded, resulting in the regulation of collagen fibrillogenesis, and the second, involving an inverse correlation between collagen synthesis and hyaluronan degradation and vice versa. We suggest that associations between collagens and hyaluronan play an important role in the initiation and maintenance of angiogenesis and put forward a model of cartilage vascularisation which relies on these associations.     

Localization of basement membrane glycoproteins in rat kidney during foetal development

The distribution of basement membrane glycoproteins (type IV collagen, laminin, fibronectin, and proteoglycans) was studied in foetal rat kidney by immunohistochemical techniques using polyclonal antibodies. From the first stages of nephron differentiation, all these glycoproteins were detectable by immunofluorescence in the tubular and glomerular basement membranes and in the mesangial matrix. As differentiation proceeded, labelling of glycoproteins progressively intensified, except for that of fibronectin, which gradually decreased in the glomerular basement membrane (GBM) and was barely observable at full differentiation. With immunoperoxidase staining in electron microscopy, all glycoproteins were seen to be widely dispersed in the spaces between the epithelial and endothelial glomerular cells so long as the GBM remained a loose structure. However, after it became a compact, 3-layered formation, type IV collagen and laminin were distributed throughout the GBM, whereas proteoglycans and anionic sites appeared as 2 rows of granules confined to the laminae rarae.     

Synthesis, pharmacological evaluation and electrochemical studies of novel 6-nitro-3,4-methylenedioxyphenyl-N-acylhydrazone derivatives: Discovery of LASSBio-881, a new ligand of cannabinoid receptors

We describe herein the discovery of LASSBio-881 (3c) as a novel in vivo antinociceptive, anti-inflammatory, and in vitro antiproliferative and antioxidant compound, with a cannabinoid ligand profile. We observed that LASSBio-881 (3c) was able to bind to CB1 receptors (71% at 100microM) and also to inhibit T-cell proliferation (66% at 10microM) probably by binding to CB2 receptors, in a non-proapoptotic manner, different from anandamide (1). It was also demonstrated that LASSBio-881 (3c) had an important antioxidant profile toward free radicals (DPPH and hydroxyl), probably due to its particular redox behavior, which reflects the presence of both nitro and 3,5-di-tert-butyl-4-hydroxyphenyl sub-units, as demonstrated by cyclic voltammetry studies. In addition, we showed that these structural sub-units are essential for the observed pharmacological activity.     

Metabolic labeling of glycosylphosphatidylinositol-anchor of heparan sulfate proteoglycans in rat ovarian granulosa cells.

The glycosylphosphatidylinositol (GPI)-anchor of the plasma membrane-associated heparan sulfate (HS) proteoglycan was metabolically radiolabeled with [3H]myristic acid, [3H]palmitic acid, [3H]inositol, [3H]ethanolamine, or [32P]phosphate in rat ovarian granulosa cell culture. Cell cultures labeled with [3H]myristic acid or [3H]palmitic acid were extracted with 4 M guanidine HCl buffer containing 2% Triton X-100 and the proteoglycans were purified by ion exchange chromatography after extensive delipidation. Specific incorporation of 3H into GPI-anchor was demonstrated by removing the label with a phosphatidylinositol-specific phospholipase C (PI-PLC). Incorporation of 3H activity into glycosaminoglycans and core glycoproteins was also demonstrated. However, the specific activity of 3H in these structures was approximately 2 orders of magnitude lower than that in the GPI-anchor, suggesting that 3H label was the result of the metabolic utilization of catabolic products of the 3H-labeled fatty acids. PI-PLC treatment of cell cultures metabolically labeled with [3H]inositol, [3H]ethanolamine, or [32P]phosphate specifically released radiolabeled cell surface-associated HS proteoglycans indicating the presence of GPI-anchor in these proteoglycans. GPI-anchored HS proteoglycans accounted for 20-30% of the total cell surface-associated HS proteoglycans and virtually all of them were removed by PI-PLC. These results further substantiate the presence of GPI-anchored heparan sulfate proteoglycan in ovarian granulosa cells and its cell surface localization.     

A novel role for 3-O-sulfated heparan sulfate in herpes simplex virus 1 entry.

Herpes simplex virus type 1 (HSV-1) binds to cells through interactions of viral glycoproteins gB and gC with heparan sulfate chains on cell surface proteoglycans. This binding is not sufficient for viral entry, which requires fusion between the viral envelope and cell membrane. Here, we show that heparan sulfate modified by a subset of the multiple D-glucosaminyl 3-O-sulfotransferase isoforms provides sites for the binding of a third viral glycoprotein, gD, and for initiation of HSV-1 entry. We conclude that susceptibility of cells to HSV-1 entry depends on (1) presence of heparan sulfate chains to which virus can bind and (2) 3-O-sulfation of specific glucosamine residues in heparan sulfate to generate gD-binding sites or the expression of other previously identified gD-binding receptors.     

Complex carbohydrates of cultured PC12 pheochromocytoma cells. Effects of nerve growth factor and comparison with neonatal and mature rat brain

The composition and biosynthesis of glycoproteins, proteoglycans, and gangliosides have been studied in a clonal line of rat pheochromocytoma (PC12) cells. Glycoproteins account for approximately 78% of the glucosamine-labeled complex carbohydrates found in the culture medium, together with 17% chondroitin sulfate and 5% heparan sulfate. 10% of the glycoproteins but less than 1% of the proteoglycans are released by trypsin treatment of the cells, whose complex carbohydrates are composed of 93% glycoproteins, 1.3% chondroitin sulfate, 3.4% heparan sulfate, and 2.6% of mono- and disialogangliosides. Sequential lectin affinity chromatography and alkali treatment of glycopeptides prepared from the medium, trypsin-releasable, membrane, and cell-soluble glycoproteins demonstrated that in all of the subfractions large tri- and tetraantennary complex oligosaccharides account for 82 to 97% of those present in PC12 cell glycoproteins. Biantennary oligosaccharides account for approximately 2-6% of those in medium and trypsinate, as compared to 10-13% in the membrane and cell soluble glycoproteins, and there were large differences (ranging from 7 to 60%) in the proportions of biantennary oligosaccharides which are substituted by fucose on the core N-acetylglucosamine which is linked to asparagine. High mannose oligosaccharides are present predominantly in the cell membrane and soluble glycoproteins, where they account for 4 to 5% of the total glycoprotein labeling. In response to nerve growth factor (NGF), the PC12 cells extend long processes and acquire other properties similar to those of differentiated sympathetic neurons. Significant alterations were also observed in the complex carbohydrates of NGF-treated cells, the most striking of which were an almost 3-fold increase in labeled gangliosides and a 75% increase in trypsin-releasable glycoproteins. Cellular heparan sulfate decreased by 70% in response to NGF and increased by an equivalent amount in the culture medium, whereas an NGF-induced increase in chondroitin sulfate labeling occurred specifically in the cell membranes.     

Structural glycoprotein from the media of pig aorta. Aggregation of the S-carboxamidomethyl subunits.

Media of pig aorta was extracted with 1 M NaCl and 2 M MgCl2 to remove most of the soluble collagen, proteoglycans and glycoproteins. The glycoproteins remaining in the residue were extracted with 6 M urea-0.1 M mercaptoethanol. The urea soluble proteins were precipitated by dialysis, redissolved in 4 M guanidine-0.05 M DTT and were S-carboxamidomethylated (CM-guanidine extract). This extract was further fractionated by a variety of methods in order to separate a glycoprotein from collagen and proteoglycans. Caesium chloride density-gradient ultracentrifugation of the CM-guanidine extract separated a minor proteoglycan peak from a major glycoprotein fraction still containing some hydroxyproline. This major glycoprotein fraction was excluded as a single peak from Sephadex G 100 and G 200 in 4 M guanidinium chloride or in 6 M urea-0.2 per cent SDS. Sodium dodecylsulphate gel electrophoresis separated this high molecular weight Sephadex fraction into a major low molecular weight (approximately 35000 daltons) component and a minor high molecular weight component. This glycoprotein fraction could also be separated from a collagenous fraction and from proteoglycans by ion exchange chromatography on DEAE cellulose or by gelfiltration on Sepharose 4 B in 6 M urea-0.02 M EDTA-0.2 per cent SDS at pH 7.0. The isolated glycoprotein fraction is rich in dicarboxylic amino acids, contains galactose, mannose, (glucose), N-acetylglucosamine and sialic acid. The S-carboxamidomethyl glycoprotein preparation interacts with acid soluble calf skin collagen on isoelectric focusing in sucrose gradient in urea. This interaction is in favour of the biological role claimed for structural glycoproteins during fibrogenesis and differentiation.