Aminoquinolines as fluorescent labels for hydrophilic interaction liquid chromatography of oligosaccharides

Abstract In this study, we investigated the potential of four different aminoquinoline (AQ) compounds as fluorescent labels for glycan analysis using hydrophilic interaction liquid chromatography (HILIC) and fluorescence detection (FLD). We confirmed the optimal excitation and emission wavelengths of 3-AQ and 6-AQ conjugated to glycan standards using three-dimensional fluorescent spectral scanning. The optimal excitation and emission wavelengths for 6-AQ were confirmed at λex=355 nm and λem=440 nm. We concluded that the optimal wavelengths for 3-AQ were λex=355 nm and λem=420 nm, which differed considerably from the wavelengths applied in previous reports. HILIC-FLD chromatograms using experimentally determined wavelengths were similar to 2-aminobenzamide controls, but the peak capacity and resolution differed significantly when published 3-AQ λex/em values were applied. Furthermore, we found that 5-AQ and 8-AQ labeled maltohexaose did not display any fluorescent pro\xadperties when used as a carbohydrate tag for HPLC analysis. Finally, we applied experimentally determined wavelengths to 3-AQ labeled N-glycans released from human IgG to illustrate changes in retention time as well as to demonstrate that AQ labeling is applicable to complex sample analysis via exoglycosidase sequencing.     

Involvement of Hydroxyl Radical Formation and Dna Strand Breakage in the Cytotoxicity of Anthraquinone Antitumour Agents

Four 9,10-anthraquinones (AQ) mono- or bis-substituted with the -NH(CH₂)₂ NH(CH₂)₂OH group were studied. 1-AQ, 1,5-AQ and 1,8-AQ but not 1,4-AQ (100°M) generated pBR322 plasmid DNA single strand breaks in the presence of purified NADPH dependent cytochrome P450 reductase. 1-AQ, 1,5-AQ and 1,8-AQ (at 100 °M) stimulated hydroxyl radical formation in MCF-7 S9 cell fraction (as measured by dimethyl pyrolline N-oxide spin trapping) and MCF-7 DNA strand breaks as measured by alkaline filter elution. In contrast 1,4-AQ did not stimulate hydroxyl radical formation and produced considerably less strand breaks in MCF-7 cells compared to the other AQ's. It would appear that the position of the -NH(CH₂)₂ NH(CH₂)₂OH groups on the chromophore is an important determinant in the metabolic activation of cytotoxic anthraquinones. This may contribute to the cytotoxicity (ID₅₀ values) of 1-AQ (0.06 °M), 1-8-AQ (0.5 °M) and 1,5-AQ (12.3 °M) but not the 1,4-AQ (1.2 °M).     

Redox Cycling in MCF-7 Human Breast Cancer Cells by Antitumor Agents Based on Mitozantrone

In a series of hydroxyethylaminoalkylaminoanthraquinones (AQ's) based on mitozantrone, 1-AQ (340%) and 1,8-AQ (137%) stimulated basal rate NADPH oxidation (72 + 18pmol min^-lmg S9 protein^-1) whilst 1,4-AQ, 1,5-AQ and mitozantrone had no effect. A similar trend was observed for O_2? generation (measured as nmol acet. cyt c reduction min-¹ mg protein^-1) by these compounds in MCF-7 S9 fraction: 1-AQ (9.5) and 1,8-AQ (7.9), whilst 1,5-AQ, 1,4-AQ and mitozantrone showed no significant effect. All the AQs including mitozantrone were cytotoxic to MCF-7 cells in a dose dependent manner with EC₅₀ values as follows: 1-AQ (0.01 μm) > doxorubicin (0.4μM) > mitozantrone (0.6μM) > 1,8-AQ (2.O μM) > 1,5-AQ (4.0μM) > 1,4-AQ (8,0 μM). Thus the redox active AQs were also the most cytotoxic. Mitozantrone however was not redox active but was more cytotoxic than all but 1-AQ hence it would appear that factors other than free radical generation contribute to the antitumor activity of this group of compounds.     

Involvement of Hydroxyl Radical Formation and Dna Strand Breakage in the Cytotoxicity of Anthraquinone Antitumour Agents

Four 9,10-anthraquinones (AQ) mono- or bis-substituted with the -NH(CH₂)₂ NH(CH₂)₂OH group were studied. 1-AQ, 1,5-AQ and 1,8-AQ but not 1,4-AQ (100°M) generated pBR322 plasmid DNA single strand breaks in the presence of purified NADPH dependent cytochrome P450 reductase. 1-AQ, 1,5-AQ and 1,8-AQ (at 100 °M) stimulated hydroxyl radical formation in MCF-7 S9 cell fraction (as measured by dimethyl pyrolline N-oxide spin trapping) and MCF-7 DNA strand breaks as measured by alkaline filter elution. In contrast 1,4-AQ did not stimulate hydroxyl radical formation and produced considerably less strand breaks in MCF-7 cells compared to the other AQ's. It would appear that the position of the -NH(CH₂)₂ NH(CH₂)₂OH groups on the chromophore is an important determinant in the metabolic activation of cytotoxic anthraquinones. This may contribute to the cytotoxicity (ID₅₀ values) of 1-AQ (0.06 °M), 1-8-AQ (0.5 °M) and 1,5-AQ (12.3 °M) but not the 1,4-AQ (1.2 °M).     

Structure-mutagenicity relationship among aminoquinolines, aza-analogues of naphthylamine, and their N-acetyl derivatives

The mutagenicity of 7 positional isomers of aminoquinolines (AQ) and their N-acetyl derivatives (AcAQ) was tested in Salmonella typhimurium TA100 and TA98 in the presence and absence of S9 mix. In a series of aminoquinolines, the order of mutagenic potency in the presence of S9 mix is: 5-AQ greater than 8-AQ greater than 7-AQ greater than 3-AQ greater than 2-AQ much greater than 4-AQ, 6-AQ. The alpha-positional isomers, 5-AQ and 8-AQ, are more mutagenic than the beta-isomer, 2-, 3-, 6-, 7-AQ's. These results are in contrast to the finding that beta-naphthylamine is more mutagenic than alpha-naphthylamine. In a series of N-acetylaminoquinolines, the order of mutagenic potency in the presence of S9 mix is: 7-AcAQ greater than 6-AcAQ greater than 8-AcAQ much greater than all the others. It is suggested that the AQ and AcAQ series might exert their mutagenicity through different molecular mechanisms (i.e., metabolic activation) from each other. The rate of metabolic activation does not seem to be correlated with the mutagenic potency of the compounds. It is noteworthy that 7-AQ and 8-AQ are mutagenic in both the strains tested in the absence of S9 mix.     

Characterization of 4-methyl-2-oxo-1,2-dihydroquinolin-6-yl acetate as an effective antiplatelet agent

We have studied earlier a membrane bound novel enzyme Acetoxy Drug: protein transacetylase identified as Calreticulin Transacetylase (CRTAase) that catalyzes the transfer of acetyl groups from polyphenolic acetates (PAs) to the receptor proteins and thus modulating their biological activities. In this communication, we have reported for the first time that acetoxy quinolones are endowed with antiplatelet action by virtue of causing CRTAase catalyzed activation of platelet Nitric Oxide Synthase (NOS) by way of acetylation leading to the inhibition of ADP/Arachidonic acid (AA)-dependent platelet aggregation. The correlation of specificity of platelet CRTAase to various analogues of acetoxy quinolones with intracellular NO and consequent effect on inhibition of platelet aggregation was considered crucial. Among acetoxy quinolones screened, 6-AQ (4-methyl-2-oxo-1,2-dihydroquinolin-6-yl acetate/6-acetoxyquinolin-2-one, 22) was found to be the superior substrate to platelet CRTAase and emerged as the most active entity to produce antiplatelet action both in vitro and in vivo. 6-AQ caused the inhibition of cyclooxygenase-1 (Cox-1) resulting in the down regulation of thromboxane A2 (TxA2) and the inhibition of platelet aggregation. Structural modification of acetoxy quinolones positively correlated with enhancement of intracellular NO and antiplatelet action.     

The use of fluoro- and deoxy-substrate analogs to examine binding specificity and catalysis in the enzymes of the sorbitol pathway

The carbohydrate specificity of the two enzymes that catalyze the metabolic interconversions in the sorbitol pathway, aldose reductase and sorbitol dehydrogenase, has been examined through the use of fluoro- and deoxy-substrate analogs. Hydrogen bonding has been shown to be the primary mode of interaction by which these enzymes specifically recognize and bind their respective polyol substrates. Aldose reductase has broad substrate specificity, and all of the fluoro- and deoxysugars that were examined are substrates for this enzyme. Unexpectedly, both 3-fluoro- and 4-fluoro-D-glucose were found to be better substrates, with significantly lower K(m) and higher Kcat/K(m) values than those of D-glucose. A more discriminating pattern of substrate specificity is observed for sorbitol dehydrogenase. Neither the 2-fluoro nor the 2-deoxy analogs of D-glucitol were found to be substrates or inhibitors, suggesting that the 2-hydroxyl group of sorbitol is a hydrogen bond donor. The 4-fluoro and 4-deoxy analogs are poorer substrates than sorbitol, also implying a binding role for this hydroxyl group. In contrast, both 6-fluoro- and 6-deoxy-D-glucitol are very good substrates for sorbitol dehydrogenase, indicating that the primary hydroxyl group at this position is not involved in substrate recognition by this enzyme.     

Fluorescent phosphatidylinositol 4,5-bisphosphate derivatives with modified 6-hydroxy group as novel substrates for phospholipase C

The capacity to monitor spatiotemporal activity of phospholipase C (PLC) isozymes with a PLC-selective sensor would dramatically enhance understanding of the physiological function and disease relevance of these signaling proteins. Previous structural and biochemical studies defined critical roles for several of the functional groups of the endogenous substrate of PLC isozymes, phosphatidylinositol 4,5-bisphosphate (PIP(2)), indicating that these sites cannot be readily modified without compromising interactions with the lipase active site. However, the role of the 6-hydroxy group of PIP(2) for interaction and hydrolysis by PLC has not been explored, possibly due to challenges in synthesizing 6-hydroxy derivatives. Here, we describe an efficient route for the synthesis of novel, fluorescent PIP(2) derivatives modified at the 6-hydroxy group. Two of these derivatives were used in assays of PLC activity in which the fluorescent PIP(2) substrates were separated from their diacylglycerol products and reaction rates quantified by fluorescence. Both PIP(2) analogues effectively function as substrates of PLC-δ1, and the K(M) and V(max) values obtained with one of these are similar to those observed with native PIP(2) substrate. These results indicate that the 6-hydroxy group can be modified to develop functional substrates for PLC isozymes, thereby serving as the foundation for further development of PLC-selective sensors.     

Evaluation of alpha-cyano ethers as fluorescent substrates for assay of cytochrome P450 enzyme activity

We have previously reported the synthesis of four alpha-cyano-containing ethers based on 2-naphthaldehyde (2-NA) as cytochrome P450 (P450) fluorescent substrates. Activity detection was based on the formation of fluorescent 2-NA following substrate hydrolysis. A major limitation of these substrates was the need to remove NADPH, a required cofactor for P450 oxidation, before measuring 2-NA fluorescence. In this article, we report the synthesis of a new series of novel P450 substrates using 6-dimethylamino-2-naphthaldehyde (6-DMANA), which has a green fluorescent emission that is well separated from the NADPH spectrum. A major advantage of the 6-DMANA substrates is that NADPH removal is not required before fluorescence detection. We used eight alpha-cyano ether-based substrates to determine the O-dealkylation activity of human, mouse, and rat liver microsomes. In addition, substrate activities were compared with the commercial substrate 7-ethoxyresorufin (7-ER). The catalytic turnover rates of both the 6-DMANA- and 2-NA-based substrates were in some cases threefold faster than the catalytic turnover rate of 7-ER. The 2-NA-based substrates had greater turnover than did the 6-DMANA-based substrates. Murine and rat liver microsomes prepared from animals that had been treated with various P450 inducers were used to examine for isozyme-selective turnover of the substrates. The vastly improved optical properties and synthetic flexibility of the alpha-cyano ether compounds suggest that they are possibly good general P450 substrates.     

New ribose-modified fluorescent analogs of adenine and guanine nucleotides available as substrates for various enzymes

The synthesis of fluorescent derivatives of nucleosides and nucleotides, by reaction with isatoic anhydride in aqueous solution at mild pH and temperature, yielding their 3'-O-anthraniloyl derivatives, is here described. The N-methylanthraniloyl derivatives were also synthesized by reaction with N-methylisatoic anhydride. Upon excitation at 330-350 nm these derivatives exhibited maximum fluorescence emission at 430-445 nm in aqueous solution with quantum yields of 0.12-0.24. Their fluorescence was sensitive to the polarity of the solvent; in N,N-dimethylformamide the quantum yields were 0.83-0.93. The major differences between the two fluorophores were the longer wavelength of the emission maximum of the N-methylanthraniloyl group and its greater quantum yield in water. All anthraniloyl derivatives, as well as the N-methylanthraniloyl ones, had virtually identical fluorescent properties, regardless of their base structures. The ATP derivatives showed considerable substrate activity as a replacement of ATP with adenylate kinase, guanylate kinase, glutamine synthetase, myosin ATPase and sodium-potassium transport ATPase. The ADP derivatives were good substrates for creatine kinase and glutamine synthetase (gamma-glutamyl transfer activity). The GMP and adenosine derivatives were substrates for guanylate kinase and adenosine deaminase, respectively. All derivatives had only slightly altered Km values for these enzymes. While more fluorescent in water, the N-methylanthraniloyl derivatives were found to show relatively low substrate activities against some of these enzymes. The results indicate that these ribose-modified nucleosides and nucleotides can be versatile fluorescent substrate analogs for various enzymes.     

Multiple proteolytic action of rat liver cathepsin B: specificities and pH-dependences of the endo- and exopeptidase activities.

Dipeptidylcarboxypeptidase, endopeptidase, and carboxypeptidase activities of rat liver cathepsin B were investigated using soluble denatured protein substrates, reduced and S-(3-trimethylammonio)propylated proteins and their derivatives. It was found that the soluble denatured proteins were degraded mainly by the dipeptidylcarboxypeptidase activity and in a few cases by the endopeptidase and carboxypeptidase activities. The eipeptidylcarboxypeptidase activity showed broad substrate specificity with broad pH optimum at 4-6. A peptide having the alpha-carboxyl group amidated with methylamine could also be a good substrate for this activity. These results suggest that this activity is dependent not upon the dissociated alpha-carboxyl group at the P2' site but upon the hydrogen-bonding abilities of the alpha-imino moiety and the protonated or amidated alpha-carboxyl moiety at P2'. On the other hand, the endopeptidase and carboxypeptidase activities were observed in a few cases, suggesting that special amino acid sequences in the substrates are responsible for these activities. These activities showed sharp pH optima at 6 and seemed to prefer basic amino acid residues at P1 site. Therefore, we suppose that cathepsin B has a carboxyl group with a pKa of about 5.5 at the S1 subsite which more effectively interacts with a positive charge at the P1 site of the substrate at pH 6 than at pH 5. Based on these results, a model of the binding subsites of this enzyme is proposed.     

6-Aminoquinoline as a fluorogenic leaving group in peptide cleavage reactions: A new fluorogenic substrate for chymotrypsin

A new fluorogenic substrate capable of measuring the amidolytic activity of chymotrypsin and based upon the enzyme-catalyzed release of a highly fluorescent aromatic amine, 6-aminoquinoline, was prepared. The substrate, 6-(N-glutaryl-l-phenylalanylamido)quinoline, was found to have at pH 8.0 and 25°C K_m = 1.77 mm and k_cat = 1.4 × 10^?1 s^?1. The aminoquinoline is a unique leaving group in that its appearance can be measured fluorometrically at its excitation and emission maxima, while, under these conditions, fluorescence associated with unhydrolyzed substrate is negligible.     

Studies on the catalytic function of aromatase: aromatization of 6-alkoxy-substituted androgens

To gain insight into the catalytic function of aromatase, we studied aromatization of a series of 6alpha- and 6beta-ether-substituted (methoxy, ethoxy, and n-butoxy) androst-4-ene-3,17-dione (AD) steroids (1 and 2) and their androsta-1,4-diene-3,17-dione (ADD) derivatives (3 and 4) with human placental aromatase by gas chromatography-mass spectrometry (GC-MS). Among the steroids examined, 6beta-methoxy and 6beta-ethoxyADDs (4a and 4b) are suicide substrates of aromatase. All of the steroids were found to be converted into the corresponding 6-alkoxy estrogens. Introduction of the alkoxy groups at C-6 of AD or ADD decreased the ability of these to serve as a substrate of aromatase. In 6alpha-alkoxy steroid series, compounds 1 and 3, the aromatization rate increased by elongating the 6-methoxy group up to the n-butoxy group whereas, in the 6beta-isomers series, 2 and 4, the rate decreased due to this structural modification. 6beta-Alkoxy steroids, 2 and 4, including the suicide substrates, were extremely poor substrates for the aromatization reaction. Apparent K(m) values obtained for 6alpha-alkoxy compounds 1 and 3 were similar to each other, ranging from 92 to 111nM, as shown by their previously-obtained K(i) values. The findings indicate that the stereochemistry as well as the bulkiness of the 6-ether-substituent play an important role in the ability to serve as a substrate. It is also predicted that the aromatization reaction and the mechanism-based inactivation reaction would be related and have a definite partition number which is characteristic to the compound in a series of suicide substrates.     

Human liver N-acetylglucosamine-6-sulphate sulphatase. Catalytic properties.

Kinetic parameters (Km and kcat.) of the two major forms (A and B) and a minor form (C) of human liver N-acetylglucosamine-6-sulphate sulphatase [Freeman, Clements & Hopwood (1987) Biochem. J. 246, 347-354] were determined with a variety of substrates matching structural aspects of the physiological substrates in vivo, namely heparin, heparan sulphate and keratan sulphate. Enzyme activity is highly specific towards glucosamine 6-sulphate or glucose 6-sulphate residues. More structurally complex substrates, in which several aspects of the aglycone structure of the natural substrate were maintained, are hydrolysed with catalytic efficiencies up to 3900 times above that observed for the monosaccharide substrate N-acetylglucosamine 6-sulphate. Forms A and B both desulphate substrates derived from keratan sulphate and heparin. Aglycone structures that influence substrate binding and/or enzyme activity were penultimate-residue 6-carboxy and 2-sulphate ester groups for heparin-derived substrates and penultimate-residue 6-sulphate ester groups for keratan sulphate-derived substrates. The 4-hydroxy group of the N-acetylglucosamine 6-sulphate or the 2-sulphaminoglucosamine 6-sulphate under enzymic attack is involved in the catalytic mechanism. The presence of a 2-amino group in place of a 2-acetamido or a 2-sulphoamino group considerably decreases the catalytic efficiency of the sulphatase, particularly in the absence of a penultimate-aglycone-residue 6-carboxy group. Both forms A and B are exo-enzymes, since activity towards internal sulphate ester bonds was not observed. The effect of incubation pH on enzyme activity towards the variety of substrates evaluated was complex and dependent on substrate aglycone structure. The presence of aglycone 2-sulphate ester, 6-carboxy group and 6-sulphate ester groups on the glucosamine 6-sulphate residue under attack considerably affects the pH response. Sulphate and phosphate ions are potent inhibitors of enzyme activity.     

Cellular Response to Substrate Rigidity Is Governed by Either Stress or Strain

Cells sense the rigidity of their substrate; however, little is known about the physical variables that determine their response to this rigidity. Here, we report traction stress measurements carried out using fibroblasts on polyacrylamide gels with Young’s moduli ranging from 6 to 110 kPa. We prepared the substrates by employing a modified method that involves N-acryloyl-6-aminocaproic acid (ACA). ACA allows for covalent binding between proteins and elastomers and thus introduces a more stable immobilization of collagen onto the substrate when compared to the conventional method of using sulfo-succinimidyl-6-(4-azido-2-nitrophenyl-amino) hexanoate (sulfo-SANPAH). Cells remove extracellular matrix proteins off the surface of gels coated using sulfo-SANPAH, which corresponds to lower values of traction stress and substrate deformation compared to gels coated using ACA. On soft ACA gels (Young’s modulus <20 kPa), cell-exerted substrate deformation remains constant, independent of the substrate Young’s modulus. In contrast, on stiff substrates (Young’s modulus >20 kPa), traction stress plateaus at a limiting value and the substrate deformation decreases with increasing substrate rigidity. Sustained substrate strain on soft substrates and sustained traction stress on stiff substrates suggest these may be factors governing cellular responses to substrate rigidity.     

On the substrate specificity of bovine liver dihydrofolate reductase: new unconjugated dihydropterin substrates

The substrate specificity of dihydrofolate reductase from cells of different origin has been thought to be quite narrow, and unconjugated dihydropterins such as 6-methyl-dihydropterin are known to be very poor substrates. We have reinvestigated the substrate specificity of several dihydropterins and, in addition, have observed that in a new series of unconjugated dihydropterins of the general structure 6-CH2O(CH2)nCH3 several compounds are excellent substrates for the bovine liver enzyme, but none of them bind as well as dihydrofolate. The substrate activity (apparent Vmax) of these compounds increases from 17 to 110% that of the natural substrate, dihydrofolate, as n is increased from 0 to 3. In contrast, these unconjugated dihydropterins are very poor substrates for the Escherichia coli enzyme.     

Molecular Docking Simulations Provide Insights in the Substrate Binding Sites and Possible Substrates of the ABCC6 Transporter

The human ATP-binding cassette family C member 6 (ABCC6) gene encodes an ABC transporter protein (ABCC6), primarily expressed in liver and kidney. Mutations in the ABCC6 gene cause pseudoxanthoma elasticum (PXE), an autosomal recessive connective tissue disease characterized by ectopic mineralization of the elastic fibers. The pathophysiology underlying PXE is incompletely understood, which can at least partly be explained by the undetermined nature of the ABCC6 substrates as well as the unknown substrate recognition and binding sites. Several compounds, including anionic glutathione conjugates (N-ethylmaleimide; NEM-GS) and leukotriene C4 (LTC4) were shown to be modestly transported in vitro; conversely, vitamin K3 (VK3) was demonstrated not to be transported by ABCC6. To predict the possible substrate binding pockets of the ABCC6 transporter, we generated a 3D homology model of ABCC6 in both open and closed conformation, qualified for molecular docking and virtual screening approaches. By docking 10 reported in vitro substrates in our ABCC6 3D homology models, we were able to predict the substrate binding residues of ABCC6. Further, virtual screening of 4651 metabolites from the Human Serum Metabolome Database against our open conformation model disclosed possible substrates for ABCC6, which are mostly lipid and biliary secretion compounds, some of which are found to be involved in mineralization. Docking of these possible substrates in the closed conformation model also showed high affinity. Virtual screening expands this possibility to explore more compounds that can interact with ABCC6, and may aid in understanding the mechanisms leading to PXE.     

Spectroscopic and kinetic analyses reveal the pyridoxal 5'-phosphate binding mode and the catalytic features of Treponema denticola cystalysin

To obtain insight into the functional properties of Treponema denticola cystalysin, we have analyzed the pH- and ligand-induced spectral transitions, the pH dependence of the kinetic parameters, and the substrate specificity of the purified enzyme. The absorption spectrum of cystalysin has maxima at 418 and 320 nm. The 320 nm band increases at high pH, while the 418 nm band decreases; the apparent pK(spec) of this spectral transition is about 8.4. Cystalysin emitted fluorescence at 367 and 504 nm upon excitation at 320 and 418 nm, respectively. The pH profile for the 367 nm emission intensity increases above a single pK of approximately 8.4. On this basis, the 418 and 320 nm absorbances have been attributed to the ketoenamine and substituted aldamine, respectively. The pH dependence of both log k(cat) and log k(cat)/K(m) for alpha,beta-elimination reaction indicates that a single ionizing group with a pK value of approximately 6.6 must be unprotonated to achieve maximum velocity. This implies that cystalysin is more catalytically competent in alkaline solution where a remarkable portion of its coenzyme exists as inactive aldamine structure. Binding of substrates or substrate analogues to the enzyme over the pH range 6-9.5 converts both the 418 and 320 nm bands into an absorbing band at 429 nm, assigned to the external aldimine in the ketoenamine form. All these data suggest that the equilibrium from the inactive aldamine form of the coenzyme shifts to the active ketoenamine form on substrate binding. In addition, reinvestigation of the substrate spectrum of alpha,beta-elimination indicates that cystalysin is a cyst(e)ine C-S lyase rather than a cysteine desulfhydrase as claimed previously.     

Oligosaccharide library-based assessment of heparan sulfate 6-O-sulfotransferase substrate specificity

Heparan sulfate mediates numerous complex biological processes. Its action critically depends on the amount and the positions of O-sulfate groups (iduronyl 2-O-sulfates, glucosaminyl 6-O- and 3-O-sulfates) that form binding sites for proteins. The structures and distribution of these protein-binding domains are influenced by the expression and substrate specificity of heparan sulfate biosynthetic enzymes. We describe a general approach to assess substrate specificities of enzymes involved in glycosaminoglycan metabolism, here applied to 6-O-sulfotransferases involved in heparan sulfate biosynthesis. To understand how 2-O-sulfation affects subsequent 6-O-sulfation reactions, the substrate specificity of 6-O-sulfotransferase 3 was probed using substrates from a heparin-based octasaccharide library. Purified 3H-labeled N-sulfated octasaccharides from a library designed to sample 2-O-sulfated motifs were used as sulfate acceptors, 3'-phosphoadenosine 5'-phosphosulfate as sulfate donor, and cell extract from 6-O-sulfotransferase 3-overexpressing 293 cells as enzyme source in the 6-O-sulfotransferase-catalyzed reactions. The first 6-O-sulfate group was preferentially incorporated at the internal glucosamine unit of the octasaccharide substrate. As the reaction proceeded, the octasaccharides acquired three 6-O-sulfate groups. The specificities toward competing octasaccharide substrates, for 6-O-sulfotransferase 2 and 6-O-sulfotransferase 3, were determined using overexpressing 293 cell extracts and purified octasaccharides. Both 6-O-sulfotransferases showed a preference for 2-O-sulfated substrates. The specificity toward substrates with two to three 2-O-sulfate groups was three to five times higher as compared with octasaccharides with no or one 2-O-sulfate group.     

A fluorimetric study of substrate and effector binding of D-ribulose-1,5-biphosphate carboxylase/oxygenase from spinach

2-p-toluidino-naphthalene-6-sulfonate (TNS) is a sensitive fluorescent reporter group for the detection of the events at the reaction centres of the ribulose biphosphate carboxylase/oxygenase from spinach. The formation of binary complexes of the carboxylase with substrates and effectors is associated with significant changes (ΔF) of the fluorescence emission of the enzyme-TNS-complex. This indicates substrate and effector induced conformational changes of the enzyme. From the concentration dependence of ΔF the following dissociation constants for ribulose biphosphate (RuBP) and Mg²⁺ were determined: K(RuBP) = 0,5 μM and K(Mg²⁺) = 1 mM. Sugar phosphates, e.g. 6-phosphogluconate, which show regulatory effects in the carboxylation and oxygenation of RuBP, function antagonistically to RuBP, presumably by competition with RuBP for its allosteric binding site.