scholarly journals Biosynthesis of heparin. Relationship between the polymerization and sulphation processes

1989 ◽  
Vol 261 (3) ◽  
pp. 999-1007 ◽  
Author(s):  
K Lidholt ◽  
L Kjellén ◽  
U Lindahl
Keyword(s):  
Microsomal Fraction ◽  
Alkali Treatment ◽  
Deae Cellulose ◽  
Molecular Size ◽  
Chain Elongation ◽  
Gel Chromatography ◽  
Sulphated Polysaccharide ◽  
Transient Interactions ◽  
Sulphated Proteoglycan

Incubation of a mouse mastocytoma microsomal fraction with UDP-[3H]GlcA and UDP-GlcNAc yielded proteoglycans containing non-sulphated polysaccharide chains. Similar incubations performed in the presence of sulphate donor 3′-phosphoadenosine 5′-phosphosulphate (PAPS) produced both sulphated and non-sulphated proteoglycans, which were separated by chromatography on DEAE-cellulose Analysis by gel chromatography of single polysaccharide chains, released from the proteoglycans by alkali treatment, showed that the non-sulphated chains produced during incubation for 5 min or 25 min, either in the absence or in the presence of PAPS, were of fairly small molecular size, with an average peak Mr of approx. 10 x 10(3)-15 x 10(3). In contrast, the sulphated chains exceeded Mr 100 x 10(3) Pulse-chase experiments suggested that sulphated chains were capable of further elongation. These results indicate that sulphation promotes, by so far unknown mechanisms, further chain elongation. Sulphated proteoglycan (retarded on DEAE-cellulose chromatography) isolated after similar incubation of the microsomal fraction for 1 min only was found to contain a mixture of sulphated and virtually non-sulphated polysaccharide chains. However, when [35S]PAPS was included in the incubations, some 35S was found to be associated, essentially as N-sulphate groups, also with the latter type of chains, preferentially the high-Mr fraction. These results are interpreted in terms of a biosynthetic model by which the heparin proteoglycan is generated through transient interactions of macromolecular intermediates with distinctly separate complexes of membranebound enzymes.

Glycosaminoglycans from growing antlers of wapiti (Cervus elaphus)

1997 ◽  
Vol 77 (4) ◽  
pp. 715-721 ◽  
Author(s):  
H. H. Sunwoo ◽  
L. Y. M. Sim ◽  
T. Nakano ◽  
R. J. Hudson ◽  
J. S. Sim
Keyword(s):  
Hyaluronic Acid ◽  
Chondroitin Sulfate ◽  
Cervus Elaphus ◽  
Molecular Size ◽  
Enzymatic Digestion ◽  
Gel Chromatography ◽  
Cellulose Acetate Electrophoresis ◽  
Cartilaginous Tissues ◽  
Chondroitin Sulfates ◽  
Velvet Antler

The emerging wapiti industry in North America is based largely on markets for velvet antlers which are used in oriental medicine. Despite the economic opportunity, enthusiasm has been dampened by incomplete understanding of the chemical and pharmacological properties of velvet antler. This study characterizes polysaccharide constituents of glycosaminoglycans in growing antler of wapiti (Cervus elaphus). Glycosaminoglycans were isolated from four sections (tip, upper, middle and base) of growing antlers, and were studied using cellulose acetate electrophoresis, gel electrophoresis, enzymatic digestion and gel chromatography. The tip and upper sections of the antler which are rich in cartilaginous tissues contained chondroitin sulfate as a major glycosaminoglycan with small amounts of hyaluronic acid. In the middle and base sections containing bone and bone marrow, chondroitin sulfate was also a major glycosaminoglycan with small amounts of hyaluronic acid and chondroitinase-ACI resistant materials. More than half of chondroitin sulfate from the middle and base sections had larger molecular size than did the chondroitin sulfates from the tip and upper sections. Key words: Glycosaminoglycans, chondroitin sulfate, antler, wapiti

scholarly journals The isocitrate dehydrogenases of Acinetobacter lwoffi. Separation and properties of two nicotinamide–adenine dinucleotide phosphate-linked isoenzymes

1972 ◽  
Vol 130 (1) ◽  
pp. 211-219 ◽  
Author(s):  
Colin H. Self ◽  
P. David J. Weitzman
Keyword(s):  
Molecular Weight ◽  
Ph Dependence ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Molecular Size ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Low Concentrations ◽  
Stimulation Of

Two isoenzymes of NADP-linked isocitrate dehydrogenase have been identified in Acinetobacter lwoffi and have been termed isoenzyme-I and isoenzyme-II. The isoenzymes may be separated by ion-exchange chromatography on DEAE-cellulose, by gel filtration on Sephadex G-200, or by zonal ultracentrifugation in a sucrose gradient. Low concentrations of glyoxylate or pyruvate effect considerable stimulation of the activity of isoenzyme-II. The isoenzymes also differ in pH-dependence of activity, kinetic parameters, stability to heat or urea and molecular size. Whereas isoenzyme-I resembles the NADP-linked isocitrate dehydrogenases from other organisms in having a molecular weight under 100000, isoenzyme-II is a much larger enzyme (molecular weight around 300000) resembling the NAD-linked isocitrate dehydrogenases of higher organisms.

Imitation of normal plasma growth hormone profile by subcutaneous administration of human growth hormone to growth hormone deficient children

1983 ◽  
Vol 102 (1) ◽  
pp. 6-10 ◽  
Author(s):  
J. Sandahl Christiansen ◽  
H. Ørskov ◽  
C. Binder ◽  
K. W. Kastrup
Keyword(s):  
Growth Hormone ◽  
Time Course ◽  
Subcutaneous Administration ◽  
Molecular Size ◽  
Hormone Deficiency ◽  
Gel Chromatography ◽  
Plasma Growth Hormone ◽  
Normal Children ◽  
Blood Samples ◽  
Long Term Treatment

Abstract. The time course of plasma growth hormone (hGH) levels following sc and im injection of hGH was studied in 12 children with growth hormone deficiency who had received long-term treatment with im injections of highly purified hGH. Also the spontaneous diurnal GH levels in 8 normal children of comparable age were recorded. Blood samples were obtained during 24 h after im and sc injections of 4 IU/m2 hGH and analysed for immunoreactive hGH. While a median peak value of 160 ng/ml (range 135 to 475 ng/ml) was obtained 2 h after im injection, sc injection resulted in a more sustained elevation reaching 41 ng/ml (range 32 to 51 ng/ml) at 6 h subsiding slowly with a median concentration of 15 ng/ml (range 5–24 ng/ml) persisting after 14 h. Gel chromatography demonstrated that the hGH immunoreactivity of blood samples obtained as late as 14 h after sc injection had unaltered molecular size. Seven of the patients were further studied after sc injection of 2 IU/m2 at 20.00 h instead of in the morning. A plasma profile was attained during the night which roughly approximated the average nocturnal plasma pattern of the normal children.

scholarly journals Neuronal N-acetyl-β-D-glucosaminidase. Evidence for its biosynthesis in vitro

1976 ◽  
Vol 158 (3) ◽  
pp. 513-527 ◽  
Author(s):  
J A Khawaja ◽  
O Z Sellinger
Keyword(s):  
Concanavalin A ◽  
Microsomal Fraction ◽  
Deae Cellulose ◽  
Neuronal Cell ◽  
Amino Acid Mixture ◽  
Acid Mixture ◽  
Freezing And Thawing ◽  
Cellulose Acetate Electrophoresis ◽  
Fluorescent Band

Neuronal cell bodies, isolated in bulk from 8-day-old rat cerebral cortices, were incubated in the presence of a 3H-labelled amino acid mixture, and subcellular fractions isolated by differential centrifugation. The particulate fractions were frozen/thawed in 0.20 M-sucrose/0.1 M-KCl [Selling et al. (1973) Biochim. Biophys. Acta 315, 128-146] and the profiles of acid-insoluble radioactivity and N-acetyl-beta-D-glucosaminidase (glucosaminidase) activity compared in the resulting non-sedimentable fractions by DEAE-cellulose chromatography and cellulose acetate electrophoresis. Radioactivity and glucosaminidase activity co-migrated to a significant extent. Electrophoresis revealed that after 1 min of incubation 42% of the radioactivity of the non-sedimentable microsomal fraction after freezing and thawing co-migrated with an intensely fluorescent band of glucosaminidase activity. Since the pellet fraction obtained on freezing/thawing the microsomal fraction contained up to 75% of the RNA, 95% of the radioactivity and 45% of the glucosaminidase, a detailed study of the association between its radioactivity and nascent glucosaminidase activity was undertaken. After 1 and 2 min of incubation, followed by centrifugation of the microsomal pellet on 35-60% (w/v) sucrose density gradients, radioactivity and glucosaminidase activity exhibited parallel profiles in the region of heavy polyribosomes and at the top of the gradient which contains spontaneously released nascent polypeptide chains. DEAE-cellulose chromatography of these chains revealed glucosaminidase A to be the principal nascent glucosaminidase component, with glucosaminidases B and C as minor peaks. After 2 min of incubation, all of the glucosaminidase components appeared labelled, and glucosaminidase A exhibited two distinct sub-components. The pattern of glucosaminidase labelling in the soluble and microsomal fractions suggested that newly formed glucosaminidase molecules traverse both the cellular sap and the lumen of the endoplasmic reticulum. Only glucosaminidase A reacted specifically with concanavalin A and radioactive glucosaminidase A could be successfully regenerated by treatment with alpha-methyl-D-glucoside. Glucosaminidase A and a substantial portion of the radioactivity associating with it could be readily converted into glucosaminidase B by re-chromatography on DEAE-cellulose and by reaction of the concanavalin A-glucosaminidase A complex with methyl glucosides.

STRUCTURE ACTIVITY RELATIONSHIPS OF DERMATAN SULFATE : EFFECT OF MOLECULAR SIZE AND CARBOXYL GROUP ESTERIFICATION ON HEPARIN C0FACT0R-II ACTIVATION

1987 ◽  
Author(s):  
J Mardiguian ◽  
M Corgier ◽  
M Jouany
Keyword(s):  
Molecular Weight ◽  
Dermatan Sulfate ◽  
Methyl Esters ◽  
Molecular Size ◽  
Carboxyl Groups ◽  
Gel Chromatography ◽  
Carboxyl Group ◽  
Heparin Cofactor Ii ◽  
High Affinity

Dermatan is a high molecular weight glycosaminoglycan which has been shown to enhance the inhibition of thrombin by heparin-cofactor II. The aim of this study was to establish the influence of the molecular size and the role of the carboxyl group on the in vitro activity of Dermatan Sulfate. Pig skin Dermatan Sulfate was fractionated according to molecular size by gel-chromatography on Ultrogel Ac 44. Each fraction was characterized by its sulfur content and by its mean molecular weight measured on a TSK - 4000 column in reference to standard heparin fractions. Methyl esters of the unfractionated Dermatan Sulfate with varying degree of esterification, where prepared via activation of the carboxyl groups with a carbodiimide and reaction with methanol. The results of this study show that the heparin - cofactor II mediated anti-thrombin activity of Dermatan Sulfate is increasing with the molecular weight and is abolished by esterification of the carboxyl groups. Moreover, it can be speculated that each fraction contains the same amount of high affinity fraction and that, like heparin, the potency of the high affinity component is increasing with the molecular weight.

scholarly journals Detection, purification and characterization of a human cancer-associated galactosyltransferase acceptor

1979 ◽  
Vol 178 (2) ◽  
pp. 279-287 ◽  
Author(s):  
D K Podolsky ◽  
M M Weiser
Keyword(s):  
Molecular Weight ◽  
Human Cancer ◽  
Deae Cellulose ◽  
Structural Data ◽  
Low Molecular Weight ◽  
Gel Chromatography ◽  
Purification And Characterization ◽  
Peptide Moiety ◽  
Cellulose Column

A low-molecular-weight acceptor of galactosyltransferase activity was detected in sera and effusions of patients with extensive maligant disease. This substance was purified to homogeneity from both human serum and effusion by using sequential charcoal/Celite and DEAE-cellulose column chromatography. The purified acceptor was shown to act as substrate for both purified normal and cancer-associated human galactosyltransferase (EC 2.4.1.22) isoenzymes, but had a higher affinity for the cancer-associated isoenzyme (Km = 20 microM) than for the normal isoenzyme (Km = 500 microM). The substrate was found to be a glycopeptide with mol.wt. approx. 3600 determined by polyacrylamide-gel chromatography. Carbohyydate analysis demonstrated only the presence of glucosamine and mannose. Amino acid analysis revealed that the peptide moiety consisted of eight different amino acids, including two residues of asparagine and one residue of serine, but no threonine. These structural data suggest that the acceptor is a fraction of an asparagine-glucosamine type of glycoprotein.

PURIFICATION OF "INHIBIN" FROM HUMAN OVARIAN FOLLICULAR FLUID

1979 ◽  
Vol 90 (1) ◽  
pp. 157-166 ◽  
Author(s):  
S. Chari ◽  
C. R. N. Hopkinson ◽  
E. Daume ◽  
G. Sturm
Keyword(s):  
Follicular Fluid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Male Rats ◽  
Acetone Powder ◽  
Gel Chromatography ◽  
Apparent Homogeneity ◽  
Ovarian Follicular Fluid

ABSTRACT Following the earlier demonstration of inhibin-like activity in human ovarian follicular fluid a method for its purification to apparent homogeneity is described. The fluid was converted to acetone powder and subjected sequentially to ammonium sulphate fractionation, gel chromatography on Sephadex G-200, continuous gradient ion-exchange chromatography on DEAE-cellulose, first with a pH gradient from 8.0 to 4.0 and then with a NaCl gradient to 1 m at pH 5.2. The active fraction from this step was subjected to gel filtration on Sephadex G-100 and finally passed through an Amicon Centriflo membrane CF-25 (cut off point: 25 000 m.w.). The ultrafiltrate was homogeneous by SDS-polyacrylamide gel electrophoresis, had a molecular weight of the order of 23 000 and was capable of suppressing serum gonadotrophin levels in the castrated male rats in as low a dose as 25 μg/rat.

scholarly journals Degradation of heparin proteoglycan in cultured mouse mastocytoma cells

1987 ◽  
Vol 246 (2) ◽  
pp. 409-415 ◽  
Author(s):  
K G Jacobsson ◽  
U Lindahl
Keyword(s):  
Mast Cells ◽  
Substrate Specificity ◽  
Cell Cultures ◽  
Alkali Treatment ◽  
Degradation Pathway ◽  
Gel Chromatography ◽  
Pulse Labelling ◽  
Intracellular Degradation ◽  
Mastocytoma Cells ◽  
Intracellular Polysaccharide

Pulse-labelling of mouse mastocytoma cell cultures, established from ascites fluid, with inorganic [35S]sulphate for 1 h yielded labelled heparin proteoglycan containing polysaccharide chains of Mr 60,000-100,000. After chase incubation for 24 h most of the 35S appeared in intracellular polysaccharide fragments similar in size to commercially available heparin, Mr 5000-25,000, as indicated by gel chromatography. Products isolated from cultures after 6 h of chase incubation consisted of partially degraded free polysaccharide chains and, in addition, residual proteoglycans that were of smaller size than the proteoglycans initially pulse-labelled. The polysaccharide chains released by alkali treatment from the residual chase-incubated proteoglycans were of the same size as the chains derived from proteoglycans after 1 h of pulse labelling. These results suggest that the intracellular degradation of heparin proteoglycan to polysaccharide fragments is initiated by release of intact polysaccharide chains, probably by action of a peptidase, and is pursued through cleavage of these chains by an endoglycosidase. An endoglucuronidase with stringent substrate specificity [Thunberg, Bäckström, Wasteson, Ogren & Lindahl (1982) J. Biol. Chem. 257, 10278-10282] has previously been implicated in the latter step. Cultures of more purified mastocytoma cells (essentially devoid of macrophages) did not metabolize [35S]heparin proteoglycan to polysaccharide fragments, but instead accumulated free intact polysaccharide chains, i.e. the postulated intermediate of the complete degradation pathway. When such purified cells were co-cultured with adherent mouse peritoneal cells, presumably macrophages, formation of polysaccharide fragments was observed. It is tentatively proposed that the expression of endoglucuronidase activity by the mast cells depends on collaboration between these cells and macrophages.

COMPARISON OF BARLEY GUMS ISOLATED BY VARIOUS PROCEDURES

1953 ◽  
Vol 31 (7) ◽  
pp. 653-664 ◽  
Author(s):  
W. O. S. Meredith ◽  
T. A. Watts ◽  
J. A. Anderson
Keyword(s):  
Aqueous Solutions ◽  
Molecular Complex ◽  
Alkali Treatment ◽  
Molecular Size ◽  
Degree Of Polymerization ◽  
Aqueous Extraction ◽  
Water Soluble ◽  
Free Sugars ◽  
Enzyme Systems ◽  
Insoluble Form

A barley gum that is believed to be the undegraded, water-soluble, nonstarch polysaccharide of the grain has been isolated. Aqueous solutions of this gum are extremely viscous and are stable. Enzymes that degrade gum during simple aqueous extraction were inactivated first by refluxing barley grist in boiling 85% alcohol followed by extraction of the dried grist with a 1% solution of papain. Gums of lower degree of polymerization, as judged by viscosity measurements, were obtained by aqueous extraction and acid treatments. Two enzyme systems that degrade gums are thought to be present in barley. One (which is inactivated by alcohol) degrades the initially soluble gum and brings an initially insoluble form into solution. The second system (which is inactivated by papain) accompanies and degrades the initially soluble gum during aqueous extraction or in aqueous solutions of the preparation. The purest gum contains only 0.1% nitrogen, and this may be part of the molecular complex. Mild, cold, alkali treatment of this gum reduces molecular size considerably as measured by viscosity of solutions. "X"-enzyme isolated from a bacterial source cleaves the gum into two oligosaccharides of glucose and a component containing D-glucose, L-arabinose, D-xylose, and D-galactose. No free sugars are produced.

scholarly journals The carbohydrate moiety of band-3 glycoprotein of human erythrocyte membranes

1980 ◽  
Vol 187 (3) ◽  
pp. 677-686 ◽  
Author(s):  
T Tsuji ◽  
T Irimura ◽  
T Osawa
Keyword(s):  
Blood Group ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Band 3 ◽  
Carbohydrate Moiety ◽  
Erythrocyte Membranes ◽  
Gel Chromatography ◽  
Branching Points ◽  
Human Erythrocyte Membranes ◽  
Group A

Band-3 glycoprotein was purified from human blood-group-A erythrocyte membranes by selective solubilization and gel chromatography on Sepharose 6B in the presence of sodium dodecyl sulphate. The purified glycoprotein was subjected to hydrazinolysis in order to release the carbohydrate moiety. The released oligosaccharides were N-acetylated and applied to a column of DEAE-cellulose. Most of the band-3 oligosaccharides obtained were found to be free of sialic acids. When this neutral fraction was subjected to gel chromatography on a column of Sephadex G-50, two broad peaks were observed indicating that the band-3 glycoprotein was heterogeneous in the size of the oligosaccharide moieties. All fractions from gel chromatography were found to contain galactose, mannose, N-acetylglucosamine and fucose. The higher-molecular-weight (mol.wt. 3000-8000) peak consisted of fucose, mannose, galactose, N-acetylglucosamine and N-acetylgalactosamine in a molar proportion of 1.6:3.0:8.4:10.5:0.2. Most of these oligosaccharides were digested with a mixture of beta-galactosidase and beta-N-acetylhexosaminidase after alpha-L-fucosidase treatment to give a small oligosaccharide with the structure alpha Man2-beta Man-beta GlcNAc-GlcNAc. Methylation studies and limited degradation by nitrous acid deamination showed that the oligosaccharides contained the repeating disaccharide Gal beta 1→4GlcNAc beta 1→3, with branching points at C-6 of some of the galactose residues. These results indicate that a major portion of the band-3 oligosaccharide has a common core structure, with heterogeneity in the numbers of the repeating disaccharides, and contains fucose residues both in the peripheral portion and in the core portion. Haemagglutination tests were also carried out to determine the blood-group specificities of the glycoprotein and the results demonstrated the presence of both blood-group-H and I antigenic activities.

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