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PubMed2006

Food restricted schedules promote differential lipoperoxidative activity in rat hepatic subcellular fractions.

Restricted access to food (from 12:00 to 14:00 h) produces a behavioral activation known as food anticipatory activity (FAA), which is a manifestation of the food entrained oscillator (FEO). Peripheral oscillators, especially in the liver, are thought to be part of the FEO. A variety of metabolic adaptations have been detected in the liver during the expression of this oscillator, including activation of mitochondrial respiration and changes in the cytoplasmic and mitochondrial redox states. Biological clocks are regulated by redox-sensitive factors. The present study explored the lipoperoxidative activity (LP) in the liver during the activity of the FEO. Conjugated dienes (CD) and thiobarbituric acid reactive substances (TBARS), with and without Fe2+-supplementation, were quantified in six subcellular fractions: whole homogenate, plasma membrane, mitochondria, microsomes, nucleus, and cytosol. The experimental protocol involved control groups of ad libitum fed and 24-h fasted rats, and groups under the restricted food schedule (RFS) which were sampled before FAA (08:00 h), during FAA (11:00 h) and after feeding (14:00 h). Clear differences in pro-oxidant activity was observed between ad libitum fed and 24-h fasted rats in almost all the subcellular fractions studied. RFS rats presented: CD levels more similar to the fasted rats, even at 14:00 h, after food presentation, and basal and Fe2+-supplemented TBARS levels tended to be lower than both controls, suggesting an increased antioxidant capacity associated with food restriction. In addition, a microarray analysis showed that several isoforms of peroxiredoxins, a family of antioxidant and hydrogen peroxide-catabolizing enzymes, were consistently up-regulated in each and every condition in which RFS was applied. Together, these data indicate a rheostatic adaptation of the liver in the handling of pro-oxidant reactions during the activity of the FEO.

Animals↗
PubMed1981

Comparison of Ehrlich ascites tumour and mouse liver cells by analytical subcellular fractionation combined with a sensitive computational method for data analysis.

A simple method of analytical subcellular fractionation, combined with a sensitive computational method for data analysis and presentation, has been used to reinvestigate the distribution and relative amounts of several enzymes in the cytoplasmic and plasma membranes of two different cell types: one is a neoplastic, transformed cell type (Ehrlich ascites tumour cells), the other an untransformed, highly differentiated cell type (liver hepatocytes plus Kupffer and endothelial cells). In general the distribution of the enzymes in particular membranes is similar in the two cell types, however the relative amounts differ. Ehrlich ascites tumour cells have a higher specific activity of galactosyltransferase and ouabain-sensitive (Na,K)ATPase, while liver cells have higher glucose-6-phosphatase, 5'-nucleotidase and succinate dehydrogenase activity. These differences appear to be correlated with morphological and, in some cases, functional differences between the two cell types.

5'-Nucleotidase↗
PubMed1976

Distribution of phospholipids, fatty acids, and platelet factor 3 activity among subcellular fractions of human platelets.

As compared with other methods, our recently reported method for subcellular fractionation of human platelets improves the separation of mitochondria, alpha granules, and lysosomal enzyme activities. The relative purity of these fractions has led us to undertake the present study to compare the subcellular distribution of phospholipids, fatty acids, and platelet factor 3 (clot-promoting) activity. Two findings pertaining to distribution of phospholipids were entirely new. (1) In the alpha granule zone, plasmalogen phosphatidyl ethanolamine peaked at the expense of diacyl phosphatidyl ethanolamine. (2) The fatty acid composition of the membrane lysophosphatidyl choline suggested that it may have been formed by the action of platelet phospholipase A2 activity. The fatty acids of the membranes showed a markedly asymmetrical distribution in noncholine versus choline phospholipids. The latter held 94%, 72%, and 85%, respectively, of the total content of 16:0, 18:1, and 18:2 fatty acids, whereas 55% of the 18:0, 72% of 20:4, and 67% of higher polyenoic acids other than 20:4 were esterified to the noncholine group. The most important new information related to clot-promoting activity, which, on the basis of protein content, was highest in the membrane fractions, but on the basis of phospholipid content in the nonmembranous fractions. The discussion centers on possible explanations for this novel finding.

Blood Coagulation Factors↗
PubMed1998

Glutathione conjugation of perchloroethene in subcellular fractions from rodent and human liver and kidney.

Perchloroethene (Per) is a widely used industrial solvent and common environmental contaminant. In rats, long-term inhalation of Per is known to cause a small increase in the incidence of renal tubule cell tumors in males only; renal toxicity is seen in female rats and in both sexes of mice after prolonged Per exposure. The renal toxicity of Per is likely mediated by a glutathione-dependent bioactivation reaction. Glutathione S-transferase mediated formation of S-(1,2,2-trichlorovinyl)glutathione is the first step in a sequence of reactions finally resulting in the formation of reactive intermediates in the kidney. In this study, we compared the enzymatic rates of formation of S-(1,2,2-trichlorovinyl)glutathione in liver and kidney subcellular fractions from rats, mice, and from both sexes of humans (n = 11). In microsomal fractions from the liver and kidney of all three species, enzymatic formation of S-(1,2,2-trichlorovinyl)glutathione from Per could not be observed. S-(1,2,2-Trichlorovinyl)glutathione formation (the structure was confirmed by electrospray mass spectrometry) was observed in liver cytosol from both male and female rats and mice. However, the rates of S-(1,2,2-trichlorovinyl)glutathione formation in liver cytosol from male rats (84.5+/-12 pmol/mg per min) were approximately four times higher than from female rats (19.5+/-8 pmol/mg per min) and from both sexes of mice (27.9+/-6 and 26.0+/-4 pmol/mg per min). Low rates of S-(1,2,2-trichlorovinyl)glutathione formation were also seen in kidney cytosol from mice (12+/-6 pmol/mg per min), but not from rats. In human liver subcellular fractions, enzymatic formation of S-(1,2,2-trichlorovinyl)glutathione could not be detected. The human liver cytosolic fractions, however, exhibited glutathione S-transferase activity (as determined using 1-chloro-2,4-dinitrobenzene and hexachlorobutadiene as marker substrates) in the same order of magnitude as rat and mouse liver cytosol. In contrast to other marker activities for glutathione S-transferases, the ability of all human liver cytosol samples to catalyze the glutathione conjugation of 1,2-dichloro-4-nitrobenzene was three orders of magnitude lower compared to rat and mouse liver cytosol. 1,2-Dichloro-4-nitrobenzene conjugation was also four times higher in liver cytosol from male rats compared to female rats. The results suggest that the ability of the human liver to catalyze the formation of S-(1,2,2-trichlorovinyl)glutathione from Per is at least two orders of magnitude lower than that of rat liver, and that sex-specific differences in the extent of hepatic conjugation of Per with glutathione, which may be relevant for nephrotoxicity, occur in rats.

Animals↗
PubMed1993

Time dependent distribution of [203Hg] mercuric nitrate in the subcellular fractions of rat and fish liver.

Cytotoxicity of inorganic mercury to the liver of two species, Anabas testudineus and Sprague Dawley male rat was evaluated. Distribution kinetics of this metal in the different hepatic subcellular fractions were followed for 48 h after a single injection of [203Hg] mercuric nitrate at a dose of 4mg/kg b.w. Interaction of this metal with protein, DNA and RNA was also studied. In rat, nuclear and lysosomal fractions showed a significant increase in the protein content, while in fish, the amount of protein increased in all fractions except microsome. Comparatively more mercury was bound to protein in fish during the later phase of treatment. Retention of mercury in nuclear DNA of rat gradually declined from 15 min to 48 h of treatment, while, mitochondrial DNA binding to mercury increased from 15 min to 2 h of post injection and then declined in the later phase of the experiment. Such a biphasic binding pattern of mercury was shown by both the nuclear and mitochondrial DNA of the fish. The nuclear RNA of rat and mitochondrial and cytosolic RNA of both test species also showed a biphasic pattern of mercury binding, however, with a higher rate of binding in fish at the later phase of the experiment. The present study thus highlights that (a) mercury follows a definite distribution pattern in the subcellular fractions of the liver in both animal species, (b) cytosol is the major site of mercury accumulation.

Animals↗
PubMed1977

Adenylate cyclase activity in lymphocyte subcellular fractions. Characterization of non-nuclear adenylate cyclase.

Human peripheral lymphocytes were broken in a Dounce homogenizer and subcellular fractions enriched in plasma membranes or microsomal particles and mitochondria were isolated by centrifugation through a discontinuous sucrose gradient. Various agents that promote cyclic AMP accumulation in intact lymphocytes were compared in their ability to stimulate adenylate cyclase activity in the individual fractions. Plasma-membrane-rich fractions that were essentially free of other subcellular particles as judged by electron microscopy and marker enzyme measurements responded to fluoride, but weakly or not at all to prostaglandin E1 and other prostaglandins. Microsomal and mitochondrial-rich fractions responded markedly to both prostaglandin E1 and fluoride. In some, but not all, experiments phytohaemagglutinin produced a modest increase in enzyme activity in plasma-membrane-rich fractions. Catecholamines, histamine, parathyrin, glucagon and corticotropin produced little or no response. In the absence of theophylline, adenosine (1-10 micronM) stimulated basal enzyme activity, although at higher concentrations the responses to prostaglandin E1 and fluoride were inhibited. GTP (1-100 micronM) and GMP(5-1000 micronM) respectively inhibited or stimulated the response to fluoride, whereas the converse was true with prostaglandin E1.

Adenylate Kinase↗
PubMed1984

The chemical form of cadmium in subcellular fractions following cadmium exposure.

Wistar rats were given drinking water containing 250 ppm Cd for 12 months. After excision of the kidney and liver, the organs were subfractionated into nuclear, mitochondrial, microsomal and cytosol fractions, and the chemical forms of Cd in the subcellular fractions were examined. Although approximately 90% of the total Cd was present in the cytosol, in the form of metallothionein, 3-5% was also present in the mitochondrial fraction and 5-7% in the microsomal fraction from both organs. By Sephadex G-75 gel filtration, after solubilizing the particulate fractions with sodium deoxycholate, approximately 89% of Cd in the microsomal fraction and 94% in the mitochondrial fraction eluted with the same retention time as that of metallothionein in both liver and kidney, while the remainder was found in a high molecular weight protein fraction. The Cd that eluted with the high molecular protein fraction may be involved in dysfunction in subcellular organelles. For estimating the toxicity of Cd associated with the high molecular weight fraction, rat liver microsomes and CdCl2 were mixed with 1% of sodium deoxycholate and the protein-Cd complex produced was isolated by eluting with Sephadex G-75. This complex had a strong toxicity toward the alcohol dehydrogenase activity (SH-enzyme), and the Ki values of the Cd-protein complexes decreased with increased amount of Cd bound to the microsomal protein fraction. The above results suggest that loosely bound Cd increased in the case of higher Cd/protein and plays a toxic role in the living cells.

Alcohol Oxidoreductases↗
PubMed2006

Glycoproteomics of Trypanosoma cruzi trypomastigotes using subcellular fractionation, lectin affinity, and stable isotope labeling.

Herein we detail the first glycoproteomic analysis of a human pathogen. We describe an approach that enables the identification of organelle and cell surface N-linked glycoproteins from Trypanosoma cruzi, the causative agent of Chagas' disease. This approach is based on a subcellular fractionation protocol to produce fractions enriched in either organelle or plasma membrane/cytoplasmic proteins. Through lectin affinity capture of the glycopeptides from each subcellular fraction and stable isotope labeling of the glycan attachment sites with H(2)18O, we unambiguously identified 36 glycosylation sites on 35 glycopeptides which mapped to 29 glycoproteins. We also present the first expression evidence for 11 T. cruzi specific glycoproteins and provide experimental data indicating that the mucin associated surface protein family (MASP) and dispersed gene family (DGF-1) are post-translationally modified by N-linked glycans.

Amino Acid Sequence↗
PubMed1985

Subcellular fractionation of rat sciatic nerve and specific localization of ganglioside LM1 in rat nerve myelin.

Subcellular fractionation of rat sciatic nerve was developed to determine the specific localization of gangliosides in the nerve membrane fractions. Myelin, microsomal, and a plasma membrane-like fraction were isolated and purified by sucrose density gradient centrifugation. These subfractions were characterized by electron microscopy, marker enzyme assays, and their protein and lipid profile. In rat sciatic nerve myelin, 90 mol% of the total gangliosides were monosialogangliosides. LM1 (sialosyl-lactoneotetraosylceramide) (61 mol%) and GM3 (21%) were the major gangliosides of the rat nerve myelin. Two other neolacto series of gangliosides, viz., sialosyl-lactoneonorhexaosylceramide and sialosyl-lactoneooctaosylceramide, were also localized mostly in the myelin fraction. GM1 was only a minor (less than 2%) ganglioside in myelin. The ganglioside patterns of the microsomal and plasma membrane-like fractions were similar with minor quantitative differences and were entirely different from that of myelin. Monosialogangliosides were approximately 70-75 mol% of the total in these fractions. The major gangliosides of the microsomal and plasma membrane-like fractions were GM3 (approximately 40%) and GM1 (approximately 20%). LM1 in these fractions was minimal (less than approximately 5%). Significant amounts of GM3 with N-glycolylneuraminic acid (approximately 10%) and GM1b (4-14%) were also identified in the microsomal and plasma membrane-like fractions but not in myelin. These and the higher lactoneo series of gangliosides have not been previously reported to be present in the rat nervous system. Almost exclusive localization of LM1 in myelin in rat peripheral nervous system is consistent with our previous observation that deposition of LM1 in the nerve with age was very similar to that of myelin marker lipids cerebrosides and sulfatides.

Animals↗
PubMed1993

Ganglioside composition of subcellular fractions, including pre- and postsynaptic membranes, from Torpedo electric organ.

Gangliosides were isolated from four subcellular fractions of the electric organ of Torpedo marmorata: synaptosomes, presynaptic membranes, postsynaptic membranes, and synaptic vesicle membranes. This exploited a principal advantage offered by this tissue: facile separation of pre-and postsynaptic elements. Total ganglioside concentration in presynaptic membranes was approximately twice that of synaptosomes and 15 times that of postsynaptic membranes (47.7, 24.4, and 3.21 micrograms of lipid sialic acid per mg protein, respectively). Synaptic vesicle membranes had the highest overall concentration (78.9) relative to protein, but a concentration approximately comparable to that of presynaptic membranes when expressed relative to phospholipid. The thin-layer patterns of these two fractions were similar, both in terms of total pattern and the specific pattern of gangliotetraose structures as revealed by overlay with cholera toxin B subunit; these were notable for the paucity of monosialo structures and the virtual absence of GM1. Postsynaptic membranes, on the other hand, had a significantly higher content of monosialogangliosides including the presence of GM1. The synaptosomal pattern resembled that of the presynaptic membranes and synaptic vesicles. Thus, a clear difference in ganglioside pattern could be discerned between the pre- and postsynaptic elements of the electric organ.

Animals↗
PubMed1982

Mechanism of luteinizing hormone-releasing hormone degradation by subcellular fractions of rat hypothalamus and pituitary.

The pathway of LH-RH degradation by two subcellular fractions (a soluble fraction and a 25 000 X g particulate fraction) of rat hypothalamus, pituitary and cerebral cortex has been studied using high performance liquid chromatography and amino acid analysis to identify the breakdown products. The primary cleavage point in the Tyr5-Gly6 bond giving [1-5] LH-RH and [6-10] LH-RH. In the presence of dithiothreitol, cleavage of LH-RH also occurred at the Pro9-Gly10 bond giving [1-9] LH-RH. The fragment [1-5] LH-RH is further degraded sequentially from the C-terminus and [1-4] LH-RH, [1-3] LH-RH, tyrosine and tryptophan were identified. The other major fragment, [6-10] LH-RH, is rapidly broken down, the only intermediate product positively identified being Arg-Pro.

Animals↗
PubMed1981

Investigation of tissue organelles by a combination of analytical subcellular fractionation and enzymic microanalysis: a new approach to pathology.

Classical approaches to the study of pathological tissue have relied mainly on morphological techniques. In an attempt to quantitate the abnormalities and to investigate the pathogenesis of tissue disorders at a subcellular level we have combined analytical subcellular fractionation by sucrose density gradient centrifugation with microanalysis of tissue enzymic activities. The methodological problems of performing these studies on milligram quantities of tissue are discussed. Details of the appropriate equipment are provided, and its application to the study of human liver specimens is described. As an example of this approach, biochemical and subcellular fractionation experiments on tissue from patients with both primary and secondary hepatic lysosomal storage diseases are discussed. Examination of the lysosomal changes reveals that increased enzyme activity is a common finding in these disorders but tissue damage occurs only when there is evidence of enhances lysosomal fragility with intracellular release of degradative enzymes. Other tissues which have proved amenable to study in this manner and in which profitable results in the investigation of their disorders have been obtained are listed.

Cell Fractionation↗
PubMed1985

Subcellular fractionation of Hymenolepis diminuta with special reference to the localization of marker enzymes.

A method for subcellular fractionation of Hymenolepis diminuta using whole worm homogenization and differential centrifugation is presented. Different fractions obtained in this study were screened for the presence of enzymes that serve as markers for plasma membrane, brush border, mitochondria, Golgi complex, endoplasmic reticulum, peroxisomes, lysosomes and cytosol. The purity of fractions was also monitored by transmission electron microscopy. The purity of fractions, particularly the brush border membranes, are compared to those obtained by previous methods for H. diminuta or other tissues.

Animals↗
PubMed1991

Proteolytic fragmentation of tetanus toxin by subcellular fractions of JY, a B lymphoblastoid cell line.

Proteolysis of 125I-labelled tetanus toxin by subcellular fractions from an Epstein-Barr-virus-transformed B lymphoblastoid cell line, JY, was investigated. Fractions enriched in lysosomes and plasma membranes cleaved the toxin molecule at several sites, with a pH optimum of 5.5. N-Terminal sequence analysis of Mr-81,000, -45,000 and -35,000 proteolytic fragments indicated cleavage of the Asp-460-Leu-461, Asp-872-Glu-873 and Ile-1013-Thr-1014 peptide bonds, all sites located within the heavy chain of the toxin molecule. Additional sites near the C-terminus of the heavy chain, giving rise to low-Mr peptides, were implicated. The toxin light chain was more resistant to proteolysis. A similar pattern of fragmentation was observed with tetanus toxin biosynthetically radiolabelled with 14C-labelled amino acids, showing that the proteolysis was not an artifact caused by iodination. The proteolytic activity was inhibited by the serine proteinase inhibitor di-isopropyl phosphorofluoridate, thiol-blocking proteinase inhibitors N-ethylmaleimide and iodoacetamide, and by EDTA. These results represent a preliminary characterization of the processing in vitro of tetanus toxin by an antigen-presenting cell line.

B-Lymphocytes↗
PubMed1982

Effect of undernutrition on DNA and RNA synthesis in subcellular fractions from different regions of the developing rat brain.

The effect of undernutrition on the incorporation of [methyl-3H] thymidine into DNA and of 5-[3H] uridine into RNA of cerebral hemispheres, cerebellum, and brain stem was studied in vivo and in vitro in rats. The labeling of DNA from nuclei and mitochondria and of RNA from nuclei, mitochondria, microsomes, and soluble fractions, was also measured in vitro. The results demonstrate that nucleic acid synthesis is impaired and delayed during undernutrition. Specific effects were observed for the different brain regions and subcellular fractions: at 10 days nuclear and mitochondrial DNA and RNA synthesis was impaired, whereas at 30 days only the mitochondrial nucleic acid synthesis was affected. The delay of DNA and RNA labeling, caused by undernutrition, was most evident in the cerebellum, probably due to its intense cell proliferation during postnatal development. The specific sensitivity of mitochondria as compared to other subcellular fractions, may by due to the intense biogenesis and/or turnover of nucleic acids in brain mitochondria not only during postnatal development, but also in the adult animal.

Animals↗
PubMed1992

Comparative metabolism of methyl parathion in intact and subcellular fractions of isolated rat hepatocytes.

Metabolism of the widely used insecticide methyl parathion by isolated hepatocytes and various subcellular fractions was compared to determine the effects of cellular integrity on the metabolic profile observed. A reverse-phase ion-pair high-performance liquid chromatographic method was developed to separate and quantify methyl parathion and six of its hepatic biotransformation products: methyl paraoxon; desmethyl parathion; desmethyl paraoxon; p-nitrophenol; p-nitrophenyl glucuronide; and p-nitrophenyl sulfate. Most compounds exhibited linear responses and limits of detection below 1 nmol. The chromatographic method was used to determine metabolic profiles of methyl parathion in isolated rat hepatocytes, sonicated hepatocytes, postmitochondrial fraction, microsomes, and cytosol. Isolated hepatocytes produced significantly more desmethyl parathion and p-nitrophenyl sulfate than the subcellular preparations, demonstrating that cellular integrity significantly affects the quantitative metabolic profile observed.

Animals↗
PubMed1986

Subcellular fractionation of the human corpus luteum: distribution of GnRH agonist binding sites.

We have investigated the subcellular localization of GnRH agonist (GnRHA) binding sites in the human corpus luteum. Corpora lutea (CL) were obtained from 13 women undergoing laparotomy during the luteal phase of the menstrual cycle. CL homogenates were subjected to subcellular fractionation by either differential rate centrifugation or by sucrose density gradient fractionation with or without digitonin perturbation. Fractions were then assayed for a number of marker activities characteristic of the major intracellular organelles and cell-surface membranes, and for specific binding of [125I]GnRHA. Specific binding of [125I]GnRH agonist was greatest in regions of the gradient (density, 1.12-1.15 g/cm3) enriched in cell-surface and endoplasmic reticulum membrane markers. Little or no GnRHA binding was associated with fractions enriched in nuclei, mitochondria or lysosomes. Digitonin treatment demonstrated that the majority of GnRHA binding sites were associated with luteal cell plasma-membranes: however, some GnRHA binding was also associated with endoplasmic reticulum. These results indicate that: (i) human luteal GnRHA binding sites were not associated with lysosomal proteases, (ii) a significant fraction (59%) of GnRHA binding sites was localised on the luteal cell-surface membrane. Specific GnRHA binding to both cell-surface and endoplasmic reticulum membranes may indicate an extensive turnover of membrane binding sites.

Binding Sites↗
PubMed1978

[Catalase biogenesis in various subcellular fractions of regenerating rat liver cells].

Dynamics of the accumulation and decay of catalase in different subcellular fractions of intact and regenerating rat liver is investigated. Biological activity of cytoplasmic factors regulating the rate of enzyme synthesis at post-transcriptional stages under partial hepatectomy is also studied. Different rates of the formation and decay of different catalase isoforms in intact and regenerating rat liver are observed.

Animals↗