Liver Soluble Fraction Research Articles

Comparative proteomic analysis of mouse liver and brain isatin-binding proteins

Isatin (indol-2,3-dione) is an endogenous indole, exhibiting various biological activities that are realized via its interacts with numerous target proteins (so-called isatin-binding proteins). To date, isatin-binding proteins have been characterized in the brain of mice and rats. In this study we have performed a comparative proteomic analysis of the isatin-binding proteins of the mouse liver and brain. Proteomic profiling of clarified lysates of membrane and soluble fractions of liver and brain homogenates was performed using 5-aminocaproyl-isatin as an affinity ligand. During affinity based separation of isatin-binding proteins of soluble and membrane fractions of mouse brain homogenates lysed with Triton X-100, 63 individual proteins were identified. A similar separation of mouse liver homogenate fractions during affinity chromatography resulted in identification of 80 proteins. All identified liver and brain proteins belonged to the following functional groups: (I) Carbohydrate metabolism and energy generation; (II) Lipid metabolism; (III) Metabolism of nucleotides and amino acids; (IV) Formation of the cytoskeleton, exocytosis; (V) Regulation of gene expression, cell division and differentiation; (VI) Antioxidant and protective proteins; (VII) Signal transmission and regulation of enzyme activity. The total number of isatin-binding proteins common for the brain and liver was only 12. The most common for the brain and liver of isatin-binding proteins was found in group VI (antioxidant and protective proteins), complete absence of coincidence in group II (lipid metabolism) and group IV (formation of the cytoskeleton, exocytosis). The observed differences in the profile of isatin-binding proteins appear to play an important role in the specific effects of isatin in certain organs.

Changes in Activity and Kinetic Properties of the Proteasome in Different Rat Organs during Development and Maturation

The proteasome is considered the most important proteolytic system for removal of damaged proteins with aging. Using fluorogenic peptide substrates, the chymotrypsin-like, the trypsin-like, and the peptidylglutamyl peptidase activities of the proteasome were measured in the soluble fractions of liver, brain, and lens rat homogenates. Specific activity was significantly decreased in liver and brain homogenates with maturation of the animal, that is, from newborn (7 days old) to fertile rats (2–4 months old). Rat lens homogenate exhibited an increase in activity with maturation and also with aging. Chymotrypsin-like activity was stimulated by calcium and this proteolytic activity was significantly decreased with maturation of the rat brain. The Michaelis-Menten constant (K m) increased with age in rat liver and lens, indicating a loss of affinity for its substrates by the proteasome in the animal with maturation and aging. The present data suggest that the loss of function of the proteasome with maturation may be due to structural changes of the proteasome or a decreased content of regulatory components.

Metabolism of trans, trans-muconaldehyde, a cytotoxic metabolite of benzene, in mouse liver by alcohol dehydrogenase Adh1 and aldehyde reductase AKR1A4

The reductive metabolism of trans, trans-muconaldehyde, a cytotoxic metabolite of benzene, was studied in mouse liver. Using an HPLC-based stopped assay, the primary reduced metabolite was identified as 6-hydroxy-trans, trans-2,4-hexadienal (OH/CHO) and the secondary metabolite as 1,6-dihydroxy-trans, trans-2,4-hexadiene (OH/OH). The main enzymes responsible for the highest levels of reductase activity towards trans, trans-muconaldehyde were purified from mouse liver soluble fraction first by Q-sepharose chromatography followed by either blue or red dye affinity chromatography. In mouse liver, trans, trans-muconaldehyde is predominantly reduced by an NADH-dependent enzyme, which was identified as alcohol dehydrogenase (Adh1). Kinetic constants obtained for trans, trans-muconaldehyde with the native Adh1 enzyme showed a Vmax of 2141+/-500 nmol/min/mg and a Km of 11+/-4 microM. This enzyme was inhibited by pyrazole with a KI of 3.1+/-0.57 microM. Other fractions were found to contain muconaldehyde reductase activity independent of Adh1, and one enzyme was identified as the NADPH-dependent aldehyde reductase AKR1A4. This showed a Vmax of 115 nmol/min/mg and a Km of 15+/-2 microM and was not inhibited by pyrazole.

Peroxynitrite alters the catalytic activity of rodent liver proteasome in vitro and in vivo.

The proteasome is an important multicatalytic enzyme complex that degrades misfolded and oxidized proteins, signal transduction factors, and antigenic peptides for presentation. We investigated the in vitro effects of peroxynitrite (PN) on the peptidase activity of both crude 20S and 26S and purified 20S proteasome preparations from rat liver as well as proteasome activity in Hep G2 cells and in mouse liver. Crude and purified proteasome preparations were exposed to PN or to the PN donor, 3-morpholinosydnonimine hydrochloride (SIN-1), and then assayed for chymotrypsin-like activity. For in vivo experiments, mice were treated with molsidomine, which is metabolized to SIN-1 in liver. PN and SIN-1 dose-dependently modulated the chymotrypsin-like activity of the 20S proteasome: lower concentrations enhanced proteasome activity, and higher concentrations caused its decline. The NO donor S-nitroso-N-acetylpenicillamine (SNAP), at all concentrations, suppressed 20S proteasome activity. We observed similar results when liver soluble fractions (S-100) were treated with PN, SIN-1, or SNAP, except that enzyme activity in S-100 fractions was less sensitive than the purified enzymes to these agents. Treatment of Hep G2 cells with 0.01 or 0.1 mmol/L SIN-1 stimulated in situ proteasome activity in these cells, while 1 mmol/L SIN-1 suppressed it. SNAP treatment did not affect proteasome activity in Hep G2 cells. Mice treated with molsidomine had enhanced liver proteasome activity 6 hours after treatment, but after 24 hours enzyme activity declined below control levels. In conclusion, PN dose-dependently modulated proteasome activity, regulating protein degradation by the proteasome in liver cells.

Relationship between metallothionein and metal contents in red-blooded and white-blooded Antarctic teleosts

Metal bioaccumulation and metallothionein were investigated in different organs of the red-blooded teleost, Trematomus bernacchii and the haemoglobinless Chionodraco hamatus. Specimens of the two Antarctic fish were sampled from Terra Nova Bay (Ross Sea), and their levels of Cd, Cu and Zn in homogenates and in soluble fractions of liver, muscle, gills, heart and plasma were determined. Dosages of metallothioneins (MTs) or MT-like proteins were assayed by the silver saturation method in soluble fractions of the same organs. In both T. bernacchii and C. hamatus the highest MT contents were found in liver. Hepatic MT and Cd, Cu and Zn concentrations correlated positively in T. bernacchii, whereas in C. hamatus hepatic MT showed a positive correlation only with Cd. Positive correlations were also found between Cd and MT in gills of the two species. A metal-binding protein containing a high percentage of cysteine from C. hamatus was purified and compared with the MT from T. bernacchii and mammals.

Characterization of a soluble mouse liver enzyme capable of hydrolyzing diisopropyl phosphorofluoridate

A novel mouse liver soluble fraction DFPase which has organophosphatase activities with sarin, soman and tabun, was purified and characterized. However, it lacks paraoxonase and arylesterase activities with paraoxon and phenyl acetate, respectively. This DFPase closely resembles and may be identical with the one purified by Little et al. in 1989 from the soluble fraction of rat liver, based on its substrate specificity, size (∼39 kDa) and its stimulation by several metal ions, namely magnesium, manganese and cobalt. Sequencing of our purified mouse liver DFPase showed it to be identical in its amino acid sequence with the recently identified senescence marker protein-30 (SMP-30) by Fujita et al. in 1996. Other senescence marker proteins possessing high structural homology with the mouse SMP-30 have also been found and sequenced from human and rat livers. There is no structural homology between the senescence marker protein family and the group of mammalian paraoxonases. Thus, it is clear that there are at least two distinct, unrelated families of mammalian liver enzymes that share DFPase activity.

Role of female sex steroids in regulating cholesteryl ester transfer protein in transgenic mice

The role of sex steroids in the regulation of cholesteryl ester transfer protein (CETP) was examined in the following groups of female transgenic mice carrying the human CETP gene: (1) normal, (2) ovariectomized, (3) ovariectomized and treated with estrogen; (4) ovariectomized and treated with progesterone; (5) ovariectomized and treated with both hormones, and (6) ovariectomized and treated with tamoxifen. CETP activity was measured in the plasma, and in the particulate and the soluble fractions of liver, muscle, and adipose tissue. Human CETP specific activity was determined by taking the difference of cholesterol ester transfer in the presence and absence of an antibody (TP2) against human CETP. Ovariectomy reduced hormone levels, but did not completely abolish them from the circulation. Plasma CETP activity was significantly reduced in the tamoxifen group. There were significant reductions in CETP in liver homogenate and the soluble fraction, as well as in the particulate fraction of adipose with ovariectomy. Hormone replacement did not restore CETP activity in either the plasma or the tissues. Tamoxifin treatment resulted in a decrease in CETP activity in both fractions of liver, but had no effect on adipose. In the soluble fraction of adipose tissue and both fractions of muscle, only trace CETP activity was detected. We conclude that (1) minimal amounts of sex steroid hormones may be sufficient to affect CETP expression; (2) the effects of sex steroid hormones vary among tissues; and (3) in addition to the sex steroids, factor(s) from the ovary are needed for the full expression of CETP in this animal model.

Synthesis,In VitroBiological Stability, and Anti-HIV Activity of 5-Halo (or Methoxy)-6-Alkoxy (Azido or Hydroxy)-5,6-Dihydro-2′,3′-Didehydro-3′-Deoxythymidine Diastereomers as Potential Prodrugs of 2′,3′-Didehydro-3′-deoxythymidine (D4T)

Abstract A new class of 5-halo (or methoxy)-6-alkoxy (azido or hydroxy)-5,6-dihydro-2′,3′-didehydro-3′-deoxythymidines (4–17) were investigated as potential anti-AIDS drugs. These 5,6-dihydro derivatives, which are also potential prodmgs of 2′,3′-didehydro-3′-deoxythymidine (D4T) were designed to have properties which would enhance their duration of action, lipophilicity and cephalic delivery to the central nervous system. The 5,6-dihydro derivatives of D4T (4–15), which differ in configuration at the C-5 and C-6 positions, were synthesized by the regiospecific addition of XR (X = Br, Cl, I; R = OMe, OEt, N3, OH) to the 5,6-olefinic bond of D4T. These 5,6-disubstituted-5,6-dihydro analogs of D4T are more lipophilic (P = 0.70 – 4.0 range) than D4T (P = 0.12) and are stable to E. coli thymidine phosphorylase. Regeneration of the 5,6-olefinic bond to give D4T, upon incubation of the 5-bromo- and 5-iodo-6-methoxy-5,6-dihydro derivatives (6, 7, 10, 11) with glutathione or a mouse liver soluble enzyme fraction,...

Insulin resistance is associated with abnormal dephosphorylation of a synthetic phosphopeptide corresponding to the major autophosphorylation sites of the insulin receptor

Insulin resistance in the ob/ob mouse model is associated with a reduction in insulin-induced protein-tyrosine phosphorylation in tissues such as liver. To ascertain whether this decrease in phosphorylation may be due to increased phosphatase activity, protein-tyrosine phosphatase (PTPase) activity was determined in particulate and soluble fractions from livers of 5− to 23-week-old ob/ob mice and age-matched lean littermates. PTPase activity was measured using a synthetic phosphopeptide, TRDIY(P)ETDY(P)Y(P)RK, as the substrate, corresponding to residues 1142 to 1153 of the insulin receptor and containing the major autophosphorylation sites of the regulatory domain. The ob/ob mice were hyperinsulinemic across all age groups, but only the youngest mice (aged 5 to 7 weeks) were hyperglycemic. Most PTPase activity was present in the liver particulate fraction and was 19% to 114% greater in ob/ob mice as compared with controls. PTPase activity in the liver soluble fraction was 26% less than control values in the youngest ob/ob mice (5 to 7 weeks), but increased with age and was 41% and 131% above control values at 21 to 23 and 25 to 27 weeks of age, respectively. Oral administration of the PTPase inhibitor sodium orthovanadate (0.6 mg/mL in drinking water for 2 weeks) to young ob/ob mice caused a significant reduction in the elevated particulate PTPase activity, with concomitant decreases in plasma insulin and plasma glucose. Assessment of PTPase activity with a monophosphate form of the same synthetic peptide, TRDIY(P)ETDYYRK, showed lower PTPase activities as compared with the triphosphate form and no significant differences between ob/ob and control preparations. In conclusion, these findings demonstrate that the mechanism of dephosphorylation of insulin receptor autophosphorylation sites is altered in the liver of the ob/ob mouse. This abnormal PTPase activity may contribute to the decrease in insulin action characteristic of this insulin-resistant model.

Evaluation and Modification of an Assay Procedure for Cysteine Dioxygenase Activity: High-Performance Liquid Chromatography Method for Measurement of Cysteine Sulfinate and Demonstration of Physiological Relevance of Cysteine Dioxygenase Activity in Cysteine Catabolism

Conflicting reports in the literature of appropriate assay procedures for measurement of cysteine dioxygenase activity led us to evaluate the procedure for assay of cysteine dioxygenase activity in rat liver preparations. Cysteine dioxygenase activity was largely in the soluble fraction of liver and was stimulated by addition of NAD + and Fe 2+. The pH optimum of the enzyme was 6.1. Addition of an inhibitor of pyridoxal 5-phosphate-dependent enzymes was necessary to prevent rapid removal of the reaction product cysteine sulfinate. Cysteine sulfinate and cysteic acid were separated by anion-exchange HPLC on a polymer-based column with trimethylamino active groups, and the reaction products were quantitated by measurement of 35S radioactivity or by formation and measurement of fluorescent derivatives. This assay of cysteine dioxygenase under optimal conditions provides a physiologically relevant measure of cysteine dioxygenase activity in liver and hepatocytes based on the observation that this activity was highly correlated with the capacity for cysteine catabolism and taurine production by isolated hepatocytes.

Vascular effects of halothane and isoflurane: cGMP dependent and independent actions.

This study investigated the effects of halothane and isoflurane on cGMP-dependent and independent regulation of vascular contraction of the isolated rat aorta and on NO-stimulated soluble guanylate cyclase (sGC) isolated from the perfused rat liver. For the studies of the aorta, isometric tension of isolated rings, with and without, endothelium was recorded and cGMP content measured. ACh was used to initiate endothelial-dependent relaxation of norepinephrine (NE)-contracted rings while NO was used to directly stimulate isolated aortic ring sGC which catalyzes the isolated aortic ring formation of cGMP. Both halothane and isoflurane interfered with ACh and NO relaxations and with NO-stimulated increases in cGMP. Halothane was more potent, having significant attenuating effects at 0.34 mM (1 MAC) and 0.72 mM (2 MAC) while isoflurane had effects only at 0.53 mM (2 MAC). For the isolated sGC studies, a soluble liver fraction was prepared from perfused rat livers. In the absence of NO stimulation, neither halothane nor isoflurane modified the activity of the sGC. However, during NO-stimulation halothane produced significant, concentration-dependent, inhibition of sGC activity over a wide range of NO concentrations. Isoflurane also inhibited sGC activity, but to a lesser extent than halothane. The mechanism whereby the anesthetics could interfere with sGC from liver and blood vessels is unknown. It could result from anesthetic interaction at hydrophobic sites that may exist in GC. However, the results of both the aorta and liver sGC enzyme studies support the suggestion that these anesthetics can compete with NO for its binding site on the ferrous heme of sGC, with chemical structural differences accounting for the potency variations. Both anesthetics also had cGMP independent effects, causing concentration dependent relaxations of NE-contracted vessels without endothelium. Isoflurane was about 5 times more effective at 1 MAC than halothane. Therefore, the net effects of these anesthetics involve the sum of two opposite effects on tension of vessels with intact endothelium: 1) interference with NO-stimulated cGMP relaxation and 2) direct stimulation of relaxation (not dependent on changes in cGMP).

Quantitation of tissue- and sex-specific induction of rat GSH transferase subunits by dietary 1,2-dithiole-3-thiones.

Male and female Sprague--Dawley rats were maintained for 5 days on control diet or diet containing 0.075% (w/w) 1,2-dithiole-3-thione (D3T) or 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione (oltipraz). The content of GSH transferase (GST) subunits 1a, 1b, 2, 3, 4, 6, 7, 8, 10 and 11 in the soluble fraction of liver, kidney, small intestine, stomach and lung of control and D3T-fed animals was determined. Liver and kidney were similarly analysed for the oltipraz-fed animals. Significant induction of GST subunits by D3T was seen in all tissues, the most substantial being subunit 7 in male liver (approximately 50-fold) and subunits 1b, 3 and 10 in male and female small intestine (5- to 16-fold). Generally subunits 7, 10, 1b and 3 were most affected, while subunits 6 and 8 were hardly inducible. Oltipraz caused qualitatively similar inductions in the liver and kidney. The effect of GSH transferase induction on the hepatic capacity for GSH conjugation of aflatoxin B1 exo-8,9-oxide is assessed.

Liver mevalonate 5-pyrophosphate decarboxylase is responsible for reduced serum cholesterol in stroke-prone spontaneously hypertensive rat.

Spontaneously hypertensive rat (stroke-prone) (SHRSP) has an interestingly low serum cholesterol level due to a reduced biosynthesis of cholesterol in the liver (Iritani, N., Fukuda, E., Nara, Y., and Yamori, Y. (1977) Atherosclerosis 28, 217-222). In this study, we examined the mechanism underlying the reduction of hepatic cholesterol biosynthesis in the rat. Our initial findings in SHRSP, as compared with normotensive Wistar Kyoto rat (WKY), showed that 1) the incorporation of [14C]acetate into cholesterol in the liver slices was markedly less, 2) 3-hydroxyl-3-methylglutaryl (HMG) CoA reductase activity was not reduced, and 3) the incorporation of [3H]mevalonic acid into both cholesterol and squalene was significantly less. The above initial findings suggested that the reduction in the hepatic cholesterol biosynthesis took place in one or more enzymatic processes starting with mevalonic acid and continuing to squalene. When the incorporation of [3H]mevalonic acid into phosphomevalonate derivatives was studied using an ion exchange column, only the radioactivity incorporated into isopentenyl-pyrophosphate (isopentenyl-PP) was less in SHRSP. Furthermore, the specific activity of diphosphomevalonate (mevalonate-PP) decarboxylase in the liver-soluble fractions was reduced 50% in SHRSP as compared with WKY. Kinetic studies using liver crude extracts indicated a lower Vmax value in SHRSP (SHRSP, 0.47; WKY, 2.05 nmol/min/mg), and an unchanged Km value (SHRSP, 18.2; WKY, 19.6 microM). The activity of mevalonate-PP decarboxylase was also found to be reduced in other tissues, including the brain, testis, small intestine, and cultured vascular smooth muscle cells. From the above observations, we concluded that the lower activity of mevalonate-PP decarboxylase was responsible for the reduced cholesterol biosynthesis in the liver of SHRSP.

O-095 - Purification of two MEKA like proteins from bovine liver soluble fraction

O-095 - Purification of two MEKA like proteins from bovine liver soluble fraction

Acetaldehyde-collagen adducts in CCL 4-induced liver injury in rats

Circulating AC levels as well as antibodies against AC-protein adducts are increased in non-alcoholic liver injury. To identify the adducts, we used rats with CCl4-induced cirrhosis. Liver subcellular fractions were analyzed by immunochemical staining of protein slot blots and of electrophoretically separated proteins, transferred to nitrocellulose, using AC-protein adduct-specific antibodies. One reactive protein of about 200 kD was detected in the liver soluble fraction and in the cytosol of isolated hepatocytes and, to a lesser extent in the liver microsomes of CCl4-treated rats; in control animals, this reactivity was much weaker. The immunopositive AC adduct co-migrated with the beta 1,2 dimer of rat collagen type I; it was sensitive to digestion by a highly purified collagenase and also reacted with anti-rat collagen type I-specific IgG. In addition, comparison of peptides of the CNBr-digested, immunoprecipitated AC adduct with those of rat collagen type I revealed a high degree of similarity. Thus, AC adduct formation occurs in liver injury of non-alcoholic origin, and a target protein appears to be related to collagen type I, most likely the procollagen precursor.

Metabolism of trans,trans-muconaldehyde, a microsomal hematotoxic metabolite of benzene, by purified yeast aldehyde dehydrogenase and a mouse liver soluble fraction

Aldehyde dehydrogenase (ALDH) from yeast, in the presence of NAD +, oxidizes trans,trans-muconaldehyde (MUC) in a biphasic manner with apparent K m values of 0.48 and 3.2 μ m and corresponding V max values of 604 and 1227 nmol/min/mg protein. Concentrations above 10 μ m, trans,trans-muconaldehyde produce an inhibition of enzyme activity. Removal of NAD +, MUC, or use of boiled enzyme results in no oxidation. Using thin-layer chromatography and high-pressure liquid chromatography techniques, a product with polarity intermediate between that of the dialdehyde trans,trans-muconaldehyde and the diacid trans,trans-muconic acid (MA) was detected in the ALDH incubation mixtures. The same product was also detected in a DBA/2J mouse liver soluble fraction supplemented with NAD + and incubated with MUC. This product was isolated from a scaled-up incubation mixture containing trans,trans-muconaldehyde, purified yeast aldehyde dehydrogenase, and NAD +. Mass spectral analysis of the compound indicates a molecular weight of 126 and an empirical formula C 6H 6O 3 containing four double bonds. This product also tested positive with reagents specific for carboxylic and aldehydic functional groups. In the presence of the mouse liver soluble fraction supplemented with NAD +, this intermediate was metabolized to MA. These findings indicate that MUC is oxidized by yeast aldehyde dehydrogenase to the monocarboxylic acid derivative which is an intermediate in the conversion of MUC to MA. The role of the monocarboxylic acid α,β-unsaturated aldehyde in benzene hematotoxicity remains to be explored.

The in vitro reduction of dimethylpropion

A study of the in vitro metabolism of dimethylpropion using different tissue homogenates of certain animals showed that: 1. 1. Only the two major metabolites of dimethylpropion, i.e., methylpseudoephedrine and monomethylpropion were formed. 2. 2. Species variation was observed with regard to the reductive ability of the liver homogenates. 3. 3. Anaerobic conditions favored in vitro reduction to form methylpseudoephedrine. 4. 4. The liver-soluble fraction exhibited the highest enzymatic reductive activity toward dimethylpropion. 5. 5. Significant metabolic reduction of dimethylpropion was exhibited by the soluble fraction of the kidney.

Intracellular distribution and chemical forms of arsenic in rabbits exposed to arsenate

Inhibition of the methylation of arsenic in rabbits by ip injection of periodate-oxidized adenosine (PAD) prior to an iv injection of 74As-arsenate (AsV; 0.4 mg As/kg body wt) caused a marked increase in the retention of 74As in both the cellular organelles and the soluble fractions of liver and kidney. One day after exposure, almost 30% of the arsenic in the liver and about 40% of the arsenic in the kidney was recovered in the nuclear fraction. In the liver nuclei, the inhibition of the methylation increased the 74As content of the insoluble fraction and most of this arsenic was protein-bound. The major part of the soluble intranuclear 74As was in the form of AsIII, formed by reduction of the administered AsV. In the liver, PAD also caused a pronounced increase in the 74As content of the microsomal fraction. In the kidneys, where most of the arsenic was present as AsV, there was a marked accumulation of arsenic in the mitochondria.

Identity of alanine: glyoxylate aminotransferase with alanine: 2-oxoglutarate aminotrasferase in rat liver cytosol

Rat liver soluble fraction contained 3 forms of alanine: glyoxylate aminotransferase. One with a p I of 5.2 and an M t of approx. 110 000 was found to be identical with cytosolic alanine:2-oxoglutarate aminotransferase. The p I 6.0 enzye with an M t of approx. 220 000 was suggested to be from broken mitochondrial alanine:glyoxylate aminotransferase 2 and the p I 8.0 enzyme with an M t of approx. 80 000 enzyme from broken peroxisomal and mitochondrial alanine:glyoxylate aminotransferase 1. These results suggest that the cytosolic alanine:glyoxylate aminotransferase activity is due to cytosolic alanine: 2-oxoglutarate aminotransferase.

Detoxification of DNA hydroperoxide by glutathione transferases and the purification and characterization of glutathione transferases of the rat liver nucleus.

DNA peroxidized by exposure to ionizing radiation in the presence of oxygen is a substrate for the Se-independent GSH peroxidase activity of several GSH transferases, GSH transferases 5-5, 3-3 and 4-4 being the most active in the rat liver soluble supernatant fraction (500, 35 and 20 nmol/min per mg of protein respectively) and GSH transferases mu and pi the most active, so far found, in the human liver soluble supernatant fraction (80 and 10 nmol/min per mg respectively). Although the GSH transferase content of the rat nucleus was found to be much lower than that of the soluble supernatant, nuclear GSH transferases are likely to be more important in the detoxification of DNA hydroperoxide produced in vivo. Two nuclear fractions were studied, one extracted with 0.075 M-saline/0.025 M-EDTA, pH 8.0, and the other extracted from the residue with 8.5 M-urea. The saline/EDTA fraction contained subunits 1, 2, 3, 4 and a novel subunit, similar but not identical to 5, provisionally referred to as 5*, in the proportions 40:25:5:5:25 respectively. The 8.5 M-urea-extracted fraction contained principally subunit 5* together with a small amount of subunit 6 in the proportion 95:5 respectively. GSH transferase 5*-5* purified from the 8.5 M-urea extract has the highest activity towards DNA hydroperoxide of any GSH transferase so far studied (1.5 mumol/min per mg). A Se-dependent GSH peroxidase fraction from rat liver was also active towards DNA hydroperoxide; however, since this enzyme accounts for only 14% of the GSH peroxidase activity detectable in the nucleus, GSH transferases may be the more important source of this activity. The possible role of GSH transferases, in particular GSH transferase 5*-5*, in DNA repair is discussed.