scholarly journals Inhibitory Effects of Glycyrrhetic Acid and Its Related Compounds on 3.ALPHA.-Hydroxysteroid Dehydrogenase of Rat Liver Cytosol.

1992 ◽  
Vol 40 (5) ◽  
pp. 1208-1210 ◽  
Author(s):  
Teruaki AKAO ◽  
Taiko AKAO ◽  
Masao HATTORI ◽  
Tsuneo NAMBA ◽  
Kyoichi KOBASHI
Keyword(s):  
Rat Liver ◽  
Hydroxysteroid Dehydrogenase ◽  
Glycyrrhetic Acid ◽  
Inhibitory Effects ◽  
Liver Cytosol ◽  
Related Compounds ◽  
Rat Liver Cytosol

scholarly journals Electrophoretic and immunochemical characterization of 3α-hydroxysteroid/dihydrodiol dehydrogenases of rat tissues

1988 ◽  
Vol 254 (3) ◽  
pp. 715-721 ◽  
Author(s):  
T E Smithgall ◽  
T M Penning
Keyword(s):  
Western Blot ◽  
Rat Liver ◽  
Hydroxysteroid Dehydrogenase ◽  
Rat Tissues ◽  
Sprague Dawley ◽  
Liver Cytosol ◽  
Immunoreactive Protein ◽  
Sprague Dawley Rat ◽  
Dihydrodiol Dehydrogenase ◽  
Rat Liver Cytosol

The properties of 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase from Sprague-Dawley rat liver cytosol have been re-examined in light of several reports which suggest that multiple forms of the enzyme may exist in this tissue. During enzyme purification, chromatography on DE-52 cellulose and chromatofocusing columns indicated the existence of only one form of the protein. Re-chromatography of the purified enzyme by either of these techniques failed to resolve the protein into additional forms. When the purified enzyme was subjected to SDS/polyacrylamide-gel electrophoresis a single band corresponding to Mr 34,000 was detected. Two-dimensional gels showed one predominant protein with a pI of 5.9. Using the homogeneous enzyme as antigen, high-titre polyclonal antibody was raised in rabbits. Western-blot analysis of cytosolic proteins prepared from male and female Sprague-Dawley rat liver indicated the presence of a single immunoreactive band with an Mr of 34,000 in both sexes. All of the 3 alpha-hydroxysteroid dehydrogenase activity present in rat liver cytosol could be immunotitrated with the antibody and the resulting titration curve was superimposable on the titration curve obtained with the purified enzyme. Western-blot analysis of cytosolic proteins prepared from livers of male Wistar and Fischer rats also revealed the presence of a single immunoreactive protein with an Mr of 34,000. These data indicate that, contrary to previous reports, only one form of the dehydrogenase may exist in liver cytosols prepared from a variety of rat strains. Although 3 alpha-hydroxysteroid dehydrogenase activity is known to be widely distributed in male Sprague-Dawley rat tissues, Western blots indicate that only the liver, lung, testis and small intestine contain immunoreactive protein with an Mr of 34,000. The levels of immunoreactive protein in these tissues follow the distribution of dihydrodiol dehydrogenase.

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 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 Sulphation of p-hydroxyphenylpyruvic acid and related compounds by the rat liver cytosol

1975 ◽  
Vol 150 (2) ◽  
pp. 175-181 ◽  
Author(s):  
P M Hext ◽  
F A Rose
Keyword(s):  
Rat Liver ◽  
Main Product ◽  
Homogentisic Acid ◽  
Liver Cytosol ◽  
Liver And Kidney ◽  
Related Compounds ◽  
Spontaneous Oxidation ◽  
Hydroxybenzyl Alcohol ◽  
Rat Liver Cytosol ◽  
Mammalian System

Cytosol preparations of rat liver and kidney were examined for their ability to transfer sulphate from adenosine 3′-phosphate 5′-sulphatophosphate to p-hydroxyphenylpyruvic acid. Little activity towards this substrate was observed, and the main product detected in the reaction mixtures was identified as p-hydroxybenzyl alcohol O-sulphate. This was not formed from p-hydroxybenzaldehyde, a spontaneous oxidation product of p-hydroxyphenylpyruvic acid, by sulphation followed by a rapid enzyme-catalysed reduction of the intermediate phydroxybenzaldehyde O-sulphate. This product was formed mainly by this sequence of reactions, but the reverse, reduction followed by sulphation, also appeared possible. p-Hydroxybenzyl alcohol itself was very readily sulphated by both preparations, and the liver also produced a sulpho-conjugate of homogentisic acid. These observations are important in calculating the turnover of L-tyrosine O-sulphate in the mammalian system, and establish that p-hydroxyphenylpyruvic acid O-sulphate is an end product of its metabolism, rather than an intermediate in its synthesis by reversed transamination.

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
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