DNA-polymerase activity of the liver of adult and old rats

The DNA-polymerase activity was determined in the cytosol of the intact and regenerating liver of adult and old rats under conditions of free passage of enzymes from nuclei and mitochondria. The DNA-polymerase activity of the intact liver is significantly increased in adult rats. The regeneration results in about 2-fold and 10-fold increase of the activity in the liver of adult and old rats, respectively. As a result, the DNA-polymerase activity in the regenerating liver of old rats significantly increased as compared to that of adult rats. The revealed age-related changes in the DNA-polymerase activity of the liver do not correlate with the decrease in the replication rate in the process of aging.     

Effect of chronic ethanol treatment on lipid peroxidation in rat liver homogenate and subcellular fractions

1. The effect of chronic ethanol treatment on the level of lipid peroxidation in rat liver homogenate and subcellular fractions was measured using chemiluminescence technique and malondialdehyde formation. 2. It was shown that after chronic ethanol treatment the level of Fe/ADP-ascorbate-induced lipid peroxidation was decreased in the whole and "postnuclear" liver homogenates. Dilution of the homogenates prevented depressive effect of ethanol on lipid peroxidation. 3. Chronic ethanol treatment did not affect the intensity of the Fe/ADP-ascorbate-induced process in rat liver mitochondria and microsomes. 4. Peroxidative alteration of the liver lipids in vivo was evaluated by measurement of conjugated dienes (absorbance at 233 nm). It was shown that ethanol did not increase the level of u.v. absorption of lipids from mitochondria and microsomes. Chronic alcohol treatment did not influence the steady-state concentration of malonic dialdehyde in the whole liver homogenate. 5. The data obtained indicate that cytosol from the ethanol treated rat liver contains a factor(s) which prevents Fe/ADP-ascorbate-dependent lipid peroxidation in biological membranes.     

Effect of denervation of the liver on the system of microsomal oxidation in adult and old rats

The role of adrenergic and cholinergic neural regulation in the functional activity of the liver microsomal oxidation enzymes has been studied. The experiments on adult and old rats using surgical denervation of the liver (vagotomy and sympathectomy++) have revealed changes in the monooxygenase activity (aminopyrine demethylase and aniline hydroxylase), in isoform composition and inductive synthesis of cytochrome P-450. The neural control over detoxication function of the liver is found to weaken in old age.     

Effect of clofibrate treatment on lipid peroxidation in rat liver homogenate and subcellular fractions

1. A study was made of the effect of hypolipidemic drug clofibrate on the level of lipid peroxidation in homogenates and subcellular fractions of rat liver. The intensity of lipid peroxidation was measured using chemiluminescence technique and malondialdehyde formation. 2. It was shown that under the action of clofibrate the levels of Fe/ADP-ascorbate-, as well as t-butyl hydroperoxide (Bu'OOH)-induced lipid peroxidation were decreased in the whole and "post-nuclear" liver homogenates. Dilution of the homogenates prevented depressing effect of clofibrate on lipid peroxidation. 3. Clofibrate significantly decreased the level of the Bu'OOH-dependent lipid peroxidation, but did not affect the activity of the Fe/ADP-ascorbate-induced reaction in rat liver mitochondria and microsomes. 4. Peroxidative alteration of membrane lipids in vivo was evaluated by determining the extent of conjugated dienes formation (absorption at 233 nm). It was shown that clofibrate did not increase the level of ultraviolet absorption of lipids from rat liver subcellular fractions. 5. The data obtained indicate that cytosol from the clofibrate treated rat liver contains a factor(s) which prevents lipid peroxidation in the mitochondria and microsomes.     

Effect of clofibrate treatment on carnitine acyltransferases in different subcellular fractions of rat liver

The subcellular distribution of carnitine acetyl-, octanoyl-, and palmitoyltransferase in the livers of normal and clofibrate-treated male rats was studied with isopycnic sucrose density gradient fraction.In normal liver 48% of total carnitine acetyltransferase activity was peroxisomal, 36% of the activity located in mitochondria and 16% in a membranous fraction containing microsomes. Carnitine octanoyltransferase and carnitine palmitoyltransferase were confined almost totally (77–81%) to mitochondria in normal liver.Clofibrate treatment increased the total activity of carnitine acetyltransferase over 30 times, whereas the total activities of the other two transferases were increased only 5-fold.From the three different subcellular carnitine acetyltransferases the mitochondrial one was not responsive to clofibrate treatment, i.e. the rise in mitochondrial activity was over 70-fold as contrasted to the 6- and 14-fold rises in peroxisomal and microsomal activities, respectively. After treatment mitochondria contained 79% of total activity.It is concluded that the clofibrate-induced increase of carnitine acetyltransferase activity is not due to the peroxisomal proliferation that occurs during clofibrate treatment. The rise in peroxisomal activity contributed only 8% to the total increase.After clofibrate treatment the greatest part of carnitine octanoyl- and palmitoyltrnasferase activities were located in mitochondria but a considerable amount of both activities was found also in the soluble fraction of liver.     

Effect of chronic ethanol consumption on the cellular and subcellular distribution of gamma-glutamyltransferase in rat liver

After 6 weeks of chronic ethanol consumption hepatic gamma-glutamyl-transferase and -hydrolase activities increased compared with pair-fed controls. There was no change in 5'-nucleotidase activity. It was found that the increase in gamma-glutamyltransferase activity occurred exclusively in the parenchymal cells although the principal cellular localisation for this enzyme is the biliary tract in both control and ethanol-fed rats. In both groups of animals the gamma-glutamyltransferase activities were localised by analytical subcellular fractionation techniques to soluble, plasma membrane and canalicular fractions, but the plasma membrane activity was selectively increased in the ethanol-fed rats.     

Effect of cycloheximide on lipid metabolism in cells, nuclei and subcellular fractions in the rat liver

Well coordinated stages of inhibition, restoration and stimulation of protein, DNA and RNA synthesis were observed after administration of cycloheximide (3 mg/kg). The changes in lipid synthesis and composition in the nuclei and intranuclear structures were studied at different stages of cycloheximide action. The accumulation and stimulation of lipid synthesis in the nuclei during the inhibition and restoration of protein and DNA syntheses were followed by electron microscopy and labeled precursors methods. Dramatic changes were observed in the phospholipid composition of chromatin and nuclear matrix. The accumulation of minor phospholipid fractions in intranuclear structures was observed during DNA synthesis. The sphingomyelin concentration was predominant and commensurable with those of phosphatidylcholine and phosphatidylethanolamine.     

Lysophospholipase activity in rat brain subcellular fractions

Lysophospholipase activity in brain subcellular fractions was measured by the release of myristic acid from 1-myristoylglycerophosphocholine or through the formation of [³²P]glycerophosphocholine from [³²P]lysophosphatidylcholine. Although the lysophospholipase activity was highest in microsomes, considerable enzyme activity was also found in other subcellular membrane fractions. The pH optimum for the microsomal enzyme was around 7, whereas the synaptosomes and non-synaptic plasma membranes exhibited a pH maximum around 8. Although the enzyme did not require divalent cations for activity, divalent cations (1 mM) such as Hg²⁺, Cu²⁺, and Zn²⁺ inhibited potently the enzyme activity. Enzyme activity was also partially inhibited by both saturated and polyunsaturated fatty acids (25–200 M), and the inhibition seemed to be greater in the membrane than in the cytosolic fractions. Ionic detergents such as deoxycholate and taurocholate inhibited the lysophospholipase. On the other hand, the effect of Triton X-100 was biphasic, i.e., stimulation at concentrations below 100 g/mg protein and inhibition at higher concentrations. Addition of cholesterol (50–250 g/ml), but not cholesteryl esters, also potently inhibited enzyme activity. The presence of active lysophospholipase(s) in brain is probably an important mechanism for preventing unnecessary accumulation of lysophospholipids which may exert a deleterious effect on the membranes because, of their detergent properties.     

Distribution of lecithin-retinol acyltransferase activity in different types of rat liver cells and subcellular fractions

It is now well documented that lecithin-retinol acyltransferase (LRAT) is the physiologically important enzyme activity involved in the esterification of retinol in the liver. However, no information regarding the cellular distribution of this enzyme in the liver is presently available. This study characterizes the distribution of LRAT activity in the different types of rat liver cells. Purified preparations of isolated parenchymal, fat-storing, and Kupffer + endothelial cells were isolated from rat livers and the LRAT activity present in microsomes prepared from each of these cell fractions was determined. The fat-storing cells were found to contain the highest level of LRAT specific activity (383 +/- 54 pmol retinyl ester formed min-1.mg-1 versus 163 +/- 22 pmol retinyl ester formed min-1.mg-1 for whole liver microsomes). The level of LRAT specific activity in parenchymal cell microsomes (158 +/- 53 pmol retinyl ester formed min-1.mg-1) was very similar to LRAT levels in whole liver microsomes. The Kuppfer + endothelial cell microsome fractions were found to contain LRAT, at low levels of activity. These results indicate that the fat-storing cells are very enriched in LRAT but the parenchymal cells also posses significant levels of LRAT activity.     

Mechanism of the ATP-dependent phosphatidylserine synthesis in liver subcellular fractions

It has been shown that the ATP-dependent incorporation of [14C]serine into phosphatidylserine in rat liver mitochondrial and microsomal fractions is prevented by EGTA. On the other hand, at low (microM) Ca2+ concentrations, serine incorporation is strongly stimulated by ATP and Mg2+. This stimulatory effect is reduced by calcium ionophore A23187. It is therefore suggested that the ATP-dependent process is that of serine base-exchange reaction, stimulated by endogenous Ca2+ accumulated inside the microsomal vesicles by Ca2+,Mg2+-ATPase. The mitochondrial activity can be accounted for by contamination by the endoplasmic reticulum.     

Effect of clofibrate treatment on carnitine acyltransferases in different subcellular fractions of rat liver.

The subcellular distribution of carnitine acetyl-, octanoyl-, and palmitoyl- transferase in the livers of normal and clofibrate-treated male rats was studied with isopycnic sucrose density gradient fractionation. In normal liver 48% of total carnitine acetyltransferase activity was peroxisomal, 36% of the activity located in mitochondria and 16% in a membranous fraction containing microsomes. Carnitine octanoyltransferase and carnitine palmitoyltransferase were confined almost totally (77--81%) to mitochondria in normal liver. Clofibrate treatment increased the total activity of carnitine acetyltransferase over 30 times, whereas the total activities of the other two transferases were increased only 5-fold. From the three different subcellular carnitine acetyltransferases the mitochondrial one was most responsive to clofibrate treatment, i.e. the rise in mitochondrial activity was over 70-fold as contrasted to the 6- and 14-fold rises in peroxisomal and microsomal activities, respectively. After treatment mitochondria contained 79% of total activity. It is concluded that the clofibrate-induced increase of carnitine acetyltransferase activity is not due to the peroxisomal proliferation that occurs during clofibrate treatment. The rise in peroxisomal activity contributed only 8% to the total increase. After clofibrate treatment the greatest part of carnitine octanoyl- and palmitoyltransferase activities were located in mitochondria but a considerable amount of both activities was found also in the soluble fraction of liver.