Rat Liver Cytosolic Fraction Research Articles

Transblot identification of biotin-containing proteins in rat liver

Peroxidase-conjugated avidin was used to detect biotin-containing carboxylases in rat liver. By a transblot method, avidin-peroxidase interacted with liver proteins with estimated molecular masses of 120 and 74 kDa. The proteins were identified as pyruvate carboxylase (120 kDa, 6.4 p I) and methylcrotonyl-CoA carboxylase (74 kDa, 7.2 p I) by two-dimensional gel electrophoresis and transblot method. An additional band with estimated molecular mass of 220 kDa was detected in the cytosol fraction of rat liver, compatible with acetyl-CoA carboxylase. Rat liver proteins were prepared and treated with avidin and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transblot with avidin-peroxidase. A 190-kDa band was found with a parallel decrease in the 120-kDa band determined by Coomassie blue staining; however, these proteins did not stain by the transblot avidin-peroxidase method. When the transblot of parallel proteins was incubated with biotin and subsequently with avidin-peroxidase, two additional bands, namely 190 and 145 kDa, were detected while the 74-kDa band disappeared correlated with decreased staining of the 120-kDa band. The present procedure is a simple, rapid, and inexpensive method for detecting biotin-containing proteins in various tissues and organs and in determining the occurrence of nonspecific staining with the avidin-biotin complex method of immunoblot.

Protein Kinase C Located in Rat Liver Nuclei: Partial purification and biochemical and immunochemical characterization

In the rat liver homogenate, maximal protein kinase C activity was found at two calcium concentrations (1.75 and 3.5 mM). Subcellular fractionation of the liver homogenate revealed that the protein kinase C activity requiring 1.75 mM calcium was present only in the cytosolic and particulate subcellular fractions. The protein kinase C activity requiring 3.5 mM calcium concentration was mainly located in the rat liver nuclei preparation. About 19% of the liver homogenate protein kinase C activity requiring 3.5 mM calcium was present in the nuclei. Goat anti-rat brain protein kinase C antibodies revealed a single immunoreactive band at 80-82 kDa in the rat liver nuclear, particulate, or cytosolic fractions. Based on the ratio of plasma membrane marker enzyme activity determined in the nuclear preparation, the purity of the isolated nuclei was ascertained. Rat liver nuclear protein kinase C activity has been partially purified. The purification steps sequentially employed were Triton X-100 extraction of isolated nuclei, DEAE-cellulose chromatography, Phenyl-Superose, and Mono Q (fast protein liquid) chromatography. The final purification step revealed, by silver nitrate staining on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two protein bands at 80 and 66 kDa, respectively. These findings provide definitive data regarding the nuclear location of protein kinase C. The nuclear location of protein kinase C may lead to an understanding of the molecular pathway involved in signal transduction from the plasma membrane to the nucleus.

Application of thermospray/high-performance liquid chromatography/mass spectrometry to the identification of glutathione conjugates derived from bioactive epoxides.

Thermospray high-performance liquid chromatography/mass spectrometry (HPLC/MS) in the positive ion mode has proven to be a useful technique for analysing different synthetic models of potential metabolites of 3,4-epoxyprecocene II (1), the postulated bioactive epoxide responsible for the cytotoxic activity exhibited by precocenes on a variety of insect and mammal tissues. Thus, whereas standards of the glutathione adducts 3a afforded poor responses, the corresponding cysteine (3b) and N-acetylcysteine (3c) derivatives gave defined spectral patterns and good sensitivity levels, particularly when analysed as methyl esters. Likewise, 3,4-dihydrodiols (2) also afforded satisfactory responses. In all cases, the peak at m/z 237, assigned to the stabilized electrophilic oxybenzopyranyl species which results from the loss of the sulphur or oxygen substituent at C(4), was present. Finally, the incubation of racemic epoxide (1) with rat liver cytosolic fraction, followed by enzymatic degradation and further thermospray HPLC/MS analysis of the resulting methylated cysteine derivatives, allowed the identification, for the first time in a biological matrix, of the pairs of cis and trans glutathione conjugates (3a).

Lack of evidence for a hepatic peroxisome proliferator receptor and an explanation for the binding of hypolipidaemic drugs to liver homogenates

The existence of a postulated hepatic receptor responsible for the peroxisomal proliferation induced in rodents by hypolipidaemic drugs has been investigated. [ 3H]-nafenopin and [ 3H]-ciprofibrate were used as labelled ligands and two competitive binding assays, using either a charcoal-dextran or a hydroxylapatite method, were developed to investigate potential binding. In both assay systems, specific displaceable binding of either nafenopin or ciprofibrate to whole homogenate, microsomal and cytosolic fractions of rat liver could not be detected in a variety of buffer systems. A positive control of ligand binding to bovine serum albumin indicated the validity of the binding assays used. In addition, both nafenopin and ciprofibrate exhibited displaceable binding to serum albumin using the hydroxylapatite binding assay and a Scatchard analysis of the binding of [ 3H]-nafenopin to fatty acid free rat serum albumin yielded a dissociation constant of 5.2 × 10 −7 M and 86 pmol of ligand bound per mg protein. Taken collectively, our data strongly argues against the existence of a specific hepatic peroxisome proliferation receptor and indicates that the peroxisome proliferating hypolipidaemic drugs bind to serum albumin and possibly to other cellular proteins not involved in the activation of genes necessary for peroxisome proliferation.

An assay for the α-tocopherol binding protein mediated transfer of vitamin E between membranes

A model system consisting of donor membrane (egg lecithin liposomes) and acceptor membrane (human erythrocyte ghosts or rat liver mitochondria) were used to investigate the α-tocopherol binding protein (αTBP) mediated transfer of α-tocopherol. Liposomes containing RRR-[α- 3H]tocopherol ([α- 3H]T) were incubated with acceptor membrane at 37°C for 0–45 min in the presence or absence of rat liver cytosol or a dialyzed 30–60% saturated ammonium sulfate precipitated fraction of rat liver cytosol (Fraction B). Erythrocyte ghosts and liver mitochondria were compared and found to behave similarly in the presence of Fraction B. α-Tocopherol transfer activity (αTTA) typically varied 0- to 27-fold greater than buffer blanks, depending upon type and concentration of protein preparation. Gel filtration of Fraction B yielded one αTTA peak (liver mitochondria as acceptor) with an estimated M r of 39,000. [α- 3H]T recovered from erythrocyte ghosts pellets by HPLC suggest that the [α- 3H]T was transferred intact. αTTA of Fraction B in the presence of varying concentrations of erythrocyte ghosts and liposomal [α- 3H]T followed saturation kinetics. Optimal concentrations gave αTTA responses directly proportional to rat liver cytosol concentration. αTTA was inhibited only 5% in the presence of a 32-fold excess of cold liposomal α-tocopheryl acetate suggesting that the free hydroxyl group on the chromanol ring of α-tocopherol is needed for transfer. Coefficient of variation of repeated measures of αTTA in rat liver cytosol was 2.9%. Thus, the intermembrane transfer phenomenon of α-tocopherol can be studied uantitatively and can be used to compare liver protein preparations exhibiting transfer activity.

Liquid chromatographic determination of diastereomeric glutathione conjugates and further derivatives of α-bromoisovalerylurea in rat bile and urine by electrochemically generated bromine

To study the glutathione conjugation of α-bromoisovalerylurea in the rat in vivo, a reversed-phase liquid chromatographic assay of the thioether metabolites in bile and urine was developed. Since α-bromoisovalerylurea has a chiral centre, two diastereomeric glutathione conjugates (in bile) and two diastereomeric mercapturates (in urine) can be expected. The separation characteristics of these metabolites and the corresponding cysteine conjugates were investigated. Whereas all thioether metabolites could be separated in one run, optimal separation of the diastereomers required different mobile phases for the glutathione conjugates (in bile) and the mercapturates (in urine). The glutathione conjugates were analysed with the ion-pairing agent sodium decanesulphonate in the mobile phase, but the mercapturates were analysed without an ion-pair-forming agent. For detection, online generation of a constant bromine level (100%) was used; bromine-reactive compounds result in a decrease of the amperometric response from the 100% baseline. This technique could be used in continuous automated operation and required little clean-up of the sample. Thus, the diastereomeric glutathione conjugates and mercapturates were quantified in rat bile and urine samples, respectively, by direct injection of the (centrifuged and diluted) samples on the column. The limit of determination of the respective metabolites was 9 and 2.6 ng in bile and urine, respectively. Incubation mixtures of α-bromoiaovalerylurea with a rat liver cytosolic fraction or with isolated rat hepatocytes were chromatographed after deproteinization with a double volume of methanol. The limit of determination of the diastereomeric glutathione conjugates in the deproteinized incubation samples was 2.0 ng.

Purification and characterization of a neutral protease from rat-liver cytosolic fraction.

Rat liver cytosol contains a neutral protease which degrades acetylated hemoglobin and some urea-denatured proteins maximally at pH 7.5. The enzyme was purified to homogeneity by conventional chromatographic techniques. It appears to be a metalloprotease since it is inhibited by EDTA and o-phenanthroline, the metal-depleted enzyme can be reactivated by Co2+, Zn2+, Mn2+, or Mg2+, and it is not inhibited by reagents specific for carboxyl, seryl, or thiol proteases. The enzyme has an apparent molecular weight of 200,000 as determined on Sephacryl S-200 column chromatography, and electrophoresis in sodium dodecyl sulfate showed 3 protein bands corresponding to the molecular weights of 110,000, 74,000, and 40,000.

Purification and Partial Characterization of Rat Liver Lipoxygenase

Abstract Lipoxygenase was purified from rat liver cytosolic fraction by a method involving two successive chromatographic steps on Sephacryl S-200 and Phenyl Sepharose CL-4B. The enzyme has a molecular weight of 96 Kdal and it seems to be composed of two identical subunits. Chromatofocusing of the enzyme revealed a single band of activity at pi 6.3. The enzyme activity of the purified fraction showed maximum activity at pH 7.0 with a Km for linoleic acid of 1.4 μM and is competitively inhibited by the specific lipoxygenase inhibitor nordihydroguaiaretic acid. The purified enzyme shows absorption and fluorescence spectra similar to those of lipoxygenase from other sources. However, the molecular weight of lipoxygenase purified from liver is found to be different from that of the enzyme from polymorphonuclear leukocytes. It is suggested that there are different isoenzymes of lipoxygenases in mammals.

Effects of levonorgestrel on enzymes responsible for synthesis of triacylglycerols in rat liver

The effects of levonorgestrel treatment (4 μg/day per kg body weight 0.75 for 18 days) on rate-limiting enzymes of hepatic triacylglycerol synthesis, namely glycerol-3-phosphate acyltransferase and phosphatidic acid phosphatase were investigated in microsomal, mitochondrial and cytosolic fractions of rat liver. Levonorgestrel treatment resulted in a significant reduction (26%) of hepatic microsomal glycerol-3-phosphate acyltransferase specific activity. Hepatic mitochondrial glycerol-3-phosphate acyltransferase specific activity was unchanged. Levonorgestrel treatment also significantly reduced (by 20%) the specific activity of hepatic microsomal magnesium-independent phosphatidic acid phosphatase. However, magnesium-dependent phosphatic acid phosphatase specific activities in microsomal and cytosolic fractions were unaffected. Cytosolic magnesium-independent phosphatidic acid phosphatase activity was also unchanged. These studies are consistent with the view that levonorgestrel lowers serum triacylglycerol levels, at least in part, by inhibition of the glycerol-3-phosphate acyltransferase (EC 2.3.1.15) step in hepatic triacylglycerol synthesis.

Metabolism of 2,4-dinitrotoluene, 2,4-dinitrobenzyl alcohol and 2,4-dinitrobenzaldehyde by rat liver microsomal and cytosol fractions.

The metabolism of 2, 4-dinitrotoluene (2, 4-DNT), 2, 4-dinitrobenzyl alcohol (2, 4-DNB) and 2, 4-dinitrobenzaldehyde (2, 4-DNAl) in rat liver microsomal and cytosol fractions was investigated. The objectives of these studies were to determine whether 2, 4-DNAI, a potent mutagen, is produced in the oxidation of 2, 4-DNB to 2, 4-DNBA and to clarify the nature of the enzymes responsible for the oxidation of 2, 4-DNT to 2, 4-DNB, 2, 4-DNAl and 2, 4-dinitrobenzoic acid (2, 4-DNBA). Data obtained from high-performance liquid chromatography indicated that the major products of 2, 4- DNT, 2, 4-DNB and 2, 4-DNAl in the microsomal and cytosol preparations were 2, 4-DNB, 2, 4- DNAl, and 2, 4-DNBA and 2, 4-DNB, respectively. The results indicate that 2, 4-DNAl is an intermediate in the oxidation of 2, 4-DNB to 2, 4-DNBA. In addition, data obtained by incubating 2, 4-DNT, 2, 4-DNB or 2, 4-DNAl with microsomal or cytosol fraction under air, nitrogen and various concentrations of CO in oxygen, using cofactors nicotinamide adenine dinucleotide phosphate and reduced nicotinamide adenine dinucleotide phosphate [NAD (P) and NAD (P) H] and inhibitors (SKF-525A, dimethyl sulfoxide, chloral hydrate, allopurinol, pyrazole and ο- phenanthroline) suggest that : (a) oxidation of 2, 4-DNT to 2, 4-DNB is mediated by microsomal P-450; (b) oxidation of 2, 4-DNB to 2, 4-DNAl is mediated mainly by cytochrome P-450 and NAD-dependent alcohol dehydrogenase; (c) oxidation of 2, 4-DNAl to 2, 4-DNBA and reduction of 2, 4-DNAl to 2, 4-DNB may be mediated by NAD (P) -dependent aldehyde dehydrogenases and NAD (P) H-dependent aldehyde reductases, respectively. These results indicate that 2, 4-DNT is metabolized stepwise to 2, 4-DNB, 2, 4-DNAl and 2, 4-DNBA in the rat liver and suggest that the oxidation of 2, 4-DNB to 2, 4-DNAl is a metabolic activation of 2, 4-DNT and that the microsomal cytochrome P-450 and alcohol dehydrogenase may play an important role in the metabolic activation of 2, 4-DNT.

Mechanism of synthesis and localization of mitochondrial and cytosolic fumarases in rat liver

Fumarases in the mitochondrial and cytosolic fractions of rat liver were separately purified and crystallized. These two fumarases were not distinguishable in physicochemical, catalytic, or immunochemical properties. The sequences of seven amino acids in the C-terminal portions of the two fumarases were shown using carboxypeptidase P to be identical, i.e.-Val-Asp-Glu-Thr-Ala-Leu-Lys-. The amino acid sequence of the N-terminal portion of the mitochondrial fumarase was determined by the Edman method as Ala-Gln-Gln-Asn-Phe-Glu-Ile-Pro-Asp-, but that of the cytosolic fumarase could not be determined by the Edman method, since the N-terminal amino acid was blocked. The N-terminal amino acid of the cytosolic fumarase was identified as N-acetyl-alanine by analysis of the acidic amino acid produced by digestion of the enzyme protein with pronase E, carboxypeptidase A and B. Then the sequence of five amino acids in the N-terminal portion was determined by analyzing the acidic peptide obtained by limited proteolysis of the enzyme protein with carboxypeptidase A as Ac-Ala-Ser-Gln-Asn-Ser-. Peptide mapping of the tryptic peptides obtained from the mitochondrial and cytosolic fumarases showed no difference in the amino acid sequences of the two except in their N-terminal portions. The turnover rates of the mitochondrial and cytosolic fumarases were determined by injecting l-[U- 14C]leucine into rat and following the decay of specific radioactivity incorporated into immunoprecipitates from the partially purified enzyme. The half-life of the cytosolic fumarase was estimated as 4.8 days from the decay curve of its specific radioactivity. The decay curve of the specific radioactivity of the mitochondrial fumarase, obtained after a single injection of l-[U- 14]leucine, was quite unusual: its specific radioactivity remained constant for about 7 days after pulse labeling, and then decreased exponentially with a half-life of 9.7 days. Similar amounts of cytosolic and mitochondrial fumarase were found in the livers of the rat, mouse, rabbit, dog, chicken, snake, frog, and carp, respectively. Similar subcellular distributions of the enzyme were also found in the kidney, heart, and skeletal muscle of rats, and in hepatoma cells (AH-109A). However, in rat brain no fumarase activity was detected in the cytosolic fraction. Two putative precursor polypeptides of rat liver fumarase were synthesized when rat liver RNA was translated in vitro in a rabbit reticulocyte lysate system. One of these putative precursor polypeptides (P 1) had a molecular mass of about 5,000 daltons more than the mature subunit of fumarase (45,000 daltons), while the other (P 2) had the same molecular mass as the mature enzyme. When the 35S-labeled cell-free translation products were incubated with rat liver mitochondria at 30°C, P 1 and the 35S-labeled mature size fumarase became associated with mitochondria. The 35S-labeled mature size fumarase was resistant to externally added protease, but P 1 was not, indicating that the 35S-labeled mature size fumarase was located in the mitochondrial matrix. The following observations strongly suggested that the 35S-labeled mature size fumarase in mitochondria was derived from P 1, which was imported energy-dependently and concomitantly processed to the mature size. 1) The amount of the 35S-labeled mature size fumarase recovered from the mitochondria increased with the duration of incubation, while the amount of P 1 recovered from the post-mitochondrial and mitochondrial fractions decreased with the duration of incubation. 2) Only P 1 could bind with the mitochondrial outer membrane at 0°C even in the presence of an uncoupler of oxidative phosphorylation. 3) P 1 bound to the mitochondrial outer membrane at 0°C was imported into the matrix, when the mitochondria were reisolated and incubated at 30°C in the presence of an energy-generating system. These results suggest that the precursor of the mitochondrial enzyme has a larger molecular weight than the mature enzyme, whereas the precursor for the cytosolic enzyme has the same molecular weight as the mature enzyme and that these two precursors of fumarase are coded by two different mRNAs.

Reduction of the metallochromic indicators murexide and tetramethylmurexide to their free radical metabolites by cytoplasmic enzymes and reducing agents

Murexide underwent reduction by rat liver cytosolic fraction or a hypoxanthine-xanthine oxidase system to produce a free radical metabolite. Reduction of murexide by the freshly prepared cytosolic fraction depended upon the presence of ascorbic acid. N 1-Methylnicotinamide, xanthine or hypoxanthine, in that order, could also serve as a source of reducing equivalents for the production of that free radical by the cytosolic fraction. Several thiol compounds (GSH, cysteine, and cysteamine), pyridine nucleotides (NADH, NADPH) and ascorbic acid were also effective in generating the murexide-derived free radical. Tetramethyl murexide was also reduced to its free radical derivative by a hypoxanthine-xanthine oxidase system.

Rat liver subcellular fractions catalyze aerobic binding of 1-nitro[14C]pyrene to DNA.

The recently characterized environmental mutagen and potential carcinogen 1-nitropyrene (NP) is known to bind DNA in Salmonella typhimurium, and also in anaerobic incubations catalyzed by purified xanthine oxidase. In this study we show that rat liver S9 supernatant, microsomal and cytosolic subcellular fractions are also able to catalyze the binding of 1-nitropyrene labeled with 14C to calf thymus DNA in vitro. In incubations conducted under air, S9 and microsomes from Charles River CD rats were the most active fractions, and NADPH was required for maximum activity (25-100 pmole NP bound/mg DNA/mg protein in 1 hr). S9 and microsomes had about one-fourth the activity under nitrogen, although less of this activity was NADPH-dependent. Binding in cytosolic incubations was generally low (1 to 5 pmole NP/mg DNA/mg protein in 1 hr), was somewhat enhanced under N2, and was more extensive in the absence of NADPH. Treatment of rats (Harlan Sprague-Dawley) with the inducing agents phenobarbital (PB), Aroclor 1254 (A), or 3-methylcholanthrene (3-MC) enhanced NADPH-dependent binding in aerobic S9 (2- to 5-fold) and microsomal (10- to 20-fold) incubations. The effects of induction regimen on binding assays conducted under N2 were more equivocal: 3-MC produced a 2-fold increase in binding in both S9 and microsomes, while the other two agents decreased binding from 50 to 75%. These results indicate that classic cytochrome P-450 inducers were able to stimulate activation of NP, but that this activation is not mediated solely by cytochrome P-450.

Study of bilirubin metabolism by high-performance liquid chromatography: Stability of bilirubin glucuronides

The stabilities of bilirubin (BR) glucuronide, monoglucuronide (BMG), and diglucuronide (BDG) were studied under various conditions by HPLC. In aqueous media, BMG showed a pronounced lability and was easily transformed into equimolar BDG and BR. It was proved by direct analysis of tetrapyrrole isomers that BDG and BR were formed from dipyrrole exchange of BMG molecules. All reducing agents examined (sodium ascorbate, cysteine, GSH, dithiothreitol, NADH, and NADPH) suppressed the transformation of BMG into BDG and BR. Bovine serum albumin and rat liver cytosol fractions also stabilized BMG strongly. BDG was fairly stable in aqueous media as compared with BMG. When BMG was incubated both with and without liver plasma membranes (N 2 fraction) from Wistar rats, the formation rates of BDG and BR in both incubation mixtures were exactly the same. The composition of BDG and BR isomers was the same in both mixtures. Also, heat denaturation of the plasma membranes did not affect formation rates. Moreover, the reaction was completely inhibited by sodium ascorbate. These findings indicate that rat liver plasma membranes have no enzyme activity for BDG formation from BMG.

Acid-catalyzed formation of isoprene from a mevalonate-derived product using a rat liver cytosolic fraction.

Isoprene formation in a rat liver cytosolic fraction is shown to be increased 146-fold by acid treatment. This acid catalysis is dependent upon prior incubation of the cytosolic fraction with DL-mevalonate and is stimulated when the incubation also contains ATP. Formation of isoprene proceeds linearly through 5 h of acid treatment and is nearly complete at 10 h. These results suggest that the acid-catalyzed isoprene formation arises from the decomposition of dimethylallyl pyrophosphate via a carbonium ion mechanism. Chemical model studies using 3-methyl-2-buten-1-ol and 3-methyl-3-buten-1-ol (the alcohols corresponding to dimethylallyl pyrophosphate and isopentenyl pyrophosphate, respectively) confirm this hypothesis. At a pH less than or equal to 1, an 85% decomposition of 3-methyl-2-buten-1-ol to isoprene occurred after 24 h, while 3% of 3-methyl-3-buten-1-ol was converted to isoprene under identical conditions and time. It is concluded that the predominant immediate precursor of isoprene is dimethylallyl pyrophosphate and at low pH the ultimate fate of dimethylallyl pyrophosphate is complete conversion to isoprene. These conclusions have important biochemical and methodological implications.

Reduction of the metallochromic indicators arsenazo III and antipyrylazo III to their free radical metabolites by cytoplasmic enzymes

At a concentration much lower than that usually employed for measuring cytosolic ionized Ca 2+ concentrations, arsenazo III underwent a one-electron reduction by rat liver cytosolic fraction or a hypoxanthinexanthine oxidase system to produce an azo anion radical metabolite. NADH, NADPH, N 1-methylnicotinamide, hypoxanthine, and xanthine, in that order, could serve as a source of reducing equivalents for the production of this free radical by the cytosolic fraction. The steady-state concentration of the azo anion radical and the arsenazo III-stimulated O 2 consumption were enhanced by calcium and magnesium. Antipyrylazo III was ineffective in increasing O 2 consumption by rat liver cytosolic fraction and gave a much weaker ESR signal of an azo anion radical with both the liver cytosolic fraction, in the presence of NADH, and the hypoxanthine-xanthine oxidase system.

Copper increases superoxide dismutase activity in rat liver

Superoxide dismutase (SOD) activity in rat liver cytosol and submitochondrial fractions was characterized as enzymatic and nonenzymatic (due to the SOD-like activity of copper) by four approaches: (i) aerobic NBT 2+ (nitroblue tetrazolium) photoreduction in the absence of EDTA; (ii) aerobic NBT 2+ photoreduction in the presence of 10 −4 m EDTA; (iii) anaerobic NBT 2+ photoreduction; and (iv) o-dianisidine photooxidation. Under normal conditions nonenzymatic SOD activity has been observed only in the intermembrane space. The single subcutaneous injection of rats with CuSO 4 solution (5 mg Cu/kg body wt) led to (i) an elevation of the copper level in all submitochondrial fractions; (ii) an increase in enzymatic SOD activity in only cytosol and intermembrane spaces; (iii) the appearance of a new electrophoretic SOD activity band in the intermembrane space preparations; and (iv) the appearance of nonenzymatic SOD-like activity in the outer and inner mitochondrial membranes, and a twofold increase in lipid hydroperoxides. This suggests that the increased nonenzymatic copper in vivo has a prooxidant effect, and does not catalyze the dismutation of O 2 − as it has been shown in in vitro experiments [ E. M. Russanov S. G. Ljutakova, and S. I. Leutcher (1982) Arch. Biochem. Biophys. 215, 220–229 ]. The peculiarities of the SOD activity in the intermembrane space are explained by the lysosomal localization of the granular CuZnSOD.

Reduction of 2,4-dinitrotoluene by Wistar rat liver microsomal and cytosol fractions

2,4-Dinitrotoluene (2,4-DNT) is an important industrial nitroaromatic compound. 2,4-Diaminotoluene (2,4-DAT), one of the urinary metabolites of 2,4-DNT, is carcinogenic when fed to rats. The objectives of these studies were to determine whether 2,4-DAT was formed from 2,4-DNT in rat liver and to clarify the nature of enzymes responsible for reduction of 2,4-DNT to 2,4-DAT. Data obtained from thin-layer and high-pressure liquid chromatography indicated that metabolites produced by microsomal preparations were 2-amino-4-nitrotoluene (2A4NT) and its isomer (4A2NT). This microsomal activity is probably mediated by cytochrome P-450 because the reduction is blocked by carbon monoxide and primary amines [aniline, n-octylamine, and 2,4-dichloro-6-phenylphenoxyethylamine (DPEA)]. In contrast, 2,4-DNT was metabolized via 2A4NT and 4A2NT to 2,4-DAT by cytosolic preparations. The greatest part of the reduction activity was due to cytosolic xanthine oxidase because the reduction was blocked by allopurinol. The results of this investigation suggest that reduction of 2,4-DNT to 2,4-DAT by cytosolic xanthine oxidase may play a role in 2,4-DNT hepatocarcinogenicity.

Formula omitted] biosynthesis of isoprene from mevalonate utilizing a rat liver cytosolic fraction

The in vitro biosynthesis of isoprene from DL-mevalonate in the cytosolic fraction of rat liver is described. Evidence is provided suggesting a non-enzymatic formation of isoprene from isopentenyl pyrophosphate and/or dimethylallyl pyrophosphate. Furthermore, the data establish an alternate fate of the mevalonate carbon skeleton providing the first evidence that breath isoprene is linked to cholesterol biosynthesis.

Reductive metabolism and activation of benznidazole

Benznidazole (Bz) ( N-benzyl-2-nitro-1-imidazole-acetamide) is a drug used against Chagas' disease. Rat liver microsomal and cytosolic fractions, but not mitochondria, exhibited Bz nitroreductase activity under anaerobic conditions in the presence of NADPH. Microsomal nitroreductase activity was enhanced by FAD and was inhibited totally by oxygen and partially by carbon monoxide. Liver cystosol fraction was able to reduce Bz nitrogroups in the presence of either N-methylnicotinamide of hypoxanthine as substrates. These enzyme activities were inhibited by menadione or allopurinol respectively. Under every experimental condition leading to enzymatic reduction of Bz nitrogroups and its inhibition or enhancement, reactive metabolites that bind covalently to proteins were also produced. This covalent binding was effectively prevented by reduced glutathione. Results suggest the participation of cytochrome P-450 and cytochrome c reductase in liver microsomal processes and of xanthine oxidase and aldehyde oxidase in liver cytosolic processes of Bz nitroreduction and activation to reactive metabolites that bind covalently to proteins. Possible pharmacological and toxicological implications of the described observations were discussed.