scholarly journals A novel aldehyde reductase with activity towards a metabolite of aflatoxin B1 is expressed in rat liver during carcinogenesis and following the administration of an anti-oxidant

1993 ◽  
Vol 292 (1) ◽  
pp. 13-18 ◽  
Author(s):  
D J Judah ◽  
J D Hayes ◽  
J C Yang ◽  
L Y Lian ◽  
G C K Roberts ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Rat Liver ◽  
Aflatoxin B1 ◽  
British Library ◽  
Aldehyde Reductase ◽  
Ph Values ◽  
Development Of Resistance ◽  
Neoplastic Lesions ◽  
Anti Oxidant ◽  
Rat Livers

In contrast with fractions from control animals, an aldehyde reductase, which catalyses the reduction of aflatoxin B1-dihydrodiol, in the dialdehyde form at physiological pH values, to aflatoxin B1-dialcohol, is expressed in cytosolic fractions prepared from rat livers bearing pre-neoplastic lesions, or following treatment with the anti-oxidant ethoxyquin. This expression parallels the development of resistance to the toxin. Unlike the aflatoxin B1-dihydrodiol, the dialcohol does not undergo binding to protein. This enzyme activity could play a mechanistic role in hepatocarcinogenesis and chemoprotection in the rat. Correlated n.m.r. and m.s. spectra are provided in Supplementary Publication SUP 50171 (3 pages), which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1993) 289, 9.

scholarly journals Purification from rat liver of a novel constitutively expressed member of the aldo-keto reductase 7 family that is widely distributed in extrahepatic tissues

2000 ◽  
Vol 348 (2) ◽  
pp. 389-400 ◽  
Author(s):  
Vincent P. KELLY ◽  
Linda S. IRELAND ◽  
Elizabeth M. ELLIS ◽  
John D. HAYES
Keyword(s):  
Rat Liver ◽  
Aflatoxin B1 ◽  
Single Gene ◽  
Reversed Phase ◽  
Cross Reactivity ◽  
Rat Tissues ◽  
Succinic Semialdehyde ◽  
Aldehyde Reductase ◽  
Cibacron Blue ◽  
Sds Page

Antiserum raised against human aflatoxin B1 aldehyde reductase 1 (hAFAR1) has been used to identify a previously unrecognized rat aldo-keto reductase (AKR). This novel enzyme is designated rat aflatoxin B1 aldehyde reductase 2 (rAFAR2) and it characteristically migrates faster during SDS/PAGE than does the archetypal ethoxyquin-inducible rAFAR protein (now called rAFAR1). Significantly, rAFAR2 is essentially unreactive with polyclonal antibodies raised against rAFAR1. Besides its distinct electrophoretic and immunochemical properties, rAFAR2 appears to be regulated differently from rAFAR1 as it is expressed in most rat tissues and does not appear to be induced by ethoxyquin. Multiple forms of rAFAR2 have been identified. Anion-exchange chromatography on Q-Sepharose, followed by adsorption chromatography on columns of Matrex Orange A and Cibacron Blue, have been employed to purify rAFAR2 from rat liver cytosol. The Q-Sepharose chromatography step resulted in the resolution of rAFAR2 into three peaks of AKR activity, two of which were purified and shown to be capable of catalysing the reduction of 2-carboxybenzaldehyde, succinic semialdehyde, 4-nitrobenzaldehyde and 9,10-phenathrenequinone. The two most highly purified rAFAR2-containing preparations eluted from the Cibacron Blue column were 91 and 98% homogeneous. Analysis of these by SDS/PAGE indicated that the least anionic (peak CBA5) comprised a polypeptide of 37.0 kDa, whereas the most anionic (peak CBA6) contained two closely migrating polypeptides of 36.8 and 37.0 kDa; by contrast, in the present study, rAFAR1 was estimated by SDS/PAGE to be composed of 38.0 kDa subunits. Final purification of the 37 kDa polypeptide in CBA5 and CBA6 was accomplished by reversed-phase HPLC. Partial proteolysis of the two preparations of the 37 kDa polypeptide with Staphylococcus aureus V8 protease yielded fragments of identical size, suggesting that they represent the product of a single gene. Furthermore, the peptide maps from CBA5 and CBA6 differed substantially from that yielded by rAFAR1, indicating that they are genetically distinct from the inducible reductase. A peptide generated by CNBr digestion of the 37 kDa polypeptide from CBA6 was shown by Edman degradation to share 88% sequence identity with residues Tyr168-Leu183 of rAFAR1. This provides evidence that the rat protein identified by its cross-reactivity with anti-hAFAR1 serum is an additional member of the AKR7 family.

scholarly journals Substrate specificity of an aflatoxin-metabolizing aldehyde reductase

1995 ◽  
Vol 312 (2) ◽  
pp. 535-541 ◽  
Author(s):  
E M Ellis ◽  
J D Hayes
Keyword(s):  
Substrate Specificity ◽  
High Activity ◽  
Rat Liver ◽  
Aflatoxin B1 ◽  
Succinic Semialdehyde ◽  
Aldehyde Reductase ◽  
Aliphatic Aldehydes ◽  
Alpha Beta ◽  
Low Activity

The enzyme from rat liver that reduces aflatoxin B1-dialdehyde exhibits a unique catalytic specificity distinct from that of other aldo-keto reductases. This enzyme, designated AFAR, displays high activity towards dicarbonyl-containing compounds with ketone groups on adjacent carbon atoms; 9,10-phenanthrenequinone, acenaphthenequinone and camphorquinone were found to be good substrates. Although AFAR can also reduce aromatic and aliphatic aldehydes such as succinic semialdehyde, it is inactive with glucose, galactose and xylose. The enzyme also exhibits low activity towards alpha, beta-unsaturated carbonyl-containing compounds. Determination of the apparent Km reveals that AFAR has highest affinity for 9,10-phenanthrenequinone and succinic semialdehyde, and low affinity for glyoxal and DL-glyceraldehyde.

scholarly journals Treatment of rats with glucagon or mannoheptulose increases mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase activity and decreases succinyl-CoA content in liver

1989 ◽  
Vol 262 (1) ◽  
pp. 159-164 ◽  
Author(s):  
P A Quant ◽  
P K Tubbs ◽  
M D Brand
Keyword(s):  
Enzyme Activity ◽  
Rat Liver ◽  
Ketone Bodies ◽  
Liver Mitochondria ◽  
Mitochondrial Activity ◽  
Acetyl Coa ◽  
Total Enzyme Activity ◽  
Liver Homogenates ◽  
Rat Livers ◽  
Total Enzyme

1. The activity of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase (EC 4.1.3.5) in extracts of rapidly frozen rat livers was doubled in animals treated in various ways to increase ketogenic flux. 2. Some 90% of the activity measured was mitochondrial, and changes in mitochondrial activity dominated changes in total enzyme activity. 3. The elevated HMG-CoA synthase activities persisted throughout the isolation of liver mitochondria. 4. Intramitochondrial succinyl-CoA content was lower in whole liver homogenates and in mitochondria isolated from animals treated with glucagon or mannoheptulose. 5. HMG-CoA synthase activity in mitochondria from both ox and rat liver was negatively correlated with intramitochondrial succinyl-CoA levels when these were manipulated artificially. Under these conditions, the differences between mitochondria from control and hormone-treated rats were abolished. 6. These findings show that glucagon can decrease intramitochondrial succinyl-CoA concentration, and that this in turn can regulate mitochondrial HMG-CoA synthase. They support the hypothesis that the formation of ketone bodies from acetyl-CoA may be regulated by the extent of succinylation of mitochondrial HMG-CoA synthase.

scholarly journals Purification from rat liver of an enzyme that catalyses the sulphurylation of phenols

1971 ◽  
Vol 123 (5) ◽  
pp. 901-906 ◽  
Author(s):  
F. A. McEvoy ◽  
J. Carroll
Keyword(s):  
Enzyme Activity ◽  
Methyl Ester ◽  
Rat Liver ◽  
The Other ◽  
Male Rat ◽  
Thiol Compounds ◽  
Km Value ◽  
Rat Livers

1. An enzyme (EC 2.8.2.1) that catalyses the transfer of sulphate from adenosine 3′-phosphate 5′-sulphatophosphate to phenols was purified approx. 2000-fold from male rat livers. 2. The purified preparation did not catalyse the sulphurylation of dehydroepiandrosterone, butan-1-ol, l-tyrosine methyl ester, 1-naphthylamine or serotonin. 3. At pH8.0 and 37°C the Km values of the enzyme for p-nitrophenol and adenosine 3′-phosphate 5′-sulphatophosphate are 51 and 14μm respectively. The Km value for either substrate is independent of the concentration of the other. 4. The sulphurylation of phenol is inhibited by thiol compounds and glutathione at a concentration of 3mm caused an approx. 50% decrease in enzyme activity. 5. The Km of the enzyme for adenosine 3′-phosphate 5′-sulphatophosphate is unaffected by the presence of added glutathione but at a concentration of 5mm-glutathione the Km of the enzyme for its phenolic substrate is decreased.

Inhibition of Aminoacyl-tRNA Synthetases From Rat Liver by Reducing Agents

1973 ◽  
Vol 51 (11) ◽  
pp. 1537-1541 ◽  
Author(s):  
Rocío Vargas ◽  
Mario Castaneda
Keyword(s):  
Enzyme Activity ◽  
Rat Liver ◽  
Enzyme Preparation ◽  
Reducing Agents ◽  
Trna Synthetases ◽  
Ph Values ◽  
Aminoacyl Trna Synthetases ◽  
Ethanesulfonic Acid ◽  
Hydroxyethyl Piperazine

Commonly used reducing agents 2-mercaptoethanol and dithiothreitol inhibit in vitro homologous aminoacylation of rat liver systems. The extent of inhibition depends on the buffer and is greater in Tris than in N-2-hydroxyethyl piperazine-N′-2-ethanesulfonic acid (HEPES). Variations in the values of pH and the concentrations of Mg2+, ATP, tRNA, enzyme preparation, and buffers were ineffective to reverse the inhibition produced by these reducing agents. Enzyme activity with no reducing agent is, in general, the same or higher in Tris than in HEPES, although it depends on the pH values and the concentrations of buffer and Mg2+ in the reaction mixture.

scholarly journals Regulation of aflatoxin B1-metabolizing aldehyde reductase and glutathione S-transferase by chemoprotectors

1994 ◽  
Vol 300 (1) ◽  
pp. 117-124 ◽  
Author(s):  
L I McLellan ◽  
D J Judah ◽  
G E Neal ◽  
J D Hayes
Keyword(s):  
Rat Liver ◽  
Aflatoxin B1 ◽  
Resistance Mechanism ◽  
Constitutive Expression ◽  
Butylated Hydroxytoluene ◽  
Detoxification Enzymes ◽  
Rat Tissues ◽  
Aldehyde Reductase ◽  
Glutathione S Transferase ◽  
Adaptive Resistance

Ingestion of aflatoxin B1 (AFB1) represents a major risk factor in the aetiology of human hepatocellular carcinoma. In the rat, the harmful effects of AFB1 can be prevented by the administration of certain drugs which induce hepatic detoxification enzymes. We have previously shown that treatment of rats with the chemoprotector ethoxyquin (EQ) results in a marked increase in expression of the Alpha-class glutathione S-transferase (GST) Yc2 subunit which has high activity towards AFB1-8,9-epoxide [Hayes, Judah, McLellan, Kerr, Peacock and Neal (1991) Biochem. J. 279, 385-398]. To allow an assessment of whether the increased expression of GST Yc2 represents a general adaptive resistance mechanism to chemical stress, that is invoked by both chemoprotectors and carcinogens, we have examined the effects of EQ, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), phenobarbital (PB), AFB1, 3-methylcholanthrene (3-MC) and clofibrate on the AFB1-glutathione-conjugating activity and the GST subunit levels in rat liver. In addition, the effect of these drugs on the hepatic levels of an aldehyde reductase (AFB1-AR) that metabolizes the cytotoxic dialdehydic form of AFB1 has been studied as this enzyme also appears to be important in chemoprotection. Administration of the antioxidants EQ, BHA or BHT, as well as PB, led to a marked increase in levels of the GST Yc2 subunit in rat liver, and this increase coincided with a substantial rise in the GST activity towards AFB1-8,9-epoxide; neither AFB1, 3-MC nor clofibrate caused induction of Yc2 or any of the GST subunits examined. Among the xenobiotics studied, EQ was found to be the most effective inducing agent for the Yc2 subunit as well as Yc1, Yb1 and Yf. However, PB was equally as effective as EQ in increasing levels of the Ya-type subunits, although it was not found to be as potent an inducer of the other GST subunits, including Yc2. In addition to induction of GST, EQ caused a substantial increase in the hepatic content of AFB1-AR. Both BHA and BHT were also able to induce this enzyme but, by contrast, PB was found to be a poor inducer of AFB1-AR. AFB1, 3-MC and clofibrate were unable to serve as inducers of this reductase. The presence of Alpha-class GST, including the Yc2 subunit, was examined in various rat tissues. Constitutive expression of Yc2 was found in the epididymis at levels comparable with that observed in the liver from EQ-treated rats.(ABSTRACT TRUNCATED AT 400 WORDS)

Hepatic uptake of intact glutathione S-conjugate, inhibition by organic anions, and sinusoidal catabolism

1993 ◽  
Vol 265 (3) ◽  
pp. G547-G554
Author(s):  
C. A. Hinchman ◽  
A. T. Truong ◽  
N. Ballatori
Keyword(s):  
Guinea Pig ◽  
Rat Liver ◽  
Marked Decrease ◽  
Hepatic Uptake ◽  
Half Time ◽  
High Concentrations ◽  
Rat Livers ◽  
Dose Dependent ◽  
Uptake And Metabolism ◽  
Guinea Pig Liver

To identify potential mechanisms for hepatic removal of circulating glutathione (GSH) conjugates, uptake and metabolism of S-2,4-dinitrophenylglutathione (DNP-SG) were examined in isolated perfused livers from rat and guinea pig. Guinea pig livers perfused with 5 mumol of DNP-SG in a recirculating system (50 microM initial concn) rapidly cleared the conjugate from the perfusate (half time 3.7 min), whereas clearance was considerably slower in rat liver (half time 35 min). Disappearance of DNP-SG from the perfusate was accompanied by a simultaneous appearance of DNP-SG and its metabolites in bile. Addition of acivicin, an inhibitor of gamma-glutamyltransferase (gamma-GT), to the perfusate resulted in a marked decrease in DNP-SG clearance by guinea pig liver but had no effect in rat liver, suggesting that in the guinea pig this process is largely dependent on sinusoidal gamma-GT activity. However, even in the presence of acivicin, rat and guinea pig livers removed nearly one-half of the administered DNP-SG from the recirculating perfusate over 30 min. High concentrations of DNP-SG were found in bile (up to 3.7 mM), indicating that the liver is capable of transporting the intact conjugate from the circulation. When rat livers were perfused with higher concentrations of DNP-SG (100 and 250 microM), biliary excretion of DNP-SG increased dose dependently, with concentrations in bile reaching 10 mM at the higher dose. This was accompanied by a dose-dependent choleresis.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of nitric oxide on Sertoli cell microtubule of piglets

2009 ◽  
Vol 6 (3) ◽  
pp. 257-263 ◽  
Author(s):  
Yang Li ◽  
Wang Xian-zhong ◽  
Yang Meng-bo ◽  
Zhang Jia-hua
Keyword(s):  
Nitric Oxide ◽  
Enzyme Activity ◽  
Protein Kinase ◽  
Cell Viability ◽  
Sodium Nitroprusside ◽  
Sertoli Cell ◽  
Sertoli Cells ◽  
High Concentration ◽  
Treatment Results ◽  
Anti Oxidant

AbstractTo illustrate the effect of nitric oxide (NO) on the microtubules of Sertoli cells (SC), SCs of piglets were treated with sodium nitroprusside (SNP). Changes in cell viability, anti-oxidant activity, enzyme activity and p38 mutagen-activated protein kinase (p38MAPK) activation were detected. The results were as follows. A low concentration of NO can keep SC microtubule and cell viability normal, and a high concentration of NO could increase p38MAPK activation, decrease anti-oxidant activity and transferrin secretion, and destroy the structure and distribution of the microtubules. The results suggest that SNP treatment results in an increase in NO in SCs and decreased cell anti-oxidant activity. The high concentration of NO destroys cell microtubules by activating p38MAPK.

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