Hydroperoxide specificity of plant and human tissue lipoxygenase: an in vitro evaluation using N-demethylation of phenothiazines

Since hydroperoxide specificity of lipoxygenase (LO) is poorly understood at present, we investigated the ability of cumene hydroperoxide (CHP) and tert-butyl hydroperoxide (TBHP) to support cooxidase activity of the enzyme toward the selected xenobiotics. Considering the fact that in the past, studies of xenobiotic N-demethylation have focused on heme-proteins such as P450 and peroxidases, in this study, we investigated the ability of non-heme iron proteins, namely soybean LO (SLO) and human term placental LO (HTPLO) to mediate N-demethylation of phenothiazines. In addition to being dependent on peroxide concentration, the reaction was dependent on enzyme concentration, substrate concentration, incubation time, and pH of the medium. Using Nash reagent to estimate formaldehyde production, the specific activity under optimal assay conditions for the SLO mediated N-demethylation of chlorpromazine (CPZ), a prototypic phenothiazine, in the presence of TBHP, was determined to be 117±12 nmol HCHO/min/mg protein, while that of HTPLO was 3.9±0.40 nmol HCHO/min/mg protein. Similar experiments in the presence of CHP yielded specific activities of 106±11 nmol HCHO/min/mg SLO, and 3.2±0.35 nmol HCHO/min/mg HTPLO. As expected, nordihydroguaiaretic acid and gossypol, the classical inhibitors of LOs, as well as antioxidants and free radical reducing agents, caused a marked reduction in the rate of formaldehyde production from CPZ by SLO in the reaction media fortified with either CHP or TBHP. Besides chlorpromazine, both SLO and HTPLO also mediated the N-demethylation of other phenothiazines in the presence of these organic hydroperoxides.     

Lipoxygenase-mediated hydrogen peroxide-dependent N-demethylation of N,N-dimethylaniline and related compounds

To date, studies of xenobiotic N-demethylation have focused on heme-proteins such as P450 and peroxidases. In this study we investigated the ability of non-heme iron proteins, namely soybean lipoxygenase (SLO) and human term placental lipoxygenase (HTPLO) to mediate N-demethylation of N,N-dimethylaniline (DMA) and related compounds in the presence of hydrogen peroxide. In addition to being hydrogen peroxide dependent, the reaction was also dependent on incubation time, concentration of enzyme and DMA and the pH of the medium. Using Nash reagent to estimate formaldehyde production, we determined the specific activity for SLO mediated N-demethylation of DMA to be 200 + 18 nmol HCHO/min per mg protein or 23 +/- 2 nmol/min per nmol of enzyme, while that of HTPLO was 33 +/- 4 nmol HCHO/min per mg protein. Nordihydroguaiaretic acid (NDGA), a classical inhibitor of lipoxygenase (LO), as well as antioxidants and free radical reducing agents, caused a marked reduction in the rate of production of formaldehyde from DMA by SLO. Besides N,N-dimethylaniline, N-methylaniline, N,N,N',N'-tetramethylbenzidine, N,N-dimethyl-p-phenylenediamine, N,N-dimethyl-3-nitroaniline and N,N-dimethyl-p-toluidine were also demethylated by SLO. The formation of a DMA N-oxide was not detected. Preliminary experiments suggested SLO-mediated hydrogen peroxide-dependent S-dealkylation of methiocarb or O-dealkylation of 4-nitroanisole does not occur.     

UV-A induced DNA nicking activities of skin photosensitive drugs: phenothiazines, benzothiadiazines and afloqualone

Plasmid pBR 322 was subjected to UV-A irradiation in the presence of photosensitive drugs, i.e., phenothiazines [chlorpromazine hydrochloride (CPZ), promethazine hydrochloride (PMZ) and mequitazine (MQZ)], benzothiadiazines [penflutizide (PFZ), hydrochlorothiazide (HCT) and methyclothiazide (MCT)] and afloqualone (AQ). The distribution of the closed-circular and the open-circular form of the plasmid DNA was analyzed by means of neutral agarose gel electrophoresis. All the drugs used induced more or less DNA nicking to yield the open-circular form. The nicking activities of the phenothiazines were in the order: CPZ greater than PMZ greater than MQZ. CPZ elicited extensive degradation of the DNA by photosensitization. The nicking activities of the benzothiadiazines and AQ were much weaker than CPZ and PMZ.     

Amphetamine and chlorpromazine modify cerebral insulin levels in rats

Rats treated with chlorpromazine (CPZ) (1 mg/kg/day i.p.) experienced a marked decline in cerebral insulin levels (0.057 +/- 0.01 ng/g wet weight) with respect to a control group (0.38 +/- 0.05 ng/g wet weight), while rats given D-amphetamine bitartrate (AMPH) chronically (20 mg/kg/day p.o.) showed a rise in cerebral insulin (0.55 +/- 0.04 ng/g wet weight). Combined treatment with both drugs at the same dosages produced lower cerebral insulin levels (0.46 +/- 0.10 ng/g wet weight) than in the AMPH animals. In the groups of rats treated with CPZ and with AMPH + CPZ, there was a slight elevation in serum insulin levels. Serum glucose values did not vary.     

Characterization and modulation by drugs of sheep liver microsomal flavin monooxygenase activity

The flavin monooxygenases (FMO) catalyse the NADPH and oxygen-dependent oxidation of a wide range of nucleophilic nitrogen-, sulfur-, phosphorus-, and selenium heteroatom-containing chemicals, drugs, and agricultural agents. In the present study, sheep liver microsomal FMO activity was determined by measuring the S-oxidation rate of methimazole and the average specific activity obtained from different microsomal preparations was found to be 3.8 +/- 1.5 nmol methimazole oxidized min(-1) mg(-1) microsomal protein (mean +/- SE, n = 7). The presence of 0.1% Triton X-100 in the reaction mixture caused an increase of specific sheep liver microsomal FMO activity towards methimazole to 6.1 +/- 1.4 nmol methimazole oxidized min(-1) mg(-1) microsomal protein (mean +/- SE, n = 6). Metabolism of imipramine and chlorpromazine was measured by following the oxidation of cofactor NADPH spectrophotometrically at 340 nm. Sheep liver microsomal FMO activity towards imipramine and chlorpromazine was found to be 10.7 and 12.3 nmol NADPH oxidized min(-1) mg(-1) microsomal protein, respectively. Characterization of sheep liver enzyme was carried out using methimazole as substrate and the maximum FMO enzyme activity was detected at 37 degrees C and at pH 8.0. The apparent K(m) value of sheep liver microsomal FMO for methimazole was 0.118 mM. Effects of the detergents Triton X-100, Cholate, and Emulgen 913, on FMO activity were determined and FMO activity was found to increase with the addition of detergents to the reaction medium. Sheep liver microsomal FMO-catalysed methimazole oxidation was inhibited by imipramine and chlorpromazine when these drugs were used at high concentrations. Western blot-immunochemical analysis revealed the presence of FMO3 in sheep liver microsomes.     

31P NMR saturation-transfer study of the in situ kinetics of the mitochondrial adenine nucleotide translocase

The exchange of intramitochondrial ATP (ATP(in)) for extramitochondrial ATP (ATP(out)) was measured by using 31P NMR spectroscopy over a range of temperatures in isolated rat liver mitochondria oxidizing glutamate and succinate in the presence of external ATP but no added ADP (state 4). The rate of this exchange is more than an order of magnitude faster than rates reported previously that were determined by using isotopic techniques in the presence of oligomycin, the potent ATPase inhibitor. Differences are ascribed in part to the low levels of matrix ATP present in oligomycin-treated mitochondria. The addition of oligomycin to mitochondrial suspensions decreases intramitochondrial ATP levels from 17 +/- 3 (SEM) nmol/mg of protein in state 4 to 1.51 +/- 0.1 nmol/mg of protein in the presence of inhibitor at 8 degrees C. Simultaneously, transporter flux falls from 960 +/- 55 nmol/min.mg to undetectable levels (less than 300 nmol/min.mg). Although transport rates are much faster when measured by saturation-transfer than by conventional isotopic methods, the enthalpy values obtained by determining the effect of temperature on flux are very similar to those reported in the past that were determined by using isotopic techniques. Intramitochondrial ATP content regulates the rate of the ATP(in)/ATP(out) exchange. At 18 degrees C, the concentration of internal ATP that produces half-maximal transport rate is 6.6 +/- 0.12 nmol/mg of mitochondrial protein. The relationship between substrate concentration and flux is sigmoidal and is 90% saturated at 11.3 +/- 0.18 nmol/mg of mitochondrial protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Catalyzation of cocaine N-demethylation by cytochromes P4502B,P4503A,and P4502D in fish liver

Cocaine N-demethylation by microsomal cytochrome P450s is the principal pathway in cocaine bioactivation and hepatotoxicity. P450 isozymes involved in N-demethylation of cocaine have not been elucidated yet and they differ from species to species. In humans and mice, P4503A contributes to cocaine N-demethylase activity, whereas in rats, both P4503A and P4502B participate. In the present study, contribution of different P450 isozymes to cocaine N-demethylase activity was studied in vitro with fish liver microsomes. The specific cocaine N-demethylase activity was found to be 0.672 +/- 0.22 nmol formaldehyde formed/min/mg protein (mean +/- SD, n = 6). Cocaine N-demethylase exhibited biphasic kinetics, and from the Lineweaver-Burk plot, two K(m) values were calculated as 0.085 and 0.205 mM for the high- and low-affinity enzyme. These results indicate that N-demethylation of cocaine in mullet liver microsomes is catalyzed by at least two cytochrome P450 isozymes. Inhibitory effects of cytochrome P450 isozyme-selective chemical inhibitors, ketoconazole, cimetidine, SKF-525A, and quinidine, on cocaine N-demethylase activity were studied at 50, 100, and 500 micro M concentrations of these inhibitors. At 100 micro M final concentrations, ketoconazole (P4503A inhibitor), SKF-525A (inhibitor of both P4502B and P4503A), and cimetidine (P4503A inhibitor) inhibited N-demethylation activity by 73, 69, and 63%, respectively. Quinidine, P4502D-specific inhibitor, at 100 micro M final concentration, reduced N-demethylation activity down to 64%. Aniline, a model substrate for P4502E1, did not alter N-demethylase activity in the final concentration of 100 micro M. IC(50) values were calculated to be 20 micro M for ketoconazole, 48 micro M for cimetidine (both specific P4503A inhibitors), 164 micro M for quinidine (P4502D inhibitor), and 59 micro M for SKF-525A (inhibitor of both P4503A and P4502B). The contribution of P4502B to cocaine N-demethylase activity in mullet liver microsomes was further explored by the use of purified mullet cytochrome P4502B in the reconstituted system containing purified mullet P450 reductase and lipid. The turnover number was calculated as 4.2 nmol HCOH/(min nmol P450). Overall, these results show that P4503A and P4502B are the major P450s responsible for N-demethylation of cocaine, whereas contribution of P4502D is a minor one, and P4502E1 is not involved in the N-demethylation of cocaine in mullet liver microsomes.     

Peroxidatic catecholestrogen production by human breast cancer tissue in vitro

The ability of breast cancer tissues from postmenopausal women to form catechol estrogens was examined by using a product isolation assay. Initial assays were carried out in the presence of either: (a) NADPH, the co-factor for monooxygenase mediated catecholestrogen (CE) formation or; (b) light-activated Tween 80 (LAT-80), a putative organic hydroperoxide co-factor for peroxidatic activity. Under monooxygenase conditions, CE formation by homogenates of 10 tumors did not exceed that obtained with heat denatured tissue. In contrast, 17 of 20 tumors incubated with LAT-80 synthesized significant amounts of CE (8.5 +/- 1.17 2-hydroxyestradiol [2-OH-E2] and 12.8 +/- 2.4 nmol/g protein/10 min 4-hydroxyestradiol [4-OH-E2]). Substitution of cumene hydroperoxide, an organic hydroperoxide, for LAT-80 enhanced estrogen 2/4 hydroxylase (E-2/4-H) activity over 200-fold, making it possible to characterize systematically the peroxidatic activity. The properties of peroxidatic E-2/4-H activity were similar to those of soluble peroxidases isolated from brain, including an acidic pH optimum, localization in the soluble fraction, an apparent Km in the range of 80 microM and an apparent Vmax in the range of 4000 nmol/g/protein/10 min for both 2- and 4-OH-E2. Under optimal assay conditions, peroxidatic E-2/4-H activity was identified in 10 of 13 tumors (2480 +/- 580 nmol/g protein/10 min for 2-OH-E2 and 2790 +/- 600 for 4-OH-E2). The level of activity detected suggests a biological relevance for CE formation by breast cancer tissue.     

Hyperoxia enhances lung and liver nuclear superoxide generation

Porcine lung and liver nuclei generated superoxide (O-2) at a rate which increased with increasing oxygen concentration. NADH-dependent O-2 generation increased from 0 to 2.21 +/- 0.11 nmol/min per mg protein for lung nuclei and from 0.16 +/- 0.09 to 1.34 +/- 0.14 nmol/min per mg protein for liver nuclei, when oxygen concentration increased from 0 to 100%. NADPH-dependent O-2 generation increased similarly in liver nuclei (from 0.20 +/- 0.09 to 1.20 +/- 0.12 nmol/min per mg protein), while lung nuclei produced only 0.45 +/- 0.09 nmol/min per mg protein at 100% oxygen. NADH and NADPH had an additive effect on O-2 generation by liver nuclei, yielding 2.58 +/- 0.21 nmol/min per mg protein at 100% oxygen. Very little or no superoxide dismutase activity was present in washed nuclear preparations. The oxygen-dependence of nuclear O-2 generation shows that nuclear-derived partially reduced species of oxygen may affect nuclear function during hyperoxia or other metabolic situations where overproduction of oxygen radicals is problematic.     

Lipoxygenase-another pathway for glutathione conjugation of xenobiotics: A study with human term placental lipoxygenase and ethacrynic acid.

In this study, we examined the ability of human term placental lipoxygenase (HTPLO) to catalyze glutathione (GSH) conjugate formation from ethacrynic acid (EA) in the presence of linoleic acid (LA) and GSH. HTPLO purified by affinity chromatography was used in all the experiments. The results indicate that the process of EA-SG is enzymatic in nature. The reaction shows dependence on pH, the enzyme, and the concentration of GSH, LA, and EA. The optimal assay conditions to observe a maximal rate of EA-SG formation required the presence of 0.3 mM LA, 0.2 mM EA, 2.0 mM GSH, and approximately 300 microg HTPLO in the reaction medium buffered at pH 9.0. Under the experimental conditions employed, the reaction exhibited K(m) values of 1.1 mM, 200 microM, and 130 microM for GSH, LA, and EA, respectively. The estimated specific activity of HTPLO-catalyzed EA-GS formation was approximately 4.4 +/- 0.4 micromol/min/mg protein. This rate is more than twofold greater than the rate noted for the reaction mediated by the purified human term placental glutathione transferase. Under physiologically relevant conditions (20 microM LA, 2.0 mM GSH, at pH 7.4), HTPLO produced EA-SG at 56% of the maximal rate noted under optimal assay conditions. Nordihydroguaiaretic acid, the classical inhibitor of different lipoxygenases, significantly blocked the reaction. It is proposed that free radicals are involved in the process of EA-SG formation by HTPLO. The evidence gathered in this in vitro study suggests for the first time that lipoxygenase present in the human term placenta is capable of EA-SG formation.     

Stimulation of CTP: phosphocholine cytidylyltransferase and phosphatidylcholine synthesis by incubation of rat hepatocytes with phospholipase A2

The effect of phospholipase A2 treatment of rat hepatocytes on CTP: phosphocholine cytidylyltransferase and phosphatidylcholine synthesis was investigated. Cytidylyltransferase is recovered from the cytosol and in a membrane-bound form with the microsomes. Digitonin treatment of cells causes rapid release into the medium of the cytosolic, but not the microsomal form of the cytidylyltransferase. Incubation of hepatocytes for 10 min with phospholipase A2 (0.9 units/dish) in the medium, resulted in a 33% decrease in the cytidylyltransferase activity released by digitonin treatment (2.5 +/- 0.15 nmol/min per mg compared to 3.9 +/- 0.10 nmol/min per mg in the control). In agreement with the digitonin experiments, incubation with 0.9 units/dish of phospholipase A2 resulted in a decrease in the cytidylyltransferase activity in the cytosol (from 4.3 +/- 0.10 nmol/min per mg to 2.6 +/- 0.14 nmol/min per mg) and a corresponding increase in the microsomal fraction (from 0.9 +/- 0.16 nmol/min per mg to 1.8 +/- 0.20 nmol/min per mg). The effect of phospholipase A2 on cytidylyltransferase translocation was concentration- and time-dependent. Incubation of hepatocytes in the presence of phospholipase A2 (0.9 units/dish) for 10 min prior to pulse-chase experiments resulted in an increase in radiolabel incorporation into phosphatidylcholine (from 2.4 +/- 0.02.10(-5) dpm/dish to 3.1 +/- 0.1.10(-5) dpm/dish) and a corresponding decrease in radiolabel associated with the choline (from 2.5 +/- 0.05.10(-5) to 1.4 +/- 0.03.10(-5) dpm) and phosphocholine fractions (from 8.5 +/- 0.07.10(-5) to 6.9 +/- 0.05.10(-5) dpm). We conclude that phospholipase A2 can cause a stimulation of CTP: phosphocholine cytidylyltransferase activity and phosphatidylcholine synthesis in cultured rat hepatocytes.     

Chlorpromazine induced aggregation of normal human hemoglobin. Electron microscopic evidence.

Normal human hemoglobin exceeding a certain minimum concentration (called critical aggregation concentration) undergoes aggregation in presence of the psychotherapeutic drug chlorpromazine (CPZ). The critical aggregation concentration decreases with the increase of CPZ concentration. Electron micrographs of CPZ-treated hemoglobin clearly indicate that the aggregates of hemoglobin are in filamentous form of average width 75 +/- 8 A. A possible mechanism for such aggregation has been discussed.     

Direct effect of the luteinizing hormone releasing hormone analog D-Trp6-Pro9-Net-LHRH on rat ovarian steroidogenesis

The luteinizing hormone releasing hormone analog D-Trp6-Pro9-Net-LHRH (LHRHa) inhibits rat ovarian estradiol secretion. To determine whether LHRHa decreases serum estradiol concentrations solely by inhibiting gonadotropin secretion or, in addition, by influencing directly ovarian estradiol biosynthesis, we examined the effects of LHRHa on the activities of 5 key ovarian steroidogenic enzymes. Fifty hypophysectomized, gonadotropin-treated rats were given either LHRHa (1 microgram/day) or saline sc during 7 days. The LHRHa treated animals exhibited a significant decrease in serum estradiol when compared with the control group (461 +/- 30 vs 31 +/- 5 pg/ml, mean +/- SE, P less than 0.001). The changes in estradiol concentration were associated with decreases in ovarian weight (372 +/- 19 vs 185 +/- 11 mg, P less than 0.001) and in the microsomal enzyme activities of 3 beta-hydroxysteroid dehydrogenase (156 +/- 5 vs 53 +/- 4 nmol/mg prot/min, P less than 0.001), 17 hydroxylase (4.7 +/- 0.8 vs 3.7 +/- 0.7 nmol/mg prot/min, P less than 0.002), 17,20 desmolase (279 +/- 14 vs 50 +/- 7 pmol/mg prot/min, P less than 0.001), 17 keto-steroid reductase (132 +/- 11 vs 6 +/- 1 nmol/mg prot/min, P less than 0.001), and aromatase (19 +/- 1.5 vs 0.9 +/- 0.1 nmol/mg prot/min, P less than 0.001) in LHRHa treated animals. These findings indicate that LHRHa can inhibit directly rat ovarian estradiol biosynthesis.     

Evidence for the presence of active cytochrome P450 systems in Schistosoma mansoni and Schistosoma haematobium adult worms

Extracts of the adult worms of both Schistosoma mansoni and Schistosoma haematobium can metabolise some typical P450 substrates but to differing degrees. S. mansoni worm extracts displayed a approximately 12-fold higher specific activity for an aminopyrine substrate than rat liver microsomes. At 4 mM substrate concentration the demethylation reaction with N-nitrosodimethylamine (NDMA) (5 nmol HCHO/mg protein/min) was only half that of rat liver microsomes, whereas in extracts of S. haematobium, no detectable activity was found towards NDMA. Using ethylmorphine as substrate the demethylation activity of S. mansoni extracts (1.82 nmol HCHO/mg protein/min) was 5.5-fold lower than that of rat liver microsomes. Benzphetamine demethylase activity was also readily detectable in S. mansoni worm extracts at 6.79 nmol HCHO/mg protein/min compared with 10.20 nmol HCHO/mg protein/min in the case of rat liver microsomes. When aniline was used as substrate, surprisingly, no activity was found in worm extracts of either S. mansoni or S. haematobium, whereas rat liver microsomes showed high activity towards this amine. The anti-P450 2E1 and 2B1/2 cross-reacted with both worm homogenates and gave a specific band corresponding to a protein of molecular weight of approximately 50.0 kDa. A study with anti-P450 IVA antibody revealed that while this protein was strongly expressed in S. haematobium worm extracts, no immunoreactivity was observed with extracts of S. mansoni. Immunoblotting analyses with anti-P450 IIIA and P450 1A1 did not detect immunoreactive protein in either S. mansoni or S. haematobium.     

Changes in serum testosterone and estradiol concentrations following acute androstenedione ingestion in young women.

The effect of androstenedione intake on serum hormone concentrations in women is equivocal. Therefore, we examined the hormonal response to androstenedione intake in healthy young (22.1 +/- 0.4 y) women for 4 hours. On day 3 of the follicular phase, subjects ingested placebo, 100, or 300 mg androstenedione in a random, double-blind, cross-over manner. Blood samples were collected before and every 30 min for 240 min after intake. Serum androstenedione concentrations (means +/- SE) increased above basal (6.2 +/- 0.8 nmol/l) from 60-240 min for both 100 mg (22.6 +/- 1.0 nmol/l at 240 min) and 300 mg (28.1 +/- 1.3 nmol/l at 210 min). Androstenedione intake increased serum total testosterone concentrations above basal (1.2 +/- 0.2 nmol/l) from 120-240 min (5.5 +/- 0.9 nmol/l at 210 min) with 100 mg and from 60-240 with 300 mg (10.2 +/- 1.6 nmol/l at 210 min). Androstenedione intake also increased serum estradiol concentrations (basal 191 +/- 24 pmol/l) at 150 min with 100 mg (237 +/- 35 pmol/l) and from 150-240 min with 300 mg (reaching 260 +/- 32 pmol/l at 240 min). These data indicate that, in contrast to men, androstenedione intake in women increases serum testosterone concentrations.     

Saturation mutagenesis of Burkholderia cepacia R34 2,4-dinitrotoluene dioxygenase at DntAc valine 350 for synthesizing nitrohydroquinone, methylhydroquinone, and methoxyhydroquinone

Saturation mutagenesis of the 2,4-dinitrotoluene dioxygenase (DDO) of Burkholderia cepacia R34 at position valine 350 of the DntAc alpha-subunit generated mutant V350F with significantly increased activity towards o-nitrophenol (47 times), m-nitrophenol (34 times), and o-methoxyphenol (174 times) as well as an expanded substrate range that now includes m-methoxyphenol, o-cresol, and m-cresol (wild-type DDO had no detectable activity for these substrates). Another mutant, V350M, also displays increased activity towards o-nitrophenol (20 times) and o-methoxyphenol (162 times) as well as novel activity towards o-cresol. Products were synthesized using whole Escherichia coli TG1 cells expressing the recombinant R34 dntA loci from pBS(Kan)R34, and the initial rates of product formation were determined at 1 mM substrate by reverse-phase high-pressure liquid chromatography. V350F produced both nitrohydroquinone at a rate of 0.75 +/- 0.15 nmol/min/mg of protein and 3-nitrocatechol at a rate of 0.069 +/- 0.001 nmol/min/mg of protein from o-nitrophenol, 4-nitrocatechol from m-nitrophenol at 0.29 +/- 0.02 nmol/min/mg of protein, methoxyhydroquinone from o-methoxyphenol at 2.5 +/- 0.6 nmol/min/mg of protein, methoxyhydroquinone from m-methoxyphenol at 0.55 +/- 0.02 nmol/min/mg of protein, both methylhydroquinone at 1.52 +/- 0.02 nmol/min/mg of protein and 2-hydroxybenzyl alcohol at 0.74 +/- 0.05 nmol/min/mg of protein from o-cresol, and methylhydroquinone at 0.43 +/- 0.1 nmol/min/mg of protein from m-cresol. V350M produced both nitrohydroquinone at a rate of 0.33 nmol/min/mg of protein and 3-nitrocatechol at 0.089 nmol/min/mg of protein from o-nitrophenol, methoxyhydroquinone from o-methoxyphenol at 2.4 nmol/min/mg of protein, methylhydroquinone at 1.97 nmol/min/mg of protein and 2-hydroxybenzyl alcohol at 0.11 nmol/min/mg of protein from o-cresol. The DDO variants V350F and V350M also exhibited 10-fold-enhanced activity towards naphthalene (8 +/- 2.6 nmol/min/mg of protein), forming (1R,2S)-cis-1,2-dihydro-1,2-dihydroxynaphthalene. Hence, mutagenesis of wild-type DDO through active-site engineering generated variants with relatively high rates toward a previously uncharacterized class of substituted phenols for the nitroarene dioxygenases; seven previously uncharacterized substrates were evaluated for wild-type DDO, and four novel monooxygenase-like products were found for the DDO variants V350F and V350M (methoxyhydroquinone, methylhydroquinone, 2-hydroxybenzyl alcohol, and 3-nitrocatechol).     

Effects of chlorpromazine and methylprednisolone on perfusion preservation of rabbit livers

The isolated perfused rabbit liver was used to determine how continuous hypothermic perfusion affected liver function. Rabbit livers were perfused for 0, 24, 48, and 72 hr at 5 degrees C with the UW perfusate containing hydroxyethyl starch (5 g%) dissolved in a solution containing gluconate (80 mM), adenosine (5 mM), glutathione (3 mM), phosphate (25 mM), and additives as described previously, and they were used successfully for kidney preservation. At the end of preservation the livers were perfused in an isolated circuit with a Krebs-Henseleit solution with addition of 4 g% bovine serum albumin and 10 mM glucose at 38 degrees C for 120 min. Bile was collected from the cannulated common duct. Biliary excretions of indocyanine green and liver enzymes lactate dehydrogenase, aspartate aminotransferase, and alanine aminotransferase, were determined both in the cold perfusate and the normothermic perfusate. Livers were also studied after pretreatment of the donor with chlorpromazine (CPZ) and/or methylprednisolone (MP). Bile production (ml/120 min, 100 g liver) upon reperfusion produced the most interesting data and decreased from a control value of 10.3 +/- 2.6 to 9.3 +/- 1.0 (24 hr), 5.3 +/- 0.7 (48 hr), and 4.1 +/- 1.5 (72 hr). Enzyme release was not predictive of the degree of preservation-induced damage. Pretreatment of rabbits with a combination of CPZ/MP improved bile flow at 48 and 72 hr (8.3 +/- 3.0 and 7.0 +/- 1.3, P less than 0.05). Pretreatment with either drug alone also improved function after 72 hr of preservation (7.1 +/- 1.8, CPZ; 8.2 +/- 3.5, MP).(ABSTRACT TRUNCATED AT 250 WORDS)     

Peroxidase-catalyzed N-demethylation reactions. Substrate deuterium isotope effects

Deuterium isotope effects on the kinetic parameters for the hydroperoxide-supported N-demethylation of N,N-dimethylaniline catalyzed by chloroperoxidase and horseradish peroxidase were determined using N,N-di-(trideuteromethyl)aniline. The isotope effect on the Vmax for the chloroperoxidase-catalyzed demethylation reaction supported by ethyl hydroperoxide was 1.42 +/- 0.31. The isotope effects on the Vmax for the horseradish peroxidase-catalyzed reaction supported by ethyl hydroperoxide and hydrogen peroxide were 1.99 +/- 0.39 and 4.09 +/- 0.27, respectively. Isotope effects ranging from 1.76 to 5.10 were observed on the Vmax/Km for the hydroperoxide substrate (i.e. the second order rate constant for the reaction of the hydroperoxide with the peroxidase to form compound I) in both enzyme systems when the N-methyl groups of N,N-dimethylaniline were deuterated. These results are not predicted by the simple ping-pong kinetic model for peroxidase-catalyzed N-demethylation reactions. The data are most simply explained by a mechanism involving the transfer of deuterium (or hydrogen) from N,N-dimethylaniline to the enzyme during catalysis. The deuterium must subsequently be displaced from the enzyme by the hydroperoxide, causing the observed isotope effects.     

Presence of ectonucleotidases in cultured chromaffin cells: hydrolysis of extracellular adenine nucleotides

The granular ATP released from chromaffin cells during the secretory response can be hydrolyzed by ectonucleotidases that are present in the plasma membrane of these cells. The ecto-ATPase activity showed a Km for ATP of 250 +/- 18 microM and a VMAX value of 167 +/- 25 nmol/10(6) cells x min (1.67 mumol/mg protein x min) for cultured chromaffin cells, while the ecto-ADPase activity showed a Km value for ADP of 375 +/- 40 microM and a VMAX of 125 +/- 20 nmol/10(6) cells x min (1.25 mumol/mg protein x min). The ecto 5'-nucleotidase activity of cultured chromaffin cells was more specific for the purine nucleotides, AMP and IMP, than for the pirimidine nucleotides, CMP and TMP. The Km for AMP was 55 +/- 5 microM and the VMAX value was 4.3 +/- 0.8 nmol/10(6) cells x min (43 nmol/mg protein x min). The nonhydrolyzable analogs of ADP and ATP, alpha, beta-methylene-adenosine 5'-diphosphate and adenylyl-(beta, gamma-methylene)-diphosphonate were good inhibitors of ecto 5'-nucleotidase activity, the KI values being 73.3 +/- 3.5 nM and 193 +/- 29 nM, respectively. The phosphatidylinositol-specific phospholipase C released the ecto-5'-nucleotidase from the chromaffin cells in culture, thus suggesting an anchorage through phosphatidylinositol to plasma membranes. The presence of ectonucleotidases in chromaffin cells may permit the recycling of the extracellular ATP exocytotically released from these neural cells.     

Kinetics of hydroperoxide degradation by NADP-glutathione system in mitochondria

Hydroperoxide decomposition by the NADP-glutathione system in rat liver mitochondria was analyzed. Mitochondria were found to contain high concentrations of the reduced form of glutathione (GSH) (4.32 +/- 0.50 nmol/mg) and NADPH (4.74 +/- 0.64 nmol/mg), and high activities of glutathione peroxidase and reductase. In the initial phase of the reaction, the rate of hydroperoxide decomposition was proportional to both the GSH level and the activity of GSH peroxidase. However, in the later steady state, the step of NADP reduction was rate-limiting, and the overall reaction rate was independent of the initial concentration of GSH, and activities of glutathione peroxidase and reductase. Some GSH was released from mitochondria during incubation, but the rate of the decomposition could be simply expressed as kappa [GSH]/2, where kappa is the first-order rate constant of the peroxidase and [GSH] is the intramitochondrial level of GSH in the steady state. The rate of the reaction in the steady state was also dependent on the NADPH level, its reciprocal being linearly correlated with [NADPH]-1. The rate of decomposition of hydroperoxide was influenced by the respiratory state. During state 3 respiration, the rate was greatly depressed, but was still considered to exceed by far the rate of physiological generation of hydroperoxide.