Kinetic dissection of pre-crRNA binding and processing by CRISPR-Cas12a.

CRISPR-Cas12a binds and processes a single pre-crRNA during maturation, providing a simple tool for genome editing applications. Here, we constructed a kinetic and thermodynamic framework for pre-crRNA processing by Cas12a in vitro, and we measured the contributions of distinct regions of the pre-crRNA to this reaction. We find that the pre-crRNA binds rapidly and extraordinarily tightly to Cas12a (Kd = 0.6 pM), such that pre-crRNA binding is fully rate limiting for processing and therefore determines the specificity of Cas12a for different pre-crRNAs. The guide sequence contributes 10-fold to the binding affinity of the pre-crRNA, while deletion of an upstream sequence has no significant effect. After processing, the mature crRNA remains very tightly bound to Cas12a with a comparable affinity. Strikingly, the affinity contribution of the guide region increases to 600-fold after processing, suggesting that additional contacts are formed and may pre-order the crRNA for efficient DNA target recognition. Using a direct competition assay, we find that pre-crRNA binding specificity is robust to changes in the guide sequence, addition of a 3' extension, and secondary structure within the guide region. However, stable secondary structure in the guide region can strongly inhibit DNA targeting, indicating that care should be taken in crRNA design. Together our results provide a quantitative framework for pre-crRNA binding and processing by Cas12a and suggest strategies for optimizing crRNA design in genome editing applications.

Kinetic dissection of pre-crRNA binding and processing by CRISPR-Cas12a.

CRISPR-Cas12a binds and processes a single pre-crRNA during maturation, providing a simple tool for genome editing applications. Here, we constructed a kinetic and thermodynamic framework for pre-crRNA processing by Cas12a in vitro, and we measured the contributions of distinct regions of the pre-crRNA to this reaction. We find that the pre-crRNA binds rapidly and extraordinarily tightly to Cas12a (Kd = 0.6 pM), such that pre-crRNA binding is fully rate limiting for processing and therefore determines the specificity of Cas12a for different pre-crRNAs. The guide sequence contributes 10-fold to the affinities of both the precursor and mature forms of the crRNA, while deletion of an upstream sequence had no significant effect on affinity of the pre-crRNA. After processing, the mature crRNA remains very tightly bound to Cas12a, with a half-life of ~1 day and a Kd value of 60 pM. Addition of a 5'-phosphoryl group, which is normally lost during the processing reaction as the scissile phosphate, tightens binding of the mature crRNA by ~10-fold by accelerating binding and slowing dissociation. Using a direct competition assay, we found that pre-crRNA binding specificity is robust to other changes in RNA sequence, including tested changes in the guide sequence, addition of a 3' extension, and secondary structure within the guide region. Together our results provide a quantitative framework for pre-crRNA binding and processing by Cas12a and suggest strategies for optimizing crRNA design in some genome editing applications.

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

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 ß-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 ß-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.

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

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.

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.

Purification and characterization of paraoxonase 1 (PON1) from Swiss Black, Holstein, and Montofon bovines.

Paraoxonase 1 (PON1: EC 3.1.8.1) is a calcium-dependent enzyme associated with high-density lipoproteins (HDLs) and has a protective effect against oxidation of low-density lipoproteins (LDLs) in mammals. PON1 is the best-studied member of a family of enzymes called serum paraoxonases, or PONs, identified in mammals and other vertebrates as well as in invertebrates. PONs exhibit a range of important activities, including drug metabolism and detoxification of organophosphates such as nerve agents. This study reports, for the first time, purification and biochemical characterization of serum PON1 from different bovine breeds namely Swiss Black, Holstein, and Montofon. Bovine serum PON1s were purified using ammonium sulfate precipitation followed by Sepharose-4B-L-tyrosine-1-naphthylamine hydrophobic interaction chromatography. SDS-polyacrylamide gel electrophoresis of the purified enzymes indicates a single band with an apparent MW of 43 kDa. The purified enzymes had a specific activity of 10.78, 27.00, and 22.38 U/mg for Swiss Black, Holstein, and Montofon bovines, respectively. The overall purification rates of our method were 262.47-, 2,476.90-, and 538.06-fold for Swiss Black, Holstein, and Montofon bovines, respectively. Furthermore, using phenyl acetate as a substrate, we determined the K M and V max values of the purified enzymes, as 0.80 mM, 1428.5 U/ml for Swiss Black; 0.40 mM, 714.3 U/ml for Holstein; and 0.50 mM, 1,111.1 U/ml for Montofon bovine. The present study has revealed that there is no substantial difference in PON1 activities among the studied bovine breeds.

Purification and characterization of ß-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-ß-d-fucopyranoside (p-NPF), para/ortho-nitrophenyl ß-d-galactopyranosides (p-/o-NPGal) and p-nitrophenyl 1-thio-ß-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 vitro inhibition effects of some coumarin derivatives on human erythrocytes glucose-6-phosphate dehydrogenase activities.

Inhibitory effects of some synthesized dihydroxycoumarin compounds on purified G6PD were investigated. For this purpose, initially human erythrocyte G6PD was purified 7069-fold in a yield of 33.6% by using ammonium sulfate precipitation and affinity chromatography which includes 2',5'-ADP Sepharose 4B. The purified enzyme showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Enzyme activity was determined spectrophotometrically according to Beutler method at 340 nm. 6,7-Dihydroxy-3-(2-methylphenyl)-2H-chromen-2-one (OPC), 6,7-dihydroxy-3-(3-methylphenyl)-2H-chromen-2-one (MPC) and 6,7-dihydroxy-3-(4-methylphenyl)-2H-chromen-2-one (PPC) were used as dihydroxycoumarin compounds. This study has demonstrated that G6PD activity is very highly sensitive to study coumarin derivatives.

In vitro inhibition effect of some dihydroxy coumarin compounds on purified human serum paraoxonase 1 (PON1).

Human serum paraoxonase 1 (PON1; EC 3.1.8.1) is a high-density lipoprotein associated, calcium-dependent enzyme that hydrolyses aromatic esters, organophosphates and lactones and can protect the low-density lipoprotein against oxidation. In this study, in vitro inhibition effect of some dihydroxy coumarin compounds namely 6,7-dihydroxy-3-(2-methylphenyl)-2H-chromen-2-one (A), 6,7-dihydroxy-3-(3-methylphenyl)-2H-chromen-2-one (B) and 6,7-dihydroxy-3-(4-methylphenyl)-2H-chromen-2-one (C) on purified PON1 were investigated by using paraoxon as a substrate. PON1 was purified using two-step procedures, namely ammonium sulphate precipitation and Sepharose-4B-L-tyrosine-1-naphthylamine hydrophobic interaction chromatography. The purified enzyme had a specific activity of 11.76 U/mg. The dihydroxy coumarin derivatives of A and B compounds inhibited PON1 enzyme activity in a noncompetitive inhibition manner with K(i) of 0.0080±0.256 and 0.0003±0.018 mM values, respectively. C compound exerted an uncompetitive inhibition of PON1 enzyme activity with K(i) of 0.0010±0.173 mM. Moreover, dihydroxy coumarin derivatives of A, B and C compounds were effective inhibitors on purified human serum PON1 activity with IC(50) of 0.012, 0.022 and 0.003 mM values, respectively. IC(50) value of unsubstituted 6,7 dihydroxy coumarin was found as 0.178 mM. The present study has demonstrated that PON1 activity is very highly sensitive to studied coumarin derivatives.

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.

The Azoarcus group I intron ribozyme misfolds and is accelerated for refolding by ATP-dependent RNA chaperone proteins.

Structured RNAs traverse complex energy landscapes that include valleys representing misfolded intermediates. In Neurospora crassa and Saccharomyces cerevisiae, efficient splicing of mitochondrial group I and II introns requires the DEAD box proteins CYT-19 and Mss116p, respectively, which promote folding transitions and function as general RNA chaperones. To test the generality of RNA misfolding and the activities of DEAD box proteins in vitro, here we measure native folding of a small group I intron ribozyme from the bacterium Azoarcus by monitoring its catalytic activity. To develop this assay, we first measure cleavage of an oligonucleotide substrate by the prefolded ribozyme. Substrate cleavage is rate-limited by binding and is readily reversible, with an internal equilibrium near unity, such that the amount of product observed is less than the amount of native ribozyme. We use this assay to show that approximately half of the ribozyme folds readily to the native state, whereas the other half forms an intermediate that transitions slowly to the native state. This folding transition is accelerated by urea and increased temperature and slowed by increased Mg(2+) concentration, suggesting that the intermediate is misfolded and must undergo transient unfolding during refolding to the native state. CYT-19 and Mss116p accelerate refolding in an ATP-dependent manner, presumably by disrupting structure in the intermediate. These results highlight the tendency of RNAs to misfold, underscore the roles of CYT-19 and Mss116p as general RNA chaperones, and identify a refolding transition for further dissection of the roles of DEAD box proteins in RNA folding.

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.) ß-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 ß-glucosidase was effectively active on p-/o-nitrophenyl-ß-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 ß-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.

Paraoxonase 1-bound magnetic nanoparticles: preparation and characterizations.

This is most probably the first time that covalently binding of Human serum paraoxonase 1 (PON1) to superparamagnetic magnetite nanoparticles via carbodiimide activation was investigated and presented in this study. PON1 was purified from human serum using ammonium sulfate precipitation and hydrophobic interaction chromatography (Sepharose 4B, L-tyrosine, 1-Napthylamine) and magnetic iron oxide nanoparticles were prepared by co-precipitation Fe(+2) and Fe(+3) ions in an ammonia solution at room temperature. X-ray diffraction (XRD) and the magnetic measurements showed that the nanoparticles are magnetite and superparamagnetic, respectively. Direct measurements by dynamic light scattering revealed that the hydrodynamic size was 16.76 nm with polydispersity index (PDI: 0.234). The analysis of Fourier transform infrared spectroscopy revealed that the PON1 was properly bound to magnetic nanoparticles replacing the characteristic band of -NH2 at 1629 cm(-1) with the protein characteristic band at 1744 cm(-1) and 1712 cm(-1). Magnetic measurements determined that PON1-bound nanoparticles have also favorable superparamagnetic properties with zero coercivity and remanence though a slightly smaller saturation magnetization due to the decrease of magnetic moment in the volume friction. The kinetic measurements indicated the PON1-bound nanoparticles retained 70% of its original activity and exhibited an improved stability than did the free enzyme. The PON1 enzyme is seen to be quite convenient to bind superparamagnetic nanoparticles as support material.

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.

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 (IC(50)) value. Liver hPON1 activity was determined using paraoxon as a substrate. The IC(50) values of the drugs were calculated from graphs of hydratase activity (%) by plotting concentration of the drugs that showed an inhibition effect.

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.

In vivo effects of oral contraceptives on paraoxonase, catalase and carbonic anhydrase enzyme activities on mouse.

Many effects that oestrogens and progestrogens used in oral contraceptive (OC) have on enzyme physiology are of importance on homeostasis. This study was carried out in order to determine the in vivo effect of three oral contraceptives containing ethinyl estradiol in combination with desogestrel and levonorgestrel on the paraoxonase (PON), catalase (CAT) and carbonic anhydrase (CA) activities in mice, which are model organisms for humans. Serum and liver paraoxonase activities were determined spectrophotometrically by using paraoxan as a substrate according to the methods of Gan et al. and Gil et al., respectively. Catalase and carbonic anhydrase activities were determined from erythrocytes used Aebi and Maren methods, respectively. For these studies, a group of ten mice (25+/-2 g) was selected for oral administration for 21 d of each drug (0.15 mg desogestrel+0.03 mg ethinylestradiol (A); 0.15 mg levanogestrel+0.03 mg ethinylestradiol (B) and 0.15 mg desogestrel+0.02 mg ethinylestradiol (C)). A group of ten mice was included in the study for a control group, which were not subject to drug administration. For each drug, a mean of the serum and liver paraoxonase activity and erythrocytes catalase and carbonic anhydrase activities were determined and compared to the control groups. While mouse liver PON activity showed a statistically significant decrease for all three drugs, serum PON activity increased. Erythrocytes catalase activity was significantly decreased by all contraceptives used. On the other hand, these contraceptives did not change the erythrocytes carbonic anhydrase activity.

Effects of some antibiotics on paraoxonase from human serum in vitro and from mouse serum and liver in vivo.

Paraoxonase (PON1, EC 3.1.8.1) is an esterase protein which plays multifunctional role in metabolism. Therefore, in this study the effects of commonly used antibiotics, namely sodium ampicillin, ciprofloxacin, rifamycin SV and clindamycin phosphate, on human PON1 were investigated in vitro and in vivo. Human serum paraoxonase (PON1) was separately purified by ammonium sulfate precipitation and hydrophobic interaction chromatography. The in vitro effects of the antibiotics in purifying human serum paraoxonase was determined using paraoxon as a substrate, and the IC50 values of these drugs exhibiting inhibition effects were found from graphs of hydratase activity % by plotting the concentration of the drugs. It was determined that sodium ampicillin, ciprofloxacin, and clindamycin phosphate were effective inhibitors on human serum PON1, and the inhibition kinetics of interaction of sodium ampicillin, ciprofloxacin, and clindamycin phosphate with the human serum PON1 was also determined, with the Ki of sodium ampicillin, ciprofloxacin, and clindamycin phosphate being 0.00714+/-0.00068, 6.5x10(-6)+/-4.59x10(-7), 0.0291+/-0.0077 mM, respectively. The in vivo effects of the antibiotics on paraoxonase enzyme activity in mouse serum and liver PON1 were also investigated. Mouse liver PON1 activity showed a statistically significant change at 2, 4 and 6 h of drug application in vivo. Sodium ampicillin and clindamycin phosphate exhibited about 80% mouse liver PON1 at 2 or 4 h (p: 0.034, 0.003 and 0.021, respectively). In addition, ciprofloxacin and rifamycin SV only showed inhibition at 4 h incubation. Sodium ampicillin (17.12 mg/kg) lead to a significant decrease in mouse serum PON1 after 4 h drug administration. Ciprofloxacin (3.2 mg/kg), rifamycin SV (3.56 mg/kg) and clindamycin phosphate (2.143 mg/kg) did not exhibit any inhibition effect for the mouse serum PON1, in vivo.

Novel purification strategy for human PON1 and inhibition of the activity by cephalosporin and aminoglikozide derived antibiotics.

Human serum paraoxonase (PON1, EC 3.1.8.1.) is a high-density lipid (HDL)-associated, calcium-dependent enzyme; its physiological substrates are not known. In this study, a new purification strategy for human PON1 enzyme was developed using two-step procedures, namely ammonium sulfate precipitation and sepharose-4B-l-tyrosine-1-napthylamine hydrophobic interaction chromatography. SDS-polyacrylamide gel electrophoresis of the enzyme indicates a single band with an apparent MW of 43 kDa. Overall purification rate of our method was found 227-fold. The V(max) and K(m) of the purified enzyme were determined 227.27 EU and 4.16 mM, respectively. The in vitro effects of commonly used antibiotics, namely gentamycin sulfate and cefazolin sodium was also investigated on the purified human serum PON1 enzyme and human liver PON1 enzyme from human hepatoma cell (HepG2). Gentamycin sulfate and cefazolin sodium caused a dose- and time-dependent decrease on PON1 activity in HepG2 cells. Moreover, gentamycin sulfate and cefazolin sodium were effective inhibitors on purified human serum PON1 activity with IC(50) of 0.887 and 0.0084 values, respectively. The kinetics of interaction of gentamycin sulfate and cefazolin sodium with the purified human serum PON1 indicated a different inhibition pattern. Cefazolin sodium showed a competitive inhibition with K(i) of 0.012+/-0.00065 mM. However, Gentamycin sulfate was inhibited in non-competitive manner with K(i) of 0.026+/-0.015. In order to determine the inhibition statue of these drugs on a living system, the effects of same antibiotics on PON1 enzyme activity of mouse serum PON1 and liver PON1 were investigated in vivo. Gentamycin sulfate (3.2 mg/kg) and cefazolin sodium (106.25 mg/kg) leads to the significant decrease in mouse serum PON1 after 2, 4, 6 h and 2, 4 h drug administration, respectively. Cefazolin sodium did not exhibit any inhibition effect for the liver PON1, in vivo.