PubMed Health⌕ Search

Biomedical subjects

L Rodén

Publications and source records attributed to L Rodén.

At least 19 recordsLinked to original sources
PubMed2000

Dermatan is a better substrate for 4-O-sulfation than chondroitin: implications in the generation of 4-O-sulfated, L-iduronate-rich galactosaminoglycans.

The biosynthesis of dermatan sulfate is a complex process that involves, inter alia, formation of L-iduronic acid residues by C5-epimerization of D-glucuronic acid residues already incorporated into the growing polymer. It has been shown previously that this reaction is promoted by the presence of the sulfate donor 3'-phosphoadenosine-5'-phosphosulfate. In the present investigation, the role of sulfation in the biosynthesis of L-iduronic acid-rich galactosaminoglycans was examined more closely by a study of the substrate specificities and kinetic properties of the sulfotransferases involved in dermatan sulfate biosynthesis. Comparison of the acceptor reactivities of oligosaccharides from chondroitin and dermatan, in an in vitro system containing microsomes from cultured human skin fibroblasts and 3'-phosphoadenosine-5'-phosphosulfate, showed that Km values for the dermatan fragments were substantially lower than those for their chondroitin counterparts. Calculation of Vmax values likewise showed that dermatan was the better substrate. Whereas dermatan incorporated [35S]sulfate exclusively at the C4 position of N-acetylgalactosamine residues, approximately equal amounts of radioactivity were found at the C4 and C6 positions in the labelled chondroitin. Under standard assay conditions, the 4-O-sulfation of dermatan proceeded about six times faster than the 4-O-sulfation of chondroitin. On the basis of these results, we propose that L-iduronic acids, formed in the course of the biosynthesis of dermatan sulfates, enhance sulfation of their adjacent N-acetylgalactosamine residues, and will thereby be locked in the L-ido configuration.

Cells, Cultured↗
PubMed2000

Biosynthesis of heparin/heparan sulfate: kinetic studies of the glucuronyl C5-epimerase with N-sulfated derivatives of the Escherichia coli K5 capsular polysaccharide as substrates.

The D-glucuronyl C5-epimerase involved in the biosynthesis of heparin and heparan sulfate was investigated with focus on its substrate specificity, its kinetic properties, and a comparison of epimerase preparations from the Furth mastocytoma and bovine liver, which synthesize heparin and heparan sulfate, respectively. New substrates for the epimerase were prepared from the capsular polysaccharide of Escherichia coli K5, which had been labeled at C5 of its D-glucuronic and N-acetyl-D-glucosamine moieties by growing the bacteria in the presence of D-[5-(3)H]glucose. Following complete or partial ( approximately 50%) N-deacetylation of the polysaccharide by hydrazinolysis, the free amino groups were sulfated by treatment with trimethylamine.SO(3)complex, which yielded products that were recognized as substrates by the epimerase and released tritium from C5 of the D-glucuronyl residues upon incubation with the enzyme. Comparison of the kinetic properties of the two substrates showed that the fully N-sulfated derivative was the best substrate in terms of its K(m)value, which was significantly lower than that of its partially N-acetylated counterpart. The V(max)values for the E.coli polysaccharide derivatives were essentially the same but were both lower than that of the O-desulfated [(3)H]heparin used in our previous studies. Surprisingly, the apparent K(m)values for all three substrates increased with increasing enzyme concentration. The reason for this phenomenon is not entirely clear at present. Partially purified C5-epimerase preparations from the Furth mastocytoma and bovine liver, respectively, behaved similarly in terms of their reactivity towards the various substrates, but the variation in apparent K(m)values with enzyme concentration precluded a detailed comparison of their kinetic properties.

Animals↗
PubMed1997

Analysis of the Morgan-Elson chromogens by high-performance liquid chromatography.

The Morgan-Elson method for quantitative N-acetylhexosamine analysis is a two-step procedure comprising alkali treatment of the sugar and subsequent condensation of the resulting chromogens with p-dimethylaminobenzaldehyde (Ehrlich's reagent) to yield a colored product. In the present investigation, the products formed in the first step of the procedure were analyzed by high-performance liquid chromatography (HPLC) on a reversed-phase (C18) column, which was eluted with a water-methanol gradient; the absorbance of the effluent was monitored at 229 nm. The profile generated from alkali-treated N-acetylglucosamine exhibited two major peaks, in a ratio of approximately 2.5:1, which accounted for 94% of the total peak area. A third peak, accounting for 3% of the peak area, was eluted in an intermediate position, and several smaller peaks were also observed. The three predominant components, isolated by preparative HPLC, all gave a purple color on addition of Ehrlich's reagent, indicating that they were Morgan-Elson chromogens. The HPLC profile of alkali-treated N-acetylmannosamine was identical to that of the products generated from N-actylglucosamine, as was expected because of the elimination of the asymmetry at C-2 during formation of the chromogens. N-Acetylgalactosamine yielded two major peaks, which were eluted in the same positions as the two major products formed from N-acetylglucosamine, but the intermediate peak seen in the N-acetylglucosamine pattern was absent. The HPLC procedure allowed detection of as little as approximately 25 ng of N-acetylglucosamine and may therefore be of value as an alternative to the complete Morgan-Elson procedure when only small amounts of sample are available for quantitative analysis.

Acetylgalactosamine↗
PubMed1997

Alkylglycosides as artificial primers for glycogen biosynthesis.

Glycogenin is a 37 kDa self-glycosylating protein which has been demonstrated to be the initiating enzyme and primer for glycogen biosynthesis in liver, skeletal muscle and other tissues. We have recently shown that glycogenin will use alkylglucosides and alkylmaltosides as artificial acceptors in glycosyl transfer from UDP-glucose and UDP-xylose in vitro and have suggested that such substrates might be used to promote the synthesis of glycogen in vitro and in vivo. We now report that alkylglycosides can also serve as acceptors for transfer of glucose by glycogen synthase, yielding alkylmaltooligosaccharide products which may potentially be elongated to glycogen. alpha-Glucosides were better substrates than the corresponding beta-glucosides, and alkylmaltosides were preferred over alkylglucosides. The hydrophobicity of the substrates markedly affected their acceptor activity, less hydrophobic substrates being more active. This is in contrast to the behavior of glycogenin, which acted preferentially upon the more hydrophobic substrates tested. Aromatic glycosides were also substrates for glycogen synthase, e.g., naphthyl-alpha-D- and beta-D-glucoside. The substrates were active in vitro both with partially purified rabbit muscle glycogen synthase and in incubations with crude muscle and liver homogenates from rat. In vivo experiments with mice further proved that intraperitoneal administration of alkylglucosides and alkylmaltosides increased the uptake of 14C-glucose in liver. The elevated uptake was due to an increase in both hydrophobic products, isolated by adsorption to Sep-Pak C18 columns, and more hydrophilic material that co-fractionated with glycogen upon treatment of the tissue with alkali and precipitation with ethanol. These results demonstrate the ability of alkylglycosides to serve as artificial primers for glycogen biosynthesis in vivo.

Alkylation↗
PubMed1996

N-acetylglucosamine kinase and N-acetylglucosamine 6-phosphate deacetylase in normal human erythrocytes and Plasmodium falciparum.

The major pathways of glucose metabolism in the malaria parasite, Plasmodium falciparum, have now been elucidated, and the structures and properties of parasite-specific enzymes are presently being investigated. Little is known, however, about the enzymes catalysing monosaccharide interconversions in the parasite. In the present investigation we have examined the pathway of N-acetylglucosamine catabolism which, in higher organisms, involves the following reaction sequence: N-acetylglucosamine -->N-acetylglucosamine 6-phosphate-->glucosamine 6-phosphate-->fructose 6-phosphate. Assay of the specific kinase (E.C. 2.7.1.59) catalysing the phosphorylation of the sugar showed that the enzyme is present in Plasmodium extracts as well as in normal human erythrocytes; specific activities of 7.2 and 5.3 nmol/h/mg protein were measured for the parasite and erythrocyte extracts, respectively, N-Acetylglucosamine 6-phosphate deacetylase (E.C. 3.5.1.25), catalysing the second reaction, was also detected in both normal and Plasmodium-infected erythrocytes. At 75% parasitaemia, the deacetylase activity was close to 3 times higher than that of normal control cells. The erythrocyte deacetylase was purified approximately 16,000-fold by chromatography on DE52 cellulose, chromatofocusing, and size exclusion chromatography. Attempts to purify the parasite enzyme by the same procedures were unsuccessful due to loss of activity. A partially purified erythrocyte deacetylase preparation (eluted from DE52 cellulose) had a pH optimum of 7.5, a pI of 6.0, as indicated by chromatofocusing, and a K(m) of 29 microM. In conjunction with previous investigations, the present study indicated that all three enzymes required for N-acetylglucosamine utilization are present in Plasmodium parasites as well as in normal erythrocytes.

Amidohydrolases↗
PubMed1995

Inhibition of glycogenin-catalyzed glucosyl and xylosyl transfer by cytidine 5'-diphosphate and related compounds.

The self-glucosylation of beef kidney glycogenin was inhibited by the following pyrimidine nucleotides and nucleotide sugars, listed in order of decreasing effectiveness: CDP-glucose, CDP, UDP-xylose, UDP-N-acetylglucosamine, UDP-galactose, UDP, CTP, CDP-choline, UDP-glucuronic acid, beta-S-UDP-glucose, and CMP. In contrast, the purine nucleotide sugars, ADP-glucose and GDP-glucose, were essentially ineffective, as was the pyrimidine nucleoside, cytidine. UDP-Xylose may be utilized by glycogenin as an alternative sugar donor instead of UDP-glucose (Rodén, L., Ananth, S., Campbell, P., Manzella, S., and Meezan, E. (1994) J. Biol. Chem. 269, 11509-11513) and therefore presumably inhibited the glucosyl transfer reaction by being a competitive substrate. Like glucosyl transfer, xylosyl incorporation into glycogenin was also inhibited effectively by CDP. On the other hand, UDP-xylose:proteoglycan core protein xylosyltransferase (EC 2.4.2.26) was not affected by CDP, nor was it inhibited by UDP-glucose. Addition of CDP or UDP-glucose to reaction mixtures containing both enzymes therefore made it possible to assay xylosyltransferase EC 2.4.2.26 reliably without the extensive product characterization that is otherwise necessary. The CDP effect on glycogenin further allowed the development of an improved procedure for the purification of this enzyme, in which specific elution of an affinity matrix (UDP-glucuronic acid-agarose) was carried out with CDP as the eluant.

Animals↗
PubMed1995

Tritium labelling of amino sugars at C-2 by alkaline epimerization in tritiated water.

N-Acetyl-D-[2-3H]glucosamine was synthesized from N-acetyl-D-mannosamine by alkaline 2-epimerization in pyridine containing 3H2O and nickelous acetate. The reaction involves reversible formation of an enol intermediate and therefore also resulted in incorporation of tritium into N-acetylmannosamine. After completed reaction, the two N-acetylhexosamines were separated from other radioactive products and Morgan-Elson chromogens by chromatography on a column of Sephadex G-10, which was eluted with 10% ethanol, and were then separated from each other by chromatography on Sephadex G-15 in 0.27 M sodium borate (pH 7.8). The location of the incorporated tritium was established by treatment of the N-acetylhexosamines with borate under the conditions of the Morgan-Elson reaction, which converts the sugars to Kuhn's chromogen I with concomitant loss of the C-2 hydrogen. As expected, this treatment resulted in the formation of 3H2O, indicating that the tritium was located at C-2. [2-3H]Glucosamine was prepared by acid hydrolysis of the labelled N-acetylglucosamine and was converted to [2-3H]glucosamine 6-phosphate by incubation with hexokinase and ATP. The sugar phosphate was used as a substrate for glucosamine 6-phosphate deaminase (isomerase, EC 5.3.1.10) in a simple 3H2O release assay.

Acetylglucosamine↗
PubMed1995

Dodecyl-beta-D-maltoside as a substrate for glucosyl and xylosyl transfer by glycogenin.

Glycogenin is the core protein of glycogen proteoglycan and is, at the same time, a self-glucosylating enzyme which catalyses early glucosyl transfer steps in the biosynthesis of glycogen. In the course of this process, glycogenin is glucosylated progressively until an oligosaccharide containing 8-11 glucose residues has been formed. Although glycogenin, under physiological conditions, is both enzyme and acceptor in the glucosyl transfer reactions, it is also capable of utilizing p-nitrophenyl-linked malto-oligosaccharides as exogenous acceptors. In view of the potential usefulness of exogenous acceptors in the study of the mechanism of the glycogenin reaction, we have expanded the search for such compounds and report here that several alkyl glucosides and alkyl maltosides may serve as acceptors in glucosyl transfer by beef kidney glycogenin. Dodecyl-beta-D-maltoside (Km approximately 100 microM) was the most effective acceptor among the compounds tested and yielded 30 times as much product as p-nitrophenyl-alpha-maltoside. Substantial product formation was also observed with tetradecyl-beta-D-maltoside and octyl-beta-D-maltoside (39 and 22%, respectively, of the value measured for dodecyl-beta-D-maltoside). It was further demonstrated that dodecyl-beta-D-maltoside served as an acceptor in the transfer of xylose from UDP-xylose, indicating that the exogenous substrate behaved similarly to glycogenin itself in this regard. Dodecyl-beta-D-maltoside has already proven useful in the development of a simple glycogenin assay, and it is further suggested that this and related compounds may be active in vivo and in cell culture as artificial initiators of glycogen synthesis.

Animals↗
PubMed1995

Glucosamine 6-phosphate deaminase in normal human erythrocytes.

In the course of an investigation of hexosamine catabolism in the human malaria parasite, Plasmodium falciparum, it became apparent that a basic understanding of the relevant enzymatic reactions in the host erythrocyte is lacking. To acquire the necessary basic knowledge, we have determined the activities of several enzymes involved in hexosamine metabolism in normal human red blood cells. In the present communication we report the results of studies of glucosamine 6-phosphate deaminase (GlcN6-P) using a newly developed sensitive radiometric assay. The mean specific activity in extracts of fresh erythrocytes assayed within 4h of collection was 14.7 nmol/h/mg protein, whereas preparations from older erythrocytes that had been stored at 4 degrees C for up to 4 weeks had a mean specific activity of 6.2 nmol/h/mg. Characterization of the deaminase by chromatofocusing gave a pI of 8.55. The enzyme was optimally active at pH 9.0 and had a Km of 41 microM. The metal chelators EDTA and EGTA were non-inhibitory; however, inhibition was observed in the presence of metal ions, especially Cu2+, Ni2+ and Zn2+. In addition, the deaminase was also inhibited by several sugar phosphates including the reaction product, fructose 6-phosphate.

Aldose-Ketose Isomerases↗
PubMed1995

Glucosamine 6-phosphate deaminase in Plasmodium falciparum.

The pathways of glucose utilization for energy production in the malaria parasite, Plasmodium falciparum, have been studied extensively. Little is known, however, about the reactions by which glucose is converted into complex carbohydrates in the parasite, and knowledge of the catabolism of these substances is likewise scanty. The present investigation was undertaken to determine whether the parasites possess a key enzyme of glucosamine catabolism, i.e. glucosamine 6-phosphate deaminase (EC 5.3.1.40), which catalyses the conversion of the sugar phosphate to fructose 6-phosphate and ammonia. Lysates of Plasmodium-infected erythrocytes had substantially higher deaminase activity than control samples from normal erythrocytes, and an even higher specific activity was observed in extracts of isolated parasites, amounting to 20-40 times that of uninfected cells. Anion exchange chromatography indicated that the parasite deaminase eluted in a retarded position when compared to the elution profile of the erythrocyte enzyme. The charge difference suggested by these findings was established more directly by chromatofocusing, which indicated pI values of 6.85 and 8.55 for the parasite and erythrocyte deaminases, respectively. Other differences were also observed, notably a greater thermolability on the part of the parasite enzyme. These results indicated that the parasites synthesize a specific deaminase that is distinct from the normal erythrocyte enzyme. Studies on synchronized parasite cultures further indicated that the parasite deaminase is developmentally regulated, because a dramatic increase in activity levels occurred during the later stages of parasite development.

Aldose-Ketose Isomerases↗
PubMed1994

Biosynthesis of heparin/heparan sulfate. Purification of the D-glucuronyl C-5 epimerase from bovine liver.

The D-glucuronyl C-5 epimerase involved in the biosynthesis of heparin/heparan sulfate was purified from the high speed supernatant fraction of a homogenate of bovine liver by chromatography on immobilized O-desulfated heparin, red Sepharose, phenyl Sepharose, and concanavalin A-Sepharose. After close to 1 million-fold purification, in 10-15% yield, the product gave a single band on SDS-polyacrylamide gel electrophoresis with silver staining and had a mobility corresponding to an M(r) of approximately 52,000. Since the epimerase assay used in the course of purification was based on release of tritium, as [3H]H2O, from a [5-3H]uronyl-labeled substrate, it was important to establish that the purified enzyme did indeed catalyze the actual conversion of D-glucuronyl to L-iduronyl residues. Upon incubation of the purified enzyme with 3H-labeled heparosan N-sulfate, prepared by metabolic labeling (with D-[1-3H]glucose) of a capsular polysaccharide from Escherichia coli K5 and subsequent chemical partial N-deacetylation and N-sulfation, approximately 30% of the D-glucuronyl residues located between two N-sulfated glucosamine units were converted to L-iduronyl units.

Amino Acids↗
PubMed1994

Xylosyl transfer to an endogenous renal acceptor. Characteristics of the reaction and properties of the product.

In the course of a study of UDP-xylose:proteoglycan core protein xylosyltransferase (EC 2.4.2.26), another xylosyltransferase was discovered in the soluble fraction of a rat kidney homogenate. The latter enzyme catalyzed [3H]xylosyl transfer from UDP-[3H]xylose to an endogenous acceptor and yielded a product in which the xylose was bound by an alkali-stable linkage. It was therefore concluded that the acceptor was not the core protein of one of the proteoglycans containing a xylose-->serine linkage, since this linkage is cleaved by alkali. The [3H]xylose-labeled product emerged with the void volume when chromatographed on Sephadex G-50, it was precipitated by trichloroacetic acid, and it had a mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to a molecular mass of about 32,000 Da. Digestion with trypsin or alpha-amylase degraded the labeled product to small fragments, as determined by gel chromatography, suggesting that it was a glycoprotein related to glycogen. A product of similar characteristics was formed when UDP-[3H]glucose was substituted for UDP-[3H]xylose as the glycosyl donor, and the two nucleotide sugars were mutually competitive in the respective transfer reactions, indicating that they were substrates for the same enzyme. On the basis of these findings, it was tentatively concluded that the xylosyltransferase and its acceptor were the renal form of glycogenin.

Animals↗
PubMed1994

Xylosyl transfer to an endogenous renal acceptor. Purification of the transferase and the acceptor and their identification as glycogenin.

A xylosyltransferase in rat kidney, tentatively identified as glycogenin (Meezan, E., Ananth, S., Manzella, S., Campbell, P., Siegal, S., Pillion, D. J., and Rodén, L. (1994) J. Biol. Chem. 269, 11503-11508), was purified by a procedure in which affinity chromatography on UDP-glucuronic acid-agarose was a particularly useful step. The purified material was nearly homogeneous, as shown by SDS-polyacrylamide gel electrophoresis and silver staining, and had an electrophoretic mobility corresponding to a M(r) of 32,000. The purified enzyme possessed both glucosyl- and xylosyltransferase activity, and incubation with UDP-[3H]xylose or UDP-[3H]glucose yielded a single macromolecular product, which had the same electrophoretic mobility as the major silver-stained component. These results indicate that the kidney transferase was indeed glycogenin and that it was functionally analogous to the larger glycogenin species previously isolated from rabbit muscle. Further examination of the properties of the rat kidney enzyme showed, i.a., that it was inhibited strongly by cytidine 5'-diphosphate. This effect was used to advantage in an alternative purification procedure, which was applied to beef kidney and involved adsorption of the enzyme to UDP-glucuronic acid-agarose and subsequent elution with cytidine 5'-diphosphate. In contrast to glycogenin, glycogen synthase did not catalyze transfer from UDP-xylose, and it is suggested that the incorporation of xylose into glycogen observed by other investigators was due to glycogenin-catalyzed xylosyl transfer and subsequent chain elongation by glycogen synthase.

Animals↗
PubMed1994

A biphasic radiometric assay of glycogenin using the hydrophobic acceptor n-dodecyl-beta-D-maltoside.

Glycogenin is a self-glycosylating protein that catalyzes early glucosyl transfer steps in the biosynthesis of glycogen. In currently used assays of glycogenin activity, the enzyme is incubated with radioactive UDP-glucose, and the labeled reaction product is then isolated by precipitation with trichloroacetic acid. A new assay is reported here which is based on the observation that glycogenin is not only self-glycosylating but may also use exogenous alkyl maltosides as substrates. After incubation of the enzyme with n-dodecyl-beta-D-maltoside and UDP-[3H]glucose, the radioactivity in the resultant n-dodecyl-beta-D-[3H]maltotrioside is determined by any one of the following three procedures, which all rely on the hydrophobic properties conferred on the reaction product by the alkyl aglycone: (i) adsorption of the product to a Sep-Pak C18 cartridge and elution with 70% ethanol; (ii) biphasic liquid scintillation counting in ScintiLene/25% isoamyl alcohol, without isolation of the product, and (iii) precipitation with trichloroacetic acid in the presence of carrier protein. The Sep-Pak C18 procedure has the advantage that it allows essentially quantitative isolation of the reaction product, while, under the conditions chosen, only about 50% of the product is precipitated by trichloroacetic acid. For most applications, however, biphasic liquid scintillation counting is the method of choice, since close to 90% of the labeled product is extracted into the organic phase and can be counted directly without interference from the labeled nucleotide sugar which remains in the aqueous phase.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Separation of sugars by ion-exclusion chromatography on a cation-exchange resin.

A method for the separation of N-acetylmannosamine and N-acetylglucosamine is described, which consists of chromatography of the two sugars on a column (30 x 1 cm) of the cation-exchange resin, Dowex 50W-X2, in borate buffer at pH 7.8. N-Acetylmannosamine is eluted near the void volume, while N-acetylglucosamine emerges in a more retarded position. It is postulated that the separation occurs as a result of the combined effects of ion exclusion and gel permeation. Thus, in borate solution, N-acetylmannosamine presumably exists largely as a negatively charged complex and is therefore excluded from the sulfonated polystyrene matrix, while N-acetylglucosamine occurs mainly as the free sugar in the equilibrium mixture and, being a neutral compound, has free access to the porous resin. The proposed mechanism for the separation was supported by the finding that glucose and glucose 6-phosphate could also be separated on a column of the same resin, with water as the eluent.

Acetylglucosamine↗
PubMed1993

Separation of N-acetylglucosamine and N-acetylmannosamine by chromatography on Sephadex in borate buffer.

Several procedures have been used previously for the separation of N-acetylglucosamine and N-acetylmannosamine, which are based on the difference in strength of the borate complexes of the two N-acetylhexosamines and include paper chromatography on borate-treated paper, paper electrophoresis in borate buffer, and anion-exchange chromatography of the borate complexes. In the present study, we have observed that the two sugars, despite their identical size in noncomplexed form, may also be separated by gel chromatography in borate buffer. Nearly complete resolution was obtained by chromatography on a column (1.5 x 117 cm) of Sephadex G-15, which was eluted at room temperature with 0.27 M sodium borate, pH 7.8 (prepared from H3BO3 by addition of NaOH), at a flow rate of 10 ml/h. This procedure complements existing methods for the separation of N-acetylmannosamine and N-acetylglucosamine and has the advantage that it can be carried out on a relatively large preparative scale.

Acetylglucosamine↗