Isolation and partial characterization of proteoglycans from rat incisors.

Newly synthesized proteoglycans of rat incisors were labelled in vivo for 6h with [35S]-sulphate in order to facilitate their detection during purification and characterization. Proteoglycans were extracted from non-mineralized portions (predentine) of rat incisors with 4M-guanidinium chloride and subsequently from dentine by demineralization with a 0.4M-EDTA solution containing 4M-guanidinium chloride. Both extractions were performed at 4 degrees C in the presence of proteinase inhibitors. Purification of proteoglycans was achieved with a procedure involving gel-filtration chromatography, selective precipitation of phosphoproteins, affinity chromatography and ion-exchange chromatography. Two proteoglycan populations were found in the initial extract (Pd-PG I and Pd-PG II), whereas only one fraction (D-PG) was obtained after demineralization. The minor proteoglycan fraction from the first extract, Pd-PG I, although not totally characterized, differed sharply from the other proteoglycans in that it had a larger molecular size with larger glycosaminoglycan chains composed of chondroitin 4- and 6-sulphate isomers. In contrast, the major proteoglycans Pd-PG II and D-PG had smaller hydrodynamic sizes with smaller glycosaminoglycan chains (but larger than those from bovine nasal cartilage proteoglycans) composed exclusively of chondroitin 4-sulphate. The major proteoglycans were incapable of interacting with hyaluronic acid. In general, the amino acid compositions of the major proteoglycans of rat incisors resembled that of bovine nasal cartilage proteoglycans, but the former had lower proline, valine, isoleucine, leucine, and higher aspartic acid, contents.     

Isolation and partial characterization of proteoglycans from rice bran

Extraction of rice bran with hot water, followed by removal of protein and starch, yielded a proteoglycan. Successive fractionation of this proteoglycan by salting out with ammonium sulfate and by DEAE-Sephadex column chromatography yielded several fractions shown to be homogeneous by disc electrophoresis. Treatment of the fractions with alkali and a proteolytic enzyme has shown that the polysaccharide and protein of the proteoglycan are most probably linked through an O-glycosyl linkage through hydroxyproline.     

Isolation and characterization of proteoglycans from porcine lungs.

Proteoglycans were extracted from porcine lungs with 4 M guanidinium chloride. The extract was subjected to associative density gradient centrifugation, and four equal fractions, labeled A1 through A4 from the bottom to the top of the gradient, were obtained. The pooled A1 fractions containing proteoglycan aggregates were further fractionated by dissociative density gradient centrifugation to yield four equal fractions labeled A1D1 through A1D4 from the bottom to the top of the gradient. These fractions were analyzed for their protein, uronic acid, glucosamine, galactosamine, hexose, and sialic acid content. The fraction A1D1 with the highest buoyant density had the highest content of uronic acid and galactosamine, and lowest content of protein, indicating the enrichment of proteoglycan monomers at the bottom of the dissociative density gradient. As the density of the gradient decreased, the protein, hexoses, and sialic acid content increased, whereas uronic acid and galactosamine content decreased. The amino acid analysis showed similar composition for all four fractions with aspartic acid, serine, glutamic acid, proline, glycine, alanine, valine, and leucine as the major constituent amino acids. No hydroxyproline was detected in any of the fractions. As the buoyant density of the fractions decreased, the aspartic acid content increased and glycine content decreased.     

Isolation and characterization of dermatan sulphate proteoglycans from bovine sclera.

1. Proteoglycans were extracted from sclera with 4 M-guanidine hydrochloride in the presence of proteinase inhibitors and purified by ion-exchange chromatography and density-gradient centrifugation. 2. The entire proteoglycan pool was characterized by compositional analyses and by specific chemical (periodate oxidation) and enzymic (chondroitinases) degradations. The glycan moieties of the molecules were exclusively galactosaminoglycans (dermatan sulphate-chondroitin sulphate co-polymers). In addition, the preparations contained small amounts of oligosaccharides. 3. The scleral proteodermatan sulphates were fractionated into one larger (I) and one smaller (II) component by gel chromatography. Proteoglycan I was eluted in a more excluded position on gel chromatography in 0.5 M-sodium acetate than in 4.0 M-guanidine hydrochloride. Reduced and alkylated proteoglycan I was eluted in the same position (in 0.5 M-sodium acetate) as was the starting material (in 4.0 M-guanidine hydrochloride). The elution position of proteoglycan II was the same in both solvents. Proteoglycans I and II had s0 20,w values of 2.8 x 10(-13) and 2.2 x 10(-13) s respectively in 6.0 M-guanidine hydrochloride. 4. The two proteoglycans differed with respect to the nature of the protein core and the co-polymeric structure of their side chains. Also proteoglycan I contained more side chains than did proteoglycan II. The dermatan sulphate side chains of proteoglycan I were D-glucuronic acid-rich (80%), whereas those of proteoglycan II contained equal amounts of D-glucuronic acid and L-iduronic acid. Furthermore, the co-polymeric features of the side chains of proteoglycans I and II were different. The protein core of proteoglycan I was of larger size than that of proteoglycan II. The latter had an apparent molecular weight of 46 000 (estimated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis), whereas the former was greater than 100 000. In addition, the amino-acid composition of the two core preparations was different. 5. As proteoglycan I altered its elution position on gel chromatography in 4 M-guanidine hydrochloride compared with 0.5 M-sodium acetate it is proposed that a change in conformation or a disaggregation took place. If the latter hypothesis is favoured, aggregation may be due to self-association or mediated by an extrinsic molecule, e.g. hyaluronic acid.     

Isolation and characterization of high-buoyant-density proteoglycans from bovine femoral-head cartilage.

Proteoglycans were extracted from bovine (15-18 months old) femoral-head cartilage. The heterogeneity of the A1D1 proteoglycan fraction was examined by gel chromatography, sedimentation velocity, sucrose rate-zonal centrifugation and CS2SO4 isopycnic centrifugation. In all cases polydisperse but unimodal distributions were obtained. Chemical analysis of the preparation yielded a galactosamine/glucosamine molar ratio of 7:1, and 13C n.m.r. spectroscopy showed that the chondroitin sulphate comprised equal proportions of the 4- and 6-sulphate isomers. Gel chromatography of a papain and Pronase digest of the proteoglycan indicated that the chondroitin sulphate chains had a Mn of approx. 10500. The mean buoyant density of the proteoglycan in pure CS2SO4 was 1.46 g/ml. Physical characterization of the proteoglycan preparation in 4M-guanidine hydrochloride, pH 7.4, by using conventional light-scattering gave a radius of gyration of 42 nm and a Mw of 0.96 X 10(6). Quasi-elastic light-scattering in the same solvent yielded a translational diffusion coefficient, D020, of 5.41 X 10(-8) cm2 X S-1, and ultracentrifugation gave a sedimentation coefficient, S020, of 12.0S. Thus from sedimentation-diffusion studies a Mw of 1.36 X 10(6) was calculated. The possible origins for the differences in the two molecular-weight estimates are discussed. It is concluded that the high-buoyant-density proteoglycans from bovine articular cartilage are significantly smaller than those from bovine nasal septum, and that this is largely due to the smaller size of their chondroitin sulphate chains.     

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.     

Isolation of inclusion bodies from rabbit lung parenchyma

The mitochondrial-plus-lysosomal fraction of rabbit lung parenchyma was studied by equilibrium density centrifugation in continuous sucrose density gradients (specific gravity 1.035 to 1.250). High concentrations of lysosomal marker enzymes were found both in a broad band at density 1.15–1.18, a density typical for lysosomes, and in a band at density 1.06–1.07. This light density band also had the highest specific activity of phospholipid, which thin layer and gas-liquid chromatography showed to be primarily lecithin with a high content of palmitic acid residues. Electron microscopy of material from the light density band showed a homogeneous array of particles which bear a strong resemblance to the inclusion bodies of the type II alveolar epithelial cell as seen in electron micrographs of rabbit lung tissue sections. These data suggest that the light density band is an isolation of intact type II alveolar epithelial cell inclusion bodies, which previous studies have implicated as the storage site of the phospholipid moiety of pulmonary surfactant.     

Isolation and characterization of galanin from sheep brain.

Galanin is a well-known, naturally occurring peptide, which has been characterized from both cDNA sequences and direct peptide analysis. Previous structural studies have been made using intestinally derived material. This report concerns galanin isolation from sheep brain and its sequence determination. Sheep galanin shows great similarity to pig galanin, differing by one amino acid substitution, that being a histidine residue, as in cow and rat galanin, instead of tyrosine at position 26.     

Chondroitin proteoglycans from squid skin. Isolation, characterization and immunological studies

Two populations of proteochondroitins were isolated from 4 M guanidine hydrochloride extracts of squid skin by a combination of ion exchange, gel chromatography and density gradient centrifugation. The proteoglycans, Mr 4.8 x 10(5) and 2.8 x 10(5), contained four and two chondroitin chains respectively and unusual oligosaccharides with uronic acid and sulphate groups, and had different amino acid and neutral sugar composition. The chondroitin chains isolated after alkaline borohydride treatment contained varying amounts of glucose, galactose, mannose, fucose and xylose, most likely as branches. Both proteoglycans were antigenic to the rabbit and showed considerable cross-reactivity as assessed by competition experiments using the ELISA technique. The proteoglycans reacted neither with exogenous hyaluronic acid nor with each other to form aggregates.     

Isolation of in vivo radiolabeled proteoglycans from rat lung

Sulfated macromolecules of rat lung tissue were labeled in vivo with 35SO4 and extracted with a solution of 7 M urea containing 0.4% Triton X-100. DEAE-Sephacel chromatography separated sulfated macromolecules into three pools. Pool I consisted of high-molecular-weight, low-density sulfated glycoprotein, probably of mucous secretion origin. Pool II contained a mixture of proteoheparan sulfate and proteodermatan sulfate, together with core protein-free heparan sulfate chains. Pool III was very heterogeneous; its resolution into at least four proteoglycan species was achieved by CsCl density gradient centrifugation. Those included two (high- and low-density) species of proteoheparan sulfate, high-density proteochondroitin sulfate, and medium-density (1.45 less than rho less than 1.55 g/ml) proteodermatan sulfate.     

Isolation and characterization of sheep pepsin.

Sheep pepsin was isolated (approx. 120-fold purification) from aqueous abomasal homogenates by (1) pH fractionation, (2) chromatography on Sepharose 4B-poly-L-lysine columns and (3) gel filtration on Sephadex G-100. The enzyme had mol.wt. approx. 34000, N-terminal valine and C-terminal alanine. The amino acid composition of sheep pepsin was generally similar to that of pig and ox pepsins, with a very low content of basic residues and a high content of acidic and hydroxy-amino acids. The pH optimum for NN-dimethyl-casein and NN-dimethyl-haemoglobin as substrates was approx. 1.8. The Km and kcat. for NN-dimethyl-haemoglobin were 46micronM and 1100min-1 respectively, and for NN-dimethyl-casein the corresponding parameters were 50micronM and 420min-1. These values were generally similar to those for pig and ox pepsins. At the pH optimum of 4.6, the sheep pepsin was about 50% as active on benzyloxycarbonyl-L-histidyl-L-phenyl-alanyl-L-tryptophan ethyl ester as was pig pepsin. The pH optimum for the hydrolysis of N-acetyl-L-phenylalanyl-L-di-iodotyrosine by sheep, ox and pig pepsins was approx. 1.85.     

Isolation and partial characterization of trehalase from Ascaris muscle.

The tissues of female Ascaris suum were assayed for alpha,apha'-glucoside 1-D-glucohydrolase (trehalase) activity. A soluble from of the enzyme was isolated from muscle tissue and purified approximately 37-fold. The enzyme was specific for trehalose as substrate. The pH optimum for enzymatic activity was found to be 6.0, and the apparent Km for trehalose was estimated to be 2.1 x 10-4 M. The product of the reaction was identified as D-glucose by chemical, chromatographic and enzymatic methods.     

Isolation and partial characterization of cysteine proteinase from sparganum.

A proteolytic enzyme was purified from the tissue extract of spargana (plerocercoids of Spirometra erinacei) by DEAE-Trisacryl M ion exchange chromatography and thiopropyl-sepharose affinity chromatography resulted in a 21-fold purification. The proteinase activity was assayed with a synthetic fluorescent substrate, carbobenzoxy-phenylalanyl-7-amino-4-trifluoromethyl-coumarin. SDS-polyacrylamide gel electrophoresis of the purified materials revealed a single 28,000 dalton band. Inhibitor profiles of the band indicated that it belonged to cysteine endopeptidases. It exhibited identical pH curves with optimum at pH 5.5, and 50% activity from pH 4.7 to 8. It could completely degrade collagen chains to three identical products. It also showed some activity on hemoglobin. Furthermore, the band on immunoblots was reactive to the sera of sparganosis patients. These results suggest that the proteolytic enzyme belongs to cysteine proteinase which plays a role in the tissue penetration. Also it may be used as the antigen for diagnosis of active sparganosis.     

Isolation and partial characterization of mitogenic factors from cementum.

Cementum is the mineralized structure through which soft connective tissues are attached to the teeth. It is a unique calcified tissue characterized by a low metabolic turnover, lack of blood supply, and presence of very few cells. However, it contains substances that influence the biological activities of fibroblasts of adjacent soft tissues. We have partially characterized cementum proteins that have mitogenic activity toward fibroblasts. Cementum was harvested from bovine teeth, and mitogenic factors were extracted in 0.5 M CH3COOH. Heparin-Sepharose chromatography separated the mitogenic activity into a major and a minor fraction eluted by 0.5 and 2.0 M NaCl, respectively. The distribution of cementum mitogens in heparin-Sepharose fractions was different from that of alveolar bone and other bones. The cementum mitogenic factor eluting with 2.0 M NaCl from a heparin-Sepharose column was shown to be basic fibroblast growth factor (bFGF) on the basis of inhibition by anti-bFGF antibody and Western blots. The 0.5 M NaCl fraction was purified by HPLC with use of a combination of a DEAE-3W column followed by TSK-250 and C18 columns. NaDodSO4-polyacrylamide gel electrophoresis revealed that the purified fraction contained two protein bands with Mr 22,000 and 19,000, and mitogenic activity was associated with the Mr 22,000 species. The activity of this mitogen, designated as CGF, was potentiated by small quantities of plasma-derived serum or epidermal growth factor. It was heat resistant, but was destroyed by reduction. Assays of CGF preparations revealed that they contained no detectable platelet-derived growth factor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of dermatan sulphate and heparan sulphate proteoglycans from fibroblast culture.

35SO42(-)- and [3H]leucine-labelled proteoglycans were isolated from the medium and cell layer of human skin fibroblast cultures. Measures were taken to avoid proteolytic modifications during isolation by adding guanidinium chloride and proteolysis inhibitors immediately after harvest. The proteoglycans were purified and fractionated by density-gradient centrifugation, followed by gel and ion-exchange chromatography. Our procedure permitted the isolation of two major proteoglycan fractions from the medium, one large, containing glucuronic acid-rich dermatan sulphate chains, and one small, containing iduronic acid-rich ones. The protein core of the latter proteoglycan had an apparent molecular weight of 47000 as determined by polyacrylamide-gel electrophoresis, whereas the protein core of the former was considerably larger. The major dermatan sulphate proteoglycan of the cell layer was similar to the large proteoglycan of the medium. Only small amounts of the iduronic acid-rich dermatan sulphate proteoglycan could be isolated from the cell layer. Instead most of the iduronic acid-rich glycans appeared as free chains. The heparan sulphate proteoglycans found in the cell culture were largely confined to the cell layer. This proteoglycan was of rather low buoyant density and seemed to contain a high proportion of protein. The major part of the heparan sulphate proteoglycan from the medium had a higher buoyant density and contained a smaller amount of protein.     

Pyruvate decarboxylase from Zymomonas mobilis. Isolation and partial characterization

Pyruvate decarboxylase (EC 4.1.1.1) from the ethanol producing bacterium Zymomonas mobilis was purified to homogeneity. This enzyme is an acidic protein with an isoelectric point of 4.87 and has an apparent molecular weight of 200,000±10,000. The enzyme showed a single band in sodium dodecylsulfate gel electrophoresis with a molecular weight of 56,500±4,000 which indicated that the enzyme consists of four probably identical subunits. The dissociation of the cofactors Mg²⁺ and thiamine pyrophosphate at pH 8.9 resulted in a total loss of enzyme activity which could be restored to 99.5% at pH 6.0 in the presence of both cofactors. For the apoenzyme the apparent K _m values for Mg²⁺ and thiamine pyrophosphate were determined to be 24 M and 1.28 M. The apparent K _m value for the substrate pyruvate was 0.4 mM. Antiserum prepared against this purified pyruvate decarboxylase failed to crossreact with cell extracts of the reportedly pyruvate decarboxylase positive bacteria Sarcina ventriculi, Erwinia amylovora, or Gluconobacter oxydans, or with cell extracts of Saccharomyces cerevisiae.Abbreviations Tris-buffer 0,01 M tris-HCl buffer, containing 1 mM MgCl₂ 0.1 mM EDTA, 1.0 mM thiamine pyrophosphate, 2 mM mercaptopropanediol, pH 7.0