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Sulphonamide drugs (sulphamethazine, sulphamerazine, sulphadiazine, sulphathiazole) were studied in a wide potential window (between 2 and − 2 V) in acetonitrile, dimethyl sulphoxide and in 50–50 v/v% binary mixtures of acetonitrile and water. The voltammograms of the outlined compounds were very similar both in the anodic and cathodic part in each non-aqueous solvents except for sulphathiazole. These sulphonamide drugs were also investigated in presence of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and its sodium salt and the voltammograms changed due to an acid–base reaction. HEPES and its sodium salt could be investigated in acetonitrile only in their saturation concentration as they were slightly soluble in this solvent. In a separate experiment their solubilities were determined at 298 K in acetonitrile with the co-solvent calibration method using water as co-solvent. Complementary fluorescence studies in dimethyl sulphoxide did not show the presence of any interaction between sulphonamide drugs and HEPES as well as its sodium salt.

In this report, the solubility of hydroquinone (1,4-dihydroxybenzene) was determined in dichloromethane, trichloromethane and carbon tetrachloride at 298 K by using cyclic voltammetry. By the proposed cosolvent calibration method a mixed solvent was used for the determination of solubility where acetonitrile was the co-solvent. The molar ratios of the two solvents were uniform during the whole procedure both in the calibration solutions and in solutions used for solubility determinations. The solubility of hydroquinone decreased by the decrease of solvent permittivity, which were in dichloromethane 5.683 mmol·L−1, in trichloromethane 2.785 mmol·L−1 and in carbon tetrachloride 0.321 mmol·L−1.

Cell-based assays against cell surface receptor targets are essential in vitro models of target-based drug discovery. At the lead generation phase large-scale functional screening assays monitoring individual cellular readouts detect interactions between the compounds and the predefined pathways but might lack sufficient sensitivity owing to the complexity of downstream signaling pathways. Cellular label-free assays offer advantages over labeled detection approaches as they reflect whole-cell responses without the prerequisite of detecting only a single cellular analyte and introducing additional genetic manipulations in favor of the chosen detection method. The combination of a label-free assay and labeled assays might integrate the advantageous characteristics of both approaches with regards to added pharmacological information and a bigger pool of chemical starting material. Here we report multiplexing of dynamic mass redistribution label-free technology with HTRF-based cAMP detection on an alpha2c adrenergic receptor expressing cell line. Besides describing the challenging assay development work associated with the set goal, a pilot screening campaign on ca. 1600 compounds is also presented. The combined assay demonstrated the ability to detect relevant activities in both readouts. Interpretation of the results as well as an outlook for further possible opportunities and applications are also discussed.

A homogenous fluorescence polarization (FP) assay was developed to monitor the enzymatic activity of polymerases. Under the optimized conditions established in this study, the assay provides highly robust and reproducible data. Miniaturization of the assay for high-throughput screening and compound testing was also performed. The sensitivity of the newly developed assay was confirmed using 2′,3′-dideoxyadenosine-5′-triphosphate (ddATP), a chain-elongating inhibitor of the polymerase reaction. Side-by-side comparison of the presented fluorescence polarization assay with already well established PicoGreen® fluorescence intensity assay revealed that the performance of both formats is comparable with good assay sensitivity. However, the direct ratiometric readout of the presented FP assay makes it superior over existing colorimetric and fluorescence intensity based assays in terms of susceptibility to false positives. Moreover, due to its generic nature the presented FP assay can be applied to other polymerases and is compatible with identification of inhibitors and requirements of hit-to-lead programs.

Ethanol effect on the interaction of ochratoxin A (OTA) with human serum albumin (HSA) was investigated by using fluorescence spectroscopy and Raman spectroscopy. The Raman results showed that after the binding of OTA, the microenvironment of tryptophan residue on HSA became less hydrophobic. The fluorescence quenching observations revealed that the binding constant for the binding of OTA to HSA decreased as ethanol concentration increased. The thermodynamic studies showed that the binding process of OTA to HSA switched from being entropy-driven to enthalpy-driven in the presence of increasing concentrations (0.7–24.7%, vol/vol) of ethanol. Enthalpy–entropy compensation effect for the binding of OTA to HSA in the presence of different ethanol concentrations had been found. Based on the thermodynamic analyses, we concluded that the ethanol-induced variation of the shape of binding site of OTA on HSA and the solvent reorganization surrounding the OTA–HSA complex are the two dominant effects.

Albumin, the most abundant plasma protein is an approximately 67 kDa sized water-soluble macromolecule. Since several drugs and xenobiotics circulate in the blood at least partially in albumin-bound form, albumin plays a key role in the pharmacokinetics/toxicokinetics of these chemicals. Most of the drugs and xenobiotics are Sudlow's site I ligands. In numerous studies, bovine serum albumin (BSA) is used for modeling albumin–ligand interactions and the results are extrapolated to human serum albumin (HSA). Furthermore, only limited information is available related to albumin–ligand interactions of different albumin species. Therefore, in our study, we have focused on the quantification of differences between bovine, human and rat serum albumin (RSA) using four Sudlow's site I ligands (luteolin, ochratoxin A, phenylbutazone and warfarin). Interactions were analyzed by fluorescence spectroscopy. Stability constants as well as competing capacities of the ligands were determined, and thermodynamic study was also performed. Our results highlight that there could be major differences between BSA, HSA and RSA in their ligand binding properties. Based on our observations we emphasize that in molecular aspects BSA behaves considerably differently from HSA or from albumins of other species therefore, it is strongly recommended to apply at least some confirmatory measurements when data obtained from other species are attempted to be extrapolated to HSA.

A novel 'three-level' deepened cavitand featuring a significantly sizable portal has been synthesized and its interaction with some transition metal ions has been investigated in THF/H2O binary solvent using fluorescence quenching technique. The results suggest that among the used transition metal ions including Ag(+), Cd(2+), Cu(2+), Fe(3+), Cr(3+), Hg(2+), La(3+), Mn(2+), Ni(2+), Zn(2+) and Co(2+), only Fe(3+) and Cu(2+) show good quenching ability. In order to interpret the quenching mechanism, the Stern-Volmer kinetics, and the presence of both the dynamic and static quenching have been discussed. It was found that the simultaneous presence of the sphere-of-action static quenching and dynamic quenching model agrees very well with the experimental results. The limits of detection for Fe(3+) and Cu(2+) were found to be 2.1×10(-6) mol L(-1) (3σ) and 3.6×10(-6) mol L(-1) (3σ), respectively. Cations with potential interference, such as K(+), Na(+), Mg(2+), Ca(2+), Co(2+), La(3+) and Mn(2+) do not have significant effects on the determinations of Fe(3+) and Cu(2+). This cavitand can be potentially applied as optical sensor for the detection of Fe(3+) and Cu(2+).

A very efficient modular reaction protocol was developed to attach various functionalities to a rigid cavitand scaffold. In this way, aryl, iodoaryl, benzyl, pyrrolidylmethyl groups, as well as a polyethylene-glycol chain were attached to the ‘triazol-level’ of the cavitand. The palladium-catalyzed ethynylation of the iodoarene moieties, followed by the copper(I)-catalyzed azide–alkyne cycloaddition produced novel cavitands with significantly elongated binding pockets. The dimensions of these molecules are calculated to be at least 9 A×18 A, which place them amongst the largest unimolecular hosts obtained by pure covalent synthesis. A cavitand-based click conjugate is used for selective complexation of Cu 2+ and Fe 3+ , providing significant fluorescent quenching.

The catalytic amino acid residues of the β-D-xylosidase (EC 3.2.1.37; GH43), from Thermobifida fusca TM51 (TfBXyl43), were investigated by direct chemical modifications. The pH dependence curves of the kinetic parameters (k(cat) and k(cat)/K(M)) gave pK values for the free enzyme (5.55 ± 0.19; 6.44 ± 0.19), and pK values of for the enzyme-substrate complex (4.85 ± 0.23; 7.60 ± 0.28) respectively, by using an artificial substrate p-nitrophenyl-β-D-xylopyranoside (pNP-Xyl). The detailed inhibition studies demonstrated well the hydrophobic character of the glycon binding site. Carbodiimide-mediated chemical modifications of the enzyme with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) in the presence of glycine methyl ester supports the conclusion that a carboxylate residue can be fundamental in the catalytic process. We have also synthesized and tested N-bromoacetyl-β-D-xylopyranosylamine (NBAXA) for TfBXyl43 as an affinity label, which also inactivated the enzyme irreversible. The pH dependence studies in both cases of inactivation revealed that the modified group is the catalytic proton donor (NBAXA pK(A) = 6.68 ± 0,1; EDAC pK(A) = 7.42 ± 0.22) which displays its essential role in the hydrolytic process. The β-D-xylopyranosylazide as competitive inhibitor protected the enzyme in all cases against the inactivation, suggesting that the chemical modification which has an impact on the activity took place in the active center. Changing of the enzyme conformation was followed by CD spectroscopy, as a result of the NBAXA inactivation. Our study is important because to our knowledge no similar investigations were made in the case of an inverting β-D-xylosidase.

Cell-based assays for G-protein-coupled receptor (GPCR) activation applied in high-throughput screening (HTS) monitor various readouts for second messengers or intracellular effectors. Recently, our understanding of diverging signaling pathways downstream of receptor activation and the capability of small molecules to selectively modulate signaling routes has increased substantially, underlining the importance of selecting appropriate readouts in cellular functional screens. To minimize the rate of false negatives in large-scale screening campaigns, it is crucial to maximize the chance of a ligand being detected, and generally applicable methods for detecting multiple analytes from a single well might serve this purpose. The few assays developed so far based on multiplexed GPCR readouts are limited to only certain applications and usually rely on genetic manipulations hindering screening in native or native-like cellular systems. Here we describe a more generally applicable and HTS-compatible homogeneous assay based on the combination of fluorometric detection of [Ca(2+)] with subsequent homogeneous time-resolved fluorescence (HTRF) cAMP readout in the same well. Besides describing development and validation of the assay, using a cell line recombinantly expressing the human PTH1 receptor screening of a small library is also presented, demonstrating the robustness and HTS compatibility of the novel paradigm.

The subject of our investigations was a recombinant β-D-xylosidase (TfBXyl43) from Thermobifida fusca TM51 which was expressed in E. coli BL21DE3 and was purified to apparent homogeneity. The SDS-PAGE investigations demonstrated that the molecular weight of the monomer unit is 62.5 kDa but the native-PAGE studies indicated that the mass of the enzyme is 240-250 kDa which proves the presence of a characteristic homo oligomer quaternary structure in solution phase. Optimal parameters of the enzyme activity were at pH 6.0 and 50 °C. The enzyme showed little stability under pH 4.5 and above 60 °C. The substrate specificity investigations indicated that the TfBXyl43 is an exo-glycosidase, hydrolyzing only xylobiose and -triose from the nonreducing end. Besides the enzyme shows very high specificity on the glycon part of the substrate, since it can only hydrolyze β-D-xylopyranoside derivatives. The importance of hydrophobic interactions in the binding of the substrates are supported that the enzyme can hydrolize about four times more efficiently the artificial p-nitrophenyl-β-D-xylopyranoside substrate compared to the natural one, xylobiose. Furthermore we could detect transxylosidase activity both in the case of xylobiose and p-nitrophenyl-β-D-xylopyranoside donors which is the first example among the inverting β-D-xylosidases from T. fusca.

The effects of barley and oat feeding in table duck were investigated. During a 49-day growing period a corn-based diet was supplemented by 45% barley and 45% oats (isonitrogenously and iso-energetically), respectively. Daily feed intake, FCR-, and weight gain were measured. Abdominal fat, liver, and gizzard weights were determined and dry matter, protein, fat content and fatty acid composition of femoro-tibial muscles and liver fat were measured on the 35 th , 42 nd and 49 th days of age. Feeding 45% barley caused a decrease of growth rate (p≤0.05) during the first 4 weeks, which was followed by a rapid, compensatory growth from the 6 th week of age (p≤0.05). Both barley and oat supplementation increased protein (p≤0.05), while decreasing fat (p≤0.05) and dry matter (p≤0.05) content of the liver. Feeding of 45% oats in the diet decreased the monounsaturated fatty acid (p≤0,05) and increased the n-6 (p≤0,05), n-3 (p≤0,05) and total polyunsaturated (p≤0,05) fatty acid content of the intramuscular fat owing to the high proportion of soluble non-starch polysaccharides (NSP) in the diet. This might be explained by the more pronounced decrease in digestibility of saturated than unsaturated fatty acids in birds fed a soluble NSP-enriched diet. This result might be caused by the "cage effect" of soluble NSP trapping the bile salts which are more important for the absorption of saturated than polyunsaturated fatty acids.

Thermobifida fusca TM51, a thermophilic actinomycete isolated from composted horse manure, was found to produce a number of lignocellulose-degrading hydrolases, including endoglucanases, exoglucanases, endoxylanases, β-xylosidases, endomannanases, and β-mannosidases, when grown on cellulose or hemicellulose as carbon sources. β-Mannosidases (EC 3.2.1.25), although contributing to the hydrolysis of hemicellulose fractions, such as galacto-mannans, constitute a lesser-known group of the lytic enzyme systems due to their low representation in the proteins secreted by hemicellulolytic microorganisms. An expression library of T. fusca , prepared in Streptomyces lividans TK24, was screened for β-mannosidase activity to clone genes coding for mannosidases. One positive clone was identified, and a β-mannosidase-encoding gene ( manB ) was isolated. Sequence analysis of the deduced amino acid sequence of the putative ManB protein revealed substantial similarity to known mannosidases in family 2 of the glycosyl hydrolase enzymes. The calculated molecular mass of the predicted protein was 94 kDa, with an estimated pI of 4.87. S. lividans was used as heterologous expression host for the putative β-mannosidase gene of T. fusca . The purified gene product obtained from the culture filtrate of S. lividans was then subjected to more-detailed biochemical analysis. Temperature and pH optima of the recombinant enzyme were 53°C and 7.17, respectively. Substrate specificity tests revealed that the enzyme exerts only β- d -mannosidase activity. Its kinetic parameters, determined on para -nitrophenyl β- d -mannopyranoside ( p NP-βM) substrate were as follows: K m = 180 μM and V max = 5.96 μmol min −1 mg −1 ; the inhibition constant for mannose was K i = 5.5 mM. Glucono-lacton had no effect on the enzyme activity. A moderate trans -glycosidase activity was also observed when the enzyme was incubated in the presence of p NP-αM and p NP-βM; under these conditions mannosyl groups were transferred by the enzyme from p NP-βM to p NP-αM resulting in the synthesis of small amounts (1 to 2%) of disaccharides.

The catalytic amino acid residues of the extracellular β- d -xylosidase (β- d -xyloside xylohydrolase, EC 3.2.1.37) from Aspergillus carbonarius was investigated by the pH dependence of reaction kinetic parameters and chemical modifications of the enzyme. The pH dependence curves gave apparent p K values of 2.7 and 6.4 for the free enzyme, while p K value of 4.0 was obtained for the enzyme–substrate complex using p -nitrophenyl β- d -xyloside as a substrate. These results suggested that a carboxylate group and a protonated group—presumably a histidine residue—took part in the binding of the substrate but only a carboxylate group was essential in the substrate cleavage. Carbodiimide- and Woodward's reagent K-mediated chemical modifications of the enzyme also supported that a carboxylate residue, located in the active center, was fundamental in the catalysis. The pH dependence of inactivation revealed the involvement of a group with p K value of 4.4, proving that a carboxylate residue relevant for hydrolysis was modified. During modification V max decreased to 10% of that of the unmodified enzyme and K m remained unchanged, supporting that the modified carboxylate group participated in the cleavage and not in the binding of the substrate. We synthesized and tested a new, potential affinity label, N -bromoacetyl-β- d -xylopyranosylamine for β- d -xylosidase. The A. carbonarius β- d -xylosidase was irreversible inactivated by N -bromoacetyl-β- d -xylopyranosylamine. The competitive inhibitor β- d -xylopyranosyl azide protected the enzyme from inactivation proving that the inactivation took place in the active center. Kinetic analysis indicated that one molecule of reagent was necessary for inactivation of one molecule of the enzyme.

Fragment-based drug discovery has emerged as an alternative to conventional lead identification and optimization strategies generally supported by biophysical detection techniques. Membrane targets like G protein-coupled receptors (GPCRs), however, offer challenges in lack of generic immobilization or stabilization methods for the dynamic, membrane-bound supramolecular complexes. Also modeling of different functional states of GPCRs proved to be a challenging task. Here we report a functional cell-based high concentration screening campaign for the identification of adrenergic α2C receptor agonists compared with the virtual screening of the same ligand set against an active-like homology model of the α2C receptor. The conventional calcium mobilization-based assay identified active fragments with a similar incidence to several other reported fragment screens on GPCRs. 16 out of 3071 screened fragments turned out as specific ligands of α2C, two of which were identified by virtual screening as well and several of the hits possessed surprisingly high affinity and ligand efficiency. Our results indicate that in vitro biological assays can be utilized in the fragment hit identification process for GPCR targets.

β-D-Glucosidase enzymes (β-D-glucoside glucohydrolase, EC 3.2.1.21) from different Aspergillus strains (Aspergillus phoenicis, A. niger and A. carbonarius) were examined with respect to the enzyme production of the different strains using different carbon sources and to the effect of the pH and temperature on the enzyme activity and stability. An efficient and rapid purification procedure was used for purifying the enzymes. Kinetic experiments were carried out using p-nitrophenyl β-D-glucopyranoside (pNPG) and cellobiose as substrates. Two different fermentation methods were employed in which the carbon source was glucose or wheat bran. Aspergillus carbonarius proved to be the less effective strain in β-glucosidase production. Aspergillus phoenicis produced the highest amount of β-glucosidase on glucose as carbon source however on wheat bran A. niger was the best enzyme producer. Each Aspergillus strain produced one single acidic β-glucosidase with pI values in the range of pH 3.52–4.2. There was no significant difference considering the effect of the pH and temperature on the activity and stability among the enzymes from different origins. The enzymes examined have only β-glucosidase activity. The kinetic parameters showed that all enzymes hydrolysed pNPG with higher efficiency than cellobiose. This shows that hydrophobic interaction plays an important role in substrate binding. The kinetic parameters demonstrated that there was no significant difference among the enzymes from different origins in hydrolysing pNPG and cellobiose as the substrates.

A combination of 3D QSAR molecular descriptors and artificial neural networks have been used to predict fluorophilicities, the natural logarithm of the perfluoro(methylcyclohexane)/toluene partition coefficients, for a wide range of partially fluorinated organic compounds. The average error of the predictions was less than twice the 0.2 experimental error. Multiple linear regression proved to be much less efficient. To better characterise the fluorous partition phenomenon, specific fluorophilicity was defined as the product of fluorophilicity and of the ratio of the van der Waals volumes of the expelled fluorous solvent and the entering solute molecules. This dimensionless term correlates well in a compound family with the calculated Hildebrand parameters of the fluorous molecules. The trifluoromethyl group was found highly effective for increasing the fluorous phase affinities of model compounds when used in combination with longer perfluoroalkyl groups.

alpha-Mannosidase and beta-galactosidase were released from boar sperm into the medium by treatment with calcium ionophore A23187 or by 0.2% Brij-35/2% acetic acid. About half as much alpha-mannosidase activity as that in the acid extract was recovered by digestion with phosphatidylinositol-specific phospholipase C (PI-PLC), whereas the liberation rate of beta-galactosidase treated with PI-PLC was low. These results suggest that some alpha-mannosidase is anchored in the plasma membrane of the acrosomal region by attachment to the lipid phosphatidylinositol and that beta-galactosidase is localized mainly in the acrosome or integrated in the plasma membrane by a spanning stretch of hydrophobic peptides. beta-Galactosidase, which is present as an oligomers in the acid extract of sperm, dissociated into monomers under weakly alkaline conditions; under acidic conditions, the monomers associated again. No pH-sensitive association-dissociation of alpha-mannosidase was observed.

C-(2-Deoxy- d -lyxo-hex-1-enopyranosyl)formamide was prepared from acetylated C-(β- d -galactopyranosyl)formamide by a radical-mediated bromination—zinc/N-methylimidazole-induced reductive elimination—Zemplen deacetylation reaction sequence. The preparation of acetylated 5-(2-deoxy- d -lyxo-hex-1-enopyranosyl)tetrazole was improved. Methyl C-(2-deoxy- d -lyxo-hex-1-enopyranosyl)formimidate was transformed by benzylamine into N-benzyl-C-(2-deoxy- d -lyxo-hex-1-enopyranosyl)formamidine and, after hydrolysis to methyl C-(2-deoxy- d -lyxo-hex-1-enopyranosyl)formate, into N-benzyl-C-(2-deoxy- d -lyxo-hex-1-enopyranosyl)formamide. A series of C-(β- d -galactopyranosyl) and C-(2-deoxy- d -lyxo-hex-1-enopyranosyl) derivatives was comparatively investigated for E. coli β- d -galactosidase inhibitory activity. N-Benzyl-C-(2-deoxy- d -lyxo-hex-1-enopyranosyl)formamidine was the best inhibitor and had Ki = 6 μM (on the basis of its free base concentration, Ki = 8.3 nM was obtained). Basicity and hydrophobicity of the aglycon proved to be more important factors for the inhibition than the conformation of the sugar ring.

The broad-specificity cyanogenic β-D-glucosidase (β-D-glucoside glucohydrolase, EC 3.2.1.21) (linamarase) from Manihot esculenta Crantz (cassava) was irreversibly inactivated by N -bromoacetyl-β-D-glucopyranosylamine according to pseudo-first-order kinetics with a second-order efficiency constant ( k i / K i = 0.1 min −1 M −1 ) identical for p -nitrophenyl-β-D-glucopyranosidase, p -nitrophenyl-β-D-galactopyranosidase, and linamarase activities of the enzyme. The competitive inhibitor p -nitrothiophenyl-β-D-glucopyranoside protected the enzyme from inactivation. pH dependence of the pseudo-first-order rate constant of inactivation revealed the involvement of an amino acid side chain in the inactivation process with pK a 7.0, which is very similar to that of the acid catalyst group of the enzyme ( pK E 2 = 7.2). The involved amino acid, which has to be ionized for the inactivation, was identified as Glu-198 using 14 C-labeled inactivator to label the enzyme, cleaving the labeled protein into peptides and then purifying and sequencing the labeled peptide. This residue is highly conserved in the homologous family A β-glucosidases and family A 1 -A 5 cellulases and lies in a consensus Asn-Glu-Pro motif occurring in all of these enzymes.

The broad-specificity cyanogenic β-D-glucosidase (β-D-glucoside glucohydrolase, EC 3.2.1.21) (linamarase) from Manihot esculenta Crantz (cassava) was kinetically characterized in mixed substrate systems and with the transition-state analogue glucono(1-5)lactone and a series of 1-thio substrate analogues. The results indicate a common catalytic and a common sugar binding site in the enzyme for all of the investigated substrates. Kinetic parameters of the hydrolysis of linamarin and p -nitrophenyl β-D-glucopyranoside were determined over the pH range 3.5-9.0. The pH-dependence curves gave apparent p K values of 4.5 (4.6) and 7.1 (7.3) for the free enzyme, while values of 4.1 (3.7) and 9.3 were obtained for the enzyme-substrate complexes, using either linamarin or p -nitrophenyl β-D-glucopyranoside as the substrate. Kinetic analysis of the modification indicated that one molecule of water-soluble carbodiimide or Woodward′s reagent K is required to bind to the enzyme for inactivation. The enzyme was protected against inactivation by the competitive inhibitors p -nitrothhiophenyl β-D-glucopyranoside, β-D-glucopyranosylamine, and glucono(1-5)lactone. Spectrophotometric analysis at 340 nm showed that from the three carboxylate groups modified by Woodward′s reagent K essentially one was protected by p -nitrothiophenyl β-D-glucopyranoside. During modification V max decreased to 30% of that of the unmodified enzyme and K m remained unchanged. The pH dependence of inactivation showed the involvement of a group with a p K value of 4.6, indicating the modification of a carboxyl residue essential for activity. Treatment of the enzyme with the histidine-group-specific reagent diethylpyrocarbonate resulted in 80% loss of enzyme activity, in biphasic kinetics. A treatment with 0.5 M hydroxylamine at pH 7.0 regenerated 92% of the original enzyme activity. The presence of the competitive inhibitor β-D-glucopyranosylamine protected the enzyme against inactivation, preventing the modification of one histidine residue. Statistical analysis of the residual fractional activity against the number of modified residues indicated that the modification of one histidine is responsible for 40-50% of the inactivation. The pH dependence of the inactivation gave a p K value of 7.0 for the histidine group upon which the activity depends. During modification, V max decreased to 30% and K m decreased to 50% of the original values.

On treatment with N-bromosuccinimide under irradiation in refluxing carbon tetrachloride, 2,3,4,6-tetra-O-acetyl-1-S-(Z)-benzhydroximoyl-1-thio-D-glucopyranose 1 and various analogs yielded new spiro anomericoxathiazole derivatives in ≈60% total yield. After deacetylation, the tested major 1(S) epimers were found good competitive inhibitors of emulsin β-D-glucosidase whereas a 1 (R) counterpart had no effect on the enzyme.

The N-acetyl-beta-D-hexosaminidase B of germinating Lupinus luteus L. seeds (McFarlane et al. (1984) Phytochemistry 23, 2431-2433) was partially purified with a six-step purification procedure following extraction. This enzyme consists of one protein chain (Mr 69,000, as determined by SDS-PAGE and 62,500, as obtained by gel filtration on Bio-Gel P-60 Gel) and has a neutral isoelectric point (pI = 7.05, as determined by chromatofocusing). Moreover, it was found to be very sensitive to low ionic strength, especially in the presence of different gels based on Sephadex. Considering the substrate specificity, the enzyme splits both p-nitrophenyl-2-acetamido-2-deoxy-beta-D-glucosaminide and -galactosaminide substrates, but lacks N,N'-diacetylchitobiase activity. A new mixed-substrate procedure was developed and is presented here to demonstrate that a common active site is responsible for the splitting of both synthetic substrates.

The N- acetyl -β- d - hexosaminidase B of germinating yellow lupin seeds catalyzed the hydrolysis of both N- acetyl -β- d - glucosaminide and -galactosaminide substrates. The investigation of the pH dependence of the kinetic parameters ( V max and V max / K m ) demonstrated that two common ionizable groups (probably two carboxyl groups) play an essential role in the catalysis. That is, the enzyme has a lysozyme-like splitting mechanism, and the possibility of an anchimeric assistance provided by the acetamido group seems to be negligible. The presence of a deprotonated carboxyl group near the glycosidic linkage was also supported by inhibition with 1-thio substrate analogues. On the other hand, some 2-acetamido-2-deoxyaldono(1 a 4)lactones proved to be effective inhibitors of the hexosaminidase with the exception of the d -arabinose derivative, which can be explained by high stereospecificity in the binding.

By reaction with arylhydroximoyl chlorides, 2,3,4,6-tetra- O -acetyl-1-thio-β- d -galactopyranose was converted to the corresponding β- d -galactopyranosyl-thiohydroximates, which gave predominantly (1 S )- d -galactopyranosylidene-spiro-oxathiazoles on illumination in the presence of NBS. Conventional O -deacetylation of both thiohydroximates and oxathiazoles gave weak inhibitors of E. coli d -galactosidase ( K i 1.1–11.1 mM).

3-Oxagranatane derivatives have been synthesized from dialdehydes obtained by the periodate oxidation of d -glucopyranosides. Reduction of the carbonyl group and removal of the substituents afforded bicyclic iminosugar analogs.

The coding sequence of the mature cyanogenic beta-D-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) (linamarase) of Manihot esculenta Crantz (cassava) was cloned into the vector pGEX-2T and expressed in Escherichia coli. The bacterial chaperonin GroEL [Braig, Otwinowski, Hedge, Boisvert, Joachimiak, Horwich and Sigler (1994) Nature (London) 371, 578-586] was found to be tightly associated with the fusion protein and co-purified with it. In the presence of excess MgATP, release and folding of the fusion beta-glucosidase were demonstrated by a fast increase in both linamarase and p-nitrophenyl-beta-D-glucopyranosidase activity at a low protein concentration. A slow endogenous folding process was also detected by activity measurements. Michaelis constants (Km) and the ratio between the maximal velocities and efficiency constants (Vmax., Vmax./Km) for the hydrolysis of the natural substrate, linamarin, and p-nitrophenyl beta-D-glucopyranoside (PNP-Glc) by the recombinant protein were found to be almost identical with those of the native glycosylated plant enzyme [Keresztessy, Kiss and Hughes (1994) Arch. Biochem. Biophys. 314, 142-152]. Molecular dissociation constants for the free enzyme (pK(E)1, pK(E)2) obtained with linamarin and PNP-Glc, and the enzyme substrate complexes (pK(ES)1, pK(ES)2) were also in accordance with that of the original protein. The reactive substrate analogue N-bromoacetyl beta-D-glucosylamine inactivated the fusion enzyme according to pseudo-first-order kinetics with first-order rate constant (k1=0.007 min-1) and apparent inhibition constants (k1=20 mM) comparable with those of the plant protein [Keresztessy, Kiss and Hughes (1994) Arch. Biochem. Biophys. 315, 323-330]. In comparison with the native glycosylated plant protein, the recombinant protein was, however, found to be extremely sensitive to proteolysis and misfolding.

Urinary N-acetyl-beta-D-glucosaminidase activity was assayed in fullterm and preterm polycythemic neonates, in preterm infants with hypoxia, and in healthy newborns. There were no significant differences between fullterm and preterm babies or between appropriate for gestational age and small for gestational age neonates in the normal group. N-acetyl-beta-D-glucosaminidase excretion on the first day of life was significantly raised in polycythemic newborns (P less than 0.01). Fourteen days after partial plasma exchange the enzyme activity returned to normal. N-acetyl-beta-D-glucosaminidase activities in preterm babies with respiratory distress syndrome were significantly (P less than 0.01) raised on the 1st, 2nd, 4th days and fell sharply to the 14th day. N-acetyl-beta-D-glucosaminidase isoenzyme studies revealed that urine samples taken from preterm babies with respiratory distress syndrome in the first week after birth contained increased amounts of intermediate and B isoenzyme forms while there was a concomitant reduction in the amount of the A form present.

Although still there are Klebsiella strains which do not harbour plasmids and produce constitutive chromosomal β-lactamases, recently clinical isolates were found in ever increasing numbers carrying mainly TEM-, CARB- and OXA type R-factors. We selected four chromosomal cephalosporinase producing Klebsiella strains to study the p I values of the enzymes and their simultaneous separability from accompanying proteins by chromatofocusing techniques. We compared p I values of the pure and the crude preparations: K. pneumoniae Kl SC 10436: p I pure = 6.4, p I crude = 6.42; K. aerogenes Kl 1082 E: p I pure = 6.5, p I crude = 6.5; K. oxytoca 1082 E: p I pure = 6.42, p I crude = 6.4; K. oxytoca 20: p I pure = 7.62, p I crude = 7.6. Excellent agreement of the p I values among each other, but occasional differences with those obtained by analytical isoelectrofocusing are attributed to methodological diversities and to the presence of satellite enzymes, known to exist in Klebsiella .

Hydrolysis of 1,2- O -isopropylidene-3,5-di- O -methyl-α- d -glucofuranose by strong acid yielded 3,5-di- O -methyl- d -glucofuranose ( 6 ) and its 1,6-anhydride ( 10 ). The mechanism of the reaction giving 10 is discussed. On treatment with a catalytic amount of sodium methoxide, 1,2,6-tri- O -acetyl-3,5-di- O -methyl- d -glucofuranose ( 8 ) gives the 6- O -acetyl derivative, whereas complete deacetylation, and subsequent isomerization to the d -fructose derivative 16 , takes place in the presence of 0.1 m sodium methoxide. The structure of 16 was proved both chemically and spectroscopically. Reduction of 6 or 8 with a borohydride afforded 3,5-di- O -methyl- d -glucitol. 2

The role of exposed tyrosine side-chains in enzyme-catalyzed reaction by sweet almond emulsin beta-D-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) has been studied using N-acetylimidazole as the specific reagent. The changes in activity, binding affinity and kinetic parameters (Km, V) as a result of acetylation of the phenolic hydroxyl groups have been determined. The acetylation increased the Km values of both beta-glucosidase and beta-galactosidase activities, whereas V remained unchanged. Similarly, the binding affinity for immobilized phenyl beta-D-glucopyranoside decreased appreciably. After the removal of the acetyl groups the enzyme regained 96% of the original activity. It is concluded that the tyrosine moieties, located in the active centre of the enzyme, have both glucoside and galactoside binding functions.

N-Acetyl- d -(−)-penicillamine as a stable second-generation biospecific affinity ligand has previously been suggested for purification of Bacillus cereus 569/H β-lactamase I. A complex spacer arm is coupled with the matrix by using epichlorohydrin and phloroglucinol doubly activated with divinyl sulphone in the meta position. Coupling of d -(−)-penicillamine ligand resulted in an active affigel. However, we found that two affinity ligands in close proximity prevents simultaneous binding of two penicillinase molecules, therefore one ligand is superfluous. Our results show that: (1) shortening the spacer arm by direct activation of the matrix with divinyl sulphone is satisfactory to produce the affinity material with N-acetyl- d -(−)-penicillamine; (2) incorporation of 15 μmol of N-acetyl- d -(−)-penicillamine per ml of wet Sepharose 4B satisfies the maximum binding capacity requirements of the affigel (about half of the originally incorporated amount of ligand); (3) our simplified affinity adsorbent is generally applicable for large-scale purification of penicillinases to homogeneity from various bacterial sources by the convenient batch method without prior concentration of these enzymes; (4) reacetylation for four/five times can regenerate the original binding capacity of the affigel.

Partially purified linamarase from Trifolium repens (genotype Lili acac ) plants was kinetically characterized. Kinetic evidence was found to support the assumption that this cyanogenic β- d -glucosidase has a broad substrate spectrum. p -Nitrophenyl-β- d -xylopyranoside and p -nitrophenyl-α- l -arabinopyranoside substrates bound almost as tightly to the active center of the enzyme as the glucono(1 → 5)lactone transition-state analog inhibitor. Substrate specificity investigation also indicated that positions C-4 and C-6 on the pyranoside ring play an essential role in both substrate orientation and splitting. Recently very similar kinetic characteristics were reported on some mammalian cytosolic β- d -glucosidases and a possible physiological interpretation of this coincidence is discussed. Inhibition studies with glucono(1 → 5)lactone revealed that the carbohydrate moiety of each substrate attached to the same binding site in the active center. Inhibition experiments with 1-thio substrate analogs demonstrated that the aglycon and the angular arrangement around the glycosidic linkage were the major determinants in the observed substrate specificity.

1,2:5,6-Di- O -isopropylidene- d -glucitol was converted via its 1,4-dimethanesulfonate into the 1-azido-4-methanesulfonate which, after deprotection and treatment with barium hydroxide, afforded a 9:1 mixture of the corresponding 3,4- and 4,5-anhydro derivatives. Reduction of this mixture by transfer hydrogenation using ammonium formate in methanol and Pd/C as catalyst afforded 1,4-dideoxy-1,4-imino- d -glucitol ( 4 ), the structure of which was proved after acetylation by 1 H-n.m.r. spectroscopy. Compound 4 is a potent α- d -glucosidase inhibitor ( K i 7 × 10 −4 m ) and a less potent β- d -glucosidase inhibitor ( K i 1.25 × 10 −4 m ), and inhibits β- d -galactosidase non-competitively.

An isoenzyme of glucosidase- isolated from sweet almond emulsin - and composed of a β-D-glucosidase, a β-D-galactosidase and a β-D-fucosidase, has been shown to possess β-D-xylosidase activity, as well. On the basis of the following results it has been concluded that the β-D-glucosidase and β-D-galactosidase activities reside in one catalytic site, but there are two kinetically distinst binding sites in the active center: 1./D-Glucono-1,5-lactone is shown to excert competitive inhibition on the hydrolysis of β-D-glucopyranoside and non-competitive inhibition on the hydrolysis of β-D-galactopyranoside. 2./ D-galactono-1,5-lactone competitively inhibits the hydrolysis of β-D-galactopyranoside, but possesses non-competitive inhibition on the hydrolysis of β-D-glucopyranoside. 3./ When the enzyme is incubated with two p-nitrophenyl glycoside substrates at or above their respective Km values, the rate of p-nitrophenol formation is not additive but rather it is equal to the value calculated from the individual Km values and relative maximum rates.

In carbon-depleted cultures of Penicillium chrysogenum, age-related chitinases were shown to play a crucial role in both autolysis and fragmentation as indicated by in vivo enzyme inhibition experiments using allosamidin. This pseudotrisaccharide even hindered significantly the outgrowth of new hyphal tips from the surviving yeastlike fragments after glucose supplementation. The antifungal effect of allosamidin on autolyzing P. chrysogenum mycelia was fungistatic rather than fungicidal. In growing hyphae, membrane-bound microsomal chitinase zymogen(s) were detected, which may be indicative of some compartmentalization of these hydrolases. Later, during autolysis, no zymogenic chitinase was detected in any enzyme fraction studied, including microsomes. These observations may explain the different sensitivity of growing and autolyzing mycelia to allosamidin. Chitinases taking part in the age-related fragmentation of hyphae and the outgrowth of surviving hyphal fragments seem to be potent targets for future antifungal drug research.