• BMB Reports
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• v.37 no.3
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• pp.275-281
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• 2004

Rhodanese was isolated and purified from the cytosolic fraction of liver tissue homogenate of the fruit bat, Eidolon helvum, by using ammonium sulphate precipitation and CM-Sephadex C-50 ion exchange chromatography. The specific activity was increased 130-fold with a 53% recovery. The $K_m$ values for KCN and $Na_2S_2O_3$ as substrates were $13.5{\pm}2.2\;mM$ and $19.5{\pm}0.7\;mM$, respectively. The apparent molecular weight was estimated by gel filtration on a Sephadex G-100 column to be 36,000 Da. The optimal activity was found at a high pH (pH 9.0) and the temperature optimum was $35^{\circ}C$. An Arrhenius plot of the heat stability data consisted of two linear segments with a break occurring at $35^{\circ}C$. The apparent activation energy values from these slopes were 11.5 kcal/mol and 76.6 kcal/mol. Inhibition studies on the enzyme with a number of cations showed that $Mg^{2+}$, $Mn^{2+}$, $Ca^{2+}$, and $Co^{2+}$ did not affect the activity of the enzyme, but $Hg^{2+}$ and $Ba^{2+}$ inhibited the enzyme.

• BMB Reports
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• v.32 no.1
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• pp.67-71
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• 1999

We have investigated the effect of cholestasis on the closely related acyl-CoA:amino acid N-acyltransferase, benzoyltransferase, and phenylacetyltransferase activities in rat liver. Benzoyltransferase and phenylacetyltransferase activities in the liver cytosol, mitochondria, and microsome were investigated for a period of 42 d after common bile duct ligation. Both the mitochondrial and microsomal benzoyltransferases showed significant increase in their activities between the 1st and 7th day after common bile duct ligation, although the cytosolic benzoyltransferase activity did not show a significant change compared to the activities from the sham-operated control. The cytosolic phenylacetyltransferase activity showed a significant increase between the 1st and 2nd day, the mitochondrial activity showed a significant increase between the 2nd and 7th day, and microsomal activity showed a significant increase between the 1st and 7th day, respectively. Enzyme kinetic parameters of hepatic benzoyltransferase were analyzed using benzoyl coenzyme A as a substrate with the preparations from the 1st day post-ligation. Enzyme parameters of hepatic phenylacetyltransferase were also analyzed using phenylacetyl coenzyme A as a substrate with the preparations from the 2nd day post-ligation. The results indicated that although the $K_m$ values of these enzymes were about the same as the sham-operated control, the $V_{max}$ values of both enzymes increased significantly. These results, therefore, suggest that the biosynthesis of benzoyltransferase and phenylacetyltransferase has been induced in response to cholestasis.

• Archives of Pharmacal Research
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• v.20 no.6
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• pp.519-524
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• 1997

Steroid $9{\alpha}$-hydroxylase is an enzyme found in nocardioform microorganisms which can utilize steroids as a sole carbon source. After fractional centrifugation of the cell homogenates, the enzyme activity in Nocardia and Rhodococcus was found in cytoplasmic membrane fraction. On the contrary, Mycobacterium had its 9.alpha.-hydroxylation activity in cytosolic fraction. To characterize the enzyme in these microorganisms, several potential inhibitors of 9.alpha.-hydroxylase were tested and the cofactor requirement for the same enzyme was also examined. The inhibitory effect of ferrous ion chelators indicated involvement of iron containing proteins in the 9.alpha.-hydroxylase system. On the other hand, metyrapone, an inhibitor known to be specific for cytochrome P450 interfered with the enzyme in Mycobacterium, but didn't inhibit the enzyme activity in Nocardia and Rhodococcus. While the $9{\alpha}$-hydroxylase system in Nocardia and Rhodococcus required NADPH, NADH was required as an election donor in Mycobacterium.

• Bulletin of the Korean Chemical Society
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• v.25 no.5
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• pp.711-714
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• 2004

Peroxynitrite enters erythrocytes through band 3 anion exchanger and oxidizes cytosolic proteins therein. As a protein associated with band 3, carbonic anhydrase II may suffer from peroxynitrite-induced oxidative damages. Esterase activity of carbonic anhydrase II decreased as the concentration of peroxynitrite increased. Neither hydrogen peroxide nor hypochlorite affected the enzyme activity. Inactivation of the enzyme was in parallel with the release of zinc ion, which is a component of the enzyme's active site. SDS-PAGE of peroxynitrite-treated samples showed no indication of fragmentation but non-denaturing PAGE exhibited new bands with lower positive charges. Western analysis demonstrated that nitration of tyrosine residues increased with the peroxynitrite concentration but the sites of nitration could not be determined. Instead MALDI-TOF analysis identified tryptophan-245 as a site of nitration. Such modification of tryptophan residues is responsible for the decrease in tryptophan fluorescence. These results demonstrate that peroxynitrite nitrates tyrosine and tryptophan residues of carbonic anhydrase II without causing fragmentation or dimerization. The peroxynitrite-induced inactivation of the enzyme is primarily due to the release of zinc ion in the enzyme's active site.

• Preventive Nutrition and Food Science
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• v.3 no.1
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• pp.62-66
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• 1998

The effect of dietary capsaicin (8-methyl-N-vanillyl-6-nonenamide, CAP) on drug-metabolizing enzyme activities was investigated in mice. Male ICR mice were divided into 4 groups and fed diets containing 0, 5, 20, 100 ppm CAP for 4 seeks. Hepatic drug-metabolizing enzyme activities and serum alanine aminotransferase and aspartate transaminease activities were measured. There was no difference in hepatic alanine aminotransferse and aspartate transaminase activities among the groups. Hepatic microsomal cytochrome P450 in CAP fed groups, but p-nitrophenol hydroxylase and the cytosolic acitivity of glutathione S-transferase activities were decreased in the dietary CAP supplemetned groups compared to the control. These results suggest that the dietary CAP at a low dose differentially modulates drug-metabolizing enzyme acitvities without causing hepatic toxicity.

• Journal of Nutrition and Health
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• v.27 no.5
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• pp.442-450
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• 1994

The purpose of this study was to determine the effects of 2-AAF injection time on hepatic lipid peroxide metabolism and cytochrome P450 content in Sprague-Dawley rats fed diets containing high amounts of vegetable oils or animal fats(15%, w/w). Fifty mg of 2-AAF/kg of body weight/day was injected in PEG 300 intraperitonially for 3 consecutive days after 4 or 8 weeks to rats fed corn oil(CO) or lard(LA) diet. The contents of lipid peroxide and cytochrome P450, and the activities of superoxide dismutase(SOD), glutathione peroxidase(GSH-peroxidase) and glutathione S-transferase(GSH-S-transferase) were determined in hepatic microsomal or cytosolic fraction. Microsomal thiobarbituric acid reactive substances(TBARS) and cytochrome P450 contents increased in Co group injected 2-AAF after 4weeks. Cytosolic SOD activity increased in CO group injected 2-AAF after 4 weeks and in LA group injected 2-AAF after 4 or 8 weeks. Cytosolic GSH-S-transferase activity increased in LA group compared to CO group without 2-AAF injection. GSH-S-transferase activity increased in CO group injected 2-AAF after 4 or 8 weeks and in LA group injected 2-AAF after 4 weeks. Therefore, it may be suggested that 2-AAF injection increase the contents of lipid peroxide or cytochrome P450, and detoxifying enzyme activities in rats fed CO diet for short period and in rats fed LA diet for longer period.

• Journal of Microbiology
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• v.34 no.2
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• pp.206-213
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• 1996

NADH-quinone reductase was purified 22-fold from the cytosolic fraction of Streptomyces sp. Imsnu-1 to apparent hemogenity, with an overall yield of 9%, by the purification procedure consisting of ammonium, sulfate precipitation and DEAE Sephacryl S-200 and DEAE 5 PW chromatographies. Thes molecular mass of the enzyme determined by gel filtration chromatography was found to be 110 kDa. SDS-PAGE revealed that the enzyme consists of two sugunits with a molecular mass of 54 kDa. The enzyme contained 1 mol of FMN per subunit as a cofactor. The $A_{272}$ A$_{457}$ ratio was 6.14 and the molar extinction coefficients were calculated to be 20, 800 and 25, 400M$^{-1}$ $cm^{-1}$ / AT 349 AND 457 nm, respectively. The N-terminal sequence of the enzyme contained the highly conserved fingerprint of ADP-binding domain. The enzyme used NADH as an electron donor and various quinones as electron acceptors. Cytochrome c was practically inactive. Air-stable flavin semiquinone was produced by the addition of NADH to the enzyme. Also, naphthosemiquinone was detected in the reaction mixture containing the enzyme.

• The Korean Journal of Physiology and Pharmacology
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• v.1 no.2
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• pp.221-224
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• 1997

Effects of melatonin on hepatic glutathione-peroxidase (GSH-Px) and glutathione-reductase (GSH-reductase) activities were studied in Sprague-Dawley (SD) rats administered i.p. (10 mg/kg body weight) with melatonin during 15 days. The activity of cytosolic GSH-reductase in the liver was not changed by melatonin. However, melatonin injection increased significantly the activity of liver cytosolic GSH-Px activity compared with those in saline-treated rats. At the same time, plasma GSH-Px was also increased significantly in melatonin-treated rats. Since GSH-Px, a major antioxidative enzyme, removes $H_2O_2$ and lipid peroxides which are formed during lipid peroxidation from cellular membrane, such elevation of heptatic GSH-Px activity may contribute to the improvement of antioxidative effects under oxidative damage in the liver.

• BMB Reports
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• v.47 no.4
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• pp.209-214
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• 2014

Ultraviolet B (UVB) radiation induces the production of reactive oxygen species (ROS) that promote apoptotic cell death. We showed that cytosolic $NADP^+$-dependent isocitrate dehydrogenase (IDPc) plays an essential role in the control of cellular redox balance and defense against oxidative damage, by supplying NADPH for antioxidant systems. In this study, we demonstrated that knockdown of IDPc expression by RNA interference enhances UVB-induced apoptosis of immortalized human HaCaT keratinocytes. This effect manifested as DNA fragmentation, changes in cellular redox status, mitochondrial dysfunction, and modulation of apoptotic marker expression. Based on our findings, we suggest that attenuation of IDPc expression may protect skin from UVB-mediated damage, by inducing the apoptosis of UV-damaged cells.

• Toxicological Research
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• v.30 no.1
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• pp.33-38
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• 2014

The human cytochrome P450 2J2 catalyzes an epoxygenase reaction to oxidize various fatty acids including arachidonic acid. In this study, three recombinant enzyme constructs of P450 2J2 were heterologously expressed in Escherichia coli and their P450 proteins were successfully purified using a $Ni^{2+}$-NTA affinity column. Deletion of 34 amino acid residues in N-terminus of P450 2J2 enzyme (2J2-D) produced the soluble enzyme located in the cytosolic fraction. The enzymatic analysis of this truncated protein indicated the typical spectral characteristics and functional properties of P450 2J2 enzyme. P450 2J2-D enzymes from soluble fraction catalyzed the oxidation reaction of terfenadine to the hydroxylated product. However, P450 2J2-D enzymes from membrane fraction did not support the P450 oxidation reaction although it displayed the characteristic CO-binding spectrum of P450. Our finding of these features in the N-terminal modified P450 2J2 enzyme could help understand the biological functions and the metabolic roles of P450 2J2 enzyme and make the crystallographic analysis of the P450 2J2 structure feasible for future studies.