Oxidation of reduced glutathione by subcellular fractions of rat liver

1. A new method was used to diminish the autoxidation of GSH. 2. The oxidation of GSH by liver homogenates was studied with regard to concentration of homogenate, concentration of GSH, time, pH and anaerobiosis. 3. GSH was oxidized by recombinations of the supernatant with microsomes and with mitochondria. Each fraction alone caused little oxidation. 4. Proteins in the supernatant were required to obtain the effect, and low-molecular-weight compounds in the same fraction increased its effect. 5. GSH diminished the formation of malonaldehyde in homogenates. 6. GSH prevented a stimulating effect of the supernatant on the formation of malonaldehyde in microsomes and in mitochondria. 7. The malonaldehyde formation in microsomes together with the supernatant did not start until the concentration of endogenous low-molecular-weight thiols had decreased to a low level. 8. It is suggested that part of the oxidation of GSH in homogenates is coupled to a mechanism that counteracts the peroxidation of membrane lipids.     

The association of calmodulin with subcellular fractions isolated from rat liver

Calmodulin associated with rat liver mitochondria has been found to belong to a contaminant membranous fraction which contains different subcellular membranes. The concentration of calmodulin in this fraction is relatively high, about 1.6 micrograms/mg protein, and can not be decreased with EGTA. The calmodulin-rich membranous fraction seems to contain cytoskeletal proteins which could be responsible for the binding of calmodulin.     

Two distinct mechanisms for taurocholate uptake in subcellular fractions from rat liver

As part of the enterohepatic circulation, hepatocytes take up bile acids from the intestines via the hepatic portal blood using a sodium-dependent carrier mechanism and resecrete the bile acids into the bile. In order to assess whether intracellular organelles are involved in the transcellular secretion of bile acids, we measured directly the ability of purified subcellular fractions of rat liver to take up taurocholate using a Millipore filtration assay. Two distinct uptake mechanisms can be discerned, one localized in the plasma membranes and the other in the Golgi and smooth microsomal fractions. Plasma membranes prepared by the method of Fleischer and Kervina (Fleischer, S., and Kervina, M. (1974) Methods Enzymol. 31, 6) take up taurocholate in a saturable manner with an apparent Vmax of 2.4 nmol min-1 mg protein-1 and a Km of 190 microM at 37 degrees C. After preincubation of the membranes with K+ ions, a sodium gradient (100 mM outside) stimulates the uptake rate by 90% with the observed Km unchanged. The stimulation is inhibited by phalloidin but not by bromosulfophthalein. Bile canalicular plasma membranes made according to Kramer et al. (Kramer, W., Bickel, U., Buscher, H. P., Gerok, W., and Kurz, G. (1982) Eur. J. Biochem. 129, 13-24) do not take up taurocholate. The transport by Golgi vesicles and smooth microsomes differs from that in the plasma membrane fraction in that it is not stimulated by a sodium gradient, has a Vmax of 12 nmol min-1 mg protein-1 and a Km of 440 microM at 37 degrees C, and is inhibited by bromosulfophthalein but not by phalloidin. Taurocholate uptake into smooth microsomes is abolished by filipin, an antibiotic that complexes with cholesterol to disrupt the membrane. This suggests that taurocholate uptake occurs into a nonendoplasmic reticulum subfraction since endoplasmic reticulum membranes contain negligible amounts of cholesterol. Little uptake was observed using rough microsomes or mitochondria. A model of transhepatic transport compatible with our observations is that taurocholate uptake into the cytoplasm occurs via the plasma membranes on the sinusoidal side of the hepatocyte; taurocholate is then taken up into smooth vesicles and the Golgi complex and is secreted into the bile by exocytosis as the vesicles fuse with the canalicular plasma membranes.     

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.     

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 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.     

Influence of subtotal hepatectomy on peroxisomes and peroxisomal enzymes of rat liver and isolated liver cell fractions.

The activities of peroxisomal enzymes of rat liver were followed 1 to 10 days after subtotal (60-70%) hepatectomy in homogenates prepared from regenerating livers and in cell fractions isolated from them. Catalase activity was found to be depressed in the total liver homogenate (H) as well as in the mitochondrial (M) and soluble (S) fractions, while it did not change appreciably in the microsomal (Mc) and lysosomal (L) fractions. Alpha-hydroxyacid oxidase behaved in a similar fashion. In contrast to these enzymes, urate oxidase activity remained unchanged in H, whereas it was decreased in M and increased in L and Mc during the first 5 days after operation. These results agree well with the assumption that microbody proliferation is initiated by the fragmentation of large peroxisomes. The different relations of peroxisomal enzyme activities during regeneration time are discussed with respect to the possible existence of various kinds of peroxisomes with different enzyme equipments and with different turnover rates. Biochemical examinations ions were paralleled to morphological and histochemical studies. An early increase in number of peroxisomes was found to occur during the first day after partial hepatectomy, which is accompanied by decrease in particle size. During the first mitotic wave (24-36 hrs post op.) the number of peroxisomes per cell was reduced to about the half. After this time number and size of the particles began to increase. Positive staining of ribosomes was frequently observed in the vicinity of peroxisomes after the application of the cytochemical catalase reaction (alkaline diaminobenzidine medium). This phenomenon is interpreted to represent rather a diffusion artifact than the cytochemical identification of newly synthesized catalase.     

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.     

Metabolism of palmitate in perfused rat liver. Isolation of subcellular fractions containing triacylglycerol.

1. The metabolism of [1-14C]palmitate in rat liver was studied in a single-pass perfusion system at concentrations of 0.2 or 1 mM. 2. After the perfusion the liver was homogenized and the floating fat was isolated. The incorporation of [1-14C]palmitate into triacylglycerol in this pool increased 9-fold when the palmitate concentration in the medium was increased from 0.2 to 1 mM. In time studies with 1 mM-[1-14C]palmitate 75% of the total accumulation of triacylglycerol occurred in this pool. Our results support the concept that the floating-fat fraction contains the storage pool of triacylglycerol, i.e. the cytoplasmic lipid droplets. 3. In a particulate preparation consisting mainly of mitochondria and microsomal fraction the incorporation of [1-14C]palmitate into triacylglycerol was proportional to the fatty acid concentration. Triacylglycerol in the perfusate medium and in the particulate fraction was in isotopic equilibrium, which indicates that the particulate fraction contained the precursor pool for secreted triacylglycerol, i.e. the pool in endoplasmic reticulum and Golgi apparatus. 4. The oxidation to labelled water-soluble products and to CO2 was increased 14-fold by the 5-fold increase in palmitate concentration.     

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.