Inhibition of 5β-Dihydrocortisone Reduction in Rat Liver Cytosol: A Rapid Spectrophotometric Screen for Nonsteroidal Anti-Inflammatory Drug Potency

1985 ◽  
Vol 74 (6) ◽  
pp. 651-654 ◽  
Author(s):  
T.M. Penning
Keyword(s):  
Rat Liver ◽  
Liver Cytosol ◽  
Inflammatory Drug ◽  
Drug Potency ◽  
Anti Inflammatory ◽  
Rat Liver Cytosol

scholarly journals Affinity-labelling of the anti-inflammatory drug and prostaglandin-binding site of 3 α-hydroxysteroid dehydrogenase of rat liver cytosol with 17 β- and 21-bromoacetoxysteroids

1987 ◽  
Vol 245 (1) ◽  
pp. 269-276 ◽  
Author(s):  
T M Penning ◽  
K E Carlson ◽  
R B Sharp
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Rat Liver ◽  
Active Site ◽  
Hydroxysteroid Dehydrogenase ◽  
Side Chain ◽  
Liver Cytosol ◽  
Affinity Labelling ◽  
Anti Inflammatory ◽  
Rat Liver Cytosol

The homogeneous 3 alpha-hydroxysteroid dehydrogenase of rat liver cytosol binds prostaglandins with low micromolar affinity at its active site and is competitively inhibited by the non-steroidal and steroidal anti-inflammatory drugs [Penning, Mukharji, Barrows & Talalay (1984) Biochem. J. 222, 601-611]. To examine the portion of this binding site that accommodates the glucocorticoid side chain, we have synthesized 17 beta-bromoacetoxy-5 alpha-dihydrotestosterone (BrDHT) and 21-bromoacetoxydesoxycorticosterone (BrDOC) as affinity-labelling agents. Both these agents promote rapid inactivation of the purified enzyme in a time- and concentration-dependent manner. Analyses of the inactivation progress curves gave estimates of Ki for the inactivators and half-life (t1/2) for the enzyme at saturation (tau) as follows: Ki = 33 microM and tau = 18 s for BrDHT, and Ki = 10 microM and tau = 203 s for BrDOC. Under initial-velocity conditions BrDHT and BrDOC act as competitive inhibitors, yielding Ki values identical with those measured in the inactivation experiments. Both indomethacin and prostaglandin E2 can protect the enzyme from inactivation, yielding Ki values for these ligands consistent with those measured independently by competitive-inhibition studies. These data confirm that the bromoacetoxysteroids label the active site, which is coincident with the prostaglandin- and anti-inflammatory-drug-binding site. Neither gel filtration nor extensive dialysis restores activity to the enzyme inactivated with either affinity-labelling agent. Use of radioactive BrDHT or BrDOC, in which either the steroid portion is labelled with 3H or the bromoacetate portion is labelled with 14C, indicates that inactivation is accompanied by a stoichiometric incorporation of 0.7-1.0 molecules of inhibitor per enzyme monomer. The linkage that forms between the dehydrogenase with either [14C]BrDHT or [14C]BrDOC is stable to acid and base treatment. Complete acid hydrolysis of the enzyme inactivated with [14C]BrDHT, followed by amino acid analyses, indicates that 87% of the radioactivity is eluted with carboxymethylcysteine. An almost identical result is obtained with [14C]BrDOC, where at least 75% of the radioactivity is eluted with this amino acid. Thus BrDHT and BrDOC alkylate at least one reactive cysteine residue at the active site that may be of functional importance in binding the glucocorticoid side chain.

scholarly journals BENZYLIDENE CYCLOPENTANONE DERIVATIVES AS INHIBITORS OF RAT LIVER GLUTATHIONE S-TRANSFERASE ACTIVITIES

2010 ◽  
Vol 5 (1) ◽  
pp. 71-75 ◽  
Author(s):  
Sudibyo Martono
Keyword(s):  
Rat Liver ◽  
Potent Inhibitor ◽  
Inflammatory Activity ◽  
Glutathione S Transferase ◽  
Inhibitory Potency ◽  
Liver Cytosol ◽  
Curcumin Analogues ◽  
Glutathione S Transferases ◽  
Anti Inflammatory ◽  
Rat Liver Cytosol

The effect of the curcumin analogues, 2,6-bis-(4-hydroxy-3-methoxy benzylidene) cyclopentanone (B1) and two of its derivatives on m class glutathione S-transferases (GSTs) from phenobarbital-induced and uninduced rat liver cytosol has been studied to elucidate their anti-inflammatory activity. GST activity was monitored spectrophotometrically using 1,2-dichloro-4-nitrobenzene. B1 was the most potent inhibitor of GSTs, both in uninduced and in phenobarbital-induced rat liver cytosol. These inhibitory properties might be explained as part of the anti-inflammatory activity of benzylidene cyclopentanone derivatives (B1 and B12).   Keywords: curcumin; benzylidene cyclopentanone; inhibitory potency; glutathione S-transferases mesoporous

scholarly journals Purification and properties of a 3α-hydroxysteroid dehydrogenase of rat liver cytosol and its inhibition by anti-inflammatory drugs

1984 ◽  
Vol 222 (3) ◽  
pp. 601-611 ◽  
Author(s):  
T M Penning ◽  
I Mukharji ◽  
S Barrows ◽  
P Talalay
Keyword(s):  
Rat Liver ◽  
Aromatic Aldehydes ◽  
Hydroxysteroid Dehydrogenase ◽  
Liver Cytosol ◽  
Purification And Properties ◽  
Competitive Inhibitors ◽  
Dihydrodiol Dehydrogenase ◽  
Inflammatory Drugs ◽  
Anti Inflammatory ◽  
Rat Liver Cytosol

An NAD(P)-dependent 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50) was purified to homogeneity from rat liver cytosol, where it is responsible for most if not all of the capacity for the oxidation of androsterone, 1-acenaphthenol and benzenedihydrodiol (trans-1,2-dihydroxycyclohexa-3,5-diene). The dehydrogenase has many properties (substrate specificity, pI, Mr, amino acid composition) in common with the dihydrodiol dehydrogenase (EC 1.3.1.20) purified from the same source [Vogel, Bentley, Platt & Oesch (1980) J. Biol. Chem. 255, 9621-9625]. Since 3 alpha-hydroxysteroids are by far the most efficient substrates, the enzyme is more appropriately designated a 3 alpha-hydroxysteroid dehydrogenase. It also promotes the NAD(P)H-dependent reductions of quinones (e.g. 9,10-phenanthrenequinone, 1,4-benzoquinone), aromatic aldehydes (4-nitrobenzaldehyde) and aromatic ketones (4-nitroacetophenone). The dehydrogenase is not inhibited by dicoumarol, disulfiram, hexobarbital or pyrazole. The mechanism of the powerful inhibition of this enzyme by both non-steroidal and steroidal anti-inflammatory drugs [Penning & Talalay (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4504-4508] was examined with several substrates. Most non-steroidal anti-inflammatory drugs are competitive inhibitors (e.g. Ki for indomethacin, 0.20 microM for 9,10-phenanthrenequinone reduction at pH 6.0, and 0.835 microM for androsterone oxidation at pH 7.0), except for salicylates, which act non-competitively (e.g. Ki for aspirin, 650 microM for androsterone oxidation). The inhibitory potency of these agents falls sharply as the pH is increased from 6 to 9. Most anti-inflammatory steroids are likewise competitive inhibitors, except for the most potent (betamethasone and dexamethasone), which act non-competitively. The enzyme is inhibited competitively by arachidonic acid and various prostaglandins.

scholarly journals Alterations in translatable messenger RNA coding for phosphoenolpyruvate carboxykinase (GTP) in rat liver cytosol during deinduction.

1978 ◽  
Vol 253 (12) ◽  
pp. 4327-4332
Author(s):  
D. Kioussis ◽  
L. Reshef ◽  
H. Cohen ◽  
S.M. Tilghman ◽  
P.B. Iynedjian ◽  
...  
Keyword(s):  
Rat Liver ◽  
Messenger Rna ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Liver Cytosol ◽  
Rat Liver Cytosol

scholarly journals Transformation of the glucocorticoid receptor in rat liver cytosol by lysosomal enzymes.

1979 ◽  
Vol 254 (5) ◽  
pp. 1537-1539 ◽  
Author(s):  
J. Carlstedt-Duke ◽  
O. Wrange ◽  
E. Dahlberg ◽  
J.A. Gustafsson ◽  
B. Högberg
Keyword(s):  
Glucocorticoid Receptor ◽  
Rat Liver ◽  
Lysosomal Enzymes ◽  
Liver Cytosol ◽  
Rat Liver Cytosol

Development of an [3H] glucocorticoid exchange assay in rat liver cytosol

Steroids ◽  
1981 ◽  
Vol 37 (4) ◽  
pp. 409-421 ◽  
Author(s):  
Ashutosh Banerji ◽  
Mohammed Kalimi
Keyword(s):  
Rat Liver ◽  
Liver Cytosol ◽  
Rat Liver Cytosol
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