Localization and some properties of lysosomal dipeptidases in rat liver

1. 1. The rates of hydrolysis of 26 synthetic depeptides by extracts from highly purified lysosomal fractions from rat liver at pH 5.0 and by whole liver homogenates at pH 7.4 have been determined. Extracts from the lysosomal fractions hydrolysed most peptides at a lower rate per mg protein than the homogenates, and some peptides not at all.

2. 2. Properties of two dipeptidases present in the extracts from the lysosomal fractions, splitting Ile-Glu and Leu-Gly, respectively, were studied in greater detail. The enzyme that hydrolysed Ile-Glu was strongly activated by dithiothreitol, showed optimal activity at pH 4.5 and had a molecular weight of about 120 000. Leu-Gly dipeptidase did apparently not contain an essential thiol group and had a molecular weight of approx. 90 000. It showed maximal activity at pH 6.5.

3. 3. After differential centrifugation of liver homogenates, Ile-Glu and Leu-Gly-splitting activities were determined in the fractions, under the optimal conditions mentioned above. The Ile-Glu-hydrolysing enzyme activity showed about the same distribution as the lysosomal marker enzyme acid phosphatase. Leu-Gly-splitting activity, however, was largely present in the cytosol fraction, with only a small peak in the lysosomal fraction. We obtained evidence that the activities present in the lysosomal fraction and in the cytosol fraction were due to different enzymes, and that one of these enzymes was localized exclusively in lysosomes.

4. 4. It is concluded that some dipeptides originating from intralysosomal proteolysis might be split by lysosomal dipeptidases, whereas others are probably hydrolysed only in the extra-lysosomal compartment of the cell.

Short-term Effects of Ethanol Intoxication on Ascorbic Acid and Lipid Metabolism and on Drug-metabolizing Enzymes in Liver of Rats

The effects of one-time ethanol intoxication on ascorbic acid and lipid metabolism and on drug-metabolizing enzymes in liver of rats were investigated. Male Donryu rats that had been fed semi-purified feed were given 5 g/kg ethanol solution (25%, w/v) via a stomach tube and killed 16 h after intubation. The amount of ascorbic acid excreted in the urine after ethanol administration increased, but renal and adrenal concentrations of ascorbic acid decreased. The serum levels of total cholesterol, high-density-lipoprotein cholesterol, triglycerides, phospholipids, and non-esterified fatty acids were elevated in rats given ethanol, but hepatic level of total lipids, cholesterol, triglycerides, phospholipids were not. The hepatic concentrations of cytochrome P-450 and cytochrome b₅ did not increase, but this large dose of ethanol increased the activities of aminopyrine N-demethylase and cytochrome c reductase.

These results indicated that the single dose of ethanol affected the ascorbic acid and lipid metabolism of rats, and induced drug-metabolizing enzymes in their liver.     

Phytochrome and phosphotungstate-chromate-positive vesicles from Cucurbita pepo L.

The phosphotungstic acid-chromic acid (PTA-CrO₃) stain, putatively specific for the plasma membrane of plants, has been used in an attempt to monitor the distribution of this membrane in a 20,000 x g particulate fraction from Cucurbita hypocotyl hooks. On discontinuous sucrose gradients, the relative distributions of the phytochrome and PTA-CrO₃-positive vesicles present in this fraction appear to be correlated. When intact tissue is stained, however, other components, in addition to the plasma membrane, react positively to the stain. These components include prolamellar-body membranes, lipid droplets, and ribosomes. This lack of specificity calls into question the reliability of the technique for the unequivocal identification and accurate quantitation of plasma-membrane fragments in isolated particulate fractions. The present data do not, therefore, provide unambiguous evidence that phytochrome is associated with plasma membrane in tissue homogenates from Cucurbita.Abbreviations PTA-CrO₃ phosphotungstate-chromate - RNP ribonucleoprotein     

Lead inhibits type-I iodothyronine 5′-monodeiodinase in the Indian rock pigeonColumba livia: A possible involvement of essential thiol groups

A study has been made to reveal the mode of action of lead inhibiting type-I iodothyronine 5′-monodeiodinase (5′-D) activity in the Indian rock pigeon,Columba livia. Administration of lead acetate (6 mg/kg body weight/day) for 20 days decreased 5′-D activity and glutathione content in the liver and kidney homogenates. It also reduced the serum concentration of 3, 3′, 5-triiodothyronine (T₃) with a marginal increase in serum thyroxine (T4). Hepatic and renal lipid peroxidative process increased significantly following lead treatment, whereas the levels of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) were decreased. The possible involvement of lipid peroxidative process in the inhibition of 5′-D activity inColumba livia is suggested.     

Mechanisms of lipid peroxide formation in animal tissues

1. Homogenates of rat liver, spleen, heart and kidney form lipid peroxides when incubated in vitro and actively catalyse peroxide formation in emulsions of linoleic acid or linolenic acid. 2. In liver, catalytic activity is distributed throughout the nuclear, mitochondrial and microsomal fractions and is present in the 100000g supernatant. Activity is weak in the nuclear fraction. 3. Dilute (0·5%, w/v) homogenates catalyse peroxidation over the range pH5·0–8·0 but concentrated (5%, w/v) homogenates inhibit peroxidation and destroy peroxide if the solution is more alkaline than pH7·0. 4. Ascorbic acid increases the rate of peroxidation of unsaturated fatty acids catalysed by whole homogenates of liver, heart, kidney and spleen at pH6·0 but not at pH7·4. 5. Catalysis of peroxidation of unsaturated fatty acids by the mitochondrial and microsomal fractions of liver is inhibited by ascorbic acid at pH7·4 but the activity of the supernatant fraction is enhanced. 6. Inorganic iron or ferritin are active catalysts in the presence of ascorbic acid. 7. Lipid peroxide formation in linoleic acid or linolenic acid emulsions catalysed by tissue homogenates is partially inhibited by EDTA but stimulated by o-phenanthroline. 8. Cysteine or glutathione (1mm) inhibits peroxide formation catalysed by whole homogenates, mitochondria or haemoprotein. Inhibition increases with increase of pH.     

Long-chain acyl CoA synthetase in microsomes from rat brain gray matter and white matter

Long-chain acyl coenzyme A (CoA) synthetase in homogenates and microsomes from rat brain gray and white matter was studied. The formation of the thioesters of CoA was studied upon addition of [1-¹⁴C]-labeled fatty acids. The maximal activities were seen with linoleic acid, followed by arachidonic, palmitic, and docosahexaenoic acids in both gray and white matter homogenates and microsomes. The specific activities in microsomes were 3–5 times higher than in homogenates. The presence of Triton X-100 in the assay system enhanced the activity of long-chain acyl CoA synthetase in homogenates. The effect was more pronounced in palmitic and docosahexaenoic acid activation. The apparentK _m values andV _max values for palmitic and docosahexaenoic acids were much lower than for linoleic and arachidonic acids. The presence of Triton X-100 in the medium caused a definite decrease in the apparentK _m and Vmax values for all the fatty acid except palmitic acid in which case the reverse was true. There were no significant differences observed in the kinetic measurements between gray and white matter microsomes. These findings are similar to those resulting from the known interference of Triton X-100 in the measurement of kinetic variables of long-chain acyl CoA synthetase of liver microsomes. In this work, no correlation was observed between the fatty acid composition of gray and white matter and the capacity of these tissues for the activation of different fatty acids.     

In vitro formation of polyunsaturated fatty acids by desaturation in rat brain: some properties of the enzymes in developing brain and comparisons with liver

Abstract— In vitro desaturation of [1-C¹⁴]linolenic, linoleic, oleic, and icosatrienoic acids was determined using homogenates and subcellular fractions of developing rat brain and liver. Linolenic, linoleic, and oleic acids were desaturated in the δ6-position and activity was optimal in the presence of CoA, ATP, MgCl₂, and NADH in a citrate-phosphate buffer at pH 6.0. Icosatrienoic acid was desaturated in the δ5-position with a much broader pH optimum. The unstable desaturation systems of brain were protected by reduced glutathione and niacinamide and markedly inhibited by dithiothreitol, p-chloromercuribenzoate, sodium cyanide or bathophenanthroline sulfonate. With brain homogenate of neonatal rats, the relative rates of desaturation of these substrates were 18:3(n - 3) > 18:2(n - 6) > 20:3(n - 6) > 18:l (n - 9). Specific activity of brain enzymes was greatest in neonatal rats with fluctuations in activity between 3 and 6 days of age. During this period, liver enzyme appeared to alter in a reciprocal manner. Total desaturation capacity of brain was maximal and fairly constant between 4 and 20 days of age, whereas liver activity increased dramatically after weaning. The activity of crude microsomal preparations from neonatal brain, like that of liver microsomes, was stimulated by a heat-labile component of the cytosolic fraction. These results demonstrate that brain has a high capacity for desaturation of the essential fatty acids during crucial stages of brain development when liver activity is relatively low.     

Depression by a Green Tea Extract of Alcohol-Induced Oxidative Stress and Lipogenesis in Rat Liver

We determined the effects of a green tea extract with 36% alcohol on the blood alcohol content, oxidative stress, lipogenesis, inflammation and liver function of female Wistar rats. Tea alcohol significantly decreased the O₂^?, H₂O₂ and HOCl amounts via catechins and not caffeine. Thirty days of alcohol gavage improved the level of reactive oxygen species (ROS) in the liver, bile and blood, increased the 4-hydroxynonenal-protein adducts, Kupffer cell infiltration and lipid accumulation in the liver, and elevated the plasma alanine aminotransferase level. A western blot analysis showed reduced expression of the oxidative enzymes (CYP2E1 and NADPH oxidase p47phox protein) and lipogenic enzymes (SREBP-1c and fatty acid synthase) in the alcohol-treated liver. Tea alcohol significantly attenuated these elevated parameters. We conclude that the green tea extract in alcohol efficiently reduced the amounts of O₂^?, H₂O₂ and HOCl primarily due to the catechin content, and not caffeine. The developed tea liquor attenuated alcohol-induced oxidative injury and lipogenesis in the liver by the synergetic action of catechins and caffeine.     

Determination of 2-n-propylquinoline in mouse plasma and liver by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the specific determination of 2-n-propylquinoline, a new anti-leishmaniasis drug, in plasma and liver homogenates of mice. 2-n-Propylquinoline was extracted with methyl-tert.-butyl ether with quinoline as internal standard. Separation was carried out using a Nucleosil C₁₈ column. The mobile phase consisted of methanol–0.005 M ammonium acetate buffer (60:40) at pH 5.5 and 8 for plasma and liver homogenates, respectively. Detection was monitored at 233 nm. The method was validated and shown to be accurate and precise for plasma and liver homogenates. Extraction yield was 96% in plasma and 81% in liver homogenates. This method was used to determine the pharmacokinetic profile of 2-n-propylquinoline following oral administration to mice.     

A Spectrofluorimetric study of the 7-hydroxylation of coumarin by liver microsomes

1. The fluorescence characteristics of 3- and 7-hydroxycoumarin, and 7-hydroxy-and 7-methoxy-4-methylcoumarin, have been determined. 7-Hydroxycoumarin shows excited-state ionization from pH1 to 9. 2. A sensitive and specific fluorimetric method for the determination of 7-hydroxycoumarin (umbelliferone), and its application to liver homogenates and other tissue preparations, are described. 3. The enzymic hydroxylation of coumarin to 7-hydroxycoumarin has been studied by this method and the optimum conditions have been determined for rabbit-liver preparations. The enzymic activity was found in the microsomal fraction and required NADPH₂ and oxygen. Activity with NADH₂ was one-third of that with NADPH₂. 4. Addition of NADP was necessary for full activity of 10000g supernatant preparations of liver. Nicotinamide added during preparation preserved coenzymic activity in tissue stored at −12°. Glucose 6-phosphate had no effect on the activity of stored or fresh tissue. 5. Inhibition occurred with p-chloromercuribenzoate, and with the usual inhibitors of the microsomal drug-metabolizing enzymes, SKF acid, SKF 525A, and Lilly 7132, but not with 2,2′-bipyridyl. 6. Liver homogenates from rabbit, guinea pig, coypu, cat and pigeon showed activity, but preparations of rat or mouse liver, and of locust fat bodies, did not hydroxylate coumarin to umbelliferone. The enzyme system was absent from rat-liver homogenates and microsomal preparations. Moreover, rat liver also contained inhibitors of the rabbit-liver coumarin-7-hydroxylase system and of the further metabolism of umbelliferone by guinea-pig liver. Guinea-pig-liver preparations hydroxylated coumarin to umbelliferone and then converted this product into its glucuronide. 7. The coumarin-7-hydroxylase activity of female rabbit liver was two to three times that of male rabbit liver.     

Modulation of antioxidant enzyme activities, platelet aggregation and serum prostaglandins in rats fed spray-dried milk containing n-3 fatty acid

Spray-dried milk enriched with n-3 fatty acids from linseed oil or fish oil were fed to rats to study its influence on liver lipid peroxides, hepatic antioxidant enzyme activities, serum prostaglandins and platelet aggregation. Significant level of α linolenic acid, eicosapentaenoic acid and docosahexaenoic acid were accumulated at the expense of arachidonic acid in the liver of rats fed n-3 fatty acid enriched formulation. The linseed oil and fish oil enriched formulation fed group had 44 and 112% higher level of lipid peroxides in liver homogenate compared to control rats fed groundnut oil enriched formulation. Catalase activity in liver homogenate was increased by 37 and 183% respectively in linseed oil and fish oil formulation fed rats. The glutathione peroxidase activity decreased to an extent of 25–36% and glutathione transferase activity increased to an extent of 34–39% in rats fed n-3 fatty acids enriched formulation. Feeding n-3 fatty acid enriched formulation significantly elevated the n-3 fatty acids in platelets and increased the lipid peroxide level to an extent of 4.2–4.5 fold compared to control. The serum thromboxane B₂ level was decreased by 35 and 42% respectively in linseed oil and fish oil enriched formulation fed rats, whereas, 6-keto- prostaglandin F1α level was decreased by 17 and 23% respectively in linseed oil and fish oil enriched formulation fed rats. The extent and rate of platelet aggregation was decreased significantly in n-3 fatty acids enriched formulation fed rats. This indicated that n-3 fatty acids enriched formulation beneficially reduces platelet aggregation and also enhances the activities of hepatic antioxidant enzymes such as catalase and glutathione transferase. (Mol Cell Biochem xxx: 9–16, 2005)     

Ganoderma lucidum total triterpenes prevent γ-radiation induced oxidative stress in Swiss albino mice in vivo

Objectives: The in vivo radio-protective effect of total triterpenes isolated from Ganoderma lucidum (Fr.) P. Karst was evaluated using Swiss albino mice, by pre-treatment with total triterpenes for 14 days, followed by a whole body exposure to γ-radiation.

Methods: The activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), and the level of reduced glutathione (GSH) were analysed in liver and brain homogenates. The extent of lipid and protein peroxidation was also estimated in liver and brain homogenates after irradiation. Protection of radiation-induced DNA strand breaks in peripheral blood lymphocytes and bone marrow cells was assessed using the comet assay.

Results: Total triterpenes were highly effective in reducing the levels of lipid peroxidation and protein oxidation to near normal values in both liver and brain tissues. Total triterpenes, when administered in vivo, were also found to be successful in restoring the antioxidant enzyme activities and GSH level in liver and brain of irradiated mice. Administration of total triterpenes, prior to radiation exposure, significantly decreased the DNA strand breaks.

Discussion: The results of the present study thus revealed the potential therapeutic use of Ganoderma total triterpenes as an adjuvant in radiation therapy.     

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.Abbreviations BAT Brown Adipose Tissue - MDA malondialdehyde     

Effect of retinol on the hemolysis and lipid peroxidation in vitamin E deficiency

A study on the effect of retinolin vitro on the hemolysis of vitamin E deficient rat red blood cells showed that retinol enhanced the lysis of the E deficient cells as compared to the lysis of normal cells. The lipid peroxidation present during hydrogen peroxide induced lysis of E deficient cells was however markedly inhibited in the presence of retinol without affecting the rate of lysis. In an actively peroxidising system of non-enzymatic lipid peroxidation of rat liver or brain homogenates and of brain lysosomes incubated with human erythrocytes, no lysis was obtained; incorporation of retinol in such systems resulted in lysis but no peroxidation. Hydrogen peroxide generating substances almost completely inhibited the lysis of normal human erythrocytes by retinol, but linoleic acid hydroperoxide and auto-oxidised liver or brain homogenates and ox-brain liposomes increased the lysis. It is concluded that vitamin E deficient erythrocyte hemolysis may be augmented by retinol, an anti-oxidant, having a lytic function without the peroxidation of stromal lipids     

Regulation by dietary choline of hepatic fatty acid synthetase in the rat

Fatty acid synthetase activity was measured in the high-speed supernatant fraction of liver homogenates from rats fed a semisynthetic diet low in lipotropic factors. If choline was omitted from the diet, a significant increase of fatty acid synthetase activity was observed after two feedings of the deficient diet. Compared with controls, the increase of fatty acid synthetase activity was of a magnitude that could account for the amount of triglyceride accumulating in the hepatic floating lipid fraction. Gas-liquid chromatographic analysis of the floating lipid triglycerides showed an increased content of palmitic acid due to choline deficiency; this increase could be predicted from the increased fatty acid synthetase activity and its known characteristic yield of palmitic acid.     

Metabolism of a glutathione conjugate of 2-hydroxyoestradiol by rat liver and kidney preparations in vitro

Adult male rat liver and kidney preparations were incubated with (2-hydroxyoestradiol-1-yl)[(35)S]glutathione. The glutamic acid and glycine residues were removed by enzymes present in the kidney microsomal fraction; the liver preparations had no effect. The resulting 2-hydroxyoestradiol-cysteine conjugate was acetylated at the alpha-amino group by both liver and kidney homogenates fortified with acetyl-coenzyme A, but not significantly in the absence of this coenzyme, or by liver or kidney slices. These results suggest that an oestrogen-glutathione conjugate, if formed in vivo, would be converted into the corresponding mercapturic acid before excretion.     

Distribution of ciliatine (2-aminoethylphosphonic acid) and phosphonoalanine (2-amino-3-phosphonopropionic acid) in human tissues

Ciliatine (2-aminoethylphosphonic acid) was detected in the human brain, heart, kidney, liver, intestine, spleen, adrenal glands, and aorta. Phosphonoalanine (2-amino-3-phosphonopropionic acid) was found in the human liver, intestine and spleen. Tissue homogenates were extracted with trichloroacetic acid and a chloroform-methanol mixture. After hydrolysis, each fraction was subfractionated by ion-exchange chromatography and examined by paper chromatography and electrophoresis using a specific ninhydrin-molybdate staining procedure to detect the phosphonic acids. The acids were found bound either to lipid or to protein; no free phosphonic acid was detected.     

Analysis of lipids in the salivary glands of Amblyomma americanum (L.): Detection of a high level of arachidonic acid

Analysis of lipids in salivary glands of the lone star tick, Amblyomma americanum, demonstrated that arachidonic acid (20:4, n-6) comprises 8% of all fatty acids identified by gas chromatography. The occurrence of arachidonic acid and other C₂₀ polyunsaturated fatty acids in tick salivary glands was confirmed by gas chromatography-mass spectrometry. Arachidonate is located entirely in the phospholipid fraction and is associated exclusively with phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Salivary glands stored and frozen for several months had a similar lipid composition as freshly dissected salivary glands, with the exception of a small amount of free arachidonic acid and an increase in lysophosphatidylcholine. Incubation of salivary gland homogenates with snake venom phospholipase A₂ showed that most saturated fatty acids are esterified in the sn-1 position of PC and PE, with the unsaturated fatty acids in the sn-2 position. Approximately 75% of arachidonic acid is in the sn-2 position of PC and PE, adding support to the hypothesis that arachidonic acid is released into the cytoplasm after activation of a phospholipase A₂ for subsequent metabolism to prostaglandins and/or other eicosanoids. © 1993 Wiley-Liss, Inc.     

Volatile Components Identified in the Acidic Fractions of Wines from Koshu and Zenkoji Grapes

The induction and mechanism of fatty liver in the rat by the synthetic carcinogen 2-acetylaminofluorene (AAF) were investigated.

The induction of this fatty liver was dose and time dependent, being gradually increased by the intake of a 0.05% AAF diet for 3 weeks. The AAF dosage was found to increase the activity of drug-metabolizing enzymes (p-nitroanisol demethylase and aniline hydroxylase) and to decrease the activity of pyruvate kinase and α-glycerophosphate dehydrogenase. The AAF dosage had no effect on the incorporation of [l-¹⁴C]acetate into the lipid fraction during in vitro incubation of liver slices. The supplement of adenine to the AAF diet had no effect on the accumulation of liver lipid.

It is suggested from the result of treatment with Triton WR-1339 that a block in the secretion of triglyceride from the liver is a major cause of the induction of fatty liver by AAF.