Selma Sinan

Purification of beta-glucosidase from olive (Olea europaea L.) fruit tissue with specifically designed hydrophobic interaction chromatography and characterization of the purified enzyme

An olive (Olea europaea L.) β-glucosidase was purified to apparent homogeneity by salting out with ammonium sulfate and using specifically designed sepharose-4B-L-tyrosine-1-napthylamine hydrophobic interaction chromatography. The purification was 155 fold with an overall enzyme yield of 54%. The molecular mass of the protein was estimated as ca. 65 kDa. The purified β-glucosidase was effectively active on p-/o-nitrophenyl-β-D-glucopyranosides (p-/o-NPG) with K(m) values of 2.22 and 14.11 mM and V(max) values of 370.4 and 48.5 U/mg, respectively. The enzyme was competitively inhibited by δ-gluconolactone and glucose against p-NPG as substrate. The K(i) and IC(50) values of δ-gluconolactone were determined as 0.016 mM and 0.23 mM while the enzyme was more tolerant to glucose inhibition with K(i) and IC(50) values of 6.4 mM and 105.5 mM, respectively, for p-NPG. The effect of various metal ions on the purified β-glucosidase was investigated. Of the ions tested, only the Fe(2+) increased the activity while Cd(2+) Pb(2+) Cu(2+), Ni(+), and Ag(+) exhibited different levels of inhibitory effects with K(i) and IC(50) values of 4.29×10(-4) and 0.38×10(-4), 1.26×10(-2) and 5.3×10(-3), 2.26×10(-4) and 6.1×10(-4), 1.04×10(-4) and 0.63×10(-4), 3.21×10(-3) and 3.34×10(-3) mM, respectively.

In vitro inhibition of cytosolic carbonic anhydrases I and II by some new dihydroxycoumarin compounds

A new series of 6, 7-dihydroxy-3-(methylphenyl) chromenones, including three new derivatives, i.e. 6,7-dihydroxy-3-(2-methylphenyl)-2H-chromen-2-one (OPC); 6,7-dihydroxy-3-(3-methylphenyl)-2H-chromen-2-one (MPC); 6,7-dihydroxy-3-(4-methylphenyl)-2H-chromen-2-one (PPC) and one previously described, namely 6,7-dihydroxy-3-phenyl-2H-chromen-2-one (DPC), were synthesized. These compounds were investigated as inhibitors of human carbonic anhydrase I (hCA-I) and human carbonic anhydrase II (hCA-II) which had been purified from human erythrocytes on an affinity gel comprised of L-tyrosine-sulfonamide-Sepharose 4B.

Mutation of Phe91 to Asn in human carbonic anhydrase I unexpectedly enhanced both catalytic activity and affinity for sulfonamide inhibitors.

Site-directed mutagenesis has been used to change one amino acid residue considered non essential (Phe91Asn) to catalysis in carbonic anhydrase (CA, EC 4.2.1.1) isozyme I (hCA I), but which is near the substrate binding pocket of the enzyme. This change led to a steady increase of 16% of the catalytic activity of the mutant hCA I over the wild type enzyme, which is a gain of 50% catalytic efficiency if one compares hCA I and hCA II as catalysts for CO(2) hydration. This effect may be due to the bigger hydrophobic pocket in the mutant enzyme compared to the wild type one, which probably leads to the reorganization of the solvent molecules present in the cavity and to a diverse proton transfer pathway in the mutant over the non mutated enzyme. To our surprise, the mutant CA I was not only a better catalyst for the physiologic reaction, but in many cases also showed higher affinity (2.6-15.9 times) for sulfonamide/sulfamate inhibitors compared to the wild type enzyme. As the residue in position 91 is highly variable among the 13 catalytically active CA isoforms, this study may shed a better understanding of catalysis/inhibition by this superfamily of enzymes.

In Vitro Inhibition Effects of some New Sulfonamide Inhibitors on Human Carbonic Anhydrase I and II

A new series of aromatic and heterocyclic sulfonamides, including six new derivatives, 2-(3-cyclohexene-1-carbamido)-1,3,4-thiadiazole-5-sulfonamide (CCTS), 4-(3-cyclohexene-1-carbamido) methyl-benzenesulfonamide (CCBS), 2-(9-octadecenoylamido)-1,3,4-thiadiazole-5-sulfonamide (ODTS), 2-(4,7,10-trioxa-tetradecanoylamido)-1,3,4-thiadiazole-5-sulfonamide (TDTS), 2-(coumarine-3-carbamido)-1,3,4-thiadiazole-5-sulfonamide (COTS) and 2-(8-methoxycoumarine-3-carbamido)-1,3,4-thiadiazole-5-sulfonamide (MCTS), has been investigated. These sulfonamides were assayed for inhibition of human carbonic anhydrase I (hCA-I) and human carbonic anhydrase II (hCA-II) which were purified by affinity chromatography.

PURIFICATION AND CHARACTERIZATION OF β‐GLUCOSIDASE FROM GREATER WAX MOTH Galleria mellonella L. (LEPIDOPTERA: PYRALIDAE)

The greater wax moth, Galleria mellonella, is one of the most ruinous pests of honeycomb in the world. Beta-glucosidases are a type of digestive enzymes that hydrolytically catalyzes the beta-glycosidic linkage of glycosides. Characterization of the beta-glucosidase in G. mellonella could be a significant stage for a better comprehending of its role and establishing a safe and effective control procedure primarily against G. mellonella and also some other insect pests. Laboratory reared final instar stage larvae were randomly selected and homogenized for beta-glucosidase activity assay and subsequent analysis. The enzyme was purified to apparent homogeneity by salting out with ammonium sulfate and using sepharose-4B-l-tyrosine-1-naphthylamine hydrophobic interaction chromatography. The purification was 58-fold with an overall enzyme yield of 29%. The molecular mass of the protein was estimated as ca. 42 kDa. The purified beta-glucosidase was effectively active on para/ortho-nitrophenyl-beta-d-glucopyranosides (p-/o-NPG) with Km values of 0.37 and 1.9 mM and Vmax values of 625 and 189 U/mg, respectively. It also exhibits different levels of activity against para-nitrophenyl-β-d-fucopyranoside (p-NPF), para/ortho-nitrophenyl β-d-galactopyranosides (p-/o-NPGal) and p-nitrophenyl 1-thio-β-d-glucopyranoside. The enzyme was competitively inhibited by beta-gluconolactone and also was very tolerant to glucose against p-NPG as substrate. The Ki and IC50 values of δ-gluconolactone were determined as 0.021 and 0.08 mM while the enzyme was more tolerant to glucose inhibition with IC50 value of 213.13 mM for p-NPG.

In vivo effects of curcumin on the paraoxonase, carbonic anhydrase, glucose-6-phosphate dehydrogenase and β-glucosidase enzyme activities in dextran sulphate sodium-induced ulcerative colitis mice

Abstract Increases in the risk of infections and malignancy due to immune suppressive therapies of inflammatory bowel diseases (IBDs) have led the researchers to focus on more nontoxic and acceptable natural products like curcumin. Here we investigate whether prophylactic and therapeutic application of the curcumin alters the enzyme activities of paraoxonase (PON), carbonic anhydrase (CA), glucose-6-phosphate dehydrogenase (G6PD) and cytosolic β-glucosidase in dextran sulphate sodium (DSS)-induced ulcerative colitis mice. Prophylactic application of curcumin resulted in higher MPO activity, less body weight loss and longer colon lengths compared to therapeutic group indicating preventive role of curcumin in IBDs. DSS-induced decrease in liver and serum PON activities were completely recovered by prophylactic administration of curcumin. DSS-induced reduction in liver cytosolic β-glucosidase activity was not affected by curcumin neither in the prophylactic group nor in the therapeutic group. Erythrocyte CA activity was significantly increased in curcumin groups, however no remarkable change in G6PD activity was observed.

Mutation of active site residues Asn67 to Ile, Gln92 to Val and Leu204 to Ser in human carbonic anhydrase II: Influences on the catalytic activity and affinity for inhibitors

Site-directed mutagenesis has been used to change three amino acid residues involved in the binding of inhibitors (Asn67Ile; Gln92Val and Leu204Ser) within the active site of human carbonic anhydrase (CA, EC 4.2.1.1) II (hCA II). Residues 67, 92 and 204 were changed from hydrophobic to hydrophilic ones, and vice versa. The Asn67Ile and Leu204Ser mutants showed similar k(cat)/K(M) values compared to the wild type (wt) enzyme, whereas the Gln92Val mutant was around 30% less active as a catalyst for CO(2) hydration to bicarbonate compared to the wt protein. Affinity for sulfonamides/sulfamates was decreased in all three mutants compared to wt hCA II. The effect was stronger for the Asn67Ile mutant (the closest residue to the zinc ion), followed by the Gln92Val mutant (residue situated in the middle of the active site) and weakest for the Leu204Ser mutant, an amino acid situated far away from the catalytic metal ion, at the entrance of the cavity. This study shows that small perturbations within the active site architecture have influences on the catalytic efficiency but dramatically change affinity for inhibitors among the CA enzymes, especially when the mutated amino acid residues are nearby the catalytic metal ion.

Differential effects of some antibiotics on paraoxonase enzyme activity on human hepatoma cells (HepG2) in vitro

Serum paraoxonase (aryldialkylphosphatase, EC 3.1.8.1., PON1) is an esterase protein synthesised by the liver and released into the serum, where it is associated with HDL lipoproteins. In this study, we have determined the in vitro effects of the following antibiotics: sodium ampicillin, ciprofloxacin, Rifamycin SV and clindamiycin phosphate, on human hepatoma (HepG2) cells (liver hPON1). All the antibiotics caused a dose-dependent and time-dependent decrease in the paraoxonase activity while Rifamycin SV was the most effective antibiotic due to its low 50% inhibition concentration (IC50) value. Liver hPON1 activity was determined using paraoxon as a substrate. The IC50 values of the drugs were calculated from graphs of hydratase activity (%) by plotting concentration of the drugs that showed an inhibition effect.

Is there a relation between genetic susceptibility with cancer? A study about paraoxanase (PON1) enzyme activity in breast cancer cases

Abstract Human serum paraoxonase 1 (PON1, EC 3.1.1.2) is a high density lipoprotein (HDL)-associated antioxidant enzyme that not only decreases oxidative stress, but it is also implicated in development of many cancers. Genetic information provides a means of identifying people who have an increased risk of cancer, thus this knowledge of cancer genetics helps to identify the ability to characterize malignancies leading to the development of new therapeutic approaches. Because of this reason, in this preliminary study we aimed to investigate the role of human serum PON1 enzyme activity and phenotypic distribution in 32 breast cancer (BC) patients (age range 28–82) and 35 cancer free (CF) control group (age range 21–67). PON1 enzyme was prepared from the serum pool of BC patients using hydrophobic interaction chromatography on L-tyrosine-9-aminophenanthrene-coupled Sepharose 4Bgel. The PON1 enzyme activity towards paraoxon substrate was quantified spectrophotometrically. The basal activity of PON1 was statistically decreased in cancer cases compared to the control group. In addition, individuals were classified according to phenotyping of human PON1 Q and R types. In the cohort of BC patients, an increase in the frequency of the PON homozygote Q (AA) genotype was observed (31% in the BC group versus 14% in the CF controls). The frequency of the PON heterozygote QR (AB) genotype was 34.5% in the patients with BC and 37% in the CF group. The same trend was observed in PON homozygote R (BB) genotype frequency (BC cases 34.5% versus controls 49%). We determined that the kinetic parameters of the purified enzyme by Lineweaver–Burk method. We obtained Km and Vmax values of 0.227 mM and 62 U/mL min for the BC enzyme, compared with 0.775 mM and 206 U/mL min for the CF control enzyme. As a conclusion, it is clear from our results that while the PON1 AA allele frequency in BC cases is much higher, that of BB allele is much lower, in comparison with the control group. The most significant finding of this study is AA allele activity which is low in BC cases was found high. We concluded that decreased AA allele PON1 activity might have a relation with BC.

Antidepressant and antipsychotic drugs differentially affect PON1 enzyme activity

Abstract Human serum paraoxonase (PON1, EC 3.1.8.1.) is a high-density lipid (HDL)-associated, calcium-dependent enzyme. In this study, the effects of Haloperidol, Fluoxetine hydrochloride, Diazepam and Acepromazine drugs used for the therapy of antidepressant and antipsychotic diseases, on paraoxonase enzyme activity was studied in in vitro inhibition studies on purified human serum PON1. PON1 enzyme was purified from human blood using two-step procedures, namely, ammonium sulfate precipitation and sepharose-4B-l-tyrosine-1-napthylamine hydrophobic interaction chromatography. The overall purification of human serum PON1 was obtained in a activity of 109.29 U/mL and this enzyme was purified 125-fold. The SDS–polyacrylamide gel electrophoresis of the enzyme indicates a single band with an apparent MW of 43 kDa. Inhibition studies indicated that haloperidol and fluoxetine hydrocloride were effective inhibitors on purified human serum PON1 activity with IC50 of 0.187 and 3.08 mM values, respectively. The kinetics of interaction of haloperidol and fluoxetine hydrocloride with the purified human serum PON1 indicated uncompetitive inhibiton pattern with Ki of 4.15 and 0.007 mM, respectively.