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Biomedical subjects

U Lindahl

Publications and source records attributed to U Lindahl.

At least 181 records · Page 10Linked to original sources
PubMed1976

Metabolism of macromolecular heparin in mouse neoplastic mast cells.

1. Polysaccharide in a heparin-producing mouse mastocytoma was pulse-labelled in vivo with [35S] sulphate, and after various periods of time was isolated from subcellular fractions. Such fractions were recovered from tissue homogenates by consecutive centrifugations at 1000g for 10min, 20000g for 20min and 100000g for 1h. Initially the 35S-labelled polysaccharide formed occurred principally in the second centrifugal fraction (20000g precipitate), with small amounts in the first (granular) and third (microsomal) fractions. Analysis for glycosyltransferase activity confirmed that glycosaminoglycans were formed chiefly in particles sedimenting at 20000g. Molecules of this newly synthesized polysaccharide were considerably larger than those of commercially available heparin, as judged from gel chromatography. 2. Within the first hour after injection of [35S]sulphate, most of the labelled polysaccharide was redistributed from the second to the first centrifugal fraction. During, and possibly also after, this shift, the macromolecular polysaccharide was degraded, ultimately to the size of commercial heparin. The degradation process appeared complete 6h after injection of [35S]sulphate. 3. Particulate subcellular fractions were incubated with macromolecular [35S]heparin and the products were analysed by gel chromatography. Significant degradation of the substrate occurred only with the second centrifugal fraction. Further characterization of this fraction, by density-gradient centrifugation in iso-osmotic colloidal silica, revealed a single visible band of particles, at approximately the same density at lysosomes. This band contained all the beta-glucuronidase, 35S-labelled endogenous polysacchride and heparin-degrading enzyme present in the second fraction.

Animals↗
PubMed1975

Biosynthesis of heparin. Studies on the microsomal sulfation process.

Nonsulfated, partially N-deacetylated, 14C-labeled polysaccharide was formed by incubation of a mouse mastocytoma microsomal fraction with UDP-[14C]glucuronic acid and UDP-N-acetylglucosamine. After 60 min at 37 degrees the incorporation of radioactivity was interrupted, and sulfation was initiated, by the addition of unlabeled UDP-glucuronic acid and 3-phosphoadenylylsulfate, respectively. After varying periods of incubation with the nucleotide sulfate, polysaccharide was isolated and analyzed by chromatography on DEAE-cellulose. During incubation with 3-phosphoadenylylsulfate, sulfated 14C-polysaccharides were formed, while the nonsulfated 14C-polysaccharide was eliminated, each type of component retaining the same elution position throughout the entire sulfation period (60 min). No accumulation of low sulfated material was observed. Fully sulfated heparin was detected after only 0.5 min of sulfation. These results suggest that the sulfation of heparin is a rapid process, involving a limited number of polysaccharide molecules at a time. Besides heparin-like products, having both N- and O-sulfate groups, and [14C]iduronic acid as predominant uronic acid constituent, an additional sulfated component was detected, having N-sulfate but no O-sulfate groups. This novel polysaccharide contained [14C]glucuronic acid but little or no [14C]iduronic acid. Previous studies have shown that L-iduronic acid residues in heparin are formed by C-5 inversion of D-glucuronic acid units, previously incorporated into the polymer; the inversion reaction requires concomitant sulfation of the polymer. The present results demonstrate that the formation of iduronic acid residues is related to the sulfation of hydroxyl groups. Pulse-chase experiments with 3-phosphoadenylyl[35S]sulfate indicated that the N-sulfated polymer, lacking O-sulfate groups, could be converted into heparin-like products by further sulfation of hydroxyl groups only. The biosynthesis of heparin may thus involve a pathway, in which the formation of N-sulfated intermediates precedes the introduction of O-sulfate groups.

Animals↗
PubMed1975

Cleavage of macromolecular heparin by an enzyme from mouse mastocytoma.

Heparinase was isolated from a transplantable mouse mastocytoma, by salt extraction of a particulate fraction sedimenting at 20,000 times g, followed by precipitation from saturated ammonium sulfate. By use of gel chromatography through Sepharose 4B, the enzyme was shown to degrade macromolecular. 35S-labeled, mastocytomal heparin (K-av about 0.25) to products similar in size to commercial heparin (K-av about 0.85), apparently by nonrandom cleavage of a limited number of glycosidic linkages per molecule. Prolonged incubation times (up to 5 days, with repeated addition of enzyme) did not result in further degradation of the product. No significant depolymerizing activity was observed with any other glycosaminoglycan tested, including chondroitin sulfate, dermatan sulfate, hyaluronic acid, heparan sulfate, and commercial heparin. The pH optimum for degradation of macromolecular heparin was around pH 5. The nature of the linkage cleaved by the heparinase was investigated by reduction of unlabeled polysaccharide degradation products with sodium [3H]borohydride. The degraded chains (but not the macromolecular substrate) incorporated significant amounts of tritium. An essentially monodisperse fraction of the labeled, degraded heparin was subjected to meniscus depletion sedimentation equilibrium ultracentrifugation, indicating a molecular weight of 14,500. By relating the molecular weight to the specific activity of the preparation, the amount of reducible groups was calculated to be approximately one per molecule. The 3H-labeled heparin was degraded to monosaccharides by a combination of acid hydrolysis and cleavage due to deamination with nitrous acid. Analysis of the degradation products, by paper electrophoresis and paper chromatography, showed a major radioactive component which behaved like L-gulonic acid. Since [3H]gulonic acid would be the expected reduction product of a polysaccharide molecule, containing a glucuronic acid residue in terminal position, these results tentatively suggest that the heparinase is an endoglucuronidase. By direct deaminative cleavage (no hydrolysis) of the 3H-labeled heparin, the glucosamine unit in penultimate position (i.e. adjacent to the [3H]gulonic acid residue) was shown to be 52% N-sulfated and 48% N-acetylated. As only 14% of the glucosamine was N-acetylated in the macromolecular heparin substrate, it is suggested that cleavage of this polysaccharide, by the heparinase, occurs in regions more abundant in N-acetylated glucosamine residues than other portions of the molecule. The possibility that formation and degradation of macromolecular heparin occurs also in mammalian species other than rodents in discussed.

Animals↗
PubMed1975

Macromolecular properties and end-group analysis of heparin isolated from bovine liver capsule.

Glycosaminoglycans were extracted from bovine liver capsule with 4 M-guanidinium chloride, resulting in solubilization of approx. 90% of the total uronic acid-containing polysaccharide of the tissue. The extracted polysaccharide was purified and fractionated by anion-exchange chromatography on DEAE-cellulose, density-gradient ultracentrifugation in CsCl and finally gel chromatography on Sepharose 4B. By using these procedures, the two major polysaccharide components, dermatan sulphate and heparin, which constituted 55 and 30% respectively of the total glycosaminoglycan content of the tissue, were separated from each other. Analysis of the macromolecular properties of the two polysaccharides showed that heparin existed exclusively as single polysaccharide chains, whereas dermatan sulphate occurred largely as a proteoglycan (protein content, 74% dry wt.). The purified heparin preparation was subjected to sedimentation-equilibrium ultracentrifugation, indicating a molecular weight of 8800. Analysis for neutral sugars (by g.l.c.) showed 0.1 residue of xylose and 0.2 residue of galactose/polysaccharide chain; serine amounted to 0.3 residue/polysaccharide chain. Reduction of the heparin with NaB3H4 resulted in incorporation of 3H, approximately corresponding to one reducible group/polysaccharide chain. The 3H-labelled sugar residue was liberated by a combination of acid hydrolysis and deaminative cleavage of the polysaccharide with HNO2; it was subsequently identified as an aldonic acid by paper electrophoresis. Most of the heparin chains thus contained a uronic acid residue in reducing position. It is suggested that heparin isolated from bovine liver capsule is a degradation product released from larger molecules by an endo-glycuronidase.

Amino Acids↗
PubMed1975

Biosynthesis of heparin. Solubilization and partial characterization of N- and O-sulphotransferases.

Assay methods were developed enabling separate determination of N- and O-sulphotransferase activities in an enzyme preparation from mouse mastocytoma. N-Desulphoheparin and chemically N-acetylated heparan sulphate were used as specific exogenous sulphate acceptors in the transfer of [35S]sulphate residues from adenosine 3'-phosphate 5'-[35S]sulphatophosphate to amino and hydroxyl groups respectively. The resulting 35S-labelled polysaccharides were isolated as their cetylpyridinium complexes on filter paper. Sulphotransferases were solubilized from a mastocytoma microsomal fraction by treatment with detergent-alkali. The pH optimum for both enzymes was about 7.5 Km with regard to adenosine 3'-phosphate 5'-sulphatophosphate was estimated to be 2 X 10(-5) M for the N-sulphotransferase and 1 X 10(-4) M for the O-sulphotransferase(s). The enzymes required bivalent cations for maximum activity, Mn2+ stimulating both the N- and O-sulphotransferase four- to five-fold, whereas Ca2+ increased the N- but not the O-sulphotransferase activity. The O-sulphotransferase was found to be more sensitive to heat-inactivation, 60% of the activity being lost after 1 min at 50 degrees C, whereas only 15% of the N-sulphotransferase activity was lost. In contrast, the N-sulphotransferase was selectively inhibited (or inactivated) by NaCl; at 0.125 M-NaCl concentration the O-sulphotransferase activity was essentially unaffected, whereas the N-sulphotransferase activity was depressed by 80%. These results strongly indicate that N- and O-sulphate-transfer reactions should be ascribed to different enzymes, or, alternatively, to separate and independent active sites on the same enzyme molecule.

Cations, Divalent↗
PubMed1974

Distribution of sulphate and iduronic acid residues in heparin and heparan sulphate.

1. A method was developed for determination of the uronic acid composition of heparin-like glycosaminoglycans. Polymers or oligosaccharides are degraded to monosaccharides by a combination of acid hydrolysis and deamination with HNO(2). The resulting uronic acid monosaccharides (accounting for about 70% of the uronic acid contents of the starting materials) are isolated and converted into the corresponding aldono-1,4-lactones, which are separated by g.l.c. The calculated ratios of glucuronic acid/iduronic acid are reproducible within 5%. 2. Samples of heparin from pig intestinal mucosa (molar ratio of sulphate/disaccharide unit, 2.40) and heparan sulphate from human aorta (sulphate/disaccharide ratio, 0.46) were subjected to uronic acid analysis. l-Iduronic acid constituted 77% and 19% respectively of the total uronic acid contents. 3. The correlation between the contents of sulphate and iduronic acid indicated by this finding also applied to the fractionated deamination products of the two polymers. The sulphated fragments varied in size from disaccharide to octasaccharide (or larger) and showed sulphate/disaccharide molar ratios in the range of 0.05-2.0. The proportion of iduronic acid increased with increasing ester sulphate contents of the oligosaccharides. 4. Previous studies on the biosynthesis of heparin in a cell-free system have shown that l-iduronic acid residues are formed by C-5 epimerization of d-glucuronic acid units at the polymer level; the process requires concomitant sulphation of the polymer. The results obtained in the present structural study conform to these findings, and suggest further that similar mechanisms may operate in the biosynthesis of heparan sulphate. The epimerization reaction appears to be linked to the sulphation of hydroxyl groups but does not seem to require sulphation of the target uronic acid residues. The significance of sulphamino groups in relation to the formation of iduronic acid is unknown.

Animals↗