Preparation of a soluble 58kDa-3-hydroxy-3-methylglutaryl CoA reductase from liver microsomes and its inhibition by ethoxysilatrane, a hypocholesterolemic compound

On repeated thawing at room temperature of frozen preparations of heavy microsomes from rat livers, HMGCoA reductase activity was solubilized due to limited proteolysis. This soluble enzyme was partially purified by fractionation with ammonium sulfate and filtration on Sephacryl S-200 column. The active enzyme was coeluted with a major 92 kDa-protein and was identified as a 58kDa-protein after separation by SDS-PAGE and immunoblotting. Ethoxysilatrane, a hypocholesterolemic compound, which decreased the liver-microsomal activity of HMGCoA reductase on intra-peritonial treatment of animals, showed little effect on the enzyme activity with isolated microsomes or the 50kDa-soluble enzyme when added in the assay. But it was able to inhibit the activity of the soluble 58kDa-enzyme in a concentration-dependent, reversible manner. Cholesterol and an oxycholesterol were without effect whereas chlorophenoxyisobutyrate and ubiquinone showed small inhibition under these conditions. The extra region that links the active site domain (50kDa protein) to the membrane, present in the 58kDa-protein appears to be involved in mediating the inhibition by silatrane.

Adriamycin treatment increases 3-hydroxy-3-methylglutaryl CoA reductase and isoprene biosynthesis in the rat liver

Treatment of rats with Adriamycin caused an increase in the incorporation into hepatic cholesterol of [1-14C] acetate, but not of [2-14C] mevalonate. The step affected was found to be 3-hydroxy-3-methylglutaryl CoA reductase whose activity in the liver microsomes increased in Adriamycin-treated animals, but was inhibited when the drug was added in the assay medium. Also, the concentration of ubiquinone in the liver and of cholesterol in the plasma increased.

Biochemical effects of 3,5-diethoxycarbonyl-1, 4-dihydrocollidine in mouse liver

3,5-Diethoxycarbonyl-1,4-dihydrocollidine (DDC) is a porphyrinogenic agent and is a powerful inducer of δ-aminolaevulinate synthetase, the first and rate-limiting enzyme of the haem-biosynthetic pathway, in mouse liver. However, DDC strikingly inhibits mitochondrial as well as microsomal haem synthesis by depressing the activity of ferrochelatase in vivo. The drug on repeated administration to female mice has been found to elicit hypertrophic effects in the liver microsomes initially, but the effects observed at later stages denote either hyperplasia or increase in polyploidal cells. The microsomal protein concentration shows a striking decrease with repeated doses of the drug. The rate of microsomal protein synthesis in vivo as well as in vitro shows an increase with two injections of DDC but decreases considerably with repeated administration of the drug. The activities of NADPH-cytochrome creductase and ribonuclease are not affected in the liver microsomes of drug-treated animals when expressed per mg of microsomal protein. DDC has also been found to cause degradation of microsomal haem, which is primarily responsible for the decrease in cytochrome P-450 content. The drug also leads to a decrease in mitochondrial cytochrome c levels due to inhibition of haem synthesis and also due to degradation of mitochondrial haem at later stages. The biochemical effects of the drug are compared and discussed with those reported for allylisopropylacetamide and phenobarbital.

Metabolic fate of menthofuran in rats. Novel oxidative pathways

Metabolic fate of menthofuran (II) in rats was investigated. Menthofuran (II) was administered orally (200 mg/kg of the body weight/day) to rats for 3 days. The following metabolites were isolated from the urine of these animals: p-cresol (VI), 5-methyl-2-cyclohexen-1- one (VII), 3-methylcyclohexanone (VIII), 3-methylcyclohexanol (IX), 4- hydroxy-4-methyl-2-cyclohexen-1-one (V), geranic acid (XI), neronic acid (XII), benzoic acid (XIII), and 2-[2'-keto-4'- methylcyclohexyl]propionic acid (X). Incubation of menthofuran (II) with phenobarbital-induced rat liver microsomes in the presence of NADPH and oxygen resulted in the formation of a metabolite tentatively identified as 2-Z-(2'-keto-4'-methylcyclohexylidene)propanal (III; alpha,beta-unsaturated-gamma-keto-aldehyde). The structure assigned was further supported by trapping this metabolite (III) as a cinnoline derivative. Phenobarbital-induced rat liver microsomes also converted 4- methyl-2-cyclohexenone (IV) to 4-hydroxy-4-methyl-2-cyclohexenone (V) and p-cresol (VI) in the presence of NADPH and oxygen. On the basis of both in vivo and in vitro studies, a possible mechanism for the formation of p-cresol from menthofuran has been proposed.

Studies on the metabolism of l-menthol in rats

Metabolism of l-menthol in rats was investigated both in vivo and in vitro. Metabolites isolated and characterized from the urine of rats after oral administration (800 mg/kg of body weight/day) of l-menthol were the following: p-menthane-3,8-diol (II), p-menthane-3,9-diol (III), 3,8-oxy-p-menthane-7-carboxylic acid (IV), and 3,8-dihyroxy-p-menthane-7-carboxylic acid (V). In vivo, the major urinary metabolites were compounds II and V. Repeated oral administration (800 mg/kg of body weight/day) of l-menthol to rats for 3 days resulted in the increase of both liver microsomal cytochrome P-450 content and NADPH-cytochrome c reductase activity by nearly 80%. Further treatment (for 7 days total) reduced their levels considerably, although the levels were still higher than the control values. Both cytochrome b5 and NADH-cytochrome c reductase levels were not changed during the 7 days of treatment. Rat liver microsomes readily converted l-menthol to p-menthane-3,8-diol (II) in the presence of NADPH and O2. This activity was significantly higher in microsomes obtained from phenobarbital (PB)-induced rats than from control microsomal preparations, whereas 3-methylcholanthrene (3-MC)-induced microsomes failed to convert l-menthol to compound II in the presence of NADPH and O2. l-Menthol elicited a type I spectrum with control (Ks = 60.6 microM) and PB-induced (Ks = 32.3 microM) microsomes whereas with 3MC-induced microsomes it produced a reverse type I spectrum.

Evidence for the formation of a known toxin, P-cresol, from menthofuran

Menthofuran (II, 4,5,6,7-tetrahydro-3,6-dimethyl benzofuran), the proximate toxin of R-(+)-pulegone (I), was administered orally to rats (200 mg/kg of body weight/day) for three days and the urinary metabolites were investigated. Among the several metabolites formed, two of them viz. 4-Hydroxy-4-methyl-2-cyclohexenone (VII) and p-cresol (VIII) were indentified. In support of the formation of these metabolites, it has been demonstrated that phenobarbital induced rat liver microsomes readily convert 4-methyl-2-cyclohexenone (V) to 4-hydroxy-4-methyl-2-cyclohexenone (VII) and p-cresol (VIII) in the presence of NADPH and O2. Possible mechanism for the formation of these two metabolites (VII, VIII) from menthofuran (II) has been proposed.

Microsomal redox systems in brown adipose tissue: high lipid peroxidation, low cholesterol biosynthesis and no detectable cytochrome P-450

The presence of redox systems in microsomes of brown adipose tissue (BAT) in cold exposed rats was investigated and compared with liver. BAT microsomes showed high activity of lipid peroxidation measured both by the formation of malondialdehyde (MDA) and by oxygen uptake. NADH and NADPH dependent cytochrome c reductase activity were present in both BAT and liver microsomes. Aminopyrine demethylase and aniline hydroxylase activities, the characteristic detoxification enzymes in liver microsomes could not be detected in BAT microsomes. BAT minces showed very poor incorporation of [1-14C]acetate and [2-14C]-mevalonate in unsaponifiable lipid fraction compared to liver. Biosynthesis of cholesterol and ubiquinone, but not fatty acids, and the activity of 3-hydroxy-3-methyl glutaryl CoA reductase appear to be very low in BAT. Examination of difference spectra showed the presence of only cytochrome b 5 in BAT microsomes. In addition to the inability to detect the enzyme activities dependent on cytochrome P-450, a protein with the characteristic spectrum, molecular size in SDS-PAGE and interaction with antibodies in double diffusion test, also could not be detected in BAT microsomes. The high activity of lipid peroxidation in microsomes, being associated with large oxygen uptake and oxidation of NADPH, will also contribute to the energy dissipation as heat in BAT, considered important in thermogenesis.

Dexamethasone negatively regulates phenobarbitone-activated transcription but synergistically enhances cytoplasmic levels of cytochrome P-450b/e messenger RNA

Dexamethasone has a potentiating effect on phenobarbitone mediated induction of cytochrome P-450b + e mRNAs in adult rat liver. However, the glucocorticoid inhibits phenobarbitone-activated transcription of cytochrome P-450b + e mRNAs by 60-70%. This inhibitory effect is evident in run-off transcription of the endogenous genes as well as in the transcription of an added cloned gene fragment. Dexamethasone inhibits the phenobarbitone-mediated increase in the binding of a transcription factor(s) to the upstream region of the gene as evidenced by gel retardation and Southwestern blot analysis. The glucocorticoid does not stabilize the phenobarbitone-induced polyribosomal cytochrome P-450b + e mRNAs but appears to stabilize the nuclear transcripts. It is proposed that a negative element may mediate the action of dexamethasone at the level of nuclear transcription and stabilization of the nuclear transcript may account for the potentiating effect of the glucocorticoid on phenobarbitone-mediated increase in cytochrome P-450b + e mRNAs in the cytoplasm of the adult rat liver. However, the cytochrome P-450b protein levels are slightly lower in phenobarbitone + dexamethasone treatment than in phenobarbitone-treated liver microsomes.

A novel phenomenon of burst of oxygen uptake during decavanadate-dependent oxidation of NADH

Oxidation of NADH by decavanadate, a polymeric form vanadate with a cage-like structure, in presence of rat liver microsomes followed a biphasic pattern. An initial slow phase involved a small rate of oxygen uptake and reduction of 3 of the 10 vanadium atoms. This was followed by a second rapid phase in which the rates of NADH oxidation and oxygen uptake increased several-fold with a stoichiometry of NADH: O2 of 1ratio1. The burst of NADH oxidation and oxygen uptake which occurs in phosphate, but not in Tris buffer, was prevented by SOD, catalase, histidine, EDTA, MnCl2 and CuSO4, but not by the hydroxyl radical quenchers, ethanol, methanol, formate and mannitol. The burst reaction is of a novel type that requires the polymeric structure of decavanadate for reduction of vanadium which, in presence of traces of H2O2, provides a reactive intermediate that promotes transfer of electrons from NADH to oxygen.

Metabolic disposition of a monoterpene ketone, piperitenone, in rats: Evidence for the formation of a known toxin, p-cresol

It was shown earlier that the monoterpene ketone, piperitenone (I) is one of the major metabolites of R-(+)-pulegone, a potent hepatotoxin, In the present studies, the metabolic disposition of piperitenone (I) was examined in rats. Piperitenone (I) was administered orally (400 mg/kg of the b. wt./day) to rats for 5 days, The following urinary metabolites were isolated and identified by various spectral analyses: p-cresol (VI), 6,7-dehydromenthofuran (III), p-mentha-1,3,5,8-tetraen-3-ol (IX), p-mentha-1,3, 5-friene-3, 8-diol (X), 5-hydroxypiperitenone (VIII), 7-hydroxypiperitenone (XI), 10-hydroxypiperitenone (XII), and 4-hydroxypiperitenone (VII). Incubation of piperitenone (I) with phenobarbital-induced rat liver microsomes in the presence of NADPH resulted in the formation of five metabolites which have been tentatively identified as metabolites III, VII, VIII, XI, XII, on the basis of gas chromatography retention time and gas chromatography-mass spectrometry analysis. Based on these results, a probable mechanism for the formation of p-cresol from piperitenone (I) via the intermediacy of metabolite III has been proposed.

Metabolites of PPI-2458, a selective, irreversible inhibitor of methionine aminopeptidase-2: structure determination and In vivo activity

The natural product fumagillin exhibits potent antiproliferative and antiangiogenic properties. The semisynthetic analog PPI-2458, (3R,4S,5S,6R)-5-methoxy-4-(2R,3R)-2-methyl-3-(3-methylbut-2-enyl) oxiran-2-yl]-1-oxaspiro2.5]octan-6-yl] N-(2R)-1-amino-3-methyl-1-oxobutan-2-yl]carbamate, demonstrates rapid inactivation of its molecular target, methionine aminopeptidase-2 (MetAP2), and good efficacy in several rodent models of cancer and inflammation with oral dosing despite low apparent oral bioavailability. To probe the basis of its in vivo efficacy, the metabolism of PPI-2458 was studied in detail. Reaction phenotyping identified CYP3A4/5 as the major source of metabolism in humans. Six metabolites were isolated from liver microsomes and characterized by mass spectrometry and nuclear resonance spectroscopy, and their structures were confirmed by chemical synthesis. The synthetic metabolites showed correlated inhibition of MetAP2 enzymatic activity and vascular endothelial cell growth. In an ex vivo experiment, MetAP2 inhibition in white blood cells, thymus, and lymph nodes in rats after single dosing with PPI-2458 and the isolated metabolites was found to correlate with the in vitro activity of the individual species. In a phase 1 clinical study, PPI-2458 was administered to patients with non-Hodgkin lymphoma. At 15 mg administered orally every other day, MetAP2 in whole blood was 80% inactivated for up to 48 hours, although the exposure of the parent compound was only similar to 10% that of the summed cytochrome P450 metabolites. Taken together, the data confirm the participation of active metabolites in the in vivo efficacy of PPI-2458. The structures define a metabolic pathway for PPI-2458 that is distinct from that of TNP-470 ((3R, 4S, 5S, 6R)-5-methoxy-4-(2R, 3R)-2-methyl-3-(3-methylbut-2-enyl)oxiran-2-yl]-1-oxaspiro2.5]octan-6 -yl] N-(2-chloroacetyl)carbamate). The high level of MetAP2 inhibition achieved in vivo supports the value of fumagillin-derived therapeutics for angiogenic diseases.

Intracellular distribution of coenzyme Q in rat liver

Coenzyme Q was found to be distributed in rat liver cell fractions. Mitochondria accounted for only 40–60% of the total. The presence of coenzyme Q in nuclei, isolated by several methods, could always be correlated with the presence of oxidative enzymes. It has been established that coenzyme Q is a constituent of microsomes. Administered coenzyme Q10-C14 was preferentially taken up by mitochondrial and microsomal fractions. Exogenous coenzyme Q appears to be rapidly metabolized.

Importance of the amino terminus in maintenance of oligomeric structure of sheep liver cytosolic serine hydroxymethyltransferase

The Role Of The Amino And Carboxyl-Terminal Regions Of Cytosolic Serine Hydroxymethyltransferase (SHMT) In Subunit Assembly And Catalysis Was Studied Using Sis Amino-Terminal (Lacking The First 6, 14, 30, 49, 58, And 75 Residues) And Two Carboxyl-Terminal (Lacking The Last 49 And 185 Residues) Deletion Mutants. These Mutants Were Constructed From A Full Length Cdna Clone Using Restriction Enzyme/PCR-Based Methods And Overexpressed In Escherichia Coli. The Overexpressed Proteins, Des-(A1-K6) SHMT And Des-(A1-W14)-SHMT Were Present In The Soluble Fraction And They Were Purified To Homogeneity. The Deletion Clones, For Des-(A1-V30)-SHMT And Des-(A1-L49)-SHMT Were Expressed At Very Low Levels, Whereas Des-(A1-R58)-SHMT, Des-/A1-G75)-SHMT, Des-(Q435-F483)-SHMT And Des-(L299-F483)-SHMT Mutant Proteins Were Not Soluble And Formed Inclusion Bodies. Des-(A1-K6)-SHMT And Des-(A1-W14)-SHMT Catalyzed Both The Tetrahydrofolate-Dependent And Tetrahydrofolate-Independent Reactions, Generating Characteristic Spectral Intermediates With Glycine And Tetrahydrofolate. The Two Mutants Had Similar Kinetic Parameters To That Of The Recombinant SHMT (Rshmt). However, At 55 Degrees C, The Des-(A1-W14)-SHMT Lost Almost All The Activity Within 5 Min, While At The Same Temperature Rshmt And Des-(A1-K6)-SHMT Retained 85% And 70% Activity, Respectively. Thermal Denaturation Studies Showed That Des-(A1-W14)-SHMT Had A Lower Apparent Melting Temperature (52 Degrees C) Compared To Rshmt (56 Degrees C) And Des-(A1-K6)-SHMT (55 Degrees C), Suggesting That N-Terminal Deletion Had Resulted In A Decrease In The Thermal Stability Of The Enzyme. Further Urea Induced Inactivation Of The Enzymes Revealed That 50% Inactivation Occurred At A Lower Urea Concentration (1.2+/-0.1 M) In The Case Of Des-(A1-W14)-SHMT Compared To Rshmt (1.8+/-0.1 M) And Des-(A1 -K6)-SHMT (1.7+/-0.1 M). The Apoenzyme Of Des-/A1-K6)-SHMT Was Present Predominantly In The Dimer Form, Whereas The Apoenzymes Of Rshmt And Des-(A1-K6)-SHMT Were A Mixture Of Tetramers (Approximate To 75% And Approximate To 65%, Respectively) And Dimers. While, Rshmt And Des-(A1-K6)-SHMT Apoenzymes Could Be Reconstituted Upon The Addition Of Pyridoxal-5'-Phosphate To 96% And 94% Enzyme Activity, Respectively Des-(A1-W14)-SHMT Apoenzyme Could Be Reconstituted Only Upto 22%. The Percentage Activity Refined Correlated With The Appearance Of Visible CD At 425 Nm And With The Amount Of Enzyme Present In The Tetrameric Form Upon Reconstitution As Monitored By Gel Filtration. These Results Demonstrate That, In Addition To The Cofactor, The N-Terminal Arm Plays An Important Role In Stabilizing The Tetrameric Structure Of SHMT.

Purification and functional characterization of type II DNA topoisomerase from rat testis and comparison with topoisomerase II from liver

A number of studies in yeast have shown that DNA topoisomerase TI is essential for chromosome condensation and disjunction during mitosis at the metaphase/anaphase transition and meiosis I. Accordingly, kinetic and mechanistic studies have implied a role for topoisomerase rr in chromosome disjunction. As a step toward understanding the nature and role of topoisomerase II in a mammalian germline in vivo, we have purified topoisomerase II from rat testis to homogeneity and ascertained several of its catalytic activities in conjunction with that of the purified enzyme from liver. The purified enzymes appeared to be monomers under denaturing conditions; however, they differed in their relative molecular mass. Topoisomerase II from testis and liver have apparent molecular masses of 150 +/- 10 kDa and 160 +/- 10 kDa, respectively. The native molecular mass of testis topoisomerase II as assayed by immunoblot analysis of cell-foe extracts, prepared in the presence of SDS and a number of protease inhibitors, corroborated with the size of the purified enzyme. Both enzymes are able to promote decatenation and relax supercoiled DNA substrates in an ATP and Mg2+-dependent manner. However, quantitative comparison of catalytic properties of topoisomerase II from testis with that of the enzyme from liver displayed significant differences in their efficiencies. Optimal pH values for testis enzyme are 6.5 to 8.5 while they are 6 to 7.5 for the liver enzyme. Intriguingly, the relaxation activity of liver topoisomerase II was inhibited by potassium glutamate at 1 M, whereas testis enzyme required about half its concentration. These findings argue that topoisomerase II from rat testis is structurally distinct from that of its somatic form and the functional differences between the two enzymes parallels with the physiological environment that is unique to these two tissues.

Cytochrome c oxidase is preferentially synthesized in the rough endoplasmic reticulum--mitochondrion complex in rat liver

The specific activity and content of cytochrome oxidase in the rough endoplasmic reticulum--mitochondrion complex are higher than in the mitochondrial fraction. Radiolabelling studies with the use of hepatocytes and isolated microsomal and rough endoplasmic reticulum--mitochondrion fractions, followed by immunoprecipitation with anti-(cytochrome oxidase) antibody, reveal that the nuclear-coded cytoplasmic subunits of cytochrome oxidase are preferentially synthesized in the latter fraction. The results have a bearing on the mechanism of transport of these subunits into mitochondria.