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Aim: This study aimed to generate an improved method of protein extraction and purification from plant tissues containing very high amounts of phenolic compounds and other interfering biomolecules. Background: Protein extraction at proteomic studies on some plant species, including conifers, is challenging, and the yield and quality are unpredictable. Objective: Two popular protocols were combined with each other to construct a novel one with enhanced abilities to produce higher purity of samples compatible for high precision molecular systems and analysis. Method: The new method was compared with the other two for their efficiencies in classical SDS- PAGE, 2-DE and capillary chromatography applications. Results: All three methods were comparable in SDS-PAGE procedure; however, only the new method created acceptable gel images in 2-DE. Bioanalyzer results, also, demonstrated that the new method provided protein samples pure enough to be used in capillary chromatography with 2 times more peaks in electropherograms with lower noise and higher total relative protein concentrations closest to the applied amount. Conclusion: The new combined method is a successful alternative for plant proteomicists with higher yield and quality of proteins from recalcitrant tissues. Other: The new method could be preferred, especially, for high-tech, sensitive proteomic analysis.

Magnetic nanoparticles (MNPs) have been increasingly used for many years as MRI agents and for gene delivery and hyperthermia therapy, although there have been conflicting results on their safety. In this study, cobalt ferrite magnetic nanoparticles (CoFe-MNPs) were prepared by the co-precipitation method and their surfaces were modified with silica by the sol-gel method. The particle and hydrodynamic sizes, morphology and crystal structure of the bare and silica-coated CoFe-MNPs were evaluated by transmission electron microscopy (TEM), dynamic light scattering (DLS), X-ray diffraction spectroscopy (XRD) and Fourier transform infrared spectroscopy (FTIR). The size of the bare CoFe-MNPs was in the range 8-20 nm and they were homogeneously coated with 3-4 nm silica shells. The bare and silica-coated CoFe-MNPs were agglomerated at physiological pH. However, the sizes of the agglomerates were below 200 nm both in water and complete medium. The cytotoxic and genotoxic potentials of the bare and silica-coated CoFe-MNPs were evaluated in a metastatic breast cancer cell line, MDA-MB-231, as well as a noncancerous mammary epithelial cell line, MCF-10A, by using XTT cytotoxicity, single-cell gel electrophoresis (comet), and cytokinesis-blocked (CB) micronucleus (CBMN) assays. Characterization studies with TEM, inductively coupled plasma optical emission spectroscopy (ICP-OES) and Prussian blue staining indicated that the CoFe-MNPs were internalized into the cells by energy-dependent endocytosis. The highest amount of uptake was observed in the cancer cells and the uptake of the silica-coated CoFe--MNPs was higher than that of the bare ones in both cell lines. The bare CoFe-MNPs showed higher levels of both cytotoxicity and genotoxicity than the silica-coated CoFe-MNPs. Moreover, the cancer cells seemed to be more susceptible to the CoFe-MNPs' toxicity compared to the noncancerous cells. There was a concentration and time-dependent increase in DNA damage and the micronucleus (MN) frequency, which was statistically significant starting with the lowest concentration of bare CoFe-MNPs (p < 0.05), while no significance was observed below the concentration of 250 mu g mL(-1) for the silica-coated MNPs. Also, the extent of both DNA damage and MN frequency was much higher in the cancer cells compared to the noncancerous cells. According to our results, the silica coating ameliorated both the cytotoxicity and genotoxicity as well the internalization of the CoFe-MNPs.

WOS: 000363980200006, PubMed ID: 26440080, Glutathione transferases (GSTs; EC 2.5.1.18) play important roles in stress tolerance and metabolic detoxification in plants. In higher plants, studies on GSTs have focussed largely on agricultural plants. There is restricted information about molecular characterization of GSTs in gymnosperms. To date, only tau class GST enzymes have been characterized from some pinus species. For the first time, the present study reports cloning and molecular characterization of two zeta class GST genes, namely PbGSTZ1 and PbGSTZ2 from Pinus brutia Ten., which is an economically important pine native to the eastern Mediterranean region and have to cope with several environmental stress conditions. The PbGSTZ1 gene was isolated from cDNA, whereas PbGSTZ2 was isolated from genomic DNA. Sequence analysis of PbGSTZ1 and PbGSTZ2 revealed the presence of an open reading frame of 226 amino acids with typical consensus sequences of the zeta class plant GSTs. Protein and secondary structure prediction analysis of two zeta class PbGSTZs have shared common features of other plant zeta class GSTs. Genomic clone, PbGSTZ2 gene, is unexpectedly intronless. Extensive sequence analysis of PbGSTZ2, with cDNA clone, PbGSTZ1, revealed 87% identity at nucleotide and 81% identity at amino acid levels with 41 amino acids differences suggesting that genomic PbGSTZ2 gene might be an allelic or a paralogue version of PbGSTZ1., Tubitak; University of Anadolu Reseach Project, Thanks to Prof. Heinz Rennenberg and Dr Stanislav Kopriva from Forest Botanic and Tree Physiology Institute, Albert-Ludwigs University, Freiburg, Germany, for providing the initial EST sequences and their help in Northern blot and glutathione assays. Dr Elif Oztetik was supported by Tubitak short term fellowship. This work was financially supported by University of Anadolu Reseach Project.

Aim: Glutathione S-transferases (GST) take roles under stress conditions by the conjugation of Glutathione (GSH) to electrophilic substrates to increase their solubility and facilitating further metabolic processing. This is one of the main pathways functioning in stress resistance and, for dought stress, there is not yet a study on Pinus brutia Ten. which is a very important forest tree of Turkey. Materials and Methods: The needles of 30 different individuals of Pinus brutia Ten. were collected three times in the season: at the begining, in the middle and at the end of summer (June-August). The total precipitation and the temperature of the region were surveyed during sampling period. The osmotic pressure values, total GST activities and the GSH pools of the needle samples were measured and statistically analysed. Results: The osmotic pressure values showed 16% increase throughout the season. Moreover, total precipitation and temperature values demonstrated that the highest drought stress was observed at the end of sampling period. Approximately 80% of increase in total GST activity between the beginning and the end of summer probably indicated the response against drought; although there was no significant change in the GSH pool. Conclusion: The presence of drought, elevated osmotic pressure and a satistically significant (p

Aim: Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) is a polyphagous pest of a wide range of crops such as cotton, tomato and soybean. Pyrethroid insecticides have commonly used against it in agricultural areas, but excess amount applications of them result in resistance development in the field populations of H. armigera. Resistance development usually occurs with increased metabolism of certain enzymatic systems such as CYP450, GST and esterases. Therefore, expressions of selected CYP450, GST and esterase genes of H. armigera field populations (Adana and Mardin) were compared to those of a susceptible strain by real-time PCR method for analyzing role of these systems in pyrethroid resistance development of H. armigera.

Helicoverpa armigera is an insect that causes important economic losses in crops. To reduce this loss, pyrethroids have been commonly used against H. armigera in farming areas. However, excess and continuous usage of pyrethroids cause resistance in H. armigera. Therefore, expressions of midgut proteins of two H. armigera field populations were compared to those of a susceptible strain by 2-D PAGE and MALDI-ToF-MS. Our results indicate that H. armigera reacts to pyrethroid-induced stress mainly by increasing the expression of energy metabolism-related proteins, such as ATP synthase and arginine kinase. NADPH cytochrome P450 reductase, also up-regulated, could play a role in detoxification of toxic pyrethroid metabolites, such as 3-phenoxybenzaldehyde. Interestingly, while GSTs were not found up-regulated in the comparative proteome analysis, biochemical assays showed significant increases of enzyme activities in both field populations as compared to the susceptible strain. Similarly, although esterases were not found differentially expressed, biochemical assays showed significant increases of esterase activities in both field populations. Thus, esterases are also proposed to be involved in metabolic responses towards pyrethroid insecticide-induced stress. In conclusion, we suggest increased energy metabolism in the midgut tissue of H. armigera as a general prerequisite for compensating the costs of energy-consuming detoxification processes. (C) 2013 Elsevier Inc. All rights reserved.

Gundelia tournefortii L. is an important food source and a well-known medicinal plant in Eastern Anatolia. Therapeutic effects of medicinal plants are known to be closely related to their antioxidant capacities. Antioxidant activities of G. tournefortii, both for the aerial parts and seeds, were investigated by using both 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and lipid peroxidation inhibition methods. The seeds were found to have higher antioxidant potential than the aerial, with IC50 values of 0.073 mg/mL for DPPH scavenging and 0.146 mg/mL for lipid peroxidation inhibition capacities. In addition, total phenolic contents of the Gundelia tournefortii L. extracts, especially the seed extracts correlates to its high antioxidant activity with 105.1 +/- 8.7 mu g gallic acid equivalents (GAEs) per mg of seed extract. Plant extracts with high phenolics content are known to have important effects on various enzymes, as well as glutathione-S-transferases, which are important detoxification enzymes in phase 11 systems with an important role in developing multi-drug resistance to chemotherapy in tumour cells. Consequently, the effects of G. tournefortii extracts on crude cytosolic glutathione-S-transferase was also studied and the seed extracts have shown effective inhibition of cytosolic GST activity, with an IC50 of 97.51 mu g/mL. (c) 2005 Elsevier Ltd. All rights reserved.

Sister chromatid exchanges (SCEs) were analyzed in human phytohemagglutinin-activated peripheral lymphocyte cultures exposed to varying concentrations (10 −7 –10 −3 M) of cadmium chloride in vitro at two different stages of the cell cycle, G o and early S phase. When cadmium chloride was administered at the G o phase, no increase in the SCEs were observed for the doses 10 −6 and 10 −5 M. Concentrations equal to or larger than 10 −4 M cadmium chloride were lethal to human lymphocytes in our experimental conditions. A highly statistically significant increase was observed in the SCE frequency with increasing cadmium chloride concentration (10 −7 –10 −4 ) when cadmium was administered at the early S phase, which was 24 h after culture initiation. The increase in SCE frequency was higher when the cultures were terminated at 54 h, compared to termination at 72 h. In order to examine the effects of cadmium administered at the S phase on SCE frequency in different individuals, 10 −5 M concentration was used and the cultures were terminated at 54 h after culture initiation. A 2- to 3-fold increase in the SCE frequency was observed in all six individuals examined. A progressive decrease in the proliferative index was also observed by increasing cadmium chloride concentration. These results demonstrate that the genotoxicity of cadmium chloride may be changed depending on the stage of the cell cycle in human lymphocytes. This may be one of the reasons of contradictory findings in the literature.

Single-strand breaks were observed in rat lung and kidney after acute treatment of animals with CdCl2 (4 mg/kg body weight) injected intraperitoneally and NiCl2, (44.4 mg/kg body weight) injected subcutaneously. In the rat liver, no single-strand breakage was evident with those doses in single and combined metal treatments. The most susceptible tissue in rats to cadmium or nickel chloride treatment was the lung tissue. The single-strand breaks were higher in cadmium treatment than in nickel treatment in the rat lung. Also the response to cadmium treatment was obtained earlier than nickel. Rat kidney was also responsive to cadmium treatment. However, the response, although statistically significant, was much lower than the one obtained in rat lung. The combined treatment, which was done by administrating cadmium prior to nickel administration, reduced the number of single-strand breaks significantly and reversed them to control values in rat lung and kidney. This study confirms that cadmium and nickel create single-strand breaks when administered alone in the rat lung. This effect, which was seen in the single metal treatments, was reduced in the combined treatments.

Cell counts and classification of the cells play an important role in the field of microbiology and cell biology. Although there exists many counting processes for cells of interest in suspension, the most basic cell counting process is performed by a person via the microscope. For counting cells the simplest, widely used and the most economic method is the use of hemocytometer counting. In this study, the hemocytometer counting was used but the the cells were counted by a proposed image based approach. The developed technique herein uses neural network along with the Hough transform.

1. Nitrofurantoin reductase which catalyzes the bioactivation of nitrofurantoin was purified to electrophoretic homogenity from sheep liver and lung microsomes, with a yield of 15% and 35%, respectively. The specific activity of both reductases was found to be similar (140 nmol/min/mg protein). 2. The effects of nitrofurantoin and NADPH concentrations, pH, ionic strength, amount of enzyme and reaction period, on the enzyme activity were studied and the optimum conditions for maximum activity of purified liver and lung nitrofurantoin reductases were determined. 3. The enzyme concentration was found proportional with the square root of the rate of nitrofuratoin reduction up to approximately 15 micrograms protein/ml and 25 micrograms protein/ml incubation mixture for liver and lung nitrofurantoin reductases, respectively. 4. The plots of inverse of the nitrofurantoin concentration against the inverse of the square root of the velocity for the reduction of nitrofurantoin by liver and lung enzymes gave Km values as 27.78 microM and 32.25 microM, respectively. 5. The purified liver and lung enzymes were also saturated by NADPH at similar concentrations and the Km values were calculated as 29.4 microM and 35.5 microM, respectively. 6. The effects of magnesium, nickel, cadmium and copper ions on the nitrofurantoin reductase activity were examined. Magnesium ion was found to have almost no effect, whereas the other ions inhibited the activity of both liver and lung reductases.

Treatment of rabbits with benzene (880 mg/kg/day), s.c. for 3 consecutive days, caused 3.8- and 5.7-fold increases in aniline 4-hydroxylation rates of liver and kidney microsomes, respectively. Benzene treatment markedly enhanced hydroxylation rats of p-nitrophenol by liver and kidney by 7.2- and 4.2-fold, respectively. Both of these enzymes are associated with cytochrome P-450 LM3a. In contrast, the activity of benzphetamine N-demethylase, associated with P-450 LM2, was not altered significantly in either liver or kidney microsomes. Although the total cytochrome P-450 contents of liver and kidney microsomes were not altered significantly by the benzene treatment, in the case of liver microsomes, formation of a new cytochrome P-450 with an apparent Mr of 51,400 was observed on SDS-PAGE. On the other hand, in the kidney microsomes, the intensity of the bands corresponding to approximate Mr of 50,000 and 51,400 was markedly increased. The results of the present work, in combination with those of the previous work (Arinç et al. 1988), indicate the existence of tissue specificity in the induction of rabbit P-450 isozyme by benzene.

An introductory study was conducted to investigate the pyrethroid resistance of Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) strains in Turkey, collected from cotton fields in the Adana and Antalya provinces, through two different synthetic pyrethroid insecticides: lambda-cyhalothrin and esfenvalerate. In addition, the roles of glutathione S-transferases (GSTs) in this resistance mechanism were analyzed. It was found that whereas resistance ratios for lambda-cyhalothrin (LD50 levels) were 3- and 98-fold increased in the Adana and Antalya strains, respectively, esfenvalerate ratios were 3.3- and 92.3-fold increased in the Adana and Antalya strains, respectively, with respect to the susceptible strain. Furthermore, Adana and Antalya strains showed 2.4- and 2.9-fold higher GST activities than the susceptible strain, respectively. In the Antalya field strain, the minor increase in GST activity compared with the resistance levels implies that GSTs may be not greatly involved in this resistance. It also provides evidence that they could not be the only metabolic mechanism responsible for resistance to lambda-cyhalothrin and esfenvalerate in H. armigera from Turkey.

This work describes the development of a heavy-metal biosensor based on either recombinant 6His-Tag glutathione S-transferase (GST-(His)(6)) or glutathione S-transferase Theta 2-2 (GST-theta 2-2), and a capacitive transducer. The dynamic range of the pure bovine liver GST-Theta 2-2 biosensor was 1 fM to 1mM for Zn2+, and 10pM to 1mM for Cd2+. The GST-(His) 6 biosensor was able to detect Zn2+ and Cd2+ in the range of 1 fM to 10 mu M, and Hg2+ in the range of 1 fM to 10mM. The bovine liver GST Theta 2-2 biosensor displays an increased selectivity and a wider dynamic range for Zn2+ compared with the GST-(His) 6 biosensor. Therefore, by using different GST isozymes, it is possible to modulate important characteristics of capacitive biosensors for the detection of heavy metals.

Alkylphenol polyethoxylates (APEs) are widely used as nonionic surfactants. Nonylphenol (NP), one of the derivatives of APEs, has been found in the aquatic environment in ranges from nanograms per liter to milligrams per liter. In this study, juvenile rainbow trout were exposed to 0 (control), 66, 220, or 660 micro g NP/L for up to 28 days. Fish remained healthy under NP exposures of 0, 66, and 220 micro g/L for the length of the experiment. All fish died after 4 days of exposure to 660 micro g NP/L. Time-dependent NP bioaccumulation was detected in the tissues of fish exposed to 220 micro g NP/L (P0.05) and histopathological changes were observed in the livers of fish exposed to 220 micro g NP/L. Furthermore, an increase in the activity of glutathione-S-transferase (GST) was found in the liver of fish exposed to 220 micro g NP/L for 1 week (P0.05). There was an increase in GST activity in the liver of fish exposed to 66 micro g NP/L but it did not occur before 2 weeks of exposure to NP. The GST activity then decreased in a time-dependent manner in treatment groups, and this decrease was lower in the livers of fish treated with 66 and 220 micro g NP/L than in control fish after 3 weeks of exposure (P0.05). These results indicated that sublethal doses of NP were accumulating in the bodies of the fish and causing histopathological and biochemical changes in the livers of rainbow trout.

The involvement of the Y chromosome in sex determination was determined by the development and the application of techniques for karyotyping the mammalian chromosome in 1960s. There were many reports on the particular region of the Y chromosome, such as histocompatibility (H-Y) antigen, bandit krait minor satellite (Bkm) the zinc finger Y gene (ZFY) and the sex-determining region of the Y chromosome (SRY) which were believed to be the testis determining factors. However, converging experimental evidence have indicated that the sex determining region of the Y chromosome (sry) is the testis determining factor (TDF) in mammalian species since sex is determined genetically at the time of fertilization in these species. In non-mammalian vertebrates especially in fishes, amphibians and reptiles, genotypic sex can be overridden by the external application of steroid hormones and temperature. In this review paper, after reviewing the complex literature on the molecular and biochemical mechanisms of sex determination and differentiation in all vertebrates, the potential danger of environmentally induced sex determination will be focused on.

Alkylphenol polyethoxylates (APEs) called environmental endocrine disruptors has been shown to accumulate in water around the world. In this study, the pollution level of alkylphenolic compounds was measured and quantified in water, sediment, and the tissues of fishes collected in two rivers, Sakarya and Degirmendere Rivers, Turkey. Butylphenol (BP) were detected in sediment samples at one sampling stations of both rivers with 1.68 and 3.15 μg/g sediment, while nonylphenol (NP) were detected with the amount of 4.46 μg/g sediment in one sampling station in Degirmendere river. Fish samples also showed the presence of alkylphenolic compounds in both rivers. The level of alkylphenol pollution in two rivers of Turkey was determined to be in the range of alkylphenol level reported in Europe but lower than that of in the USA.

Rheum species are medicinally important plants due to the presence of anthracene derivatives. This study was designed to determine the antioxidative, cytotoxic and apoptotic properties of Rheum ribes shoot and root ethyl acetate extracts using human promyelocytic leukemia (HL-60) cell line as a model system. R. ribes shoot and root dry powder samples were prepared and extracted with ethyl acetate. The extracts were revealed to be a potential scavenger of DPPH radicals (IC50 value of 206.28 μg/ml for shoot and 10.92 μg/ml for root) and the chemical composition of the extracts was quantified by colorimetric determination of total phenol (GAE) and flavonoid (CAE) contents. HL–60 cells were cultured in the presence of various concentrations of extracts up to 72 h. R. ribes inhibited the survival of HL-60 cells in a concentration- and time-dependent manner, shown by XTT assay. R. ribes caused HL-60 cells apoptosis via formation of phosphatidylserine externalization, as evidenced by flow cytometry. Exposure of HL-60 cells to higher concentrations of extracts for 72 h resulted in a shift of 87% of the cell population from normal to the early/late apoptotic stage. These findings suggest that Rheum ribes ethyl acetate root extracts exhibits potential antioxidant and cytotoxic properties against HL-60 cells better than shoot extracts and exert their toxicity via induction of apoptosis.

Humans are chronically exposed to differing doses of complex chemical mixtures from their environment. This exposure has become so severe that it increases with every technological innovation and, unfortunately, reflects itself in the high incidence of cancer. Cd2+ and Ni2+ are widely found metals in our environment and they have been classified as human carcinogens by the International Agency for Research on Cancer [1–2]. Exposure to them could be occupational or non-occupational via inhalation (major route), ingestion, dermal or oral ways. In those not occupationally exposed, food represents the most important source, with tobacco an additional important source in smokers [3–5].

When male rats were given either a single dose of cadmium (3.58 mg CdCl 2 ·6H 2 O kg , i.p.) 72 h prior to sacrifice or a single dose of nickel (59.5 mg NiCl 2 ·6H 2 O kg , s.c.) 16 h prior to sacrifice, the activities of ethylmorphine N-demethylase, aminopyrine N-demethylase and aniline 4-hydroxylase, and the levels of cytochrome P-450 and microsomal heme were significantly decreased. Cadmium decreased the cytochrome b5 level significantly, whereas it did not alter the NADPH-cytochrome c reductase activity significantly. In contrast, Ni did not alter the cytochrome b5 level significantly but decreased the NADPH-cytochrome c reductase activity significantly. For the combined treatment, animals received the single dose of nickel 56 h after the single dose of cadmium and then they were killed 16 h later. In these animals ethylmorphine N-demethylase, aminopyrine N-demethylase and NADPH-cytochrome c reductase activities and cytochromes P-450 and b5 levels increased significantly as compared to those of controls, whereas aniline 4-hydroxylase activity and microsomal heme level remained unaltered. In concordance with the increase in the enzyme activities, certain P-450 protein bands were observed to be elevated when studied on SDS-polyacrylamide gel electrophoresis. Furthermore, when the monooxygenase activities and SDS-polyacrylamide gel electrophoresis profiles of combined metal-treated animals were compared with those of the animals treated with classic inducers such as phenobarbital (75 mg kg i.p., 72, 48 and 24 h prior to sacrifice) and 3-methylcholanthrene (20 mg kg i.p., 72, 48 and 24 h prior to sacrifice), the combination of metals seemed to have tendency to stimulate certain phenobarbital and 3-methylcholanthrene inducible cytochrome P-450 isozymes.

The sulphation of bile acids is an important pathway for the detoxification and elimination of bile acids during cholestatic liver disease. A dehydroepiandrosterone (DHEA) sulphotransferase has been purified from male and female human liver cytosol using DEAE-Sepharose CL-6B and adenosine 3′,5′-diphosphate-agarose affinity chromatography [Falany, Vazquez & Kalb (1989) Biochem. J. 260, 641-646]. Results in the present paper show that the DHEA sulphotransferase, purified to homogeneity, is also reactive towards bile acids, including lithocholic acid and 6-hydroxylated bile acids, as well as 3-hydroxylated short-chain bile acids. The highest activity towards bile acids was observed with lithocholic acid (54.3 +/- 3.6 nmol/min per mg of protein); of the substrates tested, the lowest activity was detected with hyodeoxycholic acid (4.2 +/- 0.01 nmol/min per mg of protein). The apparent Km values for the enzyme are 1.5 +/- 0.31 microM for lithocholic acid and 4.2 +/- 0.73 microM for taurolithocholic acid. Lithocholic acid also competitively inhibits DHEA sulphation by the purified sulphotransferase (Ki 1.4 microM). No evidence was found for the formation of bile acid sulphates by sulphotransferases different from the DHEA sulphotransferase during purification work. The above results suggest that a single steroid sulphotransferase with broad specificity encompassing neutral steroids and bile acids exists in human liver.

1. 1. Reductase was purified to electrophoretic homogeneity from sheep liver and lung microsomes. The specific activity of both enzymes ranged from 55 to 66 μ mol cytochrome c reduced/min/mg protein. 2. 2. Liver and lung reductases appeared to have similar kinetic and spectral properties. K m , (NADPH) and K m (cytochrome c ) values were calculated to be 14.3 ± 1.23 μ M and 22.2 ± 2.78 μ M for liver and 11.1 ±0.70 μ M and 20.0 ± 2.15 μ M for lung reductase, respectively. Kinetic studies showed that cytoehrome c can bind the oxidized form of the enzyme as well as its reduced form and both reductases operated through a ping-pong type mechanism. 3. 3. These reductases cannot be distinguished on the basis of monomer molecular weights (M r 78,000) except that the liver reductase was found to be more susceptible to proteolytic attack. 4. 4. Both reductases supported aniline 4-hydroxylation and ethylmorphine N -demethylation reactions to the same extent in the reconstituted systems. However, sheep lung reductase appeared only 36.5 and 14.8% as effective in catalyzing benzo[ a ]pyrene reaction as an equivalent amount of reductase from liver in the presence of liver cytochrome P -450 and 3MC-treated rat liver cytochrome P -448, respectively.

1. 1. NADPH-cytochrome c reductase was purified to electrophoretic homogeneity from detergent solubilized sheep lung microsomes. 2. 2. The specific activity of the purified enzyme ranged from 56 to 67 μmol cytochrome c reduced/min/mg protein and the yield was 48–52% of the initial activity in lung microsomes. 3. 3. The reductase had Mr of 78,000 and contained 1 mol each of FAD and FMN. 4. 4. Km values obtained in 0.3 M phosphate buffer, pH 7.8 at 37°C for NADPH and cytochrome c were 11.1±0.70 μM and 20.0±2.15 μM. Lung reductase was inhibited by its substrate, cytochrome c when its concentration was above 160 μM. 5. 5. The lung reductase exhibited a ping-pong type kinetic mechanism for NADPH mediated cytochrome c reduction. 6. 6. Purified lung reductase was biocatalytically active in supporting benzo(a)pyrene hydroxylation reaction when coupled with lung cytochrome P-450 and lipid.

1. The effects of eight newly synthesized 2-aryl substituted benzimidazole derivatives on control and phenobarbital (PB) treated rat liver microsomal aniline 4-hydroxylase and ethylmorphine N-demethylase activities, and their binding to control and PB-treated rat liver microsomal oxidized cytochrome P-450 are presented. 2. All compounds inhibited ethylmorphine N-demethylase activity with I50 values ranging from 8.50 × 10−4 M to 27.83 × 10−4 M in control and ranging from 2.80 × 10−4 M to 15.79 × 10−4 M in PB-treated rats. 3. Aniline 4-hydroxylase activity was inhibited by all of the compounds tested having I50 values in the range of 7.04 × 10−4 M-31.37 × 10−4 M in PB-treated rats, but only five of the compounds showed inhibitory activity in control rats. 4. Only a few significant regression coefficients could be found between the parameters of the chemicals studied and their inhibitory patterns. 5. No correlation has been observed between the binding of the derivatives and their inhibitory pattern.

1. The specific activity of the aniline 4-hydroxylase which catalyses hydroxylation of aniline to p-aminophenol was found to be 0.65 (N = 10) and 0.15 (N = 13) nmol p-aminophenol formed/mg protein, min. in sheep liver and lung microsomes, respectively. 2. The effects of aniline concentration. pH. cofactors. amount of enzyme and incubation period, on enzyme activity were studied, and the optimum conditions for maximum activity of liver and lung microsomes were determined. 3. Liver and lung microsomal aniline 4-hydroxylase activity was found to be completely dependent on the presence of cofactor NADPH. 4. The Lineweaver-Burk and Eadie-Hofstee plots of the liver enzyme were found to be curvilinear, suggesting that the enzyme did not follow the Michaelis-Menten kinetics. From these graphs, two different Km values were calculated for the liver enzyme as 3.21 and 0.072 mM aniline. Km of the lung enzyme was calculated to be 1.43 mM aniline from its Lineweaver-Burk graph. 5. The effects of magnesium, nickel and cadmium ions on the liver and lung aniline 4-hydroxylase activity were examined. Magnesium ion was found to have stimulatory effect, whereas nickel and cadmium ions inhibited the activity of the both liver and lung enzyme.