Effects of some antibiotics on human erythrocyte glutathione reductase: an in vitro study

Inhibitory effects of some antibiotics on purified human erythrocyte glutathione reductase were investigated. Human erythrocyte glutathione reductase was purified 2800-fold (29% yield) at 4 degrees C using 2', 5'-ADP Sepharose 4B affinity gel and Sephadex G-200 gel filtration chromatography. SDS polyacrylamide gel electrophoresis showed a single band for the enzyme. Imipenem, rifamycin, sulfanylacetamide, ceftazidime, chloramphenicol, seftriaxon, vancomycin, cefuroxime and ornidazole exhibited inhibitory effects but clindamycin, lincomycin, amoxicillin, amikacin exhibited activatory effects on the enzyme in vitro. The IC(50) values of imipenem, rifamycin, sulfanylacetamide, ceftazidime, chloramphenicol, seftriaxon, vancomycin, cefuroxime and ornidazole were 0.030, 0.146, 0.59, 2.476, 2.36, 2.88, 4.83, 15.43 and 19.632 mM, respectively, and the K(i) constants were 0.06 +/- 0.01, 0.275 +/- 0.10, 0.85 +/- 0.05, 3.59 +/- 0.51, 3.85 +/- 0.40, 3.71 +/- 0.60, 15.11 +/- 2.50, 23.50 +/- 2.94 and 28.49 +/- 6.50 mM, respectively. While imipenem, rifamycin, sulfanylacetamide, ceftazidime, chloramphenicol and seftriaxon cefuroxime and ornidazole showed competitive inhibition, vankomycine displayed noncompetitive inhibition.     

Effects of some antibiotics on human erythrocyte glutathione reductase: an in vitro study

Inhibitory effects of some antibiotics on purified human erythrocyte glutathione reductase were investigated. Human erythrocyte glutathione reductase was purified 2800-fold (29% yield) at 4°C using 2′, 5′-ADP Sepharose 4B affinity gel and Sephadex G-200 gel filtration chromatography. SDS polyacrylamide gel electrophoresis showed a single band for the enzyme. Imipenem, rifamycin, sulfanylacetamide, ceftazidime, chloramphenicol, seftriaxon, vancomycin, cefuroxime and ornidazole exhibited inhibitory effects but clindamycin, lincomycin, amoxicillin, amikacin exhibited activatory effects on the enzyme in vitro. The IC₅₀ values of imipenem, rifamycin, sulfanylacetamide, ceftazidime, chloramphenicol, seftriaxon, vancomycin, cefuroxime and ornidazole were 0.030, 0.146, 0.59, 2.476, 2.36, 2.88, 4.83, 15.43 and 19.632 mM, respectively, and the K_i constants were 0.06 ± 0.01, 0.275 ± 0.10, 0.85 ± 0.05, 3.59 ± 0.51, 3.85 ± 0.40, 3.71 ± 0.60, 15.11 ± 2.50, 23.50 ± 2.94 and 28.49 ± 6.50 mM, respectively. While imipenem, rifamycin, sulfanylacetamide, ceftazidime, chloramphenicol and seftriaxon cefuroxime and ornidazole showed competitive inhibition, vankomycine displayed noncompetitive inhibition.     

Effects of some analgesic anaesthetic drugs on human erythrocyte glutathione reductase: an in vitro study

Inhibitory effects of some analgesic and anaesthetic drugs on human erythrocyte glutathione reductase were investigated. For this purpose, human erythrocyte glutathione reductase was initially purified 2139-fold in a yield of 29% by using 2', 5'-ADP Sepharose 4B affinity gel and Sephadex G-200 gel filtration chromatography. SDS polyacrylamide gel electrophoresis confirmed the purity of the enzyme by sharing a single band. A constant temperature (+4 degrees C) was maintained during the purification process. Diclofenac sodium, ketoprofen, lornoxicam, tenoxicam, etomidate, morphine and propofol exhibited inhibitory effects on the enzyme in vitro using the Beutler assay method. K(i) constants and IC(50) values for drugs were determined from Lineweaver-Burk graphs and plotting activity % versus [I] graphs, respectively. The IC(50) values of diclofenac sodium, ketoprofen, lornoxicam, propofol, tenoxicam, etomidate and morphine were 7.265, 6.278, 0.3, 0.242, 0.082, 0.0523 and 0.0128 mM and the K(i) constants were 23.97 +/- 2.1, 22.14 +/- 7.6, 0.42 +/- 0.18, 0.418 +/- 0.056, 0.13 +/- 0.025, 0.0725 +/- 0.0029 and 0.0165 +/- 0.0013 mM, respectively. While diclofenac sodium, ketoprofen, lornoxicam, tenoxicam etomidate and morphine showed competitive inhibition, propofol displayed noncompetitive inhibition.     

Effects of some analgesic anaesthetic drugs on human erythrocyte glutathione reductase: an in vitro study

Inhibitory effects of some analgesic and anaesthetic drugs on human erythrocyte glutathione reductase were investigated. For this purpose, human erythrocyte glutathione reductase was initially purified 2139-fold in a yield of 29% by using 2′, 5′-ADP Sepharose 4B affinity gel and Sephadex G-200 gel filtration chromatography. SDS polyacrylamide gel electrophoresis confirmed the purity of the enzyme by sharing a single band. A constant temperature (+4°C) was maintained during the purification process. Diclofenac sodium, ketoprofen, lornoxicam, tenoxicam, etomidate, morphine and propofol exhibited inhibitory effects on the enzyme in vitro using the Beutler assay method.

K_i constants and IC₅₀ values for drugs were determined from Lineweaver-Burk graphs and plotting activity % versus [I] graphs, respectively. The IC₅₀ values of diclofenac sodium, ketoprofen, lornoxicam, propofol, tenoxicam, etomidate and morphine were 7.265, 6.278, 0.3, 0.242, 0.082, 0.0523 and 0.0128 mM and the K_i constants were 23.97 ± 2.1, 22.14 ± 7.6, 0.42 ± 0.18, 0.418 ± 0.056, 0.13 ± 0.025, 0.0725 ± 0.0029 and 0.0165 ± 0.0013 mM, respectively. While diclofenac sodium, ketoprofen, lornoxicam, tenoxicam etomidate and morphine showed competitive inhibition, propofol displayed noncompetitive inhibition.     

In vitro effects of some drugs on human erythrocyte glutathione reductase

The effects of ketotifen, meloxicam, phenyramidol-HCl and gadopentetic acid on the enzyme activity of GR were studied using human erythrocyte glutathione reductase (GR) enzymes in vitro. The enzyme was purified 209-fold from human erythrocytes in a yield of 19% with 0.31?U/mg. The purification procedure involved the preparation of haemolysate, ammonium sulphate precipitation, 2',5'-ADP Sepharose 4B affinity chromatography and Sephadex G-200 gel filtration chromatography. Purified enzyme was used in the in vitro studies. In the in vitro studies, IC(50) values and K(i) constants were 0.012?mM and 0.0008?±?0.00021?mM for ketotifen; 0.029?mM and 0.0061?±?0.00127?mM for meloxicam; 0.99?mM and 0.4340?±?0.0890?mM for phenyramidol-HCl; 138?mM and 28.84?±?4.69?mM for gadopentetic acid, respectively, showing the inhibition effects on the purified enzyme. Phenyramidol-HCl showed competitive inhibition, whereas the others showed non-competitive inhibition.     

In vitro effects of some drugs on human erythrocyte glutathione reductase

The effects of ketotifen, meloxicam, phenyramidol–HCl and gadopentetic acid on the enzyme activity of GR were studied using human erythrocyte glutathione reductase (GR) enzymes in vitro. The enzyme was purified 209-fold from human erythrocytes in a yield of 19% with 0.31?U/mg. The purification procedure involved the preparation of haemolysate, ammonium sulphate precipitation, 2′′,5′-ADP Sepharose 4B affinity chromatography and Sephadex G-200 gel filtration chromatography. Purified enzyme was used in the in vitro studies. In the in vitro studies, IC₅₀ values and K_i constants were 0.012?mM and 0.0008?±?0.00021?mM for ketotifen; 0.029?mM and 0.0061?±?0.00127?mM for meloxicam; 0.99?mM and 0.4340?±?0.0890?mM for phenyramidol–HCl; 138?mM and 28.84?±?4.69?mM for gadopentetic acid, respectively, showing the inhibition effects on the purified enzyme. Phenyramidol–HCl showed competitive inhibition, whereas the others showed non-competitive inhibition.     

Effects of some metal ions on rainbow trout erythrocytes glutathione S-transferase enzyme: an in vitro study

Glutathione S-transferase enzyme (GST) (EC 2.5.1.18) was purified from rainbow trout erythrocytes, and some characteristics of the enzyme and effects of some metal ions on enzyme activity were investigated. For this purpose, erythrocyte glutathione S-transferase enzyme which has 16.54 EU/mg protein specific activities was purified 11,026-fold by glutathione-agarose affinity chromatography with a yield of 59%. Temperature was kept under control (+4°C) during purification. Enzyme purification was checked by performing SDS-PAGE. Optimal pH, stable pH, optimal temperature, and K_M and V_max values for GSH and 1-chloro-2, 4-dinitrobenzene (CDNB) were also determined for the enzyme. In addition, IC₅₀ values, K_i constants and the type of inhibition were determined by means of Line-Weaver-Burk graphs obtained for such inhibitors as Ag⁺; Cd²⁺, Cr²⁺ and Mg²⁺.     

Comparative in vitro effects of some metal ions on bovine kidney cortex glutathione reductase

Heavy metal pollution can arise from many sources and damage many organisms. Exposure to the metal ions can leads to a reduction in cellular antioxidant enzyme activities and lowers cellular defense against oxidative stress. In this study we have tested effects of the some metal ions on the purified bovine kidney cortex glutathione reductase (GR). Cadmium (Cd2+), nickel (Ni2+), and zinc (Zn2+) showed inhibitory effect on the enzyme. The obtained IC?? values of Cd2+, Ni2+, and Zn2+ are 0.027, 0.8, and 1 mM, respectively. Kinetic characterization of the inhibition is also investigated. Cd2+ inhibition is noncompetitive with respect to both oxidized glutathione (GSSG) (Ki(GSSG) 0.060 ± 0.005 mM) and NADPH (Ki(NADPH) 0.025 ± 0.002 mM). Ni2+ inhibition is noncompetitive with respect to GSSG (Ki(GSSG) 0.329 ± 0.016 mM) and uncompetitive with respect to NADPH (Ki(NADPH) 0.712 ± 0.047 mM). The effect of Zn2+ on GR activity is consistent with noncompetitive inhibition pattern when the varied substrate is the GSSG (Ki(GSSG) 0.091 ± 0.005 mM) and the NADPH (Ki(NADPH) 0.226 ± 0.01 mM), respectively. GR inhibition studies may be useful for understanding the mechanisms for oxidative damage associated with heavy metal toxicity.     

In vitro inhibition of human erythrocyte glutathione reductase by some new organic nitrates

Glutathione reductase (GR), is responsible for the existence of GSH molecule, a crucial antioxidant against oxidative stress reagents. The antimalarial activities of some redox active compounds are attributed to their inhibition of antioxidant flavoenzyme glutathione reductase, and inhibitors are therefore expected to be useful for the treatment of malaria. Twelve organic nitrate derivatives were synthesized and treated with human erythrocyte GR. The molecules were identified as strong GR inhibitors and novel antimalaria candidates.     

Effects of some drugs on human erythrocyte 6-phosphogluconate dehydrogenase: an in vitro study

Inhibitory effects of some drugs were investigated on human erythrocyte 6-phosphogluconate dehydrogenase obtained with a 6552-fold purification in a yield of 78% using 2', 5'-ADP Separose 4B affinity gel. Which on SDS polyacrylamide gel electrophoresis showed a single band. Larnoxicam, metronidazole, imipenem, ornidazole, vancomycin, clindamycin, and amoxicillin exhibited inhibitory effects on the enzyme in vitro with IC50 values of 0.17, 0.23, 0.43, 21.79, 46.39, 117.43 and 287.35 mM, and the Ki constants 0.40 +/- 0.04, 0.57 +/- 0.06, 0.77 +/- 0.11, 42.40 +/- 2.89, 65.60 +/- 4.03, 130.22 +/- 9.21, and 287.58 +/- 10.56 mM, respectively. While vancomycin, clindamycin and amoxicillin showed competitive inhibition the other drugs displayed noncompetitive inhibition.     

Effects of some drugs on human erythrocyte 6-phosphogluconate dehydrogenase: an in vitro study

Inhibitory effects of some drugs were investigated on human erythrocyte 6-phosphogluconate dehydrogenase obtained with a 6552-fold purification in a yield of 78% using 2′, 5′-ADP Separose 4B affinity gel. Which on SDS polyacrylamide gel electrophoresis showed a single band. Larnoxicam, metronidazole, imipenem, ornidazole, vancomycin, clindamycin, and amoxicillin exhibited inhibitory effects on the enzyme in vitro with IC₅₀ values of 0.17, 0.23, 0.43, 21.79, 46.39, 117.43 and 287.35 mM, and the Ki constants 0.40 ± 0.04, 0.57 ± 0.06, 0.77 ± 0.11, 42.40 ± 2.89, 65.60 ± 4.03, 130.22 ± 9.21, and 287.58 ± 10.56 mM, respectively. While vancomycin, clindamycin and amoxicillin showed competitive inhibition the other drugs displayed noncompetitive inhibition.     

The inhibition kinetics of yeast glutathione reductase by some metal ions

Glutathione reductase (GR, type IV, Baker's yeast, E.C 1.6.4.2) is a flavoprotein that catalyzes the NADPH-dependent reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH). In this study some metal ions have been tested on GR; lithium, manganese, molybdate, aluminium, barium, zinc, calcium, cadmium and nickel. Cadmium, nickel and calcium showed a good to moderate inhibitory effect on yeast GR. GR is inhibited non-competitively by Zn2+ (up to 2 mM) and activated above this concentration. Ca2+ inhibition was non-competitive with respect to GSSG and uncompetitive with respect to NADPH. Nickel inhibition was competitive with respect to GSSG and uncompetitive with respect to NADPH. The inhibition constants for these metals on GR were determined. The chelating agent EDTA recovered 90% of the GR activity inhibited by these metals.     

Inhibition of purified bovine liver glutathione reductase with some metal ions

Glutathione reductase (GR; E.C. 1.6.4.2) is a flavoprotein that catalyzes the NADPH-dependent reduction of oxidized glutathione (GSSG). In this study we tested the effects of Al³⁺, Ba²⁺, Ca²⁺, Li⁺, Mn²⁺, Mo⁶⁺, Cd²⁺, Ni²⁺, and Zn²⁺ on purified bovine liver GR. In a range of 10?μM–10?mM concentrations, Al³⁺, Ba²⁺, Li⁺, Mn²⁺, and Mo⁶⁺, and Ca²⁺ at 5?μM–1.25?mM, had no effect on bovine liver GR. Cadmium (Cd²⁺), nickel (Ni²⁺), and zinc (Zn²⁺) showed inhibitory effects on this enzyme. The obtained IC₅₀ values of Cd²⁺, Ni²⁺, and Zn²⁺ were 0.08, 0.8, and 1?mM, respectively. Cd²⁺ inhibition was non-competitive with respect to both GSSG (Ki_GSSG 0.221?±?0.02?mM) and NADPH (Ki_NADPH 0.113?±?0.008?mM). Ni²⁺ inhibition was non-competitive with respect to GSSG (Ki_GSSG 0.313?±?0.01?mM) and uncompetitive with respect to NADPH (Ki_NADPH 0.932?±?0.03?mM). The effect of Zn²⁺ on GR activity was consistent with a non-competitive inhibition pattern when the varied substrates were GSSG (Ki_GSSG 0.320?±?0.018?mM) and NADPH (Ki_NADPH 0.761?±?0.04?mM), respectively.     

The inhibition kinetics of yeast glutathione reductase by some metal ions

Glutathione reductase (GR, type IV, Baker's yeast, E.C 1.6.4.2) is a flavoprotein that catalyzes the NADPH-dependent reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH). In this study some metal ions have been tested on GR; lithium, manganese, molybdate, aluminium, barium, zinc, calcium, cadmium and nickel. Cadmium, nickel and calcium showed a good to moderate inhibitory effect on yeast GR. GR is inhibited non-competitively by Zn^{2 +} (up to 2 mM) and activated above this concentration. Ca^{2 +} inhibition was non-competitive with respect to GSSG and uncompetitive with respect to NADPH. Nickel inhibition was competitive with respect to GSSG and uncompetitive with respect to NADPH. The inhibition constants for these metals on GR were determined. The chelating agent EDTA recovered 90% of the GR activity inhibited by these metals.     

Effects of some drugs on human erythrocyte glucose 6-phosphate dehydrogenase: an in vitro study

Inhibitory effects of some drugs on glucose 6-phosphate dehydrogenase from the erythrocytes of human have been investigated. For this purpose, at the beginning, erythrocyte glucose 6-phosphate dehydrogenase was purified 2256 times in a yield of 44.22% by using ammonium sulphate precipitation and 2', 5'-ADP Sepharose 4B affinity gel. Temperature of +4°C was maintained during the purification process. Enzyme activity was determined with the Beutler method by using a spectrophotometer at 340?nm. This method was utilized for all kinetic studies. Ketotifen, dacarbazine, thiocolchicoside, meloxicam, methotrexate, furosemide, olanzapine, methylprednizolone acetate, paricalcitol, ritodrine hydrochloride, and gadobenate-dimeglumine were used as drugs. All the drugs indicated the inhibitory effects on the enzyme. Ki constants for glucose 6-phosphate dehydrogenase were found by means of Lineweaver-Burk graphs. While methylprednizolone acetate showed competitive inhibition, the others displayed non-competitive inhibition. In addition, IC(50) values of the drugs were determined by plotting Activity% vs [I].     

Effects of some antibiotics on human erythrocyte 6-phosphogluconate dehydrogenase: an in vitro and in vivo study

The in vitro and in vivo effects of some antibiotics on human erythrocyte 6-phosphogluconate dehydrogenase were investigated. Human erythrocyte 6-phosphogluconate dehydrogenase was purified with ammonium sulphate precipitation, 2',5' ADP-Sepharose 4B affinity and gel filtration chromatography. Some antibiotics (netilmicin sulphate, cefepime, amikacin, isepamycin, chloramphenicol, ceftazidim, teicoplanin, ampicillin, ofloxacin, levofloxacin, cefotaxime, penicillin G, gentamicin sulphate, ciprofloxacin) inhibited enzyme activity in vitro but others (cefozin, decefin, streptomycin, combisid, and meronem) were devoid of inhibitory effects. For the drugs having low IC50 values (netilmicin sulphate and cefepime), in vivo studies were performed in rats. Netilmicin sulphate at 15-mg/kg inhibited enzyme activity significantly (p < 0.001) 1 h, 2 h, and 3 h after dosing and cefepime at 200-mg/kg very significantly (p < 0.001) inhibited the enzyme 1 h and 2 h after dosing. Netilmicin sulphate and cefepime inhibited rat erythrocyte 6-phosphogluconate dehydrogenase both in vivo and in-vitro.     

Population study of erythrocyte glutathione reductase activity

Summary The activity of erythrocyte glutathione reductase (GR) was determined in a group of 87 prisoners from northern Thailand (65 with normal, 22 with deficient erythrocyte G-6-PD) without and with added FAD. The amount of stimulation by FAD was inversely related to the original activity suggesting that FAD stimulation in vivo is one of the main determinants of GR activity. 4 subjects showed insufficient stimulation by FAD. The binding of FAD to GR seemed to be closer to saturation in G-6-PD deficient subjects but the maximal stimulated activity of these subjects was higher than in the group with normal G-6-PD. This suggests that the marked increase of GR activity in G-6-PD deficient erythrocytes is due to increased binding of FAD and to a larger amount of stimulable enzyme. Original GR activity was positively correlated with the socio-economic status, and subjects who had taken riboflavin in the period prior to examination had a higher mean GR activity than those without vitamin intake. GR activity was markedly raised by administration of riboflavin and in G-6-PD deficient subjects glutathione stability was improved. In comparison to central European subjects more than 50 percent of the examined population are GR deficient. This seems to be due to a low dietary supply of riboflavin in most cases. The findings in 4 subjects with insufficient stimulation by added FAD raise the question whether hereditary forms of GR deficiency exist in this population.

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Zusammenfassung In einer Gruppe von 87 Strafgefangenen in Nordthailand (65 mit normaler, 22 mit defizienter G-6-PD) wurde die Aktivität der Glutathion-Reductase (GR) mit und ohne Zusatz von FAD bestimmt. Das Ausmaß der Stimulierung des Enzyms durch FAD verhielt sich reziprok zur ausgangsaktivität. Bei 4 Versuchspersonen war die Stimulierung durch FAD insuffizient. Die Bindung von FAD an GR war bei Versuchspersonen mit G-6-PD-Mangel näher am Sättigungspunkt. Aber auch die gesamte stimulierbare Aktivität der GR war bei Versuchspersonen mit G-6-PD-Mangel größer als bei Gesunden. Die deutliche Erhöhung der GR-Aktivität bei G-6-PD-Mangel scheint zwei Ursachen zu haben: vermehrte Bindung von FAD an das Enzym und Vermehrung der Gesamtmenge an stimulierbarem Enzym. In einer weiteren Gruppe war die Ausgangsaktivität der GR vom sozio-ökonomischen Status der Versuchsperson abhängig. Personen, die vor der Untersuchung Riboflavin-haltige Medikamente eingenommen hatten, hatten höhere Aktivitäten. Die GR-Aktivität wurde durch Einnahme von Riboflavin unter Kontrolle stark erhöht, und bei Versuchspersonen mit G-6-PD-Mangel wurde die Glutathion-Stabilität der Erythrocyten verbessert. Im Vergleich zu Mitteleuropäern sind mehr als die Hälfte der thailändischen Versuchspersonen als GR defizient einzustufen. Dies ist wahrscheinlich auf eine geringere Zufuhr an Riboflavin in der Nahrung zurückzuführen. Die Befunde bei 4 Versuchspersonen mit insuffizienter Stimulierung der GR durch FAD könnte durch einen erblichen Mangel an GR verursacht sein.

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Established and supported by Stiftung Volkswagenwerk.     

Effects of some drugs on human cord blood erythrocyte carbonic anhydrases I and II: an in vitro study

In the present study, we purified hcbCA I and II from human cord blood erythrocytes using by Sepharose-4B-L tyrosine-sulfanilamide affinity gel chromatography. Also, it was checked the inhibition effects of ampicillin sulfate, ceftriaxone, ceftizoxime and ranitidine on hcbCA I and hcbCA II. IC(50) values for ceftriaxone, ceftizoxime and ranitidine were found to be 27.l, 79.4 and 55.5 μM for hcbCA I and of 21.0, 79.1 and 66.1 μM for hcbCA II, respectively. According to these results, Ampicillin sulfate inhibited only hcbCAII and IC(50) values of this antibiotic was found to be 56.8 μM. All these substances were found non-competitive inhibitors. It is important to study the inhibition effects of these drugs on hcbCA I and II izoenzymes. Because, pregnant woman is take all of these substance. For this reason, these drugs should be carefully used and the dosage should be very well ordered to minimize side effects.     

Low resolution structure of human erythrocyte glutathione reductase

Glutathione reductase from human erythrocytes is a dimeric flavoenzyme with a molecular weight of 100,000. X-ray diffraction analysis using the isomorphous replacement technique with four heavy-atom derivatives yielded an electron density map at 6 Å resolution with a figure of merit of 0.88. Only minor cuts had to be made in the electron density map to isolate one molecule. The dimer interface is on a crystallographic 2-fold axis. Each subunit can be subdivided into three domains: I, II and III, which are aggregated in such a way that deep clefts are formed on opposite sides of the subunit. These clefts accommodate the substrate glutathione, binding to domain III, and the oxidized cofactor NADP, binding to domain I in a similar extended conformation as NAD binds to the dehydrogenases. The shortest connection between the centres of the nicotinamide ring of NADP and the cystine of oxidized glutathione is 18 Å long and goes along the interface between domains II and III right through the centre of the subunit. Presumably, FAD binds to domain II and its isoalloxazine ring bridges the gap between NADP and glutathione.     

Effects of some antibiotics on glutathione reductase activities from human erythrocytes in vitro and from rat erythrocytes in vivo

The effects of streptomycin sulfate, gentamicin sulfate, thiamphenicol, penicillin G, teicoplanin, ampicillin, cefotaxime, and cefodizime on the enzyme activity of glutathione reductase (GR) were studied using human and rat erythrocyte GR enzymes in in vitro and in vivo studies, respectively. The enzyme was purified 5,342-fold from human erythrocytes in a yield of 29% with 50.75 U/mg. The purification procedure involved the preparation of hemolysate, ammonium sulfate precipitation, 2',5'-ADP Sepharose 4B affinity chromatography and Sephadex G-200 gel filtration chromatography. Purified enzyme was used in the in vitro studies, and rat erythrocyte hemolysate was used in the in vivo studies. In the in vitro studies, I50 and K(i) values were 12.179 mM and 6.5123 +/- 4.1139 mM for cefotaxime, and 1.682 mM and 0.7446 +/- 0.2216 mM for cefodizime, respectively, showing the inhibition effects on the purified enzyme. Inhibition types were noncompetitive for cefotaxime and competitive for cefodizime. In the in vivo studies, 300 mg/kg cefotaxime and 1000 mg/kg cefodizime when administered to rats inhibited enzyme activity during the first 2h (p < 0.01). Cefotaxime led to increased enzyme activity at 4h (p < 0.05), but neither cefotaxime nor cefodizime had any significant inhibition or activation effects over 6 h (p > 0.05).