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1.
Arch Physiol Biochem ; : 1-10, 2024 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-38988137

RESUMO

OBJECTIVE: Angiotensin-converting enzyme (ACE, EC 3.4.15.1) is a very important factor in the regulation of blood pressure. Also, the inhibition of ACE with natural compounds has been a very important research area in the treatment of high blood pressure. ACE was purified and characterized from sheep plasma. Molecular docking studies and the inhibition effect of thiamine, riboflavin, and captopril on ACE were investigated. METHODS: Herein, ACE was purified from sheep plasma by affinity chromatography. The effect of thiamine and riboflavin on ACE was researched. Molecular docking studies were performed to understand the molecular interactions between thiamine, riboflavin, and captopril with ACE. RESULTS: The purification coefficient was found to be 8636 fold. The binding energy of thiamine, riboflavin, and captopril was found to be -6.7 kcal/mol, -8.1 kcal/mol, and -5.5 kcal/mol, respectively. Thiamine conformed to three conventional hydrogen bonds with ASP:415, HIS:513, and LYS:454. Riboflavin formed four conventional hydrogen bonds with GLN:281, GLU:376, THR:282, and TYR:520. Captopril formed two conventional hydrogen bonds with ARG:124, one conventional hydrogen bond with TYR:62 and ASN:85, and one carbon-hydrogen bond with ASN:66. Molecular docking results showed that thiamine, riboflavin, and captopril interacted with ACE through hydrogen bonding and hydrophobic interactions. Thiamine and riboflavin indicated significant inhibition effects on ACE. The IC50 values of thiamine, riboflavin, and captopril were found as 960.56 µM, 11.02 µM, and 1.60 nM, respectively. Ki values for thiamine, riboflavin, and captopril were determined as 1352.04 µM, 12.30 µM, and 1.06 nM, respectively. CONCLUSION: In this work, it was concluded that thiamine and riboflavin may have preventive and therapeutical impacts against high blood pressure with their ACE inhibitor effect. Thiamine and riboflavin showed a lower inhibitory effect with a higher IC50 than captopril. However, when the inhibitory effect of thiamine and riboflavin vitamins is compared to captopril, it is concluded that they may be natural inhibitors with fewer side effects.

2.
Nat Prod Res ; : 1-7, 2024 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-38440881

RESUMO

Hawthorn plant is used among people due to its cardiovascular, anti-inflammatory, and antihistamine properties. But no scientific study has been done about Crataegus orientalis (Mill.) M.Bieb. The presented study was planned to determine the effects of ethanol and n-hexane extracts of Crataegus orientalis leaves on human plasma ACE enzyme. In the study, the effect of plant extracts on ACE was studied by the spectrophotometric method. The chemical composition of the plant extracts was determined by HPLC-DAD analyses. In addition, molecular doking and ADME prediction studies were carried out. As a result, the obtained data showed that Crataegus orientalis could have an important place in the pharmaceutical industry and drug discovery studies, as it supports the traditional use of Crataegus orientalis as hypotensive. The results of the molecular docking studies revealed that the interactions of the selected compounds with the human ACE enzyme caused inhibition.

3.
J Biomol Struct Dyn ; : 1-9, 2024 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-38247271

RESUMO

Bioactive peptides (BPs) are a natural and important alternative to synthetic angiotensin-converting enzyme (ACE) inhibitors used in the treatment of hypertension. In this study, ACE was 3575-fold purified from human serum with the affinity chromatography process in one step. The molecular weight and purity of ACE were identified using the SDS-PAGE process and seen in two bands at around 60 kDa and 70 kDa on the gel. Vmax and KM values from the Lineweaver-Burk graphic were determined as 96.15 (µmol/min) mL-1 and 0.2 mM, respectively. The effects of Gly-Pro (GP), Arg-Gly-Asp-Ser (RGDS) and Ser-Asp-Gly-Arg-Gly (SDGRG) BPs on purified ACE were researched. Also, lisinopril was used as a reference inhibitor. GP, RGDS and SDGRG on purified ACE demonstrated an inhibitory effect. IC50 values for these peptides were found as 184.71, 107.16 and 32.54 µM, respectively. Ki values and type of inhibitory for GP, RGDS and SDGRG by the Lineweaver-Burk chart were found. The type of inhibitory for these peptides was calculated as reversible-competitive inhibitory. Ki values for GP, RGDS and SDGRG were calculated to be 260.02, 63.44 and 11.42 µM, respectively. Also, the SDGRG indicated a higher inhibition effect on ACE activity than the GP and RGDS. The IC50 value of lisinopril was designated as 0.35 nM. The inhibition type of lisinopril was designated as reversible noncompetitive inhibition from the Lineweaver-Burk chart and the Ki value was 0.15 nM. Herein, it was concluded that GP, RGDS and SDGRG have ACE inhibitor potential.Communicated by Ramaswamy H. Sarma.

4.
Pak J Pharm Sci ; 35(3): 801-805, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35791479

RESUMO

Angiotensin converting enzyme (ACE, EC 3.4.15.1) is an important enzyme responsible for regulating blood pressure. Inhibition of this enzyme is an important treatment approach in the treatment of hypertension, and natural or synthetic ACE inhibitors are often used for this purpose. In this study, the preventive effect of two important antioxidant compounds, lycopene (LYC) and thymoquinone (TQ) on ACE activity in human plasma was investigated. Human plasma was used as ACE source. ACE activity was calculated absorbance at 345 nm after incubation for 30 minutes at 35°C. TQ and LYC showed inhibitory effect on ACE activity. IC50 values for TQ and LYC were determined as 314µM and 182 µM, respectively. Type of inhibition for TQ and lycopene from plot Line weaver-Burk was designated as non-competitive inhibition. The Ki constants of TQ and LYC were determined to be 707 µM and 167 µM, respectively. It was concluded that TQ and LYC may have significant potential as ACE inhibitors.


Assuntos
Benzoquinonas , Peptidil Dipeptidase A , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Benzoquinonas/farmacologia , Humanos , Licopeno/farmacologia
5.
J Biomol Struct Dyn ; 40(20): 10086-10093, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34138692

RESUMO

Glutathione reductase (GR, EC 1.8.1.7) is a specific antioxidant enzyme that catalyzes oxidized glutathione (GSSG) to reduced glutathione (GSH). GR enzyme maintains the cellular reduced GSH level and plays a central role in cell defense against reactive oxygen species. Herein, GR was purified with affinity chromatography method in one step using 2',5'-ADP Sepharose 4B from human erythrocytes. The purification rate of glutathione reductase enzyme purified from human erythrocytes was 6224 fold and specific activity was calculated as 9.586 EU/mg protein. The molecular weight of GR was determined to be 53 kDa by SDS-PAGE. The effect of thymoquinone and lycopene compounds on the GR activity purified from human erythrocytes was researched. Both compounds showed an inhibitory effect on GR activity. IC50 values for thymoquinone and lycopene were calculated as 62.12 µM and 35.79 µM, respectively. Inhibition type and Ki values were determined from the Lineveawer-Burk graph. The type of inhibition for thymoquinone and lycopene was found to be non-competitive inhibition. Ki value was calculated as 57.71 µM for thymoquinone and 46.65 µM for lycopene. In this study, it was concluded that antioxidant compounds thymoquinone and lycopene, which have an inhibitory effect on GR activity, may have a therapeutic effect on cancer disease. Communicated by Ramaswamy H. Sarma.


Assuntos
Antioxidantes , Eritrócitos , Humanos , Glutationa Redutase , Licopeno/farmacologia , Antioxidantes/farmacologia , Eritrócitos/metabolismo
6.
Biotechnol Appl Biochem ; 69(1): 273-280, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33438805

RESUMO

Angiotensin-converting enzyme (ACE) liable for the regulation of blood pressure was purified from human plasma by affinity chromatography. Impact of water and butanol extracts of Matricaria chamomilla L. on purity ACE was examined. ACE was purified using the affinity chromatography method. The enzyme activity was evaluated at 345 nm by a spectrophotometer. Extracts of M. chamomilla plant with butanol and water were prepared. Lisinopril was utilized as a specific inhibitor. ACE was purified 3,659-fold from human plasma and the specific activity was 1,350 EU/mg protein. The molecular weight and purity of ACE were found by SDS-PAGE and two bands of 60 and 70 kDa on the gel were detected. Water and butanol extracts of M. chamomilla demonstrated inhibitor impact on ACE activity. IC50 constants for water and butanol extracts of M. chamomilla were computed to be 1.292 and 0.353 mg/mL, respectively. The type of inhibition for whole inhibitors was identified as noncompetitive. IC50 and Ki constants for lisinopril were calculated to be 0.781 and 0.662 nM, respectively. These results indicate that butanol and water extracts of M. chamomilla may have an ACE inhibitor potential.


Assuntos
Matricaria , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Butanóis , Humanos , Peptidil Dipeptidase A , Extratos Vegetais/farmacologia , Água
7.
Cell Biochem Biophys ; 80(1): 115-122, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-34618304

RESUMO

Angiotensin-converting enzyme (ACE, EC 3.4.15.1) is a significant enzyme that regulates blood pressure. ACE inhibitors are often used in the treatment of hypertension. In this work, ACE was purified and characterized in one step with affinity chromatography from sheep kidneys. ACE was 10305-fold purified and specific activity was 19,075 EU/mg protein. The molecular weight and purity of ACE were found with SDS-PAGE and observed two bands at about 60 kDa and 70 kDa on the gel. The effects of reduced nicotinamide adenine dinucleotide (NADH), an antioxidant compound, on purified ACE activity were also researched. NADH on ACE activity showed an inhibition effect. The inhibition type of NADH was determined to be non-competitive inhibition by the Lineweaver-Burk chart and IC50 and Ki values for NADH were 244.33 and 175.08 µM, respectively. These results suggest that antioxidant substances might be efficient in preventing hypertension.


Assuntos
Rim/enzimologia , NAD , Peptidil Dipeptidase A , Animais , Cromatografia de Afinidade , Peptidil Dipeptidase A/isolamento & purificação , Peptidil Dipeptidase A/metabolismo , Ovinos
8.
Chem Biol Interact ; 347: 109604, 2021 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-34352275

RESUMO

Angiotensin-converting enzyme (ACE, EC 3.4.15.1) synthesized by endothelial cells and responsible for the regulation of blood pressure was purified from the bovine lung with affinity chromatography method. The purification rate of the ACE of the bovine lung was calculated as 1748- fold. Optimum pH and optimum temperature for the purified ACE were found to be 7.6 and 35-40 °C, respectively. The purity and molecular weight of the ACE were designated with SDS-PAGE. The ACE was found to have three subunits with molecular weights of 57 kDa, 66 kDa, and 190 kDa. Then, the total molecular weight of the ACE was designated as 303 kDa with gel filtration chromatography. The effects of ACE inhibitors captopril, fosinopril, lisinopril, and beta-blockers propranolol, atenolol, and diuretic triamterene on ACE activity were studied. ACE inhibitors lisinopril, captopril, fosinopril, and diuretic triamterene demonstrated an inhibition effect on ACE activity. Beta-blockers indicated no effect on ACE. IC50 values of captopril, fosinopril, lisinopril, and triamterene from the graphical equation were calculated as 0.835 nM, 1.159 µM, 4.085 nM, and 227 µM, respectively. The inhibition type and Ki values of these compounds were determined from Lineweaver-Burk plots. Captopril, fosinopril, lisinopril, and triamterene demonstrated a non-competitive inhibition effect on ACE activity. Ki constants were found as 1.057 nM, 1.675 µM, 6.449 nM, and 419.5 µM, respectively. Captopril indicated the highest inhibitor effect with an IC50 value of 0.835 nM.


Assuntos
Peptidil Dipeptidase A/isolamento & purificação , Inibidores da Enzima Conversora de Angiotensina/química , Animais , Captopril/química , Bovinos , Cromatografia de Afinidade , Fosinopril/química , Concentração de Íons de Hidrogênio , Cinética , Lisinopril/química , Pulmão/química , Peptidil Dipeptidase A/química , Estabilidade Proteica , Temperatura , Triantereno/química
9.
Mol Biol Rep ; 48(5): 4191-4199, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-34086160

RESUMO

Angiotensin-converting enzyme (ACE, EC 3.4.15.1) in the renin-angiotensin system regulates blood pressure by catalyzing angiotensin I to the vasoconstrictor angiotensin II. In this study, the ACE was purified and characterized from sheep lung. The kinetic properties of the ACE were designated. The inhibition effect of captopril, a specific ACE inhibitor, was determined. ACE was purified from sheep lung using the affinity chromatography method in one step. NHS-activated Sepharose 4 Fast Flow as column filler and lisinopril as a ligand in this method used. The molecular weight and purity of ACE were designated using the SDS-PAGE method. Optimum temperature and optimum pH were found for purified ACE. KM and Vmax values from Lineweaver-Burk charts determined. The inhibition type, IC50, and Ki values of captopril on purified ACE were identified. ACE was 6405-fold purified from sheep lung by affinity chromatography in one step and specific activity was 16871 EU/mg protein. The purity and molecular weight of ACE were found with SDS-PAGE and observed two bands at around 60 kDa and 70 kDa on the gel. Optimum temperature and optimum pH were designated for purified ACE. Optimum temperature and pH were found as 40 °C and pH 7.4, respectively. Vmax and KM values were calculated to be 35.59 (µmol/min).mL-1 and 0.18 mM, respectively. IC50 value of captopril was found as 0.51 nM. The inhibition type of captopril was determined as non-competitive from the Lineweaver-Burk graph and the Ki value was 0.39 nM. As a result, it was observed in this study that the ACE enzyme can be successfully purified from sheep lungs in one step. Also, it was determined that captopril, which is a specific ACE inhibitor, has a significant inhibitory effect with a very low IC50 value of 0.51 nM.


Assuntos
Pulmão/enzimologia , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/isolamento & purificação , Ovinos/metabolismo , Angiotensina I/metabolismo , Angiotensina II/metabolismo , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Captopril/farmacologia , Cromatografia de Afinidade/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Concentração de Íons de Hidrogênio , Concentração Inibidora 50 , Cinética , Lisinopril/farmacologia , Peso Molecular , Peptidil Dipeptidase A/metabolismo , Temperatura
10.
Biomed Chromatogr ; 33(8): e4560, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31016743

RESUMO

Glutathione reductase (GR, E.C. 1.8.1.7), a flavoenzyme, is responsible for recycling of oxidized glutathione disulfide. This study was performed in two main sections. In the first GR was purified from bovine liver by affinity column chromatography and the purification rate and specific activity of the enzyme were calculated as 1832-fold and 141 EU/mg protein, respectively. The subunit molecular weight of the enzyme was determined as 55 kDa by means of SDS-PAGE. The second section isolated natural components of Arum rupicola Boiss. var. rupicola using column chromatography. The isolation protocol for this plant was performed with a series of different-sized columns with hexane-ethyl acetate. According to the thin-layer chromatography plate, seven substances (R1-R7) were isolated. Our study's aim was to find new activators or inhibitors for GR activity. With this aim, all isolated substances were tested for GR activity. R6 showed competitive inhibition, while R4 had noncompetitive inhibition of GR activity. R1 played a role as an activator of GR activity. The inhibitory activity percentage vs. concentration graph was plotted. Values of IC50 for R4 and R6 were calculated as 0.193 mg/mL and 3.98 µg/mL, respectively, from the equation of this graph.


Assuntos
Arum/química , Glutationa Redutase , Fígado/enzimologia , Extratos Vegetais/farmacologia , Animais , Bovinos , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Cromatografia Gasosa-Espectrometria de Massas , Glutationa Redutase/química , Glutationa Redutase/efeitos dos fármacos , Glutationa Redutase/isolamento & purificação , Glutationa Redutase/metabolismo , Fígado/química , Extratos Vegetais/química
11.
Artigo em Inglês | MEDLINE | ID: mdl-30508739

RESUMO

Angiotensin converting enzyme (ACE, peptidyldipeptidase A, EC 3.4.15.1) plays an important role in the regulation of blood pressure. In this study, ACE was purified from human plasma by affinity chromatography in single step. The enzyme purified in 5367-fold from human plasma and specific activity was found to be 1208 EU/mg protein. The purity and molecular weight of ACE were determined by SDS-PAGE, which indicated two bands at around 60 kDa and 70 kDa on the gel. Effect of oxidized glutathione (GSSG) peptide and reduced glutathione (GSH) peptide on purified ACE activity were also investigated in which lisinopril was used as reference inhibitor. GSSG showed activation effect on ACE activity whereas GSH provided inhibition effect. In the lights of activity (%) versus activator graph for GSSG and activity (%) versus inhibitor graphs for GSH and lisinopril; IC50 values for GSH and lisinopril were determined to be 16.2 µM and 0.781 nM, respectively. Type of inhibition for GSH and lisinopril from graph Lineweaver-Burk was found to be reversible non-competitive inhibition and Ki constants for GSH and lisinopril were calculated as 11.7 µM and 0.662 nM, respectively.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/metabolismo , Dissulfeto de Glutationa/metabolismo , Glutationa/metabolismo , Fragmentos de Peptídeos/metabolismo , Peptidil Dipeptidase A , Inibidores da Enzima Conversora de Angiotensina/química , Cromatografia de Afinidade , Glutationa/química , Dissulfeto de Glutationa/química , Humanos , Modelos Lineares , Fragmentos de Peptídeos/química , Peptidil Dipeptidase A/sangue , Peptidil Dipeptidase A/isolamento & purificação , Peptidil Dipeptidase A/metabolismo
12.
Mater Sci Eng C Mater Biol Appl ; 90: 454-460, 2018 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-29853112

RESUMO

Herein, (3-aminopropyl)triethoxysilane functionalized cerium (IV) oxide (CeO2-NH2) supported Pd nanoparticles were synthesized. The nanocomposites were characterized using Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and High-resolution transmission electron microscopy (HRTEM). The Pd@CeO2-NH2 showed better electrocatalytic response to the reduction of H2O2 than CeO2-NH2. The fabricated sensor exhibited two linear responses to the reduction of H2O2. The first one was from 0.001 to 3.276 mM with 0.47 µM of a limit of detection (LOD) (S/N = 3) and excellent sensitivity of 440.72 µA mM-1 cm-2 and the second one was from 3.276 to 17.500 mM with the sensitivity of 852.65 µA mM-1 cm-2 in the optimum conductions. Also, the sensor exhibited 91% of electrocatalytic activity toward H2O2 after having been used for 30 days and the reproducibility was also satisfactory. The sensor response to H2O2 was not affected by ascorbic acid, fructose, glycine, dopamine, arginine, mannose, glucose, uric acid, Mg+2, Ca+2, and phenylalanine at the studied potential. Also, the fabricated sensor was used to determine H2O2 in milk samples. The results show that the constructed sensor can be a promising devise for the determination of H2O2 in real samples.


Assuntos
Peróxido de Hidrogênio/química , Nanocompostos/química , Carbono/química , Técnicas Eletroquímicas/métodos , Grafite/química , Nanopartículas Metálicas/química
13.
Biomed Chromatogr ; 32(5): e4175, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29243277

RESUMO

In the present study, one-step purification of angiotensin-converting enzyme (ACE, peptidyldipeptidase A, EC 3.4.15.1), responsible for regulation of blood pressure, was achieved using affinity chromatography from human plasma. The enzyme was purified 12,860-fold with a specific activtiy of 5080 EU/mg protein. Optimum temperature and pH were determined for the enzyme as 35-40°C and pH 7.4-7.5, respectively. The purity of ACE was determined by SDS-PAGE and the enzyme showed two bands at 60 and 70 kDa on the gel. The native molecular weight of ACE was found to be 260 kDa by gel filtration chromatography, demonstrating that the enzyme has a heterodimeric structure. Natural fatty acids of Nigella sativa (Ranunculaceae) were isolated by means of column chromatography. The structures of these compounds were determined using NMR and GC-MS. The results showed that high concentrations of linoleic, oleic and palmitic acids were isolated from the plant. The effect of six fractions (Fr 1-6) on ACE activity was examined. Fraction 3 increased the ACE activity while the other fractions decreased the enzyme activity. The concentrations of the fractions inhibiting the half-maximum activity of the enzyme were calculated as 1.597 mg/mL for Fr 1, 0.053 mg/mL for Fr 2, 0.527 mg/mL for Fr 4, 0.044 mg/mL for Fr 5 and 0.136 mg/mL for Fr 6 using a Lineweaver-Burk graph.


Assuntos
Ácidos Graxos/farmacologia , Nigella sativa/química , Peptidil Dipeptidase A , Extratos Vegetais/farmacologia , Ácidos Graxos/química , Humanos , Modelos Lineares , Peptidil Dipeptidase A/sangue , Peptidil Dipeptidase A/efeitos dos fármacos , Peptidil Dipeptidase A/isolamento & purificação , Peptidil Dipeptidase A/metabolismo , Extratos Vegetais/química
14.
Environ Sci Pollut Res Int ; 23(12): 12343-51, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26979315

RESUMO

Acetylcholinesterase (AChE) biosensor based on conducting poly([2,2̍';5̍' 2″]-terthiophene-3̍-carbaldehyde) (PTT) modified glassy carbon electrode (GCE) was constructed. AChE was immobilized on PTT film surface through the covalent bond between aldehyde and amino groups. The properties of PTT modified GCE were studied using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and scanning electron microscopy (SEM). The biosensor showed an oxidation peak at +0.83 V related to the oxidation of thiocholine, hydrolysis product of acetylthiocholine iodide (ATCI), catalyzed by AChE. The optimum current response of the biosensor was observed at pH 7.5-8.0, 40 °C and 120 U/cm(2) of AChE concentration. The biosensor showed a high sensitivity (183.19 µA/mM), a linear range from 0.015 to 1.644 mM, and a good reproducibility with 1.7 % of relative standard deviation (RSD). The biosensor showed a good stability. The interference of glycin, ascorbic acid, histidine, uric acid, dopamine, and arginine on the biosensor response was studied. An important analytical response from these inteferents that overlaps the biosensor response was not observed. The inhibition rate of malathion as a model pesticide was proportional to its concentrations from 9.99 to 99.01 nM. The detection limit was 4.08 nM.


Assuntos
Acetilcolinesterase/metabolismo , Malation/análise , Praguicidas/análise , Técnicas Biossensoriais/métodos , Carbono/química , Espectroscopia Dielétrica , Eletrodos , Limite de Detecção , Malation/metabolismo , Praguicidas/metabolismo , Reprodutibilidade dos Testes
15.
Int J Biol Macromol ; 79: 262-8, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25934105

RESUMO

In the study, the electrochemical behavior of glucose oxidase (GOx) immobilized on poly([2,2';5',2″]-terthiophene-3'-carbaldehyde) (poly(TTP)) modified glassy carbon electrode (GCE) was investigated. The biosensor (poly(TTP)/GOx/GCE) showed a pair of redox peaks in 0.1 M phosphate buffer (pH 7.4) solution in the absence of oxygen the co-substrate of GOx. In here, Poly(TTP)/GOx/GCE biosensor acts as the co-substrate instead of oxygen. Upon the addition of glucose, the reduction and oxidation peak currents increased until the active site of GOx was fully saturated with glucose. The apparent m was estimated 26.13 mM from Lineweaver-Burk graph. The biosensor displayed a good stability and bioactivity. The biosensor showed a high sensitivity (56.1 nA/mM), a linear range (from 0.5 to 20.15 mM), and a good reproducibility with 3.6% of relative standard deviation. In addition, the interference currents of glycin, ascorbic acid, histidine, uric acid, dopamine, arginine, and fructose on GOx biosensor were investigated. All that substances exhibited an interference current under 10%. It was not shown a marked difference between GOx biosensor and spectrophotometric measurement of glucose in serum examples. UV-visible spectroscopy and scanning electron microscopy (SEM) experiments of the biosensor were also performed.


Assuntos
Técnicas Biossensoriais/métodos , Glicemia/análise , Enzimas Imobilizadas/química , Glucose Oxidase/química , Aldeídos/química , Eletrodos , Humanos , Cinética , Oxirredução , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Tiofenos/química
16.
Pharm Biol ; 53(11): 1647-52, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25856719

RESUMO

CONTEXT: The effect of antibiotics (amikacin, cefazolin, ivermectin, and kanamycin) on glutathione reductase (GR) isoenzymes activity in liver and erythrocyte of the fish, Chalcalburnus tarichi (Lake Van pearl mullet, Pallas 1811) (Cyprinidae) were investigated. OBJECTIVE: This study determined the biochemical characterization of GR purified from the liver and erythrocytes of C. tarichi and the inhibition effect of the antibiotics on the GR isoenzymes. MATERIALS AND METHODS: GR was purified by affinity chromatography from the tissues of C. tarichi. The biochemical characterization of GR such as optimum temperature, optimum pH, and ionic strength were determined. The inhibition effects of the antibiotics on the isoenzymes were evaluated as IC50 and Ki values. Ki constant and 50% inhibitory concentration (IC50) value for antibiotics were determined by Lineweaver-Burk graphs and plotting activity % versus [I], respectively, at five different concentrations of antibiotics. RESULTS: Optimum temperature, pH, and ionic strength were determined for isoenzymes as 40 °C, 60 °C; 8.0, 8.0, and 50, 50 mM, respectively. Subunit molecular weights of the isoenzymes were estimated as 55 kDa by sodium dodecyl sulfate polyacrilamide gel electrophoresis (SDS-PAGE). In addition, IC50 and Ki values were calculated for amikacin, cefazolin, ivermectin, and kanamycin. The antibiotics showed non-competitive inhibition effects. IC50 values were calculated as 16.3, 36.6, 0.504, and 18.8 mM for liver and 20.0, 30.4, 0.787, and 31.8 mM for erythrocyte, respectively. Ki constants were 13.9, 18.4, 0.654, and 11.2 mM for liver and 23.2, 46.4, 1.19, and 36.4 mM for erythrocyte, respectively. DISCUSSION AND CONCLUSION: The results indicated that the antibiotics displayed non-competitive inhibition.


Assuntos
Antibacterianos/farmacologia , Cyprinidae , Eritrócitos/efeitos dos fármacos , Glutationa Redutase/antagonistas & inibidores , Lagos , Fígado/efeitos dos fármacos , Animais , Inibidores Enzimáticos/farmacologia , Eritrócitos/enzimologia , Glutationa Redutase/metabolismo , Fígado/enzimologia
17.
Bull Environ Contam Toxicol ; 91(5): 560-4, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24057299

RESUMO

In the present study, the effect of methidathion, cypermethrin, and deltamethrin pesticides on Lake Van fish (Chalcalburnus tarichii Pallas, 1811) liver 6-phosphogluconate dehydrogenase enzyme activity was investigated due to the fact that these pesticides are extensively used to improve agricultural productivity in the Van region. 2',5'-ADP Sepharose 4B affinity chromatography was used to purify 6-phosphogluconate dehydrogenase enzyme from fish liver and SDS-PAGE technique was used to control the purity of this enzyme. The in vitro effect of methidathion, cypermethrin, and deltamethrin pesticides on the enzyme activity was investigated. The enzyme was purified 1,050-fold with specific activity of 27.04 EU/mg protein. Moreover, Ki constants of methidathion, cypermethrin, and deltamethrin were to be 3.294 ± 0.215, 0.718 ± 0.095, and 0.084 ± 0.009 mM respectively. The IC50 value were estimated as 9.95 × 10(-5) ± 0.1844 × 10(-5) mM for methidathion, 1.01 × 10(-4) ± 0.01413 × 10(-4) mM for cypermethrin, and 4.43 × 10(-6) ± 0.05653 × 10(-6) mM for deltamethrin. In conclusion, deltamethrin inhibits the enzyme activity more than methidathion and cypermethrin.


Assuntos
Cyprinidae/metabolismo , Fígado/enzimologia , Praguicidas/toxicidade , Fosfogluconato Desidrogenase/metabolismo , Poluentes Químicos da Água/toxicidade , Animais , Lagos , Fígado/efeitos dos fármacos , Nitrilas/toxicidade , Compostos Organotiofosforados/toxicidade , Piretrinas/toxicidade
18.
Environ Toxicol ; 24(2): 128-32, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18442070

RESUMO

The present study was designed to understand the effects of sublethal concentrations of dichlorvos (DIC) on hematological constituent [red blood corpuscles, white blood corpuscles (WBC), mean cell volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet counts, hemoglobin and hematocrite levels] and serum damage marker enzymes (aspartate aminotransferase, alanin aminotransferase, alkaline phosphatase, and lactate dehydrogenase) in rats at subacute period under laboratory conditions. DIC at dosages of 5 and 10 ppm was administered orally to six male rats ad libitum during the tests for 4 weeks consecutively. According to the results, DIC treatments increased significantly the levels of serum marker enzyme activities, whereas they did not change hematologic constituent except for WBC number treated with both dosages of DIC. The observations presented led us to conclude that the administrations of subacute DIC induced the levels of damage marker enzymes and leukocytosis.


Assuntos
Células Sanguíneas/efeitos dos fármacos , Diclorvós/toxicidade , Enzimas/sangue , Alanina Transaminase/sangue , Fosfatase Alcalina/sangue , Animais , Aspartato Aminotransferases/sangue , Células Sanguíneas/citologia , Diclorvós/administração & dosagem , Índices de Eritrócitos/efeitos dos fármacos , L-Lactato Desidrogenase/sangue , Contagem de Leucócitos , Masculino , Contagem de Plaquetas , Ratos , Ratos Sprague-Dawley
19.
J Hazard Mater ; 143(1-2): 415-8, 2007 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-17049736

RESUMO

Inhibitory effects of some drugs on hepatic glucose 6-phosphate dehydrogenase from Lake Van fish (chalcalburnus tarischii pallas, 1811) were investigated. For this purpose, initially liver glucose 6-phosphate dehydrogenase was purified 899-fold in a yield of 46.24% by using 2',5'-ADP Sepharose 4B affinity gel. In order to control the purification of enzyme was done SDS polyacrylamide gel electrophoresis. SDS polyacrylamide gel electrophoresis showed a single band for enzyme. A constant temperature (+4 degrees C) was maintained during the purification process. Enzyme activity was determined with the Beutler method by using a spectrophotometer at 340 nm. Vankomycine, sulfanylamide, sulfanylacetamide, nidazole, ciprofloxacin, amoxicillin and KMnO(4) were used as drugs. These drugs exhibited inhibitory effects on the enzyme. IC(50) values of vankomycine, sulfanylamide, sulfanylacetamide, nidazole, ciprofloxacin, amoxicillin and KMnO(4) were 1.88, 0.037, 0.032, 1.178, 2.26, 643.5 and 0.0002 mM, and the K(i) constants 1.18+/-0.148, 0.119+/-0.021, 0.075+/-0.015, 1.15+/-0.21, 7.69+/-0.67, 1007+/-69, and 0.001+/-0.00022 mM, respectively. While vankomycine and nidazole showed competitive inhibition, others displayed noncompetitive inhibition. K(i) constants and IC(50) values for drugs were determined by Lineweaver-Burk graphs and plotting activity percentage versus [I], respectively.


Assuntos
Anti-Infecciosos/farmacologia , Cyprinidae/metabolismo , Glucosefosfato Desidrogenase/efeitos dos fármacos , Fígado/enzimologia , Animais , Concentração de Íons de Hidrogênio , Turquia
20.
Prep Biochem Biotechnol ; 33(2): 137-45, 2003 May.
Artigo em Inglês | MEDLINE | ID: mdl-12784884

RESUMO

In this study, acetylcholinesterase (AChE; EC 3.1.1.7) was purified from plasma and erythrocytes in the Lake Van fish (Chalcalburnus tarichii P.1811) by affinity chromatography. Enzymatic activity was spectrophotometrically measured according to Ellman's method, at 412 nm. Then, the optimal pH and temperature of the enzyme was determined. According to the results, the optimal pH and the optimum temperature were 8.0 and 25 degrees C, respectively. In order to control the purification of the enzyme, sodium dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was done. SDS-PAGE showed a single band for enzyme. The purification rates for plasma AChE and erythrocyte AChE are 3251.6 and 8500, respectively.


Assuntos
Acetilcolinesterase/isolamento & purificação , Acetilcolinesterase/metabolismo , Cyprinidae , Acetilcolinesterase/sangue , Animais , Cromatografia de Afinidade/métodos , Eletroforese em Gel de Poliacrilamida , Eritrócitos/enzimologia , Concentração de Íons de Hidrogênio , Temperatura
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