Inhibition of Recombinant Aldose-6-Phosphate Reductase from Peach Leaves by Hexose-Phosphates, Inorganic Phosphate and Oxidants.

Glucitol, also known as sorbitol, is a major photosynthetic product in plants from the Rosaceae family. This sugar alcohol is synthesized from glucose-6-phosphate by the combined activities of aldose-6-phosphate reductase (Ald6PRase) and glucitol-6-phosphatase. In this work we show the purification and characterization of recombinant Ald6PRase from peach leaves. The recombinant enzyme was inhibited by glucose-1-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate and orthophosphate. Oxidizing agents irreversibly inhibited the enzyme and produced protein precipitation. Enzyme thiolation with oxidized glutathione protected the enzyme from insolubilization caused by diamide, while incubation with NADP+ (one of the substrates) completely prevented enzyme precipitation. Our results suggest that Ald6PRase is finely regulated to control carbon partitioning in peach leaves.

The UDP-glucose pyrophosphorylase from Giardia lamblia is redox regulated and exhibits promiscuity to use galactose-1-phosphate.

BACKGROUND: Giardia lamblia is a pathogen of humans and other vertebrates. The synthesis of glycogen and of structural oligo and polysaccharides critically determine the parasite's capacity for survival and pathogenicity. These characteristics establish that UDP-glucose is a relevant metabolite, as it is a main substrate to initiate varied carbohydrate metabolic routes. RESULTS: Herein, we report the molecular cloning of the gene encoding UDP-glucose pyrophosphorylase from genomic DNA of G. lamblia, followed by its heterologous expression in Escherichia coli. The purified recombinant enzyme was characterized to have a monomeric structure. Glucose-1-phosphate and UTP were preferred substrates, but the enzyme also used galactose-1-phosphate and TTP. The catalytic efficiency to synthesize UDP-galactose was significant. Oxidation by physiological compounds (hydrogen peroxide and nitric oxide) inactivated the enzyme and the process was reverted after reduction by cysteine and thioredoxin. UDP-N-acetyl-glucosamine pyrophosphorylase, the other UTP-related enzyme in the parasite, neither used galactose-1-phosphate nor was affected by redox modification. CONCLUSIONS: Our results suggest that in G. lamblia the UDP-glucose pyrophosphorylase is regulated by oxido-reduction mechanism. The enzyme exhibits the ability to synthesize UDP-glucose and UDP-galactose and it plays a key role providing substrates to glycosyl transferases that produce oligo and polysaccharides. GENERAL SIGNIFICANCE: The characterization of the G. lamblia UDP-glucose pyrophosphorylase reinforces the view that in protozoa this enzyme is regulated by a redox mechanism. As well, we propose a new pathway for UDP-galactose production mediated by the promiscuous UDP-glucose pyrophosphorylase of this organism.

Allosteric regulation of the partitioning of glucose-1-phosphate between glycogen and trehalose biosynthesis in Mycobacterium tuberculosis.

BACKGROUND: Mycobacterium tuberculosis is a pathogenic prokaryote adapted to survive in hostile environments. In this organism and other Gram-positive actinobacteria, the metabolic pathways of glycogen and trehalose are interconnected. RESULTS: In this work we show the production, purification and characterization of recombinant enzymes involved in the partitioning of glucose-1-phosphate between glycogen and trehalose in M. tuberculosis H37Rv, namely: ADP-glucose pyrophosphorylase, glycogen synthase, UDP-glucose pyrophosphorylase and trehalose-6-phosphate synthase. The substrate specificity, kinetic parameters and allosteric regulation of each enzyme were determined. ADP-glucose pyrophosphorylase was highly specific for ADP-glucose while trehalose-6-phosphate synthase used not only ADP-glucose but also UDP-glucose, albeit to a lesser extent. ADP-glucose pyrophosphorylase was allosterically activated primarily by phosphoenolpyruvate and glucose-6-phosphate, while the activity of trehalose-6-phosphate synthase was increased up to 2-fold by fructose-6-phosphate. None of the other two enzymes tested exhibited allosteric regulation. CONCLUSIONS: Results give information about how the glucose-1-phosphate/ADP-glucose node is controlled after kinetic and regulatory properties of key enzymes for mycobacteria metabolism. GENERAL SIGNIFICANCE: This work increases our understanding of oligo and polysaccharides metabolism in M. tuberculosis and reinforces the importance of the interconnection between glycogen and trehalose biosynthesis in this human pathogen.

A Chimeric UDP-glucose pyrophosphorylase produced by protein engineering exhibits sensitivity to allosteric regulators.

In bacteria, glycogen or oligosaccharide accumulation involves glucose-1-phosphate partitioning into either ADP-glucose (ADP-Glc) or UDP-Glc. Their respective synthesis is catalyzed by allosterically regulated ADP-Glc pyrophosphorylase (EC 2.7.7.27, ADP-Glc PPase) or unregulated UDP-Glc PPase (EC 2.7.7.9). In this work, we characterized the UDP-Glc PPase from Streptococcus mutans. In addition, we constructed a chimeric protein by cutting the C-terminal domain of the ADP-Glc PPase from Escherichia coli and pasting it to the entire S. mutans UDP-Glc PPase. Both proteins were fully active as UDP-Glc PPases and their kinetic parameters were measured. The chimeric enzyme had a slightly higher affinity for substrates than the native S. mutans UDP-Glc PPase, but the maximal activity was four times lower. Interestingly, the chimeric protein was sensitive to regulation by pyruvate, 3-phosphoglyceric acid and fructose-1,6-bis-phosphate, which are known to be effectors of ADP-Glc PPases from different sources. The three compounds activated the chimeric enzyme up to three-fold, and increased the affinity for substrates. This chimeric protein is the first reported UDP-Glc PPase with allosteric regulatory properties. In addition, this is a pioneer work dealing with a chimeric enzyme constructed as a hybrid of two pyrophosphorylases with different specificity toward nucleoside-diphospho-glucose and our results turn to be relevant for a deeper understanding of the evolution of allosterism in this family of enzymes.

Characterization of recombinant UDP- and ADP-glucose pyrophosphorylases and glycogen synthase to elucidate glucose-1-phosphate partitioning into oligo- and polysaccharides in Streptomyces coelicolor.

Streptomyces coelicolor exhibits a major secondary metabolism, deriving important amounts of glucose to synthesize pigmented antibiotics. Understanding the pathways occurring in the bacterium with respect to synthesis of oligo- and polysaccharides is of relevance to determine a plausible scenario for the partitioning of glucose-1-phosphate into different metabolic fates. We report the molecular cloning of the genes coding for UDP- and ADP-glucose pyrophosphorylases as well as for glycogen synthase from genomic DNA of S. coelicolor A3(2). Each gene was heterologously expressed in Escherichia coli cells to produce and purify to electrophoretic homogeneity the respective enzymes. UDP-glucose pyrophosphorylase (UDP-Glc PPase) was characterized as a dimer exhibiting a relatively high V(max) in catalyzing UDP-glucose synthesis (270 units/mg) and with respect to dTDP-glucose (94 units/mg). ADP-glucose pyrophosphorylase (ADP-Glc PPase) was found to be tetrameric in structure and specific in utilizing ATP as a substrate, reaching similar activities in the directions of ADP-glucose synthesis or pyrophosphorolysis (V(max) of 0.15 and 0.27 units/mg, respectively). Glycogen synthase was arranged as a dimer and exhibited specificity in the use of ADP-glucose to elongate α-1,4-glucan chains in the polysaccharide. ADP-Glc PPase was the only of the three enzymes exhibiting sensitivity to allosteric regulation by different metabolites. Mannose-6-phosphate, phosphoenolpyruvate, fructose-6-phosphate, and glucose-6-phosphate behaved as major activators, whereas NADPH was a main inhibitor of ADP-Glc PPase. The results support a metabolic picture where glycogen synthesis occurs via ADP-glucose in S. coelicolor, with the pathway being strictly regulated in connection with other routes involved with oligo- and polysaccharides, as well as with antibiotic synthesis in the bacterium.

Lithium-mediated suppression of morphogenesis and growth in Candida albicans.

Hyphal development in Candida albicans contributes to virulence, and inhibition of filamentation is a target for the development of antifungal agents. Lithium is known to impair Saccharomyces cerevisiae growth in galactose-containing media by inhibition of phosphoglucomutase, which is essential for galactose metabolism. Lithium-mediated phosphoglucomutase inhibition is reverted by Mg(2+). In this study we have assessed the effect of lithium upon C. albicans and found that growth is inhibited preferentially in galactose-containing media. No accumulation of glucose-1-phosphate or galactose-1-phosphate was detected when yeasts were grown in the presence of galactose and 15 mM LiCl, though we observed that in vitro lithium-mediated phosphoglucomutase inhibition takes place with an IC(50) of 2 mM. Furthermore, growth inhibition by lithium was not reverted by Mg(2+). These results show that lithium-mediated inhibition of growth in a galactose-containing medium is not due to inhibition of galactose conversion to glucose-6-phosphate but is probably due to inhibition of a signaling pathway. Deletion of the Ser-Thr protein phosphatase SIT4 and treatment with rapamycin have been shown to inhibit filamentous differentiation. We observed that C. albicans filamentation was inhibited by lithium in solid medium containing either galactose as the sole carbon source or 10% fetal bovine serum. These results suggest that suppression of hyphal outgrowth by lithium could be related to inhibition of the target of rapamycin (TOR) pathway.

A glycosyltransferase with a length-controlling activity as a mechanism to regulate the size of polysaccharides.

Cyclic beta-1,2-glucans (CbetaG) are osmolyte homopolysaccharides with a cyclic beta-1,2-backbone of 17-25 glucose residues present in the periplasmic space of several bacteria. Initiation, elongation, and cyclization, the three distinctive reactions required for building the cyclic structure, are catalyzed by the same protein, the CbetaG synthase. The initiation activity catalyzes the transference of the first glucose from UDP-glucose to a yet-unidentified amino acid residue in the same protein. Elongation proceeds by the successive addition of glucose residues from UDP-glucose to the nonreducing end of the protein-linked beta-1,2-oligosaccharide intermediate. Finally, the protein-linked intermediate is cyclized, and the cyclic glucan is released from the protein. These reactions do not explain, however, the mechanism by which the number of glucose residues in the cyclic structure is controlled. We now report that control of the degree of polymerization (DP) is carried out by a beta-1,2-glucan phosphorylase present at the CbetaG synthase C-terminal domain. This last activity catalyzes the phosphorolysis of the beta-1,2-glucosidic bond at the nonreducing end of the linear protein-linked intermediate, releasing glucose 1-phosphate. The DP is thus regulated by this "length-controlling" phosphorylase activity. To our knowledge, this is the first description of a control of the DP of homopolysaccharides.

A high-throughput screening for phosphatases using specific substrates.

A high-throughput screening was developed for the detection of phosphatase activity in bacterial colonies. Unlike other methods, the current procedure can be applied to any phosphatase because it uses physiological substrates and detects the compelled product of all phosphatase reactions, that is, orthophosphate. In this method, substrates diffuse from a filter paper across a nitrocellulose membrane to bacterial colonies situated on the opposite face, and then reaction products flow back to the paper. Finally, a colorimetric reagent discloses the presence of orthophosphate in the filter paper. We validated the performance of this assay with several substrates and experimental conditions and with different phosphatases, including a library of randomly mutagenized rapeseed chloroplast fructose-1,6-bisphosphatase. This procedure could be extended to other enzymatic activities provided that an appropriate detection of reaction products is available.

Ultrasensitive behavior in the synthesis of storage polysaccharides in cyanobacteria.

The glycogen synthetic pathway operates ultrasensitively as a function of the ADPglucose pyrophosphorylase (ADPGlcPPase) allosteric effectors, 3-phosphoglycerate and Pi, in permeabilized cells of the cyanobacterium Anabaena PCC 7120. In vitro data previously showed that the ultrasensitive behavior of ADPGlcPPase depends upon cross-talk between the two allosteric effectors, the enzyme's response being additionally modulated by molecular crowding [D.F. Gómez Casatiet al. (2000) Biochem J 350:139-147]. In the present work we show, experimentally and with a mathematical model, that alpha-1,4-glucan synthesis is also ultrasensitive in cells due to the propagation of the switch-like behavior of ADPGlcPPase to the synthetic pathway. Amplifications of up to 20-fold in storage-polysaccharide synthesis can be achieved with a modest 6.7-fold increase in 3-phosphoglycerate in the presence of 5 mM Pi in contrast to the 30-fold necessary in its absence. This is the first time that this phenomenon has been reported to occur in the glycogen synthetic pathway of a photosynthetic prokaryote. The implications of the results for plant cell physiology during light-dark transitions are discussed.

Activity and expression of banana starch phosphorylases during fruit development and ripening.

Two main forms of starch phosphorylase (EC 2.4.1.1) were identified and purified from banana (Musa acuminata Colla. cv. Nanicão) fruit. One of them, designated phosphorylase I, had a native molecular weight of 155 kDa and subunit of 90 kDa, a high affinity towards branched glucans and an isoelectric point around 5.0. The other, phosphorylase II, eluted at a higher salt concentration from the anion exchanger, had a low affinity towards branched glucans, a native molecular weight of 290 kDa and subunit of 112 kDa. Kinetic studies showed that both forms had typical hyperbolic curves for orthophosphate (Pi) and glucose-1-phosphate, and that they could not react with substrates with a blocked reducing end or alpha-1,6 glucosidic bonds. Antibodies prepared against the purified type-II form and cross-reacting with the type-I form showed that there was an increase in protein content during development and ripening of the fruit. The changes in protein level were parallel to those of phosphorylase activity, in both the phosphorolytic and synthetic directions. Considering the kinetics, indicating that starch phosphorylases are not under allosteric control, it can be argued that protein synthesis makes a contribution to regulating phosphorylase activity in banana fruit and that hormones, like gibberellic acid and indole-3-acetic acid, may play a regulating role. For the first time, starch phosphorylases isoforms were detected as starch-granule-associated proteins by immunostaining of SDS-PAGE gels.

Glycogen brain branching enzyme.

Rat brain glycogen branching enzyme was partially purified in order to elucidate its mechanism of action. The alpha1,4-alpha1,6-glucan polysaccharide was synthesized using rat brain branching enzyme under two different elongation conditions: Glc-1-P and phosphorylase or UDP-Glc and glycogen synthase. The products obtained demonstrated that the cpolysaccharides synthesized (pattern of the spectra obtained in the presence of Krisman's reagent, lambda max, parameter A and R, % beta-amylolysis and degree of branching) under different incubation times are nearly constant. These results imply that the degree of branching of a polysaccharide depends only on the enzyme specificity.

Structure and catalytic mechanism of glucosamine 6-phosphate deaminase from Escherichia coli at 2.1 A resolution.

BACKGROUND: Glucosamine 6-phosphate deaminase from Escherichia coli is an allosteric hexameric enzyme which catalyzes the reversible conversion of D-glucosamine 6-phosphate into D-fructose 6-phosphate and ammonium ion and is activated by N-acetyl-D-glucosamine 6-phosphate. Mechanistically, it belongs to the group of aldoseketose isomerases, but its reaction also accomplishes a simultaneous amination/deamination. The determination of the structure of this protein provides fundamental knowledge for understanding its mode of action and the nature of allosteric conformational changes that regulate its function. RESULTS: The crystal structure of glucosamine 6-phosphate deaminase with bound phosphate ions is presented at 2.1 A resolution together with the refined structures of the enzyme in complexes with its allosteric activator and with a competitive inhibitor. The protein fold can be described as a modified NAD-binding domain. CONCLUSIONS: From the similarities between the three presented structures, it is concluded that these represent the enzymatically active R state conformer. A mechanism for the deaminase reaction is proposed. It comprises steps to open the pyranose ring of the substrate and a sequence of general base-catalyzed reactions to bring about isomerization and deamination, with Asp72 playing a key role as a proton exchanger.

Different properties of the mitochondrial and cytosolic hexokinases in maize roots.

After tissue homogenization, 43% of the total hexokinase activity found in maize radicles was recovered in the mitochondrial fraction and 35% was soluble, in the cytosol. The maize submitochondrial particles obtained after mitochondrial sonication retained a high hexokinase activity. The mitochondrial respiration (state 4 rate) was activated by glucose. This activation was blocked by carboxyatractyloside (0.5 mM) and by oligomycin (2 micrograms/ml). The affinities for ATP and glucose of both soluble and membrane-bound maize hexokinases are similar to those of yeast hexokinase. The Km for ATP of these different forms of hexokinase varied between 0.15 and 0.37 mM, and the Km for glucose between 0.05 and 0.13 mM. A major difference between the two maize hexokinase forms is that only the mitochondrial enzyme was strongly inhibited by ADP (Ki 0.04 mM). The soluble forms of hexokinase found both in the cytosol of maize radicles and in yeast are not inhibited by ADP. In a previous report [de Meis, Grieco and Galina (1992) FEBS Lett. 308, 197-201] it was shown that the mitochondrial F1-F0-ATPase can use glucose 6-phosphate and yeast hexokinase as an ATP regenerating system. We now show that the membrane-bound hexokinase and glucose 6-phosphate can also serve as an ATP regenerating system for the mitochondria of maize radicles provided that the ADP concentration is kept below 0.05 mM. Higher ADP concentrations inhibit the reverse reaction of the mitochondrial hexokinase.

Sequential assembly and polymerization of the polyprenol-linked pentasaccharide repeating unit of the xanthan polysaccharide in Xanthomonas campestris.

Lipid-linked intermediates are involved in the synthesis of the exopolysaccharide xanthan produced by the bacterium Xanthomonas campestris (L. Ielpi, R. O. Couso, and M. A. Dankert, FEBS Lett. 130:253-256, 1981). In this study, the stepwise assembly of the repeating pentasaccharide unit of xanthan is described. EDTA-treated X. campestris cells were used as both enzyme preparation and lipid-P acceptor, and UDP-Glc, GDP-Man, and UDP-glucuronic acid were used as sugar donors. A linear pentasaccharide unit is assembled on a polyprenol-P lipid carrier by the sequential addition of glucose-1-P, glucose, mannose, glucuronic acid, and mannose. The in vitro synthesis of pentasaccharide-P-P-polyprenol was also accompanied by the incorporation of radioactivity into a polymeric product, which was characterized as xanthan, on the basis of gel filtration and permethylation studies. Results from two-stage reactions showed that essentially pentasaccharide-P-P-polyprenol is polymerized. In addition, the direction of chain elongation has been studied by in vivo experiments. The polymerization of lipid-linked repeat units occurs by the successive transfer of the growing chain to a new pentasaccharide-P-P-polyprenol. The reaction involves C-1 of glucose at the reducing end of the polyprenol-linked growing chain and C-4 of glucose at the nonreducing position of the newly formed polyprenol-linked pentasaccharide, generating a branched polymer with a trisaccharide side chain.

Effects of ethanol on carbohydrate metabolism in the elderly.

We have previously reported that in young men, ethanol caused acute insulin resistance, but compensatory insulin secretion prevented deterioration of glucose tolerance (1). In this study, we tested the hypothesis that elderly men, because of their pre-existing insulin resistance and compromised insulin secretory capacity, may experience worsening of their glucose tolerance after ethanol. Nine elderly men (65.7 +/- 0.8 yr, BMI 25.8 +/- 1.4 kg/m2) received ethanol (13 mmol/kg for 30 min i.v.) or saline followed 30 min later by i.v. glucose (2.8 mmol/kg for 5 min). To determine the mechanism of the ethanol effect, six of the men underwent euglycemic-hyperinsulinemic (approximately 350 pM) clamping with simultaneous infusion of ethanol or saline. Muscle biopsies were obtained before and 1 and 4 h after insulin infusion. In all nine men, glucose concentrations after i.v. glucose were higher after ethanol than after saline, whereas insulin was the same and glucose tolerance decreased by 23% (Kg 2.41 +/- 0.2 vs. 1.86 +/- 0.1%/min, P < 0.01). Ethanol reduced insulin-stimulated glucose uptake from 40.6 +/- 3.1 to 25.6 +/- 1.9 mumol.kg-1.min-1 (-37%, P < 0.05), glucose oxidation from 11.7 +/- 1.1 to 7.0 +/- 0.7 mumol.kg-1.min-1 (-33%, P < 0.01), and glucose storage from 28.7 +/- 2.4 to 18.6 +/- 1.7 mumol.kg-1.min-1 (-35%, P < 0.01). Ethanol increased muscle lactate concentration from 0.49 +/- 0.14 to 1.99 +/- 0.99 mumol/mg protein (P < 0.05), but had no effects on muscle concentration of free glucose, G-6-P, and citrate concentrations, nor did it affect muscle GS activity.(ABSTRACT TRUNCATED AT 250 WORDS)

[In situ studies of myoinositol-1-P synthase in wild and inos- strains of Neurospora crassa]. / Estudios "in situ" de la mioinositol 1-P sintasa en cepas silvestre e inos- de Neurospora crassa.

The biosynthetic pathway for myo-inositol consist of two enzymatic steps: first, the cycloaldolization of glucose-6P to L-myo-inositol-IP followed by its hydrolysis to form free myo-inositol. The former reaction is catalyzed by myo-inositol-IP synthase (MIPS) while, a phosphatase is responsible for the hydrolysis step. Depending on its degree of purification and storage age, MIPS activity us to be, from partial to fully, dependent on added NAD. Therefore, we decided to study the kinetic properties of the enzyme within the cell, specially its requirements for free NAD. To this purpose, a method was designed for the assay of MIPS-activity in situ, using toluene permeabilized mycelia. MIPS-activity "in situ" was fully displayed in the absence of added NAD; on the contrary, the purified enzyme showed only 33% of that activity displayed when NAD was included in the assay. Thus, it seems that the native enzyme contains tightly bound NAD, instrumental for its activity, and that during purification or storage, the coenzyme is progressively lost, rendering the NAD-dependent enzyme, as was previously envisage. In addition, the in situ assay method for MIP-Synthase was applied to several mutants of N. crassa having the inosphenotype. Our results showed that only in 3 of 14 cases analyzed the phenotype could be clearly associated to the lack of MIP-synthase activity. Indeed most of the mutants analyzed showed significant levels (from 5 to 21%) of MIP-synthase, when compared to the activity shown by the RL-21 WT strain. Finally, all the mutants and WT strains were zymographically analyzed for phosphatase activity and showed close to equal strong reaction levels.

Development modulates the serum induced effect on the incorporation of [2-3H]mannose into chick optic lobe protein: the possible role of glia.

Recently, we described that serum decreases tritiated mannose incorporation into protein in the chick optic lobe at 18 days of embryonic age (Rossi et al., 1990). In this paper, we found a strikingly different response of this serum effect according to age. The data obtained showed no serum induced decrease in 6-10-day-old embryo. In addition, our results demonstrate that the differential response of the tissue to the serum is independent of the rate of sugar entry into nerve cells. Furthermore, we also report that the variation of mannose or leucine incorporation into protein coincides very closely with the pattern of protein and glycoprotein accumulation during chick optic lobe development. Finally, data were obtained to define glial cells as the cellular target of the serum induced effect. This finding may contribute to elucidate the mechanism of cellular pathogenesis of cerebral lesions that occur after the breakdown of the blood brain barrier, such as in some diseases or during bleeding after injuries.

Reversal of oxidative phosphorylation in submitochondrial particles using glucose 6-phosphate and hexokinase as an ATP regenerating system.

During steady-state, the Pi released in the medium is derived from glucose-6-phosphate which continuously regenerates the ATP hydrolyzed. A membrane potential (delta psi) can be built up in submitochondrial particles using glucose-6-phosphate and hexokinase as an ATP-regenerating system. The energy derived from the membrane potential thus formed, can be used to promote the energy-dependent transhydrogenation from NADH to NADP+ and the uphill electron transfer from succinate to NAD+. In spite of the large differences in the energies of hydrolysis of ATP (delta G degrees = -7.0 to -9.0 kcal/mol) and of glucose-6-phosphate (delta G degrees = -2.5 kcal/mol), the same ratio between Pi production and either NADPH or NADH formation were measured regardless of whether millimolar concentrations of ATP or a mixture of ADP, glucose-6-phosphate and hexokinase were used. Rat liver mitochondria were able to accumulate Ca2+ when incubated in a medium containing hexokinase, ADP and glucose-6-phosphate. The different reaction measured with the use of glucose-6-phosphate and hexokinase were inhibited by glucose concentrations varying from 0.2 to 2 mM. Glucose shifts the equilibrium of the reaction towards glucose-6-phosphate formation thus leading to a decrease of the ATP concentration in the medium.

Membrane-associated and secreted proteoglycans from a continuous cell line derived from fibrotic schistosomal granulomas.

Proteoglycans were isolated from a continuous murine cell line (GRX) established from fibrotic granulomas induced in mouse liver by schistosomal infection, representative of liver connective tissue cells. The proteoglycans were labelled with 35SO4, extracted by guanidine-HCl + Triton X-100 in the presence of proteinase inhibitors, and purified by anion-exchange, gel-filtration and affinity-column chromatography. The major fractions of cell-associated and secreted proteoglycans are heparan sulfate proteoglycans. Gel-filtration chromatography on Sephacryl S-400 revealed Kav values of 0.20 and 0.30 for the cell-associated and secreted heparan sulfate proteoglycans, respectively. About 50% of the cell-associated heparan sulfate proteoglycans contained hydrophobic regions, as evidenced by their ability to bind to octyl-Sepharose, while only about 20% of secreted proteoglycans bound to this resin. In addition, no proteoglycan was competitively displaced from the cell surface by heparin. Taken together with other reports on proteoglycan synthesis by a variety of cell types in culture, these observations suggest that cell-surface heparan sulfate proteoglycans possibly contain a hydrophobic domain that functions as a membrane anchor in their attachment to cells. Addition of beta-D-xyloside to the cultures greatly enhanced the release of 35S-dermatan sulfate to the medium. Interestingly, dermatan sulfate is the major glycosaminoglycan found in the schistosoma-induced granuloma, from which the GRX cell line is derived. These studies provide the first biochemical description of the proteoglycans produced by a liver connective tissue cell line derived from schistosomal granulomas.