Activity In Rat Liver Cytosol Research Articles

Reduction of Dimethylarsinic Acid to the Highly Toxic Dimethylarsinous Acid by Rats and Rat Liver Cytosol

Dimethylarsinic acid (DMAs(V)), the major urinary metabolite of inorganic arsenic, is weakly cytotoxic, whereas its reduced form, dimethylarsinous acid (DMAs(III)), is highly toxic. Although glutathione S-transferase omega 1 (GSTO1) and arsenic methyltransferase have been shown or thought to catalyze DMAs(V) reduction, their role in DMAs(V) reduction in vivo, or in cell extracts is uncertain. Therefore, the reduction of DMAs(V) to DMAs(III) in rats and in rat liver cytosol was studied to better understand its mechanism. To assess DMAs(V) reduction in rats, a novel procedure was devised based on following the accumulation of red blood cell (RBC)-bound dimethylarsenic (DMAs), which represents DMAs(III), in the blood of DMAs(V)-injected anesthetized rats. These studies indicated that rats reduced DMAs(V) to DMAs(III) to a significant extent, as in 90 min 31% of the injected 50 μmol/kg DMAs(V) dose was converted to DMAs(III) that was sequestered by the circulating erythrocytes. Pretreatment of rats with glutathione (GSH) depletors (phorone or BSO) delayed the elimination of DMAs(V) and the accumulation of RBC-bound DMAs, whereas the indirect methyltransferase inhibitor periodate-oxidized adenosine was without effect. Assessment of DMAs(V)-reducing activity of rat liver cytosol revealed that reduction of DMAs(V) required cytosolic protein and GSH and was inhibited by thiol reagents, GSSG and dehydroascorbate. Although thioredoxin reductase (TRR) inhibitors (aurothioglucose and Sb(III)) inhibited cytosolic DMAs(V) reduction, recombinant rat TRR plus NADPH, alone or when added to the cytosol, failed to support DMAs(V) reduction. On ultrafiltration of the cytosol through a 3 kDa filter, the reducing activity in the retentate was lost but was largely restored by NADPH. Such experiments also suggested that the reducing enzyme was larger than 100 kDa and was not GSTO1. In summary, reduction of DMAs(V) to the highly toxic DMAs(III) in rats and rat liver cytosol is a GSH-dependent enzymatic process, yet its mechanism remains uncertain.

Effect of Tea Beverages on Aldehyde Oxidase Activity

Aldehyde oxidase (AO) plays an important role in metabolizing antitumor and antiviral drugs, including methotrexate, cyclophosphamide and acyclovir. Green tea and its catechins have been shown to modulate the activities of various xenobiotic-metabolizing cytochrome P450 species, both in vivo and in vitro, but their effect on AO has not been studied. Therefore, we evaluated the effect of tea beverages on AO activity in rat and human liver cytosol. We also investigated the influence of several catechins on AO activity in rat liver cytosol. AO activity was evaluated in terms of oxidation of N-1-methylnicotinamide to N-1-methyl-2-pyridone-5-carboxamide and N-1-methyl-4-pyridone-3-carboxamide. Bottled green tea beverages at 10% (vol/vol) inhibited AO activity by 90.0-93.5%, while at 1.0% (vol/vol), they reduced AO activity by 73.9-90.0%. At 0.1% (vol/vol), green tea II and III, which have high contents of catechins and their derivatives, inhibited AO activity by 24.3% and 38.8%, respectively. Bottled mineral water had no effect. AO activity was inhibited potently by epicatechin and epicatechin gallate. These results indicate that the AO-inhibitory activity of tea beverages is predominantly due to catechins and their derivatives. Thus, consumption of tea beverages may cause a decrease of AO activity, which may result in reduced clearance of drugs that are AO substrates.

Phenylalanine hydroxylase: possible involvement in the S-oxidation of S-carboxymethyl-l-cysteine

Activated phenylalanine 4-monooxygenase, phenylalanine hydroxylase (PAH), is known to be involved in the S-oxidation of a number of sulfide compounds. One of these compounds, S-carboxymethyl-l-cysteine (SCMC), is currently used for the treatment of chronic obstructive pulmonary disease and otitis media with effusion as a mucolytic agent, and the S-oxides are the major metabolites found in urine. However, the enzyme catalyzing the S-oxidation of SCMC has yet to be identified. Here we report on the role of nonactivated phenylalanine 4-monooxygenase activity in rat liver cytosol in the S-oxidation of SCMC. Linearity of the enzyme assays was seen for both time (0–16min) and cytosolic protein concentration (0.1–0.5mg/ml). The calculated Km and Vmax values for the formation of SCMC (S) S-oxide were 3.92±0.15mM and 1.10±0.12nmol SCMC (S) S-oxide formed/mg protein/min, respectively. The calculated Km and Vmax values for the formation of SCMC (R) S-oxide were 9.18±1.13mM and 0.46±0.11nmol SCMC (R) S-oxide formed/mg protein/min, respectively. These results indicate that in the female Wistar rat, nonactivated PAH showed a stereospecific preference for the formation of the (S) S-oxide metabolite of SCMC against the (R) S-oxide metabolite of SCMC.

Regucalcin increases superoxide dismutase activity in rat liver cytosol.

The effect of regucalcin, a regulatory protein in the intracellular signaling process, on superoxide dismutase (SOD) activity in the cytosol of rat liver was investigated. The presence of zinc sulfate (10(-6) or 10(-5) m) or cupric sulfate (10(-6) m) in the enzyme reaction mixture caused a significant increase in SOD activity, indicating that Cu/Zn-SOD may be present in the liver cytosol. SOD activity was significantly increased by the addition of regucalcin (0.1, 0.25, or 0.5 microM) to the reaction mixture. The presence of dithiothreitol (DTT; 0.1, 0.5, or 1.0 mM), a protective reagent for the sulfhydryl group, caused a significant decrease in SOD activity. The effect of regucalcin (0.25 microM) in increasing SOD activity was not seen in the presence of DTT (1.0 mM). Meanwhile, SOD activity was significantly raised by the addition of N-ethylmaleimide (NEM; 0.5 or 1.0 mM), a modifying reagent for the sulfhydryl reagent. Regucalcin (0.25 microM) caused a significant increase in SOD activity in the presence of NEM (1.0 mM). The effect of regucalcin in increasing SOD activity may not involve the sulfhydryl group of SOD. This study demonstrates that regucalcin has an activatory effect on SOD in the liver cytosol of rats.

Identification of cytosolic leucyl aminopeptidase (EC 3.4.11.1) as the major cysteinylglycine-hydrolysing activity in rat liver.

Cysteinylglycine hydrolysis is a step in the metabolism of glutathione and glutathione S-conjugates. We had previously observed that in rat liver the enzymatic activity is predominantly located in the cytosol. Here we demonstrate that cytosolic leucyl aminopeptidase (EC 3.4.11.1) is the major cysteinylglycine hydrolysing activity in rat liver. Evidence was obtained from the use of peptidase inhibitors and from immunoprecipitation studies using Pansorbin-coupled antibodies raised against hog kidney cytosolic leucyl aminopeptidase. Both isolated cytosolic leucyl aminopeptidase and the cysteinylglycine-hydrolysing activity in rat liver cytosol are bound with equal efficiency to the affinity matrix. We demonstrate that cytosolic leucyl aminopeptidase exhibits leucinamidase and cysteinylglycinase activity. Cysteinylglycine, cystinyl-bis-glycine, S-nitrosocysteinylglycine, and bimane-S-cysteinylglycine are hydrolysed at high rates; low activity is seen with leukotriene D4. Our findings establish a previously unrecognised physiological function of cytosolic leucyl aminopeptidase, participating in glutathione metabolism and in the degradation of glutathione S-conjugates via the mercapturic acid pathway.

Purine nucleoside phosphorylase as a cytosolic arsenate reductase.

The findings of the accompanying paper (Németi and Gregus, Toxicol: Sci. 70, 4-12) indicate that the arsenate (AsV) reductase activity of rat liver cytosol is due to an SH enzyme that uses phosphate (or its analogue, arsenate, AsV) and a purine nucleoside (guanosine or inosine) as substrates. Purine nucleoside phosphorylase (PNP) is such an enzyme. It catalyzes the phosphorolytic cleavage of 6-oxopurine nucleosides according to the following scheme: guanosine (or inosine) + phosphate <--> guanine (or hypoxanthine) + ribose-1-phosphate. Therefore, we have tested the hypothesis that PNP is responsible for the thiol- and purine nucleoside-dependent reduction of AsV to AsIII by rat liver cytosol. AsIII formed from AsV was quantified by HPLC-hydride generation-atomic fluorescence spectrometry analysis of the deproteinized incubates. The following findings support the conclusion that PNP reduces AsV to AsIII, using AsV instead of phosphate in the reaction above: (1) Specific PNP inhibitors (CI-1000, BCX-1777) at a concentration of 1 microM completely inhibited cytosolic AsV reductase activity. (2) During anion-exchange chromatography of cytosolic proteins, PNP activity perfectly coeluted with the AsV reductase activity, suggesting that both activities belong to the same protein. (3) PNP purified from calf spleen catalyzed reduction of AsV to AsIII in the presence of dithiothreitol (DTT) and a 6-oxopurine nucleoside (guanosine or inosine). (4) AsV reductase activity of purified PNP, like the cytosolic AsV reductase activity, was inhibited by phosphate (a substrate of PNP alternative to AsV), guanine and hypoxanthine (products of PNP favoring the reverse reaction), mercurial thiol reagents (nonspecific inhibitors of PNP), as well as CI-1000 and BCX-1777 (specific PNP inhibitors). Thus, PNP appears to be responsible for the AsV reductase activity of rat liver cytosol in the presence of DTT. Further research should clarify the mechanism and the in vivo significance of PNP-catalyzed reduction of AsV to AsIII.

Both myo-inositol to chiro-inositol epimerase activities and chiro-inositol to myo-inositol ratios are decreased in tissues of GK type 2 diabetic rats compared to Wistar controls

Previous data from our and other labs demonstrated a decreased chiro-inositol content in urine and tissues of human subjects and animals with type 2 diabetes. In urine this decrease in chiro-inositol was accompanied by an increase in myo-inositol content. Decreased urine levels of chiro-inositol in monkeys were next correlated with the severity of underlying insulin resistance determined by five separate assays. To investigate the decreased chiro-inositol and the accompanying increased myo-inositol excretions in urine in humans and monkeys, we postulated a defect in the epimerization of myo-inositol to chiro-inositol. [ 3H]Myo-inositol was then shown to be converted to [ 3H]chiro-inositol in rats in vivo and in fibroblasts in vitro in a process stimulated by insulin. We next demonstrated that the conversion of [ 3H]myo-inositol to [ 3H]chiro-inositol in vivo was markedly decreased in GK type 2 diabetic rats compared to Wistar controls in liver, muscle, and fat, insulin sensitive tissues. Decreases of 20–25% conversion to baseline levels of under 5% conversion were observed. In the present work, we initially compared the total contents of myo-inositol and chiro-inositol in GK type 2 diabetic rat kidney, liver, and muscle compared to Wistar controls. We demonstrated a consistent decreased total chiro-inositol to myo-inositol ratio in kidney, liver, and muscle compared to controls. We next established the presence of a myo-inositol to chiro-inositol epimerase activity in rat liver cytosol. Enzyme activity was shown to be time and enzyme concentration dependent with a broad pH optimum. It required NADH and NADPH for full activity, which is compatible with its action via an oxido-reductive mechanism. Lastly, we demonstrated that the epimerase enzyme bioactivity was significantly decreased in muscle, liver, and fat cytosolic extracts of GK type 2 diabetic rats versus Wistar controls. Decreased myo-inositol to chiro-inositol epimerase activity may therefore play a role in explaining the decreased chiro-inositol to myo-inositol urine and tissue ratios observed here and in previous animal and human studies. Further it may also possibly play a role in the underlying insulin resistance.

Analysis of rat cytosolic 9- cis-retinol dehydrogenase activity and enzymatic characterization of rat ADHII

We report the characterization of two enzymes that catalyze NAD +-dependent 9- cis-retinol dehydrogenase activity in rat liver cytosol. Alcohol dehydrogenase class I (ADHI) contributes >80% of the NAD +-dependent 9- cis-retinol dehydrogenase activity recovered, whereas alcohol dehydrogenase class II (ADHII), not identified previously at the protein level, nor characterized enzymatically in rat, accounts for ∼2% of the activity. Rat ADHII exhibits properties different from those described for human ADHII. Moreover, rat ADHII-catalyzed rates of ethanol dehydrogenation are markedly lower than octanol or retinoid dehydrogenation rates. Neither ethanol nor 4-methylpyrazole inhibits the 9- cis-retinol dehydrogenase activity of rat ADHII. We propose that ADHII represents the previously observed additional retinoid oxidation activity of rat liver cytosol which occurred in the presence of either ethanol or 4-methylpyrazole. We also show that human and rat ADHII differ considerably in enzymatic properties.

Effect of anti-regucalcin antibody on neutral phosphatase activity in rat liver cytosol: involvement of endogenous regucalcin.

The effect of anti-regucalcin monoclonal antibody on neutral phoshatase activity in rat liver cytosol was investigated. Phosphotyrosine, phosphoserine, and phosphothreonine were used as the substrate toward phosphatase assay. Liver cytosolic phosphatase activity with three phosphoaminoacids was significantly increased in the presence of anti-regucalcin antibody (100 and 200 ng/ml) in the enzyme reaction mixture with calcium chloride (0.1 mM) or EGTA (1.0 mM). The effect of anti-regucalcin antibody was completely abolished in the presence of exogenous regucalcin (1.0 microM), indicating the involvement of endogenous regucalcin. The anti-regucalcin anti body- increased phosphatase activity was not significantly altered in the presence of trifluoperazine (20 microM), an antagonist of calmodulin, or akadaic acid ( 10 microM), an inhibitor of protein phosphatase, although these inhibitors caused a slight decrease in liver cytosolic phosphatase activity. The effect of endogenous regucalcin might be not related to calmodulin, and it was insensitive to okadaic acid. The present findings suggest that endogenous regucalcin is involved in the regulation of protein phasphatase in rat liver cytoplasm.

Inhibition of Ca2+/calmodulin-dependent phosphatase activity by regucalcin in rat liver cytosol: Involvement of calmodulin binding

The regulatory effect of regucalcin on Ca2+/calmodulin-dependent phosphatase activity and the binding of regucalcin to calmodulin was investigated. Phosphatase activity toward phosphotyrosine, phosphoserine, and phosphothreonine in rat liver cytosol was significantly increased by the addition of Ca2+ (100 microM) and calmodulin (0.30 microM). These increases were clearly inhibited by the addition of regucalcin (0.50-1.0 microM) into the enzyme reaction mixture. The cytosolic phosphoamino acid phosphatase activity was significantly elevated by the presence of anti-regucalcin monoclonal antibody (0.2 microg/ml), suggesting that endogenous regucalcin in the cytosol has an inhibitory effect on the enzyme activity. This elevation was prevented by the addition of regucalcin (0.50 microM). Purified calcineurin phosphatase activity was significantly increased by the addition of calmodulin (0.12 microM) in the presence of Ca2+ (1 and 10 microM). This increase was completely inhibited by the presence of regucalcin (0.12 microM). The inhibitory effect of regucalcin was reversed by the addition of calmodulin with the higher concentration (0.36 microM). Regucalcin has been demonstrated to bind on calmodulin-agarose beads by analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The present study demonstrates that regucalcin inhibits Ca2+/calmodulin-dependent protein phosphatase activity in rat liver cytosol, and that regucalcin can bind to calmodulin.

Polyamines Inhibit Lipopolysaccharide-Induced Nitric Oxide Synthase Activity in Rat Liver Cytosol

Liver cells can produce nitric oxide froml-arginine through either constitutive NO synthase or inducible NO synthase (NOS) detected afterin vivoorin vitrotreatment with cytokines and/or lipopolysaccharide (LPS). The effects of NO on liver cells are associated with protein synthesis and mitochondrial electron transfer inhibition.l-Arginine is also the precursor ofl-ornithine and polyamines. The latter are considered to be protective in the liver in several experimental models. The aim of the present work was to test the effects of polyamines on LPS-inducible NOS activity in rat liver cytosol using the test of radioactivel-citrulline synthesis froml-[guanido-14C]arginine. The three polyamines inhibited inducible NO synthase activity with the following hierarchy: spermine > spermidine ≈ putrescine. The 0.5 mmspermine was found to inhibit 50% of inducible NO synthase activity. The present data suggest an inhibitory interrelationship in the liver between two metabolites derived from the common precursorl-arginine.

Characterization of NADPH-dependent ubiquinone reductase activity in rat liver cytosol: effect of various factors on ubiquinone-reducing activity and discrimination from other quinone reductases.

Cytosolic NADPH-dependent ubiquinone reductase (NADPH-UQ reductase) accounted for about 68% of the total ubiquinone (UQ) reductase activity in rat liver homogenate [Takahashi, T. et al. (1995) Biochem. J. 309, 883-890]. We investigated the effects of various factors on this enzyme activity in rat liver cytosol with the aim of elucidating its physiological roles. The NADPH-UQ reductase in rat liver cytosol catalyzed the reduction of UQ to UQH2 with concomitant oxidation of equimolar NADPH. The optimal pH was around 7.4, and the optimal temperatures were about 28 degrees C for NADH and about 37 degrees C for NADPH. NADH, deamino NADH, and deamino NADPH were much less active hydrogen donors than NADPH, whereas reduced nicotinamide mononucleotide, ascorbate, erythorbate, reduced glutathione, and cysteine were inactive. As the hydrogen acceptor, UQ-9 had the highest Vmax/Km among the long-chain UQ homologues tested. FAD and FMN stimulated the activity. Anionic detergents, Mg2+ and Sr2+ also enhanced the activity. Rotenone, malonic acid, antimycin A, and KCN, which inhibit mitochondrial and microsomal electron transfer enzymes, superoxide dismutase, and acetylated cytochrome c had no effect on the NADPH-UQ reductase activity. These results indicated that the NADPH-UQ reductase in rat liver cytosol is a flavoprotein that reduces UQ-10 by a two-electron reduction mechanism and is distinguishable from known microsomal and mitochondrial enzymes, as well as DT-diaphorase [EC 1.6.99.2].

Recombinant rat and hamster N-acetyltransferases-1 and -2: relative rates of N-acetylation of arylamines and N,O-acyltransfer with arylhydroxamic acids.

Genes for the 290 amino acid, 33-34 kDa cytosolic acetyltransferases (NAT1* and NAT2*) from rat and hamster were cloned and expressed in Escherichia coli. Active clones were selected by a simple visual test for their ability to decolorize 4-aminoazobenzene in bacterial medium by acetylation. These recombinant acetyltransferases were analyzed for: (i) N-acetyltransferase, which was assayed by the rate of acetyl coenzyme A-dependent N-acetylation of 2-aminofluorene (2-AF) or 4-aminoazobenzene (AAB); (ii) arylhydroxamic acid acyltransferase, assayed by N,O-acyltransfer with N-hydroxy-N-acetyl-2-aminofluorene. Both NAT2s showed first order increases in N-acetylation rates with increasing 2-AF or AAB concentrations between 5 and 100 microM, with apparent K(m) values of 22-32 and 62-138 microM respectively. Although under the same conditions the N-acetylation rates for the two NAT1s declined by > 50%, below 5 microM 2-AF or AAB, the NAT rate data fit Michaelis-Menten kinetics, and the apparent K(m) values were 0.2-0.9 microM. For N,O-acyltransferase, the apparent K(m) values of the NAT1s were approximately 6 microM, while the K(m) values of the NAT2s were approximately 20- to 70-fold higher. SDS-PAGE/Western blot analysis of the recombinant acetyltransferases gave apparent relative molecular weights (MWr) of approximately 31 kDa for both NAT1s and rat NAT2 and approximately 29 kDa for hamster NAT2. Comparable MWr values were observed for native hamster liver NAT1 and NAT2 and for rat NAT1 under the same conditions. Although we did not detect NAT2-like activity in rat liver cytosol previously, the present data show that the rat NAT2* gene does code for a functional acetyltransferase, with properties similar to those of hamster liver NAT2. The data also indicate that at low substrate concentrations, NAT1 would apparently play the predominant role in vivo in N-acetylation and N,O-acyltransfer of aromatic amine derivatives, including their metabolic activation to DNA-reactive agents.

Characterization of regucalcin effect on proteolytic activity in rat liver cytosol: relation to cysteinyl-proteases.

The increasing effect of regucalcin, isolated from rat liver cytosol, on neutral proteolytic activity in the hepatic cytosol was characterized. The proteolytic activity was markedly elevated by the addition of regucalcin (0.1-0.5 microM) in the absence of Ca2+. This increase was not significantly altered by the presence of diisopropylfluorophosphate (DPF; 2.5 mM)--although DFP caused a significant decrease in the proteolytic activity. Regucalcin (0.25 microM) additively enhanced the dithiothreitol (DTT; 1.0 mM)--increased proteolytic activity, while the regucalcin or DTT effect was completely abolished by NEM (5 mM), indicating that regucalcin may act on the SH group in proteases. Also, regucalcin (0.25 microM) enhanced the effect of Ca2+ (10 microM) increasing liver proteolytic activity, suggesting that regucalcin does not influence on the active sites for Ca2+ in proteases. Moreover, the proteolytic activity of regucalcin (0.25 microM) was significantly decreased by the presence of calpastatin (24 micrograms/ml), an inhibitor of Ca(2+)-activated neutral protease (calpain). Now, regucalcin (0.25 microM) increased about 7-fold the activity of m-calpain isolated from rabbit skeletal muscle. These observations demonstrate that regucalcin directly activates cysteinyl-proteases. Regucalcin may have a role as a potent proteolytic activator in the cytoplasm of liver cells.

Age-dependent changes in rat liver prenyltransferases

Mevalonate pathway lipids including cholesterol, ubiquinone and dolichol, are of great importance for cellular function. Many of the enzymes of this pathway are thus strictly regulated. During development of the rat, the cellular levels of certain of these lipids vary. Prenyltransferases have been investigated and it is reported here that farnesyl pyrophosphate synthase activity in rat liver cytosol decreases after birth to a lower, steady level. This decrease is not paralleled by the level of synthase protein, which shows two maxima, one immediately after birth and the other 30 days later. cis-Prenyltransferase activity is low after birth, increases continuously up to day-54 and then decreases to a low level which was maintained throughout the remainder of the study (365 days). Squalene synthase exhibits high activity after birth, but decreases during the first 100 days thereafter, and subsequently remains at the low level thus reached. In contrast to these changes in the activities of the prenyltransferases, the level of cholesterol is constant and the dolichol concentration increases continuously throughout the entire period studied.

CDNA cloning and characterization of mitochondrial import stimulation factor (MSF) purified from rat liver cytosol.

We identified a liver cytosolic protein factor that stimulated the import of wheat germ lysate-synthesized precursor proteins into mitochondria. It was termed mitochondrial import stimulation factor or MSF [Hachiya, N. et al. (1993) EMBO J. 12, 1579-1586]. It consisted of 32-kDa (MSFL) and 30-kDa (MSFS) polypeptides as assessed by SDS-PAGE. MSF recognized the presequence portion of mitochondrial precursor proteins and catalyzed the depolymerization and unfolding of in vitro synthesized mitochondrial precursor proteins in an ATP-dependent manner. We report here the cDNA cloning and characterization of MSF. Microsequencing of MSFL and MSFS showed that they belonged to a highly conserved, widely distributed eukaryotic protein family, collectively designated as 14-3-3 proteins. We cloned the cDNA of MSFL and that of one component of MSFS (MSFS1) from a rat liver cDNA library. The cloned cDNAs were separately expressed in Escherichia coli and the expressed proteins were purified to homogeneity. The purified recombinant MSFL and MSFS1 stimulated mitochondrial import of adrenodoxin precursor (pAd) synthesized in vitro with wheat germ lysate translation system. Recombinant MSFL or MSFS1 had the ability to bind with denatured pAd and they kept the precursor in an import-competent state. Rabbit polyclonal antibodies raised against the recombinant proteins inhibited the import-stimulation activity of rat liver cytosol as well as that of authentic purified MSF. Identification of MSF as 14-3-3 proteins establishes a novel function for this family of proteins and indicates their role as cytosolic chaperones to aid many important cellular events.

Nuclear 3,5,3′-triiodothyronine receptors and malic enzyme activity in liver of rats fed fish oil or cocoa butter

The role of different fat supplements (fish oil and cocoa butter), applied intragastrically for 3 weeks, on specific binding of T 3 by rat liver nuclear receptors was examined. Fish oil (rich in n-3 polyunsaturated fatty acids) supplementation resulted in a significant ( P < 0.05) enhancement of the maximal binding capacity (MBC) for T 3 with a diminution of the apparent association constant (K a). No changes in nuclear receptor MBC or K a were observed after cocoa butter administration. Moreover, the rate of fatty acid synthesis in the liver was evaluated by measuring malic enzyme (EC. 1.1.1.40.) activity in rat liver cytosol, which was found to be decreased in both the cocoa butter- ( P < 0.002) and the fish oil- ( P < 0.001) treated rats. Interestingly, in fish oil-supplemented animals, not even a higher MBC value for the T 3 receptors in liver nuclei and specific binding of T 3 to its receptor were able to restore the activity of malic enzyme in liver cytosol. The findings of the same level of thyroid hormones—thyroxine (T 4) and triiodothyronine (T 3)—in blood of all groups showed that there was apparently no effect of fat supplementation on the thyroid function. However, it may be hypothesized that the decrease of malic enzyme activity in both fat-treated groups may have resulted from a decreased T 4 to T 3 conversion in liver cells. The data suggest the following: (a) increased dietary fat intake is accompanied by lower lipogenesis in the liver in relation to the degree of fat unsaturation; (b) although polyunsaturated dietary fat increases T 3 receptor binding to liver nuclei; (c) thus, an uncoupling between T 3 binding and action on malic enzyme activity during raised fish oil intake cannot be ruled out.

Reversible effect of calcium-binding protein regucalcin on the Ca2+-induced inhibition of deoxyuridine 5′-triphosphatase activity in rat liver cytosol

The effect of regucalcin, a calcium-binding protein isolated from rat liver cytosol, on deoxyuridine 5'-triphosphatase (dUTPase) in the cytosol of rat liver was investigated. Addition of Ca2+ up to 5.0 microM to the enzyme reaction mixture caused a significant decrease of dUTPase activity, while Zn2+, Cd2+, Co2+, Al3+, Mn2+ and Ni2+ (10 microM) did not have an appreciable effect. The Ca(2+)-induced decrease of dUTPase activity was reversed by the presence of regucalcin; the effect was complete at 1.0 microM of the protein. Regucalcin had no effect on the basal activity of the enzyme. Meanwhile, the reversible effect of regucalcin on the Ca2+ (10 microM)-induced decrease of dUTPase activity was not altered by the coexistence of Cd2+ or Zn2+ (10 microM). The present data suggest that liver cytosolic dUTPase is uniquely regulated by Ca2+ of various metals, and that the Ca2+ effect is reversed by regucalcin.

Calcium-binding protein regucalcin increases calcium-independent proteolytic activity in rat liver cytosol.

The effect of regucalcin, isolated from rat liver cytosol, on neutral proteolytic activity in the hepatic cytosol was investigated. The Ca(2+)-requiring proteinase required 5-10 microM Ca2+ for maximal activity in the presence of a protein substrate (globin). The proteinase activity was markedly elevated by the addition of regucalcin (0.25-2.0 microM) in the absence or presence of Ca2+ (5.0 microM) added. The effect of regucalcin, however, was the greater in the absence of Ca2+ than that in the presence. The pronounced effect of regucalcin on the proteinase activity was also seen in the presence of 1.0 mM EGTA with or without Ca2+ (5.0 microM). In the absence of Ca2+, the regucalcin-increased proteinase activity was clearly inhibited by the presence of anti-regucalcin antiserum (diluted to 240-fold), leupeptin (20 and 200 micrograms/ml), and heavy metals (25 microM cadmium or 25 microM zinc), although the inhibition was not complete at the concentration used. The present findings suggest that regucalcin increases proteolytic activity in rat liver cytosol, and that regucalcin may activate Ca(2+)-independent neutral cysteinyl-proteinase.

Inhibitory effects of glycyrrhetic acid derivatives on 11 beta- and 3 alpha-hydroxysteroid dehydrogenases of rat liver.

Glycyrrhetic acid (GA), an aglycone of glycyrrhizin (GL), is a potent inhibitor of 11 beta- and 3 alpha-hydroxysteroid dehydrogenases. 11 beta-Hydroxysteroid dehydrogenase activity of rat liver microsomes was potently inhibited by GA, 3-deoxyglycyrrhetic acid (3-deoxyGA), 3-ketoglycyrrhetic acid (3-ketoGA), 3-epiglycyrrhetic acid (3-epiGA) and 11-deoxoglycyrrhetic acid (11-deoxoGA), with I50 values of 2-4 x 10(-7) M. However, 18 alpha-stereoisomers (I50 = 3-7 x 10(-6) M) of GA, 3-deoxyGA and 11-deoxoGA were one tenth less inhibitory on the enzyme activity than the corresponding 18 beta-isomers. On the other hand, 18 alpha-stereoisomers of GA, 3-deoxyGA and 11-deoxoGA inhibited 3 alpha-hydroxysteroid dehydrogenase activity of rat liver cytosol more potently than the corresponding 18 beta-isomers. I50 values of 18 alpha- and 18 beta-isomers were 2 and 7 x 10(-6) M, respectively, in the case of GA, 8 and 20 x 10(-6) M in 3-deoxyGA, 3 and 20 x 10(-6) M in 11-deoxoGA. These results indicate that the 18 beta-conformation of oleanane is important for the inhibition of 11 beta-hydroxysteroid dehydrogenase but on the contrary the 18 alpha-conformation is important for the inhibition of 3 alpha-hydroxysteroid dehydrogenase.