Glycosaminoglycans of arterial basement membrane-like material from cultured rabbit aortic myomedial cells

Arterial basement membrane-like material was prepared by a sonication-differential centrifugation technique from cultures of rabbit aortic myomedial cells after metabolic labelling with [35S]sulphate and [3H]glucosamine. Labelled glycosaminoglycans were obtained from isolated basement membrane-like material by proteinase digestion and gel filtration. Glycosaminoglycans were identified by a combination of Sephadex G-50 chromatography and sequential degradation with nitrous acid, Streptomyces hyaluronidase, testicular hyaluronidase and chondroitinase ABC. The data showed that heparan sulphate and chondroitin sulphate were the predominant glycosaminoglycans of myomedial basement membrane-like material. Heparan sulphate accounted for about 55% of [3H]glucosamine-labelled glycosaminoglycans. In addition small amounts of hyaluronic acid was present. Only trace amounts of dermatan sulphate was found. The glycosaminoglycans were analysed by DEAE-cellulose chromatography. Two major peaks were found in the chromatogram consistent with the predominance of heparan sulphate and chondroitin sulphate.     

Purification of hyaluronidase from human placenta.

Hyaluronidase [EC 3.2.1.35] was isolated from human placenta and purified by ammonium sulfate fractionation, DEAE-cellulose column chromatography and gel filtration on Sephadex G-150. Its isoelectric point was at pH 5.2 and the molecular weight was 7 X 10(4) based on Sephadex G-200 gel filtration data. This enzyme was very stable at temperatures below 30 degree, but was almost completely inactivated at 60degree within 30 min. Its optimum pH was 3.9, a characteristic property of a lysosomal hyaluronidase. The Michaelis constant was 1.18 x 10(-1) mg per ml with purified hyaluronate. This enzyme depolymerized hyaluronate, chondroitin, chondroitin 4-sulfate and 6-sulfate, and the end product formed from hyaluronate was tetrasaccharide. Its biological diffusing activity was statistically significant on intracutaneous injection of 1.86 mU of the hyaluronidase into the back skine of a rabbit.     

Degradation of heparin in mouse mastocytoma tissue

1. Heparin was prepared from mouse mastocytoma tissue by mild procedures, including extraction of mast-cell granules with 2m-potassium chloride, precipitation of the extracted polysaccharide with cetylpyridinium chloride from 0.8m-potassium chloride and finally digestion of the isolated material with testicular hyaluronidase. The resulting product (fraction GE(H)) represented approx. 40% of the total heparin content of the tissue. 2. Fraction GE(H) was fractionated by gel chromatography on Sepharose 4B into three subfractions, with average molecular weights ( M(w)) of approx. 60000-70000 (highly polydisperse material), 26000 and 9000 respectively. Treatment of each of the subfractions with alkali or with papain did not affect their behaviour on gel chromatography. Amino acid and neutral sugar analyses indicated that the two low-molecular-weight fractions consisted largely of single polysaccharide chains lacking the carbohydrate-protein linkage region. It was suggested that these heparin molecules had been degraded by an endopolysaccharidase. 3. Pulse labelling in vivo of mastocytoma heparin with [(35)S]sulphate showed initial labelling of large molecules followed by a progressive shift of radioactivity toward fractions of lower molecular weight. Further, heparin-depolymerizing activity was demonstrated by incubating (35)S-labelled heparin in vitro with a mastocytoma 10000g-supernatant fraction. Appreciable degradation of the polysaccharide occurred, as demonstrated by gel chromatography. In contrast, no depolymerization was observed on subjecting (14)C-labelled chondroitin sulphate to the same procedure.     

The distribution of sulphate residues in the chondroitin sulphate chain

1. Electrophoresis of chondroitin sulphate, before and after partial degradation with testicular hyaluronidase, revealed charge heterogeneity of the degraded but not of the intact polymer. 2. Hyaluronidase-treated chondroitin sulphate was fractionated by gel chromatography. Two subfractions which were essentially monodisperse with regard to molecular weight (values of 8600 and 4800, respectively) were separated further by chromatography on Dowex 1. The resulting subfractions differed considerably with respect to their sulphate/disaccharide molar ratios. 3. Amino acid and neutral-sugar analyses of the Dowex 1 subfractions showed that the less sulphated fragments contained the carbohydrate-protein linkage region, whereas the high-sulphated fragments essentially lacked this constituent. It was concluded that chondroitin sulphate contains relatively less sulphate in the vicinity of the carbohydrate-protein linkage region than in the more peripheral portion of the polysaccharide chain.     

Immunoenzymic studies on testicular hyaluronidase from rhesus monkeys (Macaca mulatta)

Hyaluronidase from rhesus monkey testes was purified by detergent extraction, ammonium sulphate fractionation, Sephadex G-200 column chromatography and concanavalin A-Sepharose affinity chromatography. The purified hyaluronidase showed one protein band on acrylamide gel electrophoresis. Antibodies to the purified hyaluronidase were raised in rabbits and showed a single precipitin line by Ouchterlony gel diffusion. The enzyme had a molecular weight of 62,000. The Km was 0.5 mg/ml for hydrolysis of hyaluronic acid at 37 degrees C. The optimum pH for the enzyme was 5.0 but activity was present over a broad pH range. The hyaluronidase was inhibited by HgCl2, CuSO4, FeSO4 and p-chloromercuribenzoate all at a concentration of 2 x 10(-4) M. Cysteine protected the enzyme against HgCl2 inhibition.     

Evidence for the existence of a multichain proteoglycan of heparan sulphate

1. Glycosaminoglycans were extracted with 2m-potassium chloride from bovine aorta and purified by precipitation with cetylpyridinium chloride from 0.5m-potassium chloride. The yield amounted to 24% of the total glycosaminoglycan content of the tissue. 2. After removal of chondroitin sulphate by digestion with testicular hyaluronidase, the residual glycosaminoglycan material (11% of the extracted polysaccharide) was fractionated by gel chromatography on Sephadex G-200. Two peaks (I and II) were obtained, the more retarded of which (II) corresponded to single polysaccharide chains. 3. The macromolecular properties of fraction I were investigated by repeated gel chromatography, after treatment of the fraction with alkali or digestion with papain. In both cases the elution position of fraction I was shifted towards that of the single polysaccharide chains. 4. Analysis of fraction I showed approximately equal amounts of heparan sulphate and dermatan sulphate. It is concluded that these glycosaminoglycans both occur in the aortic wall as multichain proteoglycans.     

Lectin from rice

N-Acetyl-D-glucosamine-binding lectin was isolated and purified from rice by ammonium sulphate fractionation and affinity chromatography using N-acetyl-D-glucosamine linked Sepharose 6B column. It gave a single hand on Polyacrylamide disc gel. It was identified as a glycoprotein. The purified lectin dissociated into two components on Sephadex G-100 column chromatography,-a higher molecular weight fraction not containing any carbohydrate and a lower molecular weight glycoprotein fraction. The apparent molecular weights of these fractions were 85,000 and 14,500. The lectin agglutinated erythrocytes of human A,B,O groups and of several other mammals and its activity was inhibited only by N-acetyl-D-glucosamine. The glycopeptide isolated by pronase digestion of the lectin was homogeneous and did not possess agglutinating activity. It contained about 10% carbohydrate of which xylose, arabinose and glucose were the major components.     

The molecular weight-dependent distribution of urinary glycosaminoglycans in Werner's syndrome

The macromolecular AGAG in the urine of patients with Werner's syndrome were analyzed by enzymatic methods after digestion with chondroitinases and Streptomyces hyaluronidase. The molecular weight-dependent distribution of the urinary AGAG has been determined by gel filtration on a Sephadex G-100 column. The distribution of HA and HS was predominant in the macromolecular fractions. Chondroitin sulfate isomers were prominent in the low molecular weight fractions but the ratio of the 4-type to the 6-type increased with decreasing molecular weight. These observations indicated that Werner's syndrome is a metabolic disorder of the molecular weight-dependent AGAG composition.     

东亚钳蝎毒透明质酸酶的纯化和部分性质的研究

用CM-SephadexC50,CM-SephadexC25和SephadexG-75凝胶过滤,从东亚钳蝎毒中提纯蝎毒透明质酸酶,应用低pH系统不连续聚丙烯酰胺凝胶圆盘电泳,SDS-不连续聚丙烯酰胺凝胶垂直板电泳鉴定均为单一条带,活力提高34倍,产率为12％,纯品无出血活性,无神经毒性。用凝胶过滤法和SDS电泳法测得分子量为54000,PAS染色证实为糖蛋白。 纯化的透明质酸酶的最适pH为4.5～6.5,最适温度为37℃,该酶对热的稳定性比蛇毒透明质酸酶高一些,但在碱性环境中也易失活。0.15MNaCl对酶活性有明显稳定作用,Fe~(2+)、Fe~(3+)及肝素对酶活性有明显的抑制作用,Cu~(2+)对酶活力也有一定影响。     

Attempted isolation of a heparin proteoglycan from bovine liver capsule

(1) Polysaccharides were isolated from bovine liver capsule by extraction with 2m-potassium chloride followed by precipitation from 0.8m-potassium chloride with cetylpyridinium chloride. Chondroitin sulphate was eliminated by digestion with hyaluronidase. The yield of heparin was approx. 40% of that obtained after extraction of the papain-digested tissue. (2) The macromolecular properties of the hyaluronidase-digested polysaccharide were studied by gel chromatography on Sephadex G-200 of the intact, as well as of the alkali-degraded, material. The results suggested the presence of single heparin chains in addition to a dermatan sulphate proteoglycan. (3) A purified heparin preparation was analysed for amino acids and neutral sugars. Xylose, galactose and serine were found in amounts corresponding to 0.1, 0.2, and 0.4 residue/polysaccharide chain (mol.wt. 7400), respectively. It is suggested that the isolated material had been degraded by a polysaccharidase with endo-enzyme properties.     

Staphylococcal L-asparaginase: purification and properties of enzymic protein

Staphylococcal L-asparaginase has been purified 400-fold with 40% recovery. The procedure involves ammonium sulphate precipitation and a column chromatography on Sephadex G-200 gel filtration). The enzyme is composed of not identical subunits. protein (pI 4.4) with the approximate molecular weight of 125,000 (estimated by Sephadex G-200 gel filtration). The enzyme is composed of not identical subunits. The polyacrylamide-SDS gel electrophoresis indicated two subunits with molecular weight 18,000 and 22,000.     

The purification and some properties of pig liver hyaluronidase

Hyaluronidase (hyaluronate 4-glycanohydrolase, EC 3.2.1.35) has been isolated from pig liver and purified 1720-fold with an overall yield of 9.5%. The enzyme was purified using an acid-extraction technique followed by successive chromatography on DEAE-cellulose, two boronate affinity columns and Sephadex G-75. This final preparation, which was essentially homogeneous as determined by gel electrophoresis, was a single subunit enzyme of apparent molecular weight 70 000 with an isoelectric point of 5.0. No contaminant enzymes capable of degrading glycosaminoglycans could be detected in the final preparation. The substrate specificity of the enzyme was the same as for bovine testicular hyaluronidase; however, both the Km and V values were significantly lower for the pig liver enzyme with all of the substrates tested (hyaluronate, chondroitin 4-sulphate, chondroitin 6-sulphate). A full kinetic analysis of the enzyme using hyaluronate as a substrate showed that the activity of pig liver hyaluronidase was uncompetitively activated by either protons or NaCl.     

蓖麻β-半乳糖苷酶的纯化及其基本性质

蓖麻籽黄化苗中存在高活性β-半乳糖苷酶。经硫酸铵分级分离、DEAE-纤维素离子交換层析、Sephadex G-100、CM-Sephadex和DEAE-Sephadex层析纯化。活性收率为6.4％,纯化倍数达107倍。纯化了的酶经聚丙烯酰胺凝胶电泳显示单一蛋白带,SDS-PAGE显示两条蛋白带,其相应分子量分别为3.25×10~4和2.94×10~4。用Sephadex G-200分子筛层析法测得分子量为6.7×10~4。综合上述结果推测该酶是由两个不同的亚基构成。以邻硝基苯酚-β-半乳糖苷为底物测得该酶的表观Km为5.9×10~(-3)mol/L。最适pH和最适温度分别为4.5和50℃。酸碱稳定区域在pH4.6—7.5之间。不同浓度缓冲液以及不同种类缓冲液、不同金属离子对酶活性影响均进行了讨论。     

Beta-endorphin-like and adrenocorticotropin-like materials in heart tissues of the rat, gerbil, hamster and guinea pig

1. Heart tissues of several rodent species including the rat, gerbil (Meriones unguiculatus), hamster (Mesocricetus auratus) and guinea pig (Cavia porcellus) were extracted with an acetone-water-HCl mixture. An acid acetone powder was obtained by adding a copious volume of acetone to the extract. 2. Rat heart acid acetone powder was subjected to ion exchange chromatography on CM-cellulose. Gerbil heart acid acetone powder was subjected to salt fractionation, gel filtration on Sephadex G-10 and then ion exchange chromatography on CM-cellulose. Hamster and guinea pig heart acid acetone powders were subjected to gel filtration on Sephadex G-25. 3. The fractions were assayed for the ability to stimulate corticosterone production in isolated rat adrenal decapsular (zona fasciculata, zona reticularis and medulla) cells, to displace D-ala2-D-leu5-(tyrosyl-3,5-3H) enkephalin from binding to rat brain membranes, and to inhibit 125I-human beta-endorphin from binding to its antibodies. 4. The widespread occurrence of beta-endorphin-like immunoreactivity among the rat heart CM-cellulose fractions may reflect different species of beta-endorphin. The fraction with the highest beta-endorphin-like immunoreactivity and opiate receptor binding activity was strongly adsorbed on CM-cellulose. 5. In hamster and guinea pig hearts, beta-endorphin-like immunoreactivity and opiate receptor binding activity were distributed among high molecular weight and low molecular weight fractions. 6. In gerbil hearts, opiate receptor binding activity was present in fractions unretarded on Sephadex G-10 (i.e. with a molecular weight greater than 700) as well as in the retarded fractions (i.e. with a molecular weight smaller than 700).(ABSTRACT TRUNCATED AT 250 WORDS)     

Pro-enkephalin peptides possessing Met-enkephalin-Arg6-Gly7-Leu8-immunoreactivity in rat CSF, striatum and adrenal gland

Cerebrospinal fluid (CSF) taken from rats implanted with chronic cisternal cannulae and extracts prepared from rat adrenal gland and striatum were subjected to Sephadex G-50 chromatography and HPLC. Fractions were monitored using specific radioimmunoassays (RIA) for the pentapeptide methionine enkephalin (Met-Enk) and methionine enkephalin-Arg6-Gly7-Leu8 (Met-EnkRGL). In rat CSF, striatum and adrenal gland, three Met-EnkRGL-immunoreactive (IR) peaks of Mrs 8000, 5000 and 1000 daltons were detected. The same peaks were also found to possess Met-Enk-immunoreactivity after enzyme digestion of Sephadex G-50 fractions with trypsin and carboxypeptidase B (CPB), suggesting their derivation from proenkephalin. HPLC of the 8K and 5K peaks on a column of Ultrapore RPSC showed them to elute discretely with similar retention times, indicative of hydrophobic peptides of large molecular weight. Their similar hydrophobicities yet significant separation during gel filtration would suggest that the 8K and 5K peptides are structurally closely related yet different with respect to their molecular weights. HPLC of the small molecular weight material from rat striatum and adrenal gland revealed the presence of Met-EnkRGL and Met-EnkRGL sulphoxide in both tissues. In rat striatum Met-Enk and its sulphoxide were also detected. The oxidised pentapeptide was found to be present in rat CSF, together with two unidentified small molecular weight Met-Enk-IR peaks detected without prior enzyme digestion of fractions. The small molecular weight Met-EnkRGL-IR material in rat CSF was found to be comprised of two unknown peptides which were less hydrophobic than Met-EnkRGL and its sulphoxide derivative.     

Brain pyridoxal kinase. Purification and characterization

Pyridoxal kinase has been purified 9000-fold from sheep brain. The purification procedure involves ammonium sulphate fractionation, DEAE-cellulose chromatography, affinity chromatography and Sephadex G-100 gel filtration. The final chromatography step yields a homogeneous preparation of high specific activity with a pI of 5. The molecular mass of the native enzyme was estimated to be approximately 80 kDa by 10-25% gradient polyacrylamide gel electrophoresis and Sephadex G-200 gel filtration. The subunit molecular mass was determined by sodium dodecyl sulphate (SDS)/polyacrylamide gel electrophoresis to be 40 kDa compared with a series of molecular mass standards. This indicates that pyridoxal kinase is a dimeric enzyme. Further results obtained from electron microscopy, using a negative staining technique, provide evidence that pyridoxal kinase exists as a dispherical subunit structure.     

Structural studies on sialylated and sulphated O-glycosidic mannose-linked oligosaccharides in the chondroitin sulphate proteoglycan of brain.

We have previously described the structures of neutral and sialylated O-glycosidic mannose-linked tetrasaccharides and keratan sulphate polysaccharide chains in the chondroitin sulphate proteoglycan of brain. The present paper provides information on a series of related sialylated and/or sulphated tri- to penta-saccharides released by alkaline-borohydride treatment of the proteoglycan glycopeptides. The oligosaccharides were fractionated by ion-exchange chromatography and gel filtration, and their structural properties were studied by methylation analysis and fast-atom-bombardment mass spectrometry. Five fractions containing [35S]sulphate-labelled oligosaccharides were obtained by ion-exchange chromatography, each of which was eluted from Sephadex G-50 as two well-separated peaks. The apparent Mr values of both the large- and small-molecular-size fractions increased with increasing acidity (and sulphate labelling) of the oligosaccharides. The larger-molecular-size fractions contained short mannose-linked keratan sulphate chains of Mr 3000-4500, together with some asparagine-linked oligosaccharides. The smaller tri- to penta-saccharides, of Mr 800-1400, appear to have a common GlcNac(beta 1-3)Manol core, and to contain one to two residues of sialic acid and/or sulphate.     

Beta-endorphin-like and adrenocorticotropin-like materials in turtle heart and intestine

1. Turtle heart and intestine acetone powders were extracted with an acetone-water-HCl mixture. An acid acetone powder resulted by adding a copious volume of acetone to the extract. The powder was subjected to gel filtration on Sephadex G-25 and ion exchange chromatography on CM-cellulose. 2. In turtle heart, corticotropin-like bioactivity was distributed among chromatographic fractions (derived from material unretarded on Sephadex G-25) unadsorbed and adsorbed on CM-cellulose. The highest opiate receptor binding activity and beta-endorphin-like immunoreactivity were adsorbed on CM-cellulose. 3. In turtle intestine, corticotropin-like bioactivity was absent. Opiate receptor binding activity was present in fractions unretarded as well as in fractions retarded on Sephadex G-25, indicating a molecular weight of greater and smaller than 5000 respectively. 4. The highest opiate receptor binding activity and beta-endorphin-like immunoreactivity were found in a fraction adsorbed on CM-cellulose.     

The antigenic structure ofFrancisella tularensis

The ether antigen ofFrancisella tularensis was fractionated using DEAE-cellulose and CM-cellulose chromatography, and Sephadex G-200 gel filtration. The CM-cellulose chromatography did not appear to be a suitable method for separation of individual components of the complex ether antigen. Most of the polysaccharide material and substances with a high phosphorus content were eluted already in the first peak of the elution curve. This method could be used only to separate a component, yielding one immunoelectrophoretic precipitin line, localized towards the cathode. On the other hand, the DEAE-cellulose chromatography, and particularly the Sephadex G-200 gel filtration (especially when recycling was introduced), yielded a clear separation of relatively clean components of the ether antigen. The present work provides a comparison between the immunochemical (immunoelectrophoretic) properties of fractions obtained by these methods and components isolated by salting out with ammonium sulphate, ethanol or trichloracetic acid precipitation.     

IUB commission of editors of biochemical journals the citation of bibliographic references in biochemical journals recommendations (1971)

A pentose-rich acidic glycoprotein was isolated from protease digested bovine vitreous humor by fractionation on an AG1-X2 column using NaCl solution gradient.The material eluted at 0.35 M NaCl (glycoprotein) was electrophoretically heterogeneous at pH 8.6 after partial purification on Sephadex G-25. Gel filtration on G-100 resolved the glycoprotein into two fractions. These fractions differ in molecular weight; mol. wt approx. 95 000 material consisted of two components on electrophoresis and mol. wt approx. 28 000 material showed only a single component on electrophoresis. The lower molecular weight component was re-chromatographed on Sephadex G-100 yielding a single orcinol positive component which gave a homogeneous band on gel electrophoresis.Quantitative analysis of this material gave 30% protein, 7.0% pentose, 18.7% glucosamine, 9.2% galactosamine, 10.9% hexuronic acid and 16.1% hexose.Treatment with 0.5 M NaOH at 20°C for 24 h resulted in a 50% decrease in the threonine content suggesting the possible involvement of this amino acid in the protein-carbohydrate linkage group.Paper chromatography of the fraction hydrolysate demonstrated the presence of glucurone, xylose, arabinose, glucose and galactose.