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PubMed2001

One-step subcellular fractionation of rat liver tissue using a Nycodenz density gradient prepared by freezing-thawing and two-dimensional sodium dodecyl sulfate electrophoresis profiles of the main fraction of organelles.

In the present study, we describe a new procedure using freezing-thawing to density gradient solution of Nycodenz for one-step separation of organelles from the rat liver and subsequent proteome analysis of subcellular fractions. To prepare two-dimensional electrophoresis (2-D PAGE) profiles of tissue organelles, we performed one-step subcellular fractionation of rat liver homogenate using a density gradient of Nycodenz solution, which resulted in the separation of the cytosolic fraction from the postnuclear supernatant. The density gradient of Nycodenz was prepared from a 20% solution in a centrifuge tube by freezing-thawing overnight at -20 degrees C and at room temperature for a few hours without the initial centrifugation procedure. The shape of the gradient density curve was dependent on Nycodenz concentration and tube size. After fractionation, the protein profiles were examined using one-dimensional sodium dodecyl sulfate (SDS)-PAGE. The organelles were confirmed using Western blotting. Our results indicate that our procedure provides a simple method for the separation of organelle fractions from the rat liver tissue.

Animals↗
PubMed2001

The effect of cold stress on ganglioside fatty acid composition and ganglioside-bound sialic acid content of rat brain subcellular fractions.

The effect of cold stress on the ganglioside fatty acid composition and sialic acid content of brain subcellular fractions and homogenate of rats was studied, the animals were kept in a cold room with 12h light-dark cycles at 3 and 10 degrees C for 2 weeks. (1) The rat brain homogenate, synaptosomes and myelin of rats exposed to 3 degrees C contained significantly higher amounts of ganglioside-bound sialic acid per mg of protein than these fractions of control rats kept at 23 degrees C; the differences were less pronounced in rats exposed to 10 degrees C. (2) A small, but significant, diminution of relative palmitic acid content and an increase of stearic acid content was found to take place in gangliosides from rat brain synaptosomes, synaptosomal plasma membranes and homogenate as a result of the exposure of animals to 3 degrees C and to a lesser extent to 10 degrees C. (3) The content of unsaturated fatty acids in gangliosides from brain subcellular fractions was approximately the same in cold exposed and control rats.

Journal Article↗
PubMed1985

Subcellular fractionation of pig coronary artery smooth muscle.

A detailed procedure for subcellular fractionation of the smooth muscle from pig coronary arteries based on dissection of the proper tissue, homogenization, differential centrifugation and sucrose density gradient centrifugation is described. A number of marker enzymes and Ca2+ uptake in presence or absence of oxalate, ruthenium red and azide were studied. The ATP-dependent oxalate-independent azide- or ruthenium red-insensitive Ca2+ uptake, and the plasma membrane markers K+-activated ouabain-sensitive p-nitrophenylphosphatase, 5'-nucleotidase and Mg2+-ATPase showed maximum enrichment in the F2 fraction (15-28% sucrose) which was also contaminated with the endoplasmic reticulum marker NADPH: cytochrome c reductase, and to a small extent with the inner mitochondrial marker cytochrome c reductase, and also showed a small degree of oxalate stimulation of the Ca2+ uptake. F3 fraction (28-40% sucrose) was maximally enriched in the ATP- and oxalate-dependent azide-insensitive Ca2+ uptake and the endoplasmic reticulum marker NADPH: cytochrome c reductase but was heavily contaminated with the plasma membrane and the inner mitochondrial markers. The mitochondrial fraction was enriched in cytochrome c oxidase and azide- or ruthenium red-sensitive ATP-dependent Ca2+ uptake but was heavily contaminated with other membranes. Electron microscopy showed that F2 contained predominantly smooth surface vesicles and F3 contained smooth surface vesicles, rough endoplasmic reticulum and mitochondria. The ATP-dependent azide-insensitive oxalate-independent and oxalate-stimulated Ca2+ uptake comigrated with the plasma membrane and the endoplasmic reticulum markers, respectively, and were preferentially inhibited by digitonin and phosphatidylserine, respectively. This study establishes a basis for studies on receptor distribution and further Ca2+ uptake studies to understand the physiology of coronary artery vasodilation.

4-Nitrophenylphosphatase↗
PubMed1994

The activities of acyl-CoA hydrolase in lysate and subcellular fractions of human blood platelets in relation to activities of acyl-CoA:1-acyl-lysophospholipid acyltransferase.

The activities of acyl-CoA hydrolase (EC 3.1.2.2.) and acyl-CoA:1-acyl- lysophospholipid acyltransferase (EC 2.3.1.23) have been studied in subcellular fractions of human platelets. The acyl-CoA:1-acyl-lysophospholipid acyltransferase activity was higher in the 'dense-tubular-system-enriched' fraction than in the 'light-mitochondrial' fraction, using endogenously acyl-CoAs formed from labelled fatty acids, ATP, CoA and various lysophospholipids. No activity was found in the 'particle-free' fraction. No difference in specificities was observed between the incorporation of various fatty acids into different lysoPLs in the subcellular fractions compared with the platelet lysates. Generally, arachidonic, linoleic and eicosapentaenoic acids were better substrates for the acyl-CoA:1-acyl-lysophospholipid acyltransferases than oleic, docosahexaenoic and palmitic acids. The opposite was observed with the acyl-CoA hydrolase activity, palmitoyl-CoA was the substrate giving the highest activity, and eicosapentaenoyl-CoA and arachidonoyl-CoA the lowest. About 85% of the hydrolase activity was detected in the 'particle-free' fraction, with each of the six acyl-CoA derivatives tested.

1-Acylglycerophosphocholine O-Acyltransferase↗
PubMedDate not supplied

[In vivo effect of L-thyroxine on carbohydrate content of glycoproteins in subcellular fractions of the rat liver].

Effect of various doses of L-thyroxin on content of some carbohydrates (sialic acids, hexosamines, hexoses bound with proteins and L-fucose) in glycoproteins was studied in rat liver subcellular fractions. Content of these carbohydrates was altered similarly in liver tissue, whereas various doses of the hormone caused different changes of the glycoprotein components in liver tissue subcellular fractions. Within 24 days after L-thyroxin administration small doses of the hormone led to elevation of these carbohydrates in mitochondria and microsomes, while their content was unaltered in biomembranes, intermediate doses caused a decrease of the carbohydrates in mitochondria and microsomes and an increase in plasmatic mitochondrial and microsomal membranes, high doses of L-thyroxin caused a decrease of the carbohydrates in all the structures studied. Duration of normalization of the glycoprotein carbohydrate components in rat liver tissue depended on the L-thyroxin dose administered.

Animals↗
PubMed1979

Lipid composition and microviscosity of subcellular fractions from rabbit thymocytes. Differences in the microviscosity of plasma membranes from subclasses of thymocytes.

There are indications from freeze-fracture experiments that subclasses of rabbit thymocytes show different mobilities of plasma membrane components. Consequently, one would expect differences in the fluidity of the plasma membrane. For this reason, rabbit thymocytes were separated on a Ficoll/Metrizoate gradient yielding three subclasses representing various levels of cell differentiation. These thymocyte subclasses did not show any significant differences in the degree of fluorescence polarization using the probe 1,6-diphenyl-1,3,5-hexatriene. The fluorescence polarization of the plasma membrane may be overshadowed by the contribution of all cellular lipids due to penetration of the fluorescent probe into the cell. Therefore, plasma membranes were isolated from rabbit thymocytes using a cell-disrupting pump, differential centrifugation, and sucrose density gradient centrifugation. As shown by biochemical and electron microscopical analyses, plasma membranes with a high degree of purity were obtained. As expected the plasma membrane fractions showed a higher microviscosity than the other subcellular fractions. This was attributed to a higher cholesterol to phospholipid molar ratio and a higher degree of saturation of phospholipid fatty acid chains. Subsequently, the microviscosity was measured of plasma membrane preparations obtained from two main subclasses of thymocytes representing mature and immature lymphocytes. The immature thymocytes yielded two plasma membrane fractions with higher microviscosity than the mature cells. These finding is in line with earlier observed differences in the glycerol-induced clustering of intramembranous particles. Furthermore, the results of this study support the view that the fluorescence polarization technique applied to whole cells does not exclusively monitor the plasma membrane.

Animals↗
PubMed1981

Influence of prenatal hypoxia on brain development: effects on body weight, brain weight, DNA, protein, acetylcholinesterase, 3-quinuclidinyl benzilate binding, and in vivo incorporation of [14C]lysine into subcellular fractions.

The influence of prenatal hypoxia on subsequent brain development in the young rat was investigated by examining body and brain weight, cerebral cortex wet weight, protein and DNA concentrations, acetylcholinesterase (AChE) activity, 3-quinuclidinyl benzilate (QNB)-binding levels, the relative amounts of protein in various subcellular fractions, and the in vivo incorporation of [14C]lysine into the protein of homogenate and subcellular fractions. Exposure of pregnant females to a mild hypoxia (9.1% O2, 10 h per day for the 9--11 days preceding birth) resulted in a reduced body weight in the pups and days 1 and 5 after birth; total cortical DNA was reduced but brain weight and protein content were unaffected, leading to a higher protein/DNA ratio in prenatally hypoxic pups. By 10 days of age these differences between prenatally hypoxic and control animals were no longer apparent. There were no differences between prenatally hypoxic and control animals in AChE and QNB binding per milligram cortex protein. The relative amount of synaptic membrane protein from the cerebral cortex was reduced at day 1 in prenatally hypoxic animals and the synaptic membrane fraction showed a higher level of incorporation of [14C]lysine on days 1, 5, and 10. The developmental profile of [14C]lysine incorporation showed a peak on day 10 which was higher in prenatally hypoxic rats. By 46 days after birth little difference could be found between prenatally hypoxic and control animals.

Acetylcholinesterase↗
PubMed1984

The lipid composition of ovine placental tissue homogenate and its subcellular fractions.

Homogenates of the placental tissue of near term sheep were separated by differential centrifugation into mitochondrial, microsomal and cytosolic fractions. The relative proportions of the major neutral lipids and phospholipids, together with their fatty acid compositions, were determined in the homogenates and in each subcellular fraction. The cytosolic fraction contained the highest proportion of cholesteryl esters (CEs) and these possessed a fatty acid composition markedly different from the total CEs extracted from the homogenate. Both the mitochondrial and microsomal fractions contained significant proportions of solvent front phospholipid (SFP) and whereas the mitochondrial SFP displayed the relatively unsaturated fatty acid composition characteristic of diphosphatidylglycerol (cardiolipin), the fatty acids of the microsomal SFP were distinctly more saturated. These results are compared with those obtained from other mammalian tissues, both ruminant and non-ruminant, and discussed in terms of the function of the components of the subcellular fractions.

Animals↗
PubMed1976

Effects of hypolipidemic agents on lipid synthesis in subcellular fractions from Tetrahymena pyriformis.

Lipid synthesizing systems have been prepared from subcellular fractions of Tetrahymena pyriformis, GL. These fractions, the mitochondrial fraction, the microsomal fraction and the soluble cell fraction, have been characterized as to cofactors and cations required for optimal lipid synthesis. The effects of hypolipidemic agents on lipid synthesis by all fractions are presented.

Acetates↗
PubMed1976

(3H)-isoproterenol binding to subcellular fractions of mouse parotid: relationship to cyclic nucleotide formation and the stimulation of DNA synthesis.

(3H) Isoproterenol binding to subcellular fractions of mouse parotid: Relationship to cyclic nucleotide formation and the stimulation of DNA synthesis. (Unión the (3H) Isoproterenol a fracciones subcelulares de parótida de ratón y su relacón con la formacón de nucleótidos cíclicos y la estimulación de la síntesis de DNA). Arch. Biol. Med. Exper. 10: 105-114, 1976. Tritiated isoproterenol binds to all subcellular fractions of mouse parotid but 70% of the binding is to the nuclear fraction. Binding to other mouse tissues was less than to the parotid. The patterns of binding did not correlate with the distribution of adenylate cyclase, guanylate cyclase or catechol-O-methyl transferase among the fractions or tissues nor with the extent of response in stimulation of DNA synthesis among the tissues. Inhibition of (3H) Isoproterenol binding to parotid fractions by catecholamine analogs was studied. There was no correlation between their ability to inhibit binding and the ability of the analogs themselves to raise cyclic AMP levels or stimulate DNA synthesis.

Adenosine Monophosphate↗
PubMedDate not supplied

[Peptide hydrolase activity in rabbit brain subcellular fractions under conditions of adrenalectomy and administration of hydrocortisone and ACTH].

Adrenalectomy and administration of hydrocortisone and ACTH are shown to induce no changes in the total activity of neutral peptide-hydrolase in both homogenates and brain subcellular fractions. The absence of adrenalectomy and hormone administration effect on the total peptide-hydrolase activity in homogenates is established simultaneously with its essential changes in the brain subcellular fractions. A decrease in the enzymic activity of the mitochondrial-lysosomal fraction (MLF) following adrenalectomy is observed side by side with its increase in a soluble fraction. Hydrocortisone and ACTH administration cause an increase in the acid peptide-hydrolase activity in MLF and its decrease in the brain soluble and microsomal fractions of adrenalectomized rabbits. The degree of solubilization of MLF acid and neutral peptide-hydrolase by detergent triton X-100 after adrenalectomy is increased. Hydrocortisone and ACTH administration, on the contrary, decrease a degree of solubilization of the brain MLF peptide-hydrolase in adrenalectomized animals.

Adrenalectomy↗
PubMed1979

Subcellular fractionation studies of the intestinal mucosa in congenital sucrase--isomaltase deficiency.

1. Jejunal biopsy specimens from three children with congenital sucrase-isomaltase deficiency were assayed for disaccharidase activity and were subjected to analytical subcellular fractionation with enzymic microanalysis. 2. By use of the highly sensitive fluorigenic modification of the disaccharidase assay, brush-border sucrase and isomaltase activities were depressed but nevertheless detectable in each child. 3. Apart from the expected decrease in brush-border alpha-glucosidase activity, the other enterocyte marker-enzyme activities were normal. 4. There were no abnormalities in the enterocytes of any child on analytical subcellular fractionation or on electron microsocopy.

Acetylglucosaminidase↗
PubMed1984

Metabolism of cerebrosides and sulfatides in subcellular fractions of developing rat brain.

Previous studies on myelinating rat brain indicated that microsomes, Golgi-enriched and cytosol fractions may process galactolipids destined for myelin. To extend these findings we labeled brain galactolipids in vivo and determined the specific radioactivity of cerebrosides and sulfatides in several subcellular fractions. 17-day-old rats were treated by intracranial injection with [14C]galactose 60 min prior to and [3H]galactose 15 min prior to killing. Subcellular fractions were prepared from brain stem, and concentrations of cerebrosides and sulfatides were determined, their radioactivity measured and the 3H/14C ratio compared. Our results showed that the heavier Golgi-enriched fraction (designated Fraction 2) is unique in its low galactolipid content and high specific radioactivities of cerebrosides and sulfatides. The low ratio of the specific activity of cerebroside to that of sulfatide in Fraction 2 compared to other fractions indicates that it may be the site of most rapid conversion of newly synthesized cerebrosides to sulfatides. The specific radioactivities of cerebrosides and sulfatides in cytosol are intermediate between those in Golgi-enriched Fraction 2 and microsomes and those in myelin, consistent with the role postulated for cytoplasmic elements in the transport of cerebrosides and sulfatides to myelin.

Animals↗
PubMed1988

Lipoxygenase activities of human platelets and their subcellular fractions: comparison between lipoxygenase-deficient platelets and normal platelets.

Lipoxygenase activities were estimated in washed platelets (intact platelets) and their subcellular fractions obtained from 7 patients with deficient platelet lipoxygenase activities and 9 normal subjects. From sonicated platelet preparations, 12,000 g supernatant (F-I), cytosol (F-II) and microsomal fractions (F-III) were prepared by differential centrifugation. When 12-hydroxyeicosatetraenoic acid (12-HETE) produced by the incubation of arachidonic acid with intact platelets or each of their subcellular fractions from normal subjects was measured by reversed-phase high-performance liquid chromatography analysis, the lipoxygenase activities of F-I, F-II and F-III were 87%, 31% and 17%, respectively, of the enzyme activity of intact platelets. One of the patients showed no detectable lipoxygenase activity in any preparation, while the other patients showed reduced enzyme activities in all preparations. The addition of CaCl2 significantly increased 12-HETE synthesis solely by F-I from these patients. In most of these patients, contrary to normal subjects, it appeared that the lipoxygenase activity was not fully expressed in intact platelets, since the F-I produced more 12-HETE than the intact platelets.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1980

Specific binding of kainic acid to purified subcellular fractions from rat brain.

The subcellular distribution of kainic acid (KA) binding sites in rat brain has been studied using a microcentrifugation assay. KA did not bind to myelin or brain cytosol and had few or no binding sites in the nuclear fraction. However, it bound to microsomal components (Kd = 128-136 nM; 2.5-4.8 pmol/mg protein), purified synaptic plasma membranes (SPM)(Kd = 45-71 nM; 5.8-6.5 pmol/mg), and purified cell-body and intraterminal mitochondria (Kd = 11-31 nM; 0.4-1.1 pmol/ng). Bound KA could be totally displaced by L-glutamate or L-asparate, but several putative antagonists of these amino acids (nuciferin, compound HA-966, 2-amino-4-phosphonobutyrate, and 2-amino-3-phosphonoproprionate) failed to displace KA or did so at very high concentrations (greater than or equal to 4 mM). Glutamic acid diethyl ester (GDEE) and D,L-alpha-aminoadipate (alpha-AA) were more effective (IC50, 0.2-0.8 mM) and showed differential effects in their capacity to displace KA bound to the various subcellular fractions. Thus, GDEE only displaced 40-60% of the KA bound by SPM or mitochondria and did not prevent the binding of KA to microsomes. alpha-AA, on the other hand, was more effective in preventing the binding of KA at high concentrations and displaced between 80 and 100% of the drug. Both compounds showed biphasic curves of KA displacement from synaptic plasma membranes and mitochondria. The overall results indicate the presence of multiple binding sites for KA in brain cells and suggest that KA does not act exclusively as synaptic glutamate receptors. The mechanism of KA action is most likely quite complex, and the drug probably acts at multiple binding sites affecting a number of processes.

Animals↗
PubMedDate not supplied

[Effect of difenin on taurine binding by subcellular fractions of nerve cells].

The binding of 14C-taurine by subcellular fractions of rat brain cortex was studied in vitro. The binding was discovered to be more marked in the presence of phenytoin. The stimulant effect of phenytoin was found to be dose-dependent. The other anticonvulsant drug phenobarbital was not shown to have any noticeable effect on 14C-taurine binding. Analogous results were obtained during investigation of 14C-taurine incorporation into cell cultures of neuroblastoma C1300.

Animals↗
PubMed1987

Effects of triethyltin bromide on protein phosphorylation in subcellular fractions from rat and rabbit brain.

Phosphorylation of specific proteins in subcellular fractions of rat brain is affected by the presence of low concentrations (1-50 microM) of triethyltin bromide (Et3SnBr), in vitro. SDS-PAGE and autoradiography showed that Et3SnBr increased phosphorylation of an Mr = 42,000 phosphorylation of an Mr = 52,000 component. The Mr = 42,000 and 76,000-80,000 phosphoproteins have been identified as the alpha-subunit of pyruvate dehydrogenase (PDH) and synapsin, respectively. Et3SnBr-induced phosphorylation of rabbit brain PDH results in partial inactivation of the PDH complex.

Animals↗
PubMed1977

Glutathione dependent control of protein disulfide-sulfhydryl content by subcellular fractions of hepatic tissue.

The disulfide-sulfhydryl (SS/SH) ratios of subcellular fractions of rat hepatic tissue were found to vary diurnally with the ratio lowest in the early morning and highest in the early evening. These changes were found in the nuclear, microsomal and cytosol fractions. The primary reaction is the reversible formation of mixed disulfides of glutathione with proteins. This formation is controlled by the activity of thiol transferase and the level of oxidized glutathione (GSSG) as substrate. Several enzymes including mitochondrial and microsomal oxidases, glutathione reductase and peroxidase and glucose-6-phosphate dehydrogenase were found to control the levels of GSSG. An NADPH-dependent microsomal oxidase system, inhibited by GSSG, was found to produce activated oxygen which served as substrate for flutathione peroxidase. Evidence is presented for the concept that the formation of mixed disulfides of proteins with glutathione is a mechanism for maintenance of a disulfide-sulfhydryl ratio such that the integrity of particulate membranes is maintaine during oxidative and reductive stresses on the hepatic cells.

Animals↗