Subcellular Fractions Of Rat Liver Research Articles

Intracellular distribution of radiogold. Localization to large granule membranes.

The distribution of gold in Kupffer cells and in subcellular fractions of rat liver was studied at intervals following intraperitoneal injection of 195Au sodium thiomalate (Myochrysine). Kupffer cells, isolated by digestion of whole liver with Pronase, had radioactive gold counts per milligram of protein that were twice the counts in the digested liver supernatant. After fractionation of liver cells by differential centrifugation, radiogold was found predominantly in the nuclear, mitochondrial, and lysosomal fractions. When the distribution of isotope was related to the protein content, the highest gold concentration was found in the lysosomal fraction, where it was 28 times that in the soluble fraction. Most radiogold was nondialyzable, probably a result of binding to larger intracellular compounds. Approximately 95% of the nondialyzable gold was in the organelle membrane of attached to membrane-adsorbed material, as determined by treatment of dialyzed mitochondrial and lysosomal fractions with Triton X-100. These data suggest that the intracellular locus of gold action may reside in organelle membranes.

Conversion of proalbumin into serum albumin in the secretory vesicles of rat liver

The conversion site of proalbumin into serum albumin was investigated in the subcellular fractions of rat liver labeled with [ 3H] leucine in vivo. In the cisternae-rich fraction of the Golgi complex as well as in the microsomal fraction most of the labeled albumin was detected as proalbumin, while in the secretory vesicles, which were obtained in increased amount by oral administration of ethanol, more than 70% of the labeled albumin was found as serum type, indicating that conversion of proalbumin into serum albumin occurs within the secretory vesicles in rat liver. Little accumulation of albumin was observed in colchicine-treated rats.

Studies on the subcellular localization and properties of bis(monoacylglyceryl)phosphate biosynthesis in rat liver.

Phosphatidylglycerol conversion to bis(monoacylglyceryl)phosphate by rat liver homogenate was studied and maximum rates of synthesis were observed at pH 4.4. The distribution of bis(monoacylglyceryl)P synthetase in rat liver subcellular fractions was determined, and evidence is presented establishing the lysosomes as the site of bis(monoacylglyceryl)P synthesis. In addition to phosphatidylglycerol, 1-acyl- and 2-acyllysophosphatidylglycerol also served as precursors for bis(monoacylglyceryl)P with lysosomes as an enzyme source. Bis(monoacylglyceryl)P synthesis did not require high energy intermediates or cofactors. The possibility that a lysosomal phospholipase A with acyl transferase activity catalyzes the formation of bis(monoacylglyceryl)P was investigated. Heat stability and inhibitor studies suggested that this is probably not the case. Lysosomes were shown to be unable to synthesize phosphatidylglycerol, and lipid analyses showed that lysosomes do not contain phosphatidylglycerol or lysophosphatidylglycerol. Bis(monoacylglyceryl)P synthesis in the cell may require the interaction of lysosomes with a phosphatidylglycerol-containing membrane.

Subcellular localization of ornithine decarboxylase in liver of control and growth-hormone-treated rats

1. Ornithine-2-oxo acid aminotransferase activity was inhibited by amino-oxyacetate (10(-5) M). This permitted the measurement of ornithine decarboxylase in the presence of mitochondria by using the 14CO2-trapping technique. 2. Subcellular fractionation of rat liver by differential centrifugation, followed by the assay of ornithine decarboxylase in the presence of amino oxyacetate and of marker enzymes for each fraction, demonstrated that ornithine decarboxylase was located in the cytosol. 3. The greatly increased ornithine decarboxylase activity observed after growth-hormone administration was also found to be localized in the cytosol. 4. The Km of ornithine decarboxylase from rat liver for ornithine was 28 muM. Administration of growth hormone 4 h before death did not affect the apparent affinity of ornithine decarboxylase for ornithine.

Activity and Subcellular Distribution of Protein Kinase Dependent on Adenosine 3′: 5′‐Monophosphate in Liver from Normal and Adrenalectomized Rats

We have examined whether glucocorticoids control the activity and (or) the subcellular distribution of protein kinase dependent on cyclic AMP (adenosine 3':5'-monophosphate), since they influence cyclic-AMP-dependent responses to other hormones. Protein kinase activity was determined in rat liver homogenates and subcellular fractions, nuclear, large granular, microsomal and supernatant obtained by differential sedimentation in 0.25 M sucrose. 63% of the tissue protein kinase activity detected in absence of cyclic AMP reside in the particulate fractions. Upon addition of exogenous cyclic AMP, protein kinase activity is stimulated 1.8, 1.2, 1.2 and 4.5-fold in nuclear, large granular, microsomal and supernatant fractions, respectively. Under these conditions, 66% of tissue activity are found in the supernatant fraction. The activity sensitive to exogenous cyclic AMP resolves into a major (84%) cytosoluble and a minor (16%) nucleomicrosomal component. The latter activity resists elution with isotonic saline and is increased in the presence of Triton X-100. Three groups of rats were studied: control and adrenalectomized with or without cortisol treatment. In whole liver homogenates, both protein kinase activity detected in absence of exogenous cyclic AMP and sensitivity of the enzyme to cyclic AMP were comparable in all groups. Moreover, the distribution patterns of proteins kinase activity amoung the fractions were essentially the same in all groups of animals, whether or not particles had been treated with Triton X-100. Finally, in cell-free experiments, glucocorticoids alone or in combination with their intracellular receptor did not modify protein kinase activity of rat liver. Thus the results reported do not support the possibility that glucocorticoids influence cyclic AMP-dependent protein kinase in rat liver. Yet, this study provides data, not available before, on subcellular distribution of this enzyme in rat liver.

Application of nicotinamide-adenine dinucleotide analogs for clinical enzymology: alcohol dehydrogenase activity in liver injury

The activities of alcohol dehydrogease(ADH) in serum and in the subcellular fractions of rat liver were determined with n-amyl alcohol or ethanol as substrate and thionicotinamide-adenine dinucleotide as coenzyme. It was found that the enzymes's activity ratio on the amyl alcohol and ethanol(A/E value) of serum and on the particulate fractions of the liver were different, but the A/E value of the soluble fraction was similar to that of serum. The A/E value of the particulate fractions were higher than that of the soluble fraction. From the results of experimental liver damage in the rat, it seems that estimation of the A/E value of ADH activity in serum is a useful parameter for the diagnosis of active liver injury. Since the A/E values of patients' sera differed from those of the normal subjects, the estimation of the A/E value of serum may give diagnostic information on liver injury, especially in chronic liver injury.

Studies on nucleotide diphosphate diacylglycerol specificity of acidic phospholipid biosynthesis in rat liver subcellular fractions

1. (1) Cytidine diphosphate diacylglycerol, uridine diphosphate diacylglycerol, adenosine diphosphate diacylglycerol and guanosine diphosphate diacylglycerol were synthesized chemically and their purity assessed. The activity of these compounds in acidic phospholipid synthesis was examined in rat liver mitochondria and microsomes. 2. (2) Phosphatidylglycerol synthesis in rat liver mitochondria exhibited considerable activity with CDPdiacylglycerol ( V 7.0 nmol · mg −1 · h −1), UDPdiacylglycerol ( V 5.4) and ADPdiacylglycerol ( V 4.2). GDPdiacylglycerol activity was detectable but very low. 3. (3) Diphosphatidylglycerol formation in mitochondria and phosphatidyl-inositol synthesis in microsomes exhibited considerable more specificity for CDPdiacylglycerol. However, at high concentrations, measurable diphosphatidylglycerol and phosphatidylinositol synthesis was observed with the other liponucleotides. 4. (4) Although considerable phosphatidylglycerol formation was observed with UDPdiacylglycerol and ADPdiacylglycerol, it is unlikely that these compounds are of physiologic importance, at least in rat liver, since CTP:phosphatidic acid cytidyltransferase in microsomes and mitochondria was shown to be specific for cytidine triphosphate. The lack of specificity of phosphatidylglycerol synthesis for CDPdiacylglycerol is currently unexplained but may be of some importance in other tissues or in other organisms.

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.

Lysolecithinase activity in subcellular fractions of rat organs

Lysolecithinase activity was measured in subcellular fractions of rat liver, kidney, lung and intestine and compared to similar findings in brain. To obtain optimal assay conditions, each fraction was subjected to a kinetic analysis in the absence and presence of albumin. Among the particulate preparations, lysolecithinase activity of the intestine exceeded by far similar fractions of other organs. Among the soluble fractions, the 100,000xg supernatant of lung had the highest activity. Under the assay conditions used, most of the lysolecithinase activity of all organs was particulate.

The degradation of rRNA in ribosomes by ribonucleases present in rat liver sub-cellular fractions

The degradation of rRNA in ribosomes by ribonucleases present in rat liver sub-cellular fractions

Conversion of 7alpha-hydroxycholesterol and 7alpha-hydroxy-beta-sitosterol to 3alpha, 7alpha-dihydroxy- and 3alpha, 7alpha, 12alpha-trihydroxy-5beta-steroids in vitro.

The metabolism of 7alpha-hydroxycholesterol and 7alpha-hydroxy-beta-sitosterol (24alpha-ethyl-5-cholestene-3beta,7alpha-diol) has been compared in rat liver subcellular fractions. 7alpha-Hydroxy-beta-sitosterol was shown to be metabolized in the same manner as 7alpha-hydroxycholesterol. Thus, the following C29 metabolites have been identified: 24alpha-ethyl-7alpha-hydroxy-4-cholesten-3-one, 24alpha-ethyl-7alpha,12alpha-dihydroxy-4-cholesten-3-one, 24alpha-ethyl-7alpha-hydroxy-5beta-cholestan-3-one, 24alpha-ethyl-5beta-cholestane-3alpha,7alpha-diol, 24alpha-ethyl-7alpha,12alpha-dihydrozy-5beta-cholestan-3-one, and 24alpha-ethyl-5beta-cholestane-3alha,7alpha,12alpha-triol. The C29 compounds were generally less efficient substrates. The most pronounced difference was noted for the delta4-3-oxosteroid 5beta-reductase. Thus, 7alpha-hydroxy-4-cholesten-3-one was three to four times as efficiently reduced as the C29 analog. The oxidation of the 3beta,7alpha-dihydroxy-delta5-steroid to the 7alpha-hydroxy-delta4-3-oxosteroid, the 12alpha-hydroxylation of the 7alpha-hydroxy-delta4-3-oxosteroid, and the reduction of the 7alpha-hydroxy-5beta-3-oxosteroid to the 3alpha,7alpha-dihydroxy-5beta-steroid occurred in up to two times better yields for the C27 steroids.

Ontogeny of cyclic AMP-dependent protein phosphokinase during hepatic development of the rat

The ontogeny of protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) and cyclic AMP-binding activity in subcellular fractions of liver was examined during prenatal and postnatal development of the male rat. 1. 1. Protein kinase activity and cyclic AMP-binding activity were found in the nuclear, microsomal, lysosomal-mitochondrial, and soluble liver fractions. 2. 2. The protein kinase activity of the soluble (105 000 × g supernatant) fraction measured with histone F1 as substrate was stimulated by cyclic AMP. Cyclic AMP did not stimulate the protein kinase activity of the particulate fractions. 3. 3. The protein kinase activity of all subcellular fractions increased rapidly from the activity observed in prenatal liver (3–4 days before birth) to reach maximal activity in 2-day-old rats. Thereafter, the protein kinase activity declined more slowly and regained the prenatal levels at 10 days after birth. 4. 4. Considerable latent protein kinase activity was associated with liver microsomal fractions which could be activated by treatment of microsomes with Triton X-100. The latent microsomal protein kinase activity was highest in prenatal liver, at the time of birth, and 2 days after birth. During the subsequent postnatal development the latent microsomal protein kinase activity gradually declined to insignificantly low levels. 5. 5. During the developmental period examined (4 days before birth to age 60–90 days) marked alterations of the cyclic AMP-binding activity were determined in all subcellular fractions of rat liver. In general, cytosol, microsomal, and lysosomal-mitochondrial cyclic AMP-binding activity was highest in 10–11 day-old rats. Nuclear cyclic AMP-binding activity was highest 3–4 days before birth and declined at birth and during the postnatal period. There was no correlation between the developmental alteration of cyclic AMP-binding activity and cyclic AMP dependency of the protein kinase activity in any of the subcellular fractions. This suggests that the measured cyclic AMP-binding activity does not reflect developmental alterations of the cyclic AMP-binding regulatory subunit of cyclic AMP-dependent protein kinase.

Cellular binding sites for insulin in rat liver

A study of the sites of insulin binding in subcellular fractions of rat liver is reported. A method for the isolation of liver plasma membranes, which permits one to follow quantitatively the distribution of all the parameters of interest, was modified and applied to the study of the cellular topography of insulin binding. The insulin-binding capacity did not follow closely the enzyme marker (5′-nucleotidase) for plasma membranes when differential centrifugation schemes were used, and the divergence from this marker was more prominent when separations were performed on discontinuous sucrose gradients. A significant amount of insulin binding capacity was always present in fractions with higher density than those containing the majority of 5′-nucleotidase. Results of studies on linear sucrose gradients have disclosed in some of the purified membrane fractions small but consistent differences in density of the insulin binding, and plasma membrane particles. It is suggested that there may be several types of intracellular membranes to which insulin can bind besides the plasma membranes.

Distribution of silicon in the adult rat and rhesus monkey

The distribution of silicon in tissues of the rat and rhesus monkey was determined using a colorimetric method specifically designed for biological materials. Soft-tissue silicon levels in both species varied from 1 to 33 μg silicon/g dry weight (ppm) excepting the primate lung and lymph nodes, which averaged 942 ppm and 101 ppm, respectively. This distribution may reflect flow-through of silicon in the form of particulates and water-soluble silicate contaminates in the environment and food effectively masking tissue or organ-specific silicon of functional significance. The silicon content of the whole rat femur was 38 ppm as determined by emission spectroscopy, whereas the monkey femur shaft was 43 ppm and the femur head containing the epiphysis was 456 ppm. This silicon may be functionally involved in the calcification process as suggested by others. The silicon content of rat liver subcellular fractions was also determined. The supernatant, nuclei/debris, and mitochondrial portions contained 0.6–1.0 μg silicon/g liver equivalents, whereas the microsomal fraction contained only 0.2 μg silicon/g liver equivalents.

UDP glucuronyltransferase and phenolsulfotransferase in vivo and in vitro: Conjugation of harmol and harmalol

Glucuronidation and sulfation of the phenolic — OH group of harmol and harmalol by the rat in vivo, and by rat liver subcellular fractions in vitro have been studied. In vivo harmol was extensively excreted (59% of the dose in 3 hr) in bile and urine, mostly as harmol-sulfate (70%) but also as harmol-glucuronide (30%). Harmalol was also excreted in bile and urine (50% of the dose in 3 hr), mostly as harmalol-glucuronide (70%), with only a trace of harmalol-sulfate present (less than 3%). In vitro kinetic parameters of conjugating activities towards both substrates were determined. Harmol and harmalol were glucuronidated by UDP glucuronyltransferase at comparable rates. Phenolsulfotransferase converted harmol readily to its sulfate-conjugate, whereas harmalol was a very poor substrate of this enzyme. Thus, the excretory pattern of harmol and harmalol can be explained by different rates of conjugation of these substrates by UDP glucuronyltransferase and phenolsulfotransferase, as found in vitro.

The effect of fasting on the acylation of carnitine and glycerophosphate in rat liver subcellular fractions

1. 1. The effect of a 48 h fast on the acylation rates of carnitine and glycerophosphate by isolated liver mitochondria and microsomes is measured, in the presence of ATP and CoASH, with palmitate (complexed to albumin) as the acyl donor. 2. 2. During fasting the acylation rate of carnitine increases 2–3 fold in the microsomal fraction and by a small percentage in the mitochondria. It is not inhibited by palmitoyl-CoA at concentrations up to 0.1 mM. 3. 3. During fasting the acylation rate of glycerophosphate decreases by 33% in isolated mitochondria and by 13% in microsomes. High concentrations of palmitoyl-CoA (above 0.02 mM) inhibit the reaction. 4. 4. The significance of these results for the partition of long-chain fatty acids between the esterification and oxidation pathways is discussed.

Formation and metabolism in vitro of 5,6-epoxides of cholesterol and β-sitosterol

The formation of 5alpha,6alpha- and 5beta,6beta-epoxides of cholesterol and beta-sitosterol in rat liver subcellular fractions has been studied. The results show that the epoxidation seems to occur only in connection with the nonspecific tissue oxidation of the sterols. The beta-epoxides were formed in three- to fourfold excess over the alpha-epoxides. Both cholesterol epoxides were efficiently converted by a microsomal hydrolase into the 3beta,5alpha,6beta-triol. The conversion was less extensive with beta-sitosterol epoxides, especially the beta-epoxide. The possible biological significance in the formation of the sterol epoxides and the triols was evaluated by their ability to inhibit the microsomal cholesterol 7alpha-hydroxylase. Only the cholesterol epoxides and especially the beta-epoxide were active in this respect.

In vitro metabolism of atrazine by rat liver

The metabolism of atrazine and 6 possible metabolites by rat liver subcellular fractions was studied in vitro. The dealkylation reaction predominated over the conjugation reaction with glutathione; the isopropyl group being more easily dealkylated than the ethyl group. With the compounds investigated, the reactions involved dealkylation in the microsomal fraction and conjugation with glutathione in the soluble fraction. All of the chloro- s-triazines were able to form conjugates with glutathione. No evidence for the dechlorination of the chloro- s-triazines to hydroxy- s-triazines was observed in vitro.

Increased acridine orange uptake and lysosomal enzyme levels in rat liver after steroid administration

A steroid which stabilizes lysosomes in vitro and a pyrogenic steroid which labilizes lysosomes in vitro were compared with respect to their ability to modify lysosomal uptake and lysosomal enzyme levels in vivo . Cortisone acetate increased the uptake of acridine orange by rat liver lysosomes when the dye was administered by intrathoracic injection. The steroid increased and accelerated the uptake of acridine orange so that, in liver lysosomes from treated rats, the maximum uptake was double that of controls and was reached at 2h, whereas in controls the maximum uptake was at 4h after the injection of the dye. This large elevation of uptake is specific to the lysosomal fraction and is not seen in other subcellular fractions of rat liver. The specific activities of a lysosomal enzyme β-N-acetylglucosaminidase were increased in lysosomal fractions from cortisone acetate-treated rats. Etiocholanolone, a steroid which labilizes lysosome in vitro , similarly accelerated and increased acridine orange uptake by lysosomes but had little effect on lysosomal β-N-acetylglucosaminidase levels. Thus the ability of steroids to stabilize or labilize lysosomes in vitro does not correlate with their effect on lysosomal uptake of injected substances in vivo , or with their ability to induce increased specific activities of lysosomal enzymes.

Characterization and subcellular localization of lipase activities in rat liver cell. Comparison with phospholipase A

Summary u 1. – This study deals with the localization, characterization and properties of lipases present in rat liver subcellular fractions. 2. – No triglyceride lipase activity was detected in purified mitochondrial preparations. 3. – Lysosomes, isolated as tritosomes, contain a triglyceride-lipase very active against long chain fatty acid triglycerides. It is a soluble enzyme and has a pH optimum at 4.5. 4. – A triglyceride-lipase activity is also found in microsomes and plasma membranes; it is membrane-bound. In microsomes the pHopt is 8 and the apparent KM 27 mM (triolein emulsified in 5 p. cent arabic gum). The pHopt is 9–9.5 and the apparent KM 9 mM for plasma membranes (same substrate). 5. – A very active monoglyceride-lipase has been detected in smooth and rough endoplasmic reticulum membranes (pHopt 9). Monoglyceride-lipase activity with a pHopt of 9 is also present in plasma membrane. 6. – An attempt was made to determine whether the lipase and phospholipase A1 activities, which are both present in endoplasmic reticulum and in plasma membranes, are due to the same enzyme. The response to heat treatment, the different extractibility by salts or detergents, the differential inactivation of phospholipase A by removal or alteration of lipid membrane components suggest but do not prove that the triglyceride-lipase, the monoglyceride-lipase and the phospholipase A1 are different protein entities.