An oligosaccharide component in proteoglycans of articular cartilage

Two types of sialic acid-containing component are released from articular cartilage proteoglycan monomer (D1) treated with 0.05 M NaOH containing 1 M NaBH₄. The smaller component, which has not been described before, contains galactosamine, glucosamine, galactose and sialic acid (Molar ratio 1:1:1:2). It is eluted from ECTEOLA-cellulose with low molarity (0.4 M) sodium formate and has a K_av of 0.70 on Bio-gel P30. Its presence on the proteoglycan monomer was demonstrated at all stages of foetal and adult life.     

Isolation and characterization of proteoglycans from the swarm rat chondrosarcoma.

Proteoglycan monomer (D1) and aggregate (A1) preparations were isolated from 4 M guanidinium chloride extracts of the Swarm rat chondrosarcoma. When EDTA, 6-aminohexanoic acid, and benzamidine were present in the solutions, the D1 preparation contained a single component (SO = 23 S), and the A1 preparation contained 30% monomer (SO = 23 S) and 70 percent aggregate (SO = 111 S). In the absence of EDTA, 6-aminohexanoic acid, and benzamidine, the A1 preparations contained only small proteoglycan fragments, indicating that extensive enzymatic degradation had occurred. The composition of the proteoglycan monomer was different from that of proteoglycan monomer preparations from normal hyaline cartilages in that it did not contain keratan sulfate and chondroitin 6-sulfate; only chondroitin 4-sulfate was found. The A1 preparation from the chondrosarcoma contained only one link protein, which was like the smaller (molecular weight of 40,000) of the two link proteins present in A1 preparations from bovine nasal cartilage. When the A1 preparation from the chondrosarcoma was treated with chondroitinase ABC and trypsin and the digest was chromatographed on Sepharose 2B, a complex was isolated which contained the link protein and the segments of the protein core from the hyaluronic acid-binding region of the proteoglycan molecules.     

Equilibrium-binding studies of pig laryngeal cartilage proteoglycans with hyaluronate oligosaccharide fractions.

The binding of hyaluronate oligosaccharide fractions to proteoglycans from pig laryngeal cartilage has been studied by equilibrium dialysis in dilute solution. It has been shown that: (1) each proteoglycan monomer binds only one hyaluronate oligosaccharide molecule [containing about eighteen saccharide residues (HA approximately 18) and of number-average molecule weight (Mn) 37501]; (2) the dissociation constant, Kd, for interaction between proteoglycan monomer and oligosaccharide HA approximately 18 is 3 x 10(-8) M at 6 degrees C at I 0.15-0.5, pH 7.4; (3) the dissociation constant has little dependence on temperature, so that Kd at 54 degrees C is 3 x 10(-7) M under the same conditions; (4) the aggregatability is high at 6 degrees C, falls significantly at 54 degrees C, but much of it can be recovered on cooling to 6 degrees C again, demonstrating reversible denaturation; (5) a method for determining the proportion of the proteoglycan molecules capable of binding to hyaluronate by equilibrium dialysis was compared with gel-chromatographic and ultracentrifugal methods; (6) a hyaluronate oligosaccharide, HA approximately 56 (Mn 11 000), could bind more than one proteoglycan molecule; (7) consideration of ultracentrifugal data shows that when proteoglycans bind to a hyaluronate of larger size (mol..wt. 670 000), an average Kd of 12 x 10(7) M fits the data in 0.5 M-guanidine hydrochloride at 20 degrees C.     

Low-density lipoprotein binding affinity of arterial wall proteoglycans: characteristics of a chondroitin sulfate proteoglycan subfraction

The characteristics of an arterial wall chondroitin sulfate proteoglycan (CS-PG) subfraction that binds avidly to low-density lipoproteins (LDL) was studied. A large CS-PG was extracted from bovine aorta intima-media under dissociative conditions, purified by density-gradient centrifugation and gel filtration chromatography, and further subfractionated by affinity chromatography on LDL-agarose. A proteoglycan subfraction, representing 25% of the CS-PG, showed an elution profile (with dissociation from LDL-agarose occurring between 0.5 and 1.0 M NaCl) corresponding to that of heparin, heretofore considered to be the most strongly binding glycosaminoglycan with LDL. The proteoglycan subfraction which migrated as a single band on composite agarose-polyacrylamide gel electrophoresis contained chondroitin 6-sulfate, chondroitin 4-sulfate and dermatan sulfate in a proportion of 70:22:8. The core protein of the proteoglycan had an apparent molecular weight of 245,000, and contained approx. 33 glycosaminoglycan chains with an average molecular weight of 32,000. The CS-PG subfraction, like heparin, formed insoluble complexes in the presence of 30 mM Ca2+. Complexing of LDL with proteoglycan resulted in two classes of interactions with 0.1 and 0.3 proteoglycan monomer bound per LDL particle characterized by an apparent Kd of 4 and 21 nM, respectively. This indicates that multiple LDL particles bind to single proteoglycan monomers even at saturation. In contrast, LDL-heparin interactions showed a major component characterized by an apparent Kd of 151 nM and a Bmax of 9 heparin molecules per LDL particle. The occurrence of a potent LDL-binding proteoglycan subfraction within the family of arterial CS-PG may be of importance in terms of lipid accumulation in atherogenesis.     

Biosynthesis of cartilage proteoglycan and link protein by articular chondrocytes from immature and mature rabbits

Chondrocytes from immature and mature rabbits have been compared in biosynthetic studies with [3H] leucine and [35S]sulfate as precursors. The time course of incorporation of [3H]leucine into general protein, proteoglycan monomer core protein, and link protein and of [35S]sulfate into proteoglycan monomer has been examined. Proteoglycan monomer was isolated from the high buoyant density (p greater than 1.60) fractions of dissociative CsCl gradients and link protein by immunoprecipitation with antibody 8A4 followed by gel electrophoresis. Results based on the period of linear isotope incorporation showed that mature cells synthesize protein at about 40% of the rate of immature cells and both proteoglycan and link protein at about 20% of the rate of immature cells. The labeling rates obtained suggest that immature cells synthesize an approximate 1:1 molar ratio of link protein to proteoglycan monomer, and for mature cells this ratio is about 0.8:1. While cell layer retention of newly synthesized proteoglycan was markedly lower in mature relative to immature cell cultures, link protein retention was high in both immature and mature cultures; this finding provides an explanation for our previous observation (Plaas, A. H. K., and Sandy, J. D. (1984) Biochem, J. 220, 337-340) that link-free monomer accumulates in the medium of mature but not immature cultures. The link protein synthesized by both ages of cells and isolated from cell layer or medium was a single major species of apparent molecular mass 48-51 kDa. The results suggest that mature chondrocytes are less efficient than immature chondrocytes in the coordinated assembly of link-stabilized proteoglycan aggregates in this culture system.     

Selective aggregation of proteoglycans with hyaluronic acid.

Conditions were established to separate proteoglycan aggregate (AH1) from a bovine nasal septum extract by associative rate zonal sedimentation on a NaCl gradient. The AH1 has a higher protein content than the mixed aggregate-monomer (A1) isolated by conventional associative CsCl density gradient centrifugation from a portion of the same extract. The same associative rate zonal conditions separated the A1 fraction into aggregated AH1 containing hyaluronic acid and nonaggregated proteoglycan monomer (N1) essentially free of hyaluronic acid. The AH1 fraction is richer in protein and keratin sulfate than is N1. Dissociative rate zonal sedimentation of A1 under conditions which totally sedimented most of the disaggregated monomer (AH1-D1) and the nonaggregated monomer N1 separated a less sedimentable protein and keratan sulfate-rich proteoglycan monomer (AH1-D2). Chromatography on Sepharose 2B under dissociative conditions demonstrated that the nonaggregated N1 monomer is intermediate in size between the disaggregated monomers AH1-D1 and AH1-D2. N1 has a buoyant density higher than AH1 and is practically equivalent to AH1-D1. All are dense fractions so that separation by CsCl density gradient equilibration is not feasible.     

Isolation and partial characterization of low sulfated chondroitin 4-sulfate-proteoglycan in bovine blood

Bovine plasma low sulfated chondroitin sulfate-proteoglycan (34 μg/ml plasma), accounting for the main component of acidic glycosaminoglycans in blood, has been purified by isoelectric precipitation, dissociation with 4 M guanidine chloride followed by DEAE-chromatography, Sephadex G-200 chromatography and by preparative polyacrylamide gel electrophoresis. The proteoglycan, having a molecular weight of approx. 44 000, is composed of about 77% protein and 23% glycosaminoglycan at a molar ratio of 1 : 1 which could be cleaved by alkaline treatment into each component. Amino acid analysis of the proteoglycan and its glycosylpeptide has shown that the material is derived from a different origin from other tissue proteoglycans, though the amino acid residues surrounding O-glycosidic linkage to serine residue are quite similar to that of cartilage proteoglycan. Characteristic features of plasma low sulfate chondroitin sulfate-proteoglycan are discussed, compared with tissue materials.     

Isolation and partial characterization of low sulfated chondroitin 4-sulfate-proteoglycan in bovine blood.

Bovine plasma low sulfated chondroitin sulfate-proteoglycan (34 microgram/ml plasma), accounting for the main component of acidic glycosaminoglycans in blood, has been purified by isoelectric precipitation, dissociation with 4 M guanidine chloride followed by DEAE-chromatography, Sephadex G-200 chromatography and by preparative polyacrylamide gel electrophoresis. The proteoglycan, having a molecular weight of approx. 44,000, is composed of about 77% protein and 23% glycosaminoglycan at a molar ratio of 1 : 1 which could be cleaved by alkaline treatment into each component. Amino acid analysis of the proteoglycan and its glycosylpeptide has shown that the material is derived from a different origin from other tissue proteoglycans, though the amino acid residues surrounding O-glycosidic linkage to serine residue are quite similar to that of cartilage proteoglycan. Characteristic features of plasma low sulfate chondroitin sulfate-proteoglycan are discussed, compared with tissue materials.     

Isolation and characterization of a link protein from bovine aorta proteoglycan aggregate

Proteoglycan aggregates were isolated from bovine aorta by extraction with 0.5 M guanidine hydrochloride in the presence of proteinase inhibitors and purified by isopycnic CsCl centrifugation. The bottom two-fifths (A1) of the gradient contained 30% of proteoglycans in the aggregated form. The aggregate had 14.8% protein and 20.4% hexuronic acid with hyaluronic acid, dermatan sulfate and chondroitin sulfates in a proportion of 18:18:69. A link protein-containing fraction was isolated from the bottom two-fifths by dissociative CsCl isopycnic centrifugation. The link protein that floated to the top one-fifth of the gradient was purified by chromatography on Sephadex G-200 in the presence of 4 M guanidine hydrochloride. It moved as a single band in SDS-polyacrylamide gel electrophoresis with a molecular weight of 49 000. The amino acid composition of link protein resembled that of link protein from cartilage, but was strikingly different from that of the protein core of the proteoglycan monomer. The neutral sugar content of link protein was 3.5% of dry weight. Galactose, mannose and fucose constituted 21, 62 and 16%, respectively of the total neutral sugars. In aggregation studies the link protein was found to interact with both proteoglycan monomer and hyaluronic acid. Oligosaccharides derived from hyaluronic acid decreased the viscosity of link protein-free aggregates of proteoglycan and hyaluronic acid but not of link-stabilized aggregates, demonstrating that the link protein increases the stability of proteoglycan aggregates.     

A study of the interaction between cartilage proteoglycan and link protein.

The interaction between proteoglycan and link protein extracted from bovine articular cartilage (15-18-month-old animals) was investigated in 0.5 M-guanidinium chloride. The proteoglycans, radiolabelled as the aggregate (A1 fraction), were fractionated by two 'dissociative' density-gradient centrifugations (A1D1D1) followed by a rate-zonal centrifugation (S1) to yield an A1D1D1S1 preparation. At least 65% of these proteoglycans were able to bind to hyaluronate, but only 52% were able to bind to link protein as assessed by chromatography on Sepharose CL-2B. Over 80% of the [3H]link-protein preparation, radiolabelled as the aggregate, was able to interact with proteoglycan as assessed by chromatography on Sepharose CL-4B. Equilibrium-boundary-centrifugation studies performed at low link-protein concentrations (2.42 x 10(-9) M-5.93 x 10(-8) M) were analysed by Scatchard-type plots and indicated a Kd of 1.5 x 10(-8) M and a stoichiometry, n = 0.56, i.e. approx. 56% of those proteoglycans capable of binding to link protein had a strong site for link protein if a 1:1 stoichiometry were assumed. However, experiments performed at higher link-protein concentrations (3.5 x 10(-7) M and 8 x 10(-7) M) yielded stoichiometry values which were link-protein-concentration-dependent. Non-equilibrium binding studies using chromatography on Sepharose CL-2B and rate-zonal centrifugation yielded apparent stoichiometries between 0.6 and 7.5 link-protein molecules per proteoglycan monomer as a function of increasing link-protein concentration. It was concluded that a proportion of the proteoglycan molecules had a strong site for binding a single link protein (Kd 1.5 x 10(-8) M) and that at high link-protein concentrations a weaker, open-ended, process of link-protein self-association nucleated upon the strong link-protein-proteoglycan complex occurred. Hyaluronate oligosaccharides appeared to abolish a proportion of this self-association (as observed by Bonnet, Dunham & Hardingham [(1985) Biochem. J. 228, 77-85] in a study of link-protein-hyaluronate-oligosaccharide interactions) so as to leave a link protein:proteoglycan stoichiometry of 2. It is not clear whether this second link-protein molecule binds directly to the proteoglycan or to the first link protein.     

Pig vitreous gel: macromolecular composition with particular reference to hyaluronan-binding proteoglycans

The aim of this study was to examine the macromolecular composition of pig vitreous body with particular emphasis on hyaluronan-binding proteoglycans. The whole pig vitreous gel was found to contain 76 microg of hyaluronan-derived uronic acid, 700 microg of total protein and 150 microg of collagen per ml of gel. The contents of neutral hexoses and sialic acids were 80 and 22 microg/ml of vitreous gel, but only a minor proportion of them were found to be associated with the proteoglycan fraction. As estimated by gel chromatography on Sepharose CL-2B, hyaluronan presents a polydisperse hydrodynamic behavior with a lower molecular mass (M(r)) value of 220 kDa. The existence of low amounts of a hyaluronan-binding proteoglycan population with structural and immunological characteristics similar to a member of the hyalectan family, versican, has also been demonstrated. The concentration of this versican-like proteoglycan in whole vitreous accounts for 50 microg proteoglycan protein per ml of vitreous gel and represents a minor proportion (about 7%) of the total protein content. The proteoglycan has an average M(r) of 360 kDa and is substituted by chondroitin sulphate (CS) side chains. Study of the CS sulphation pattern showed that the chains were composed of both type 4- and 6-sulphated disaccharide units.     

Synthesis of a "free" form of hyaluronic acid by articular chondrocytes in monolayer culture

Primary and first passage rabbit chondrocyte cultures synthesized a "free" form of hyaluronic acid (HA-f) previously characterized in rabbit cartilage. HA-f was isolated from the [3H] glcN/35SO4-labelled cell-associated-fraction (CAF) and from the culture medium by successive equilibrium centrifugations in Cs2SO4/CsCl/Cs2SO4 under low salt conditions. The culture medium HA-f appeared in the void volume of Sepharose CL-2B eluted with low salt, (0.5M sodium acetate), and was susceptible to digestion with Streptomyces hyaluronidase. HA-f aggregated purified rabbit cartilage proteoglycan monomer. These results indicated that HA-f probably subserves hyaluronic acid already complexed with proteoglycan monomer. Newly synthesized HA-f may be required for the continual formation of proteoglycan aggregates.     

Isolation of dermatan sulfate proteoglycans from mature bovine articular cartilages

Two species of dermatan sulfate proteoglycans, called DS-PGI and DS-PGII, have been isolated from mature bovine articular cartilages. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis at low ionic strength in 0.01 M phosphate the dermatan sulfate proteoglycans appeared as a single polydisperse species whose molecular weight ranged from 80,000 to 140,000. The dermatan sulfate proteoglycans eluted as a single peak on Sepharose CL-4B chromatography in 4 M guanidine hydrochloride and showed no tendency to separate into two components. Following chondroitinase AC and ABC digestion, a core protein was obtained whose molecular weight was 45,000. However, what appeared to be a single dermatan sulfate proteoglycan was consistently separated into two species of distinctly different mobilities by sodium dodecyl sulfate-polyacrylamide gel electrophoresis at high ionic strength in 0.375 M Tris. The molecular weight of the smaller species (DS-PGII) ranged from 87,000 to 120,000. The molecular weight of the larger species (DS-PGI) ranged from 165,000 to 285,000. DS-PGI self-associates in 0.375 M Tris, while DS-PGII does not. This phenomenon was exploited to separate DS-PGI and DS-PGII by preparative electrophoresis on 5 to 20% gradient slab gels. The immunological identities of the individual species, DS-PGI and DS-PGII, were examined by enzyme-linked immunosorbent assay using polyclonal antiserum to cartilage-specific proteoglycan monomer from bovine articular cartilage and polyclonal and monoclonal antibodies to DS-PGII. The polyclonal antiserum to cartilage-specific proteoglycan monomer did not react with DS-PGI or DS-PGII, indicating that DS-PGI and DS-PGII possess different core proteins from cartilage-specific proteoglycan monomer. Polyclonal and monoclonal antibodies raised against the mixture of DS-PGI and DS-PGII reacted strongly with DS-PGII, but weakly or not at all with DS-PGI. These results suggest that DS-PGI and DS-PGII possess different core proteins and may represent two different species of dermatan sulfate proteoglycans.     

Biosynthesis of proteoglycan in vitro by cartilage from human osteochondrophytic spurs

Proteoglycan biosynthesis by human osteochondrophytic spurs (osteophytes) obtained from osteoarthritic femoral heads at the time of surgical joint replacement was studied under defined culture conditions in vitro. Osteophytes were primarily present in two anatomic locations, marginal and epi-articular. Minced tissue slices were incubated in the presence of [(35)S]sulphate or [(14)C]glucosamine. Osteophytes incorporated both labelled precursors into proteoglycan, which was subsequently characterized by CsCl-isopycnic-density-gradient ultracentrifugation and chromatography on Sepharose CL-2B. The material extracted with 0.5m-guanidinium chloride showed 78.1% of [(35)S]sulphate in the A1 fraction after centrifugation. Only 23.0% of the [(35)S]sulphate in this A1 fraction was eluted in the void volume of Sepharose CL-2B under associative conditions. About 60-80% of the [(35)S]sulphate in the tissue 4m-guanidinium chloride extract was associated with monomeric proteoglycan (fraction D1). The average partition coefficient (K(av.)) of the proteoglycan monomer on Sepharose CL-2B was 0.28-0.33. Approx. 12.4% of this monomer formed stable aggregates with high-molecular-weight hyaluronic acid in vitro. Sepharose CL-2B chromatography of fractions with lower buoyant densities (fractions D2-D4) demonstrated elution profiles on Sepharose CL-2B substantially different than that of fraction D1, indicative of the polydisperse nature of the newly synthesized proteoglycan. Analysis of the composition and chain size of the glycosaminoglycans showed the following: (1) preferential elution of both [(35)S]sulphate and [(14)C]glucosamine in the 0.5m-LiCl fraction on DEAE-cellulose; (2) the predominant sulphated glycosaminoglycan was chondroitin 6-sulphate (60-70%), with 9-11% keratan sulphate in the monomer proteoglycan; (3) K(av.) values of 0.38 on Sephadex G-200 and 0.48 on Sepharose CL-6B were obtained with papain-digested and NaBH(4)-treated D1 monomer respectively. A comparison of the synthetic with endogenous glycosaminoglycans indicated similar types. These studies indicated that human osteophytes synthesized in vitro sulphated proteoglycans with some characteristics similar to those of mature human articular cartilage, notably in the size of their proteoglycan monomer and predominance of chondroitin 6-sulphate. They differed from articular cartilage primarily in the lack of substantial quantities of keratan sulphate and aggregation properties associated with monomer interaction with hyaluronic acid.     

Correlated metabolism of proteoglycans and hyaluronic acid in bovine cartilage organ cultures

Proteoglycans exist in cartilage as complexes in which many proteoglycan molecules are bound to a central filament of hyaluronic acid. Many studies have investigated changes taking place in proteoglycan monomer structure during cartilage catabolism usually under the assumption that hyaluronic acid is a relatively inert metabolic component of the complex. In this paper we present organ culture data supporting a new hypothesis that the catabolism of proteoglycans and hyaluronic acid are coordinately regulated by chondrocytes. The data indicates that: 1) newly synthesized hyaluronate and proteoglycan maintain a nearly constant ratio, almost identical to that existing for the total chemical amounts of these two components in cartilage tissue; 2) these two components are catabolized with virtually identical kinetics; and 3) this catabolic relationship in vitro reflects the loss of hyaluronate and proteoglycans from native, undissociated aggregates as isolated from the tissue. We conclude that hyaluronate catabolism is an integral part of the overall mechanism of proteoglycan resorption in cartilage and that further understanding of this process may be key to the elucidation of the regulatory pathways for proteoglycan resorption in health and disease.     

Complexes of low-density lipoproteins and arterial proteoglycan aggregates promote cholesteryl ester accumulation in mouse macrophages

We studied the effect of complexes of low-density lipoproteins (LDL) and different proteoglycan preparations from bovine aorta on LDL degradation and cholesteryl ester accumulation in mouse peritoneal macrophages. Native proteoglycan aggregate containing proteoglycan monomers, hyaluronic acid and link protein was isolated by associative extraction of aortic tissue, while proteoglycan monomer was obtained by dissociative isopycnic centrifugation of the native proteoglycan aggregate. In vitro proteoglycan aggregates were prepared by reaction of the proteoglycan monomer with exogenous hyaluronic acid. 125I-labeled LDL-proteoglycan complexes were formed in the presence of 30 mM Ca2+ and incubated with macrophages. At equivalent uronic acid levels in the proteoglycans the degradation of 125I-labeled LDL contained in the native proteoglycan aggregate complex was 3.7-7.5-fold greater than the degradation of the lipoprotein in the proteoglycan monomer complex. Degradation of 125I-LDL in the in vitro aggregate complex, while higher than that in the monomer complex, was markedly less than that in the native aggregate complex. The larger size and the greater complex-forming ability of the native proteoglycan aggregate might account for the greater capacity of the aggregate to promote LDL degradation in macrophages. The proteoglycan-stimulated degradation of LDL produced a marked increase in cholesteryl ester synthesis and content in macrophages. The LDL-proteoglycan complex was degraded with saturation kinetics, suggesting that these complexes are internalized through high-affinity receptors. Degradation was inhibited by the lysosomotropic agent, chloroquine. Acetyl-LDL, but not native LDL, competitively inhibited the degradation of the 125I-LDL component of the complex. Polyanionic compounds such as polyinosinic acid and fucoidin, while completely blocking the acetyl-LDL-stimulated cholesteryl ester formation, had no effect on the proteoglycan aggregate-stimulated cholesterol esterification. This suggests that LDL-proteoglycan complex and acetyl-LDL are not entering the cells through the same receptor pathway. These results demonstrate that the interaction of LDL with arterial wall proteoglycan aggregates results in marked cholesteryl ester accumulation in macrophages, a process likely to favor foam cell formation. A role for arterial proteoglycans in atherosclerosis is obvious.     

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.     

Isolation and partial characterization of a tubulin-like protein from human and swine synaptosomal membranes.

Synaptic membranes from human and swine brains were solubilized with 8 M urea and the proteins were reduced and alkylated. A protein was isolated from both sources and had identical amino acid compositions and molecular weights as determined by electrophoresis on polyacrylamide-sodium dodecylsulfate gels and by ion-exchange chromatography and gel filtration on Bioglas 1000. The apparent molecular weight of the protein was 53 000 on the acrylamide-sodium dodecylsulfate gels. Neither neutral sugars nor sialic acid was a significant component of the protein. When the proteins were digested with trypsin and the resultant peptides subjected to chromatography (n-butanol/acetic acid/water) and electrophoresis (pH 3.7) the peptide maps were identical. The protein comprises 1-2 percent of the total synaptosomal protein. With regard to amino acid composition, molecular weight, peptide map characteristics, behavior on DEAE-cellulose columns, electrophoretic mobility and sugar content, the synaptic protein is quite similar to the monomer of swine tubulin.     

A minor sialoglycoprotein of the human erythrocyte membrane

The sialoglycoprotein periodate fuchsin sulfite 2 has about 8% of the sialic acid contained in the sialoglycoproteins of the human erythrocyte membrane. This polypeptide appears to have an apparent monomeric molecular weight of 35,000, somewhat smaller than the monomer of the major sialoglycoprotein (periodate fuchsin sulfite 1) as judged by sodium dodecyl sulfate-polyacry lamide gel electrophoresis, and has frequently been confused with the monomer of the major sialoglycoprotein. Periodate fuchsin sulfite 2 is not labeled by the lactoperoxidase procedure in the intact cell, although it is accessible to neuraminidase and other hydrolases. On the other hand, this component can be labeled by lactoperoxidase on the cytoplasmic surface of open membranes or resealed ghosts. Thus, it is a trans membrane protein. Although most of the other transmembrane proteins of the human erythrocyte membrane are extracted from the membrane by 0.1% Triton X-100 in 7 mm phosphate buffer, pH 7.4, this component is not removed and may be a cytoskeletal component. Trypsin, chymotrypsin, and thermolysin peptides, as well as cyanogen bromide fragments, clearly indicate that the primary sequence of this polypeptide can be distinguished from dimeric or monomeric forms of the major sialoglycoprotein (periodate fuchsin sulfite 1).