Lung microsomal p-nitrophenol hydroxylase -- characterization and reconstitution of its activity

1. Formation of catechols from benzene and nitrobenzene have been implicated in the carcinogenic activity of these chemicals. In liver, p-nitrophenol, an intermediate of p-nitrobenzene is enzymatically converted to 4-nitrocatechol. 2. For the first time in this study, the presence of a highly active enzyme catalyzing the formation of 4-nitrocatechol from p-nitrophenol was detected in lung microsomes. The average specific activity of lung p-nitrophenol hydroxylase was found to be 0.494 nmol 4-nitrocatechol formed mg prot-1 min-1. 3. The optimum conditions for sheep lung microsomal p-nitrophenol hydroxylase were established. The maximal activity was noted at pH 6.8. The rate of p-nitrophenol hydroxylation was linear up to 2 mg prot/ml of incubation mixture. The maximal rate of 4-nitrocatechol formation was observed with 0.25 mM p-nitrophenol. 4. The Lineweaver-Burk and Eadie-Hofstee plots were found to be curve-linear. Two different Km values were calculated as 11.6 and 71.4 microM from the Lineweaver-Burk plot and as 10.7 and 74.5 microM from the Eadie-Hofstee plot. This suggested that there were either two forms of enzyme or two different enzymes participating in ortho hydroxylation of p-nitrophenol in lung microsomes. 5. Lung microsomal p-nitrophenol hydroxylase activity of sheep was reconstituted in the presence of purified lung microsomal cytochrome P-450, NADPH dependent cytochrome P-450 reductase and synthetic lipid, phosphatidylcholine dilauroyl.     

对硝基苯酚冲击对UASB反应器中污泥活性和微生物种群的影响

研究了在稳定运行的上流式厌氧污泥床(Upflow Anaerobic Sludge Blanket,UASB)反应器中,对硝基苯酚(p-NP)冲击对反应器活性的影响。采用PCR-DGGE技术监测了反应器受对硝基苯酚冲击后微生物种群多样性的变化。实验结果表明,p-NP冲击对污泥的产甲烷活性和COD去除活性均有严重的抑制,污泥活性的恢复需要较长时间;高浓度冲击比低浓度冲击产生更严重的影响,20mg/L和40mg/Lp-NP冲击后污泥活性的恢复期分别为16d和27d。p-NP冲击后,真细菌和古菌的多样性均发生了显著的变化,而且p-NP冲击对真细菌的影响大于对古菌的影响。p-NP冲击后甲烷产量下降的主要与Methanosaetasp.的活性下降以及Methanomicrobia sp.丰度的下降有关。而冲击后真细菌的主要变化表现为Chloroflexisp.、Bacteroidesp.和Anaerovibrio sp.的丰度均下降;Rheinheimera sp.在受到20mg/Lp-NP冲击时丰度下降,继续受到40mg/Lp-NP冲击时该种群消失。Flavobacteria sp.是p-NP冲击后新出现的细菌种群,可能与p-NP的降解有关。     

A spectrophotometric assay for the transphosphatidylation activity of phospholipase D enzyme

We developed a specific spectrophotometric assay for the quantitative determination of phospholipase D-catalyzed transphosphatidylation activity. The assay measures p-nitrophenol liberated by phospholipase D-catalyzed reaction of phosphatidyl-p-nitrophenol and ethanol in an aqueous-organic emulsion system. The release of p-nitrophenol was linear to reaction time at an early stage of the reaction with phospholipase D from Streptomyces sp. In the spectrophotometric assay for the reaction with phospholipase D from Streptomyces chromofuscus, which has higher hydrolytic activity than transphosphatidylation activity, p-nitrophenol was not found. The advantages of this novel method for measuring the transphosphatidylation activity of phospholipase D are that (i) it does not use radioactive compounds, (ii) it can measure the initial velocity of the reaction, and (iii) it is rapid, easy, and accurate to perform.     

Phospholipid-dependence of oestrone UDP-glucuronyltransferase and p-nitrophenol UDP-glucuronyltransferase.

Hepatic UDP-glucuronyltransferase activity was resolved into two fractions, one exhibiting oestrone glucuronyltransferase activity and the other exhibiting p-nitrophenol glucuronyltransferase activity. Hydroxyapatite-column chromatography removed greater than 95% of the phospholipids from both preparations. The partially purified delipidated enzymes were essentially devoid of catalytic activity, but activities were restored by the addition of phospholipids or phosphatidylcholine mixtures containing various saturated and unsaturated fatty acids. Both oestrone and p-nitrophenol glucuronyl-transferase activities were reconstituted to similar degrees with the phosphatidylcholine mixtures. When purified phospholipids were tested, phosphatidylcholine and lysophosphatidylcholine were most effective in restoring activity, whereas phosphatidylethanolamine was the least effective. These results further suggest that oestrone and p-nitrophenol UDP-glucuronyltransferases are dependent on phospholipids for their activity.     

A method for determination of transketolase activity based on the use of a pH indicator.

A new method for assaying transketolase activity is proposed. The method consists in recording the pH changes in the course of the enzymatic reaction and is based on the use of the pH-indicator p-nitrophenol. When p-nitrophenol is added to a reaction mixture containing hydroxypyruvate and glycolaldehyde as substrates the absorbance increases. The rate of the change of absorbance is proportional to the enzyme concentration.     

Ethanol as an inducer of UDP-glucuronyltransferase: a comparison with phenobarbital and 3-methylcholanthrene induction in rabbit hepatic microsomes

In this report we have examined the ability of ethanol to induce UDP-glucuronyltransferase activity in male rabbits using p-nitrophenol as substrate. The rabbit was found to be an excellent species for studies of ethanol induction since almost 3-fold increases in activity were observed relative to controls. Ethanol induction of p-nitrophenol UDP-glucuronyltransferase activity was even greater than induction by 3-methylcholanthrene, the prototypic inducer of this type of activity. Thus, the rabbit shows promise for studies of UDP-glucuronyltransferase isozymes that are induced by chronic ethanol consumption.     

In situ structural analysis of microsomal UDP-glucuronyltransferases by radiation inactivation

The structure of the UDP-glucuronyltransferases in microsomes from guinea pig and rat liver was examined in situ by radiation inactivation analysis. The p-nitrophenol conjugating activity of guinea pig microsomes increased at lower doses of radiation; at higher doses (greater than or equal to 36 megarads), activity showed a first order decline yielding a target size of 71 +/- 9 kDa. Treating microsomes with Triton X-100 eliminated the activation seen at lower doses of radiation and yielded a simple exponential decrease in activity which gave a larger target size (95 +/- 18 kDa). A monoexponential decrease in activity was seen in sonicated microsomes, at greater than or equal to 36 megarads. The same response was obtained when the reaction was assayed in the reverse direction. The estrone conjugating activity of guinea pig microsomes was similarly activated at lower doses of radiation and declined at higher doses (greater than or equal to 36 megarads), with a target size of 57 +/- 11 kDa. Allosteric activation of the enzyme by UDP-N-acetylglucosamine was eliminated by lower doses of radiation. Thus, activation of the enzyme by radiation, detergent, sonication, and UDP-N-acetylglucosamine appear to be interdependent. These activations are postulated to be due to the existence of the enzyme in an oligomeric form which can be dissociated into monomers with higher activity. The same biphasic activation-inactivation curves were obtained for p-nitrophenol conjugation in rat liver microsomes. The target sizes were 54 +/- 8 kDa (p-nitrophenol in the forward direction) and 66 +/- 10 kDa (p-nitrophenol in the reverse direction). Thus, the enzyme appears to be smaller in rat liver as compared with guinea pig liver. Lithocholate glucuronidating activity in rat liver microsomes (at greater than 36 megarads) gave a target size of 74 +/- 1 kDa.     

Human neutrophil N-acetyl-beta-D-glucosaminidase: heparin inhibition

To determine N-acetyl-beta-D-glucosaminidase (EC 3.2.1.30) in human neutrophil granules separated by a method requiring heparin, the inhibition of this enzyme by heparin was studied. Neutrophils were purified from blood of five donors by modifications of the Hypaque-Ficoll and dextran separation methods resulting in a suspension which was 96% neutrophils. Neutrophil lysates were assayed for N-acetyl-beta-D-glucosaminidase by measuring the amount of p-nitrophenol released from p-nitrophenyl-N-acetyl-beta-D-glucosaminide. The reaction showed first-order kinetics with regard to enzyme concentration. Triton X-100, 0.1% v/v, enhanced enzyme activity. Heparin was shown to reduce neutrophil lysate N-acetyl-beta-D-glucosaminidase to a specific activity of 46% at a heparin concentration of 2 units per assay and to 43% (maximal inhibition) at 17 and 50 units of heparin per assay. Substantially higher heparin concentrations partially restored the inhibited activity, the maximal restoration being a return to 80% of the original activity at 1700 units of heparin per assay. Protamine sulfate was assessed for its ability to restore N-acetyl-beta-D-glucosaminidase activity in the presence of heparin. At 1.0 mg/10 units of heparin, protamine restores enzyme activity to its heparin-free activity. These studies of human neutrophil N-acetyl-beta-D-glucosaminidase demonstrate: (1) specific enzyme activity is 28.8 +/- 7.0 nmole p-nitrophenol released per minute per milligram of protein or 1.7 +/- 0.5 nmole p-nitrophenol released per minute per 10(6) neutrophils; (2) heparin rapidly but finitely inhibits enzyme activity at very low concentrations and paradoxically restores it toward normal at high concentrations; and (3) protamine sulfate restores enzyme activity inhibited by heparin.     

Increased catalytic activity of cytochrome P-450IIE1 in pericentral hepatocytes compared to periportal hepatocytes isolated from pyrazole-treated rats.

Cytochrome P-450IIE1 is induced by a variety of agents, including acetone, ethanol and pyrazole. Recent studies employing immunohistochemical methods have shown that P-450IIE1 was expressed primarily in the pericentral zone of the liver. In order to evaluate whether catalytic activity of P-450IIE1 is preferentially localized in the pericentral zone of the liver acinus, the oxidation of aniline and p-nitrophenol, two effective substrates for P-450IIE1, by periportal and pericentral hepatocytes isolated from pyrazole-treated rats was determined. Periportal and pericentral hepatocytes were prepared by a digitonin-collagenase procedure; the marker enzymes glutamine synthetase and gamma-glutamyl transpeptidase indicated reasonable separation of the two cell populations. Viability, yield and total cytochrome P-450 content were similar for the periportal and pericentral hepatocytes. Pericentral hepatocytes oxidized aniline and p-nitrophenol at rates that were 2-4-fold greater than periportal hepatocytes under a variety of conditions. Carbon monoxide inhibited the oxidation of the substrates with both preparations and abolished the increased oxidation found with the pericentral hepatocytes. Pyrazole or 4-methylpyrazole, added in vitro, effectively inhibited the oxidation of aniline and p-nitrophenol and prevented the augmented rate of oxidation by the pericentral hepatocytes. Western blots carried out using isolated microsomes revealed a more than 2-fold increase in immunochemical staining with microsomes isolated from the pericentral hepatocytes, which correlated to the 2-4-fold increase in the rate of oxidation of aniline or p-nitrophenol by the pericentral hepatocytes. These results suggest that functional catalytic activity of cytochrome P-450IIE1 is preferentially localized in the pericentral zone of the liver acinus, and that most of the induction by pyrazole of P-450IIE1 appears to occur within the pericentral zone.     

Estimation of Cellobiohydrolase Ⅰ Activity by Numerical Differentiation of Dynamic Ultraviolet Spectroscopy

1,4-β-D-glucan cellobiohydrolase Ⅰ （CBH Ⅰ）, p-nitrophenyl β-D-cellobioside, p-nitrophenol and cellobiose show distinct ultraviolet spectra, allowing the design of an assay to track the dynamic process of p-nitrophenyl β-D-cellobioside hydrolysis by CBH Ⅰ. Based on the linear relationship between p-nitrophenol formation in the hydrolysate and its first derivative absorption curve of AUC340-400 m （area under the curve）, a new sensitive assay for the determination of CBH Ⅰ activity was developed. The dynamic parameters of catalysis reaction, such as Vm and kcat, can all be derived from this result. The influence of β-glucosidase and endoglucanase in crude enzyme sample on the assay was discussed in detail. This approach is useful for accurate determination of the activity of CBHs.     

对硝基苯酚降解菌P3的分离、降解特性及基因工程菌的构建

分离到一株假单胞菌 (Pseudomonassp .)P3 ,该菌能够以对硝基苯酚为唯一碳源和氮源进行生长。在有外加氮源的条件下 ,P3降解对硝基苯酚并在培养液中积累亚硝酸根。P3有比较广泛的底物适应性 ,对多种芳香族化合物都有降解能力。不同金属离子对P3降解对硝基苯酚有不同的作用。葡萄糖的存在对P3降解对硝基苯酚无明显促进作用 ,而微量酵母粉可以大大促进P3对硝基苯酚的降解。以P3为受体菌 ,通过接合转移的手段将甲基对硫磷水解酶基因mpd克隆至P3菌中 ,获得了表达甲基对硫磷水解酶活性的基因工程菌PM ,PM能够以甲基对硫磷为唯一碳源进行生长。工程菌PM具有较高的甲基对硫磷降解活性及稳定性     

An ectophosphatase activity in Candida parapsilosis influences the interaction of fungi with epithelial cells

This study describes the biochemical characterization of a phosphatase activity present on the cell surface of Candida parapsilosis, a common cause of candidemia. Intact yeasts hydrolyzed p-nitrophenylphosphate to p-nitrophenol at a rate of 24.30+/-2.63 nmol p-nitrophenol h(-1) 10(-7) cells. The cell wall distribution of the Ca. parapsilosis enzyme was demonstrated by transmission electron microscopy. The duration of incubation of the yeast cells with the substrate and cell density influenced enzyme activity linearly. Values of V(max) and apparent K(m) for p-nitrophenylphosphate hydrolysis were 26.80+/-1.13 nmol p-nitrophenol h(-1) 10(-7) cells and 0.47+/-0.05 mM p-nitrophenylphosphate, respectively. The ectophosphatase activity was strongly inhibited at high pH as well as by classical inhibitors of acid phosphatases, such as sodium orthovanadate, sodium molybdate, sodium fluoride, and inorganic phosphate, the final product of the reaction. Only the inhibition caused by sodium orthovanadate was irreversible. Different phophorylated amino acids were used as substrates for the Ca. parapsilosis ectoenzyme, and the highest rate of phosphate hydrolysis was achieved using phosphotyrosine. A direct relationship between ectophosphatase activity and adhesion to host cells was established. In these assays, irreversible inhibition of enzyme activity resulted in decreased levels of yeast adhesion to epithelial cells.     

Product inhibition study on carbonic anhydrase using spectroscopy and calorimetry

Kinetic and thermodynamic studies have been made on the effect of the p-nitrophenol product on the activity of bovine carbonic anhydrase in 50 mM Tris buffer pH 7.5, at 300K using UV spectrophotometry and isothermal titration calorimetry (ITC). A competitive inhibition was observed for p-nitrophenol as a product of the enzymatic reaction. A graphical fitting method was used for determination of the binding constant and enthalpy of inhibitor binding using ITC data. The dissociation binding constant was 0.10mM by the microcalorimetric method, which is in good agreement with the value of 0.11mM for the inhibition constant obtained from the spectrophotometric method.     

Modulation of rat liver cytochrome P450 activity by prolonged red wine consumption

Cytochrome P450-dependent oxidation of lauric acid, p-nitrophenol and ethanol by liver microsomal fractions were studied in control rats and in animals given either ethanol, red wine, or alcohol-free red wine for 10 weeks. Ethanol increased the total cytochrome P450 and the isoenzyme 2E1 content, as well as the p-nitrophenol hydroxylation and ethanol oxidation. These effects of ethanol treatment were attenuated by red wine administration. Red wine increased the total antioxidant capacity of plasma, whereas the alcohol-free red wine decreased the cytochrome P450 content and decreased the oxidation of lauric acid, p-nitrophenol and ethanol to values lower than control. It is concluded that red wine administration attenuates the ethanol-induced enhancement in liver microsomal parameters dependent on cytochrome P450 2E1 activity, an affect that seems to be accomplished by the non-alcoholic constituents of red wine known to have antioxidant properties.     

Effect of carbon starvation on p-nitrophenol degradation by a Moraxella strain in buffer and river water

This study examines the effect of carbon starvation on the ability of a Moraxella sp. strain to degrade p-nitrophenol (PNP). Carbon starvation for 24 h decreased the induction time for p-nitrophenol degradation by the bacterium in a minimal salt medium from 6 to 1 h but it did not completely eliminate the induction time. Moraxella cells with 2-day carbon starvation had an induction time of 3 h and the induction time of the 3-day starved cells was 6 h. A 100% increase in density of the non-starved cells did not affect the induction time for p-nitrophenol degradation by the bacterium, indicating that the initial increase in cell density of the carbon-starved culture did not cause the faster onset of p-nitrophenol degradation. However, the initial uptake of p-nitrophenol of the 1-day carbon-starved Moraxella cells was 3-fold higher than the non-starved cells. A green fluorescent protein gene (gfp)-labelled Moraxella (M6 strain) was constructed to examine the survival of and p-nitrophenol degradation by the bacterium in non-sterile river water samples. Similar p-nitrophenol degradation behaviour was observed in the river water samples inoculated with the M6 cells. The time needed for complete degradation of p-nitrophenol by the non-starved M6 was 19-27 and 33 h in samples spiked with 80, 200 and 360 microM p-nitrophenol, respectively. However, the 1-day carbon-starved inocula required about 16 h to degrade the p-nitrophenol completely regardless of its concentration in the water samples. Survival of the carbon-starved and non-starved M6 was not significantly different from each other in the river water regardless of the p-nitrophenol concentration. In the absence of p-nitrophenol, the inoculum density decreased continuously. At 200 and 360 microM p-nitrophenol, the cell densities of M6 increased in the first two days of incubation and declined steadily afterward.     

Separation of oestrone UDP-glucuronyltransferase and p-nitrophenol UDP-glucuronyltransferase activities.

Rabbit liver UDP-glucuronyltransferase activity was resolved into two separate fractions on DEAE-cellulose, one containing most of the transferase activity toward oestrone and the other most of the activity toward p-nitrophenol. These two activities were completely separated by rechromatography of each fraction on a second DEAE-cellulose column.     

Purification of cytosolic beta-glucosidase from pig liver and its reactivity towards flavonoid glycosides

Flavonoid glycosides are common dietary components which may have health-promoting activities. The metabolism of these compounds is thought to influence their bioactivity and uptake from the small intestine. It has been suggested that the enzyme cytosolic beta-glucosidase could deglycosylate certain flavonoid glycosides. To test this hypothesis, the enzyme was purified to homogeneity from pig liver for the first time. It was found to have a molecular weight (55 kDa) and specific activity (with p-nitrophenol glucoside) consistent with other mammalian cytosolic beta-glucosidases. The pure enzyme was indeed found to deglycosylate various flavonoid glycosides. Genistein 7-glucoside, daidzein 7-glucoside, apigenin 7-glucoside and naringenin 7-glucoside all acted as substrates, but we were unable to detect activity with naringenin 7-rhamnoglucoside. Quercetin 4'-glucoside was a substrate, but neither quercetin 3, 4'-diglucoside, quercetin 3-glucoside nor quercetin 3-rhamnoglucoside were deglycosylated. Estimates of K(m) ranged from 25 to 90 microM while those for V(max) were about 10% of that found with the standard artificial substrate p-nitrophenol glucoside. The non-substrate quercetin 3-glucoside was found to partially inhibit deglycosylation of quercetin 4'-glucoside, but it had no effect upon activity with p-nitrophenol glucoside. This study confirms that mammalian cytosolic beta-glucosidase can deglycosylate some, but not all, common dietary flavonoid glycosides. This enzyme may, therefore, be important in the metabolism of these compounds.     

The submicrosomal distribution of hepatic uridine diphosphate glucuronyltransferases in the rabbit

1. Rabbit liver microsomes were subfractionated into rough- and smooth-surfaced types, and glucuronyltransferase activity in each microsomal subfraction was determined with p-nitrophenol, o-aminophenol and phenolphthalein as substrates. The glucuronyltransferase activity measured with p-nitrophenol and o-aminophenol as substrates was localized predominantly in rough-surfaced microsomes. Glucuronyltransferase measured with phenolphthalein as substrate was equally present in rough- and smooth-surfaced microsomes. 2. Phenobarbital pretreatment of rabbits did not stimulate any of the glucuronyltransferase activities measured in either rough- or smooth-surfaced microsomes. 3. Preincubation of rabbit liver microsomes for 30-60min. at 37 degrees under oxygen did not cause any loss of glucuronyltransferase activity. Such preincubation caused either no change or increased enzyme activity in both submicrosomal fractions. The relative distribution of transferase activity in rough- and smooth-surfaced microsomes was not affected by preincubation.     

Sulfating-activity and stability of cDNA-expressed allozymes of human phenol sulfotransferase, ST1A3*1 ((213)Arg) and ST1A3*2 ((213)His), both of which exist in Japanese as well as Caucasians.

We recently found single amino acid substitutions ((213)Arg/His and (223)Met/Val) in polymorphic human phenol-sulfating phenol sulfotransferase (SULT: cDNAs encoding ST1A3, P PST or HAST1/2) among Caucasians and African-Americans. In a Japanese population (n = 143), allele frequencies of (213)Arg and (213)His were 83.2 and 16. 8%, respectively, but the (223)Val allele was not found. (213)His homozygosity was reportedly associated with both very low (>7-fold) sulfating activities of p-nitrophenol (at 4 microM) and low thermostability in platelets. Sulfating-activity determinations using recombinant (213)Arg- and (213)His-forms (ST1A3*1 and ST1A3*2, respectively) did not, however, reveal appreciable deficiency in [(35)S]3'-phosphoadenosine 5'-phosphosulfate (PAPS)-dependent sulfation of p-nitrophenol (4 microM) by ST1A3*2 (7.5 vs. 10.2 nmol/min/nmol SULT for ST1A3). Kinetic parameters for p-nitrophenol for p-nitrophenol sulfation supported the slight decrease in sulfating activities at 4 microM (K(m), 0.82 vs. 1.75 microM; V(max), 13.2 vs. 13.1 nmol/min/nmol SULT, respectively, for ST1A3*1 and *2). p-Nitrophenyl sulfate-dependent 2-naphthol sulfation by ST1A3*2 was 69% of that by ST1A3*1 (p<0.05). However, ST1A3*2 was remarkably unstable at 45 and 37 degrees C as compared to ST1A3*1. The lower p-nitrophenol sulfating activity of ST1A3*2 may explain the lower platelet p-nitrophenol sulfation in ST1A3*2 homozygotes. Protein instability and ST1A3 gene regulation may be both involved in the polymorphism of p-nitrophenol sulfation in human tissues.     

New substrate specificity of modified porcine pancreatic alpha-amylase

Conversion of the substrate specificity of porcine pancreatic alpha-amylase (PPA) was studied using chemical modification of His residues. Diethyl pyrocarbonate modified His residues in PPA and the activity of the modified PPA for the hydrolysis of the alpha-D-(1,4)glucoside bond in starch or oligosaccharides decreased to less than 1% of that of the native enzyme. However, the activity for the hydrolysis of the bond between p-nitrophenol and oligosaccharides in p-nitrophenyl oligosaccharides was increased by chemical modification. When the modified PPA was incubated with a proteinaceous alpha-amylase inhibitor (Mr 60,000) purified from white kidney bean (Phaseolus vulgaris), it bound to the inhibitor. As a result, the remaining less than 1% hydrolytic activity of the modified PPA for starch disappeared completely but that for p-nitrophenyl oligosaccharides remained unaltered. The hydrolytic activity of the native PPA for the alpha-D-(1,4)glucoside bond in oligosaccharides was stronger than that between p-nitrophenyl and oligosaccharides in p-nitrophenyl oligosaccharides. Therefore, when p-nitrophenyl oligosaccharides (three to five glucose residues) were used as substrates for the native PPA, the alpha-D-(1,4)glucoside bonds in the oligosaccharides were hydrolyzed. However, the modified PPA-inhibitor complex hydrolyzed only the bond between p-nitrophenol and oligosaccharides in p-nitrophenyl oligosaccharides. The above results reveal that, by chemical modification with diethyl pyrocarbonate and biochemical modification with an amylase inhibitor, amylase can be converted to a new exo-type enzyme which hydrolyzes only the bond between p-nitrophenol and oligosaccharides in p-nitrophenyl oligosaccharides.