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PubMed1982

Induction of pole cells in sterilized Drosophila embryos by injection of subcellular fraction from eggs.

A subcellular fraction isolated from a homogenate of young Drosophila embryos was shown to be capable of inducing pole cells when injected into UV-sterilized Drosophila embryos. Most of the pole cell-inducing activity was recovered from the precipitate after centrifugation at 27,000 x g. The activity remained in this precipitate (called F-3 fraction hereafter) even after membranous structures were removed from it through centrifugation on a sucrose density gradient. Dialysis, lyophilization, and heating at 80 degrees C for 10 min did not inactivate the F-3 fraction. The pole cells, which were produced when the F-3 fraction was injected at the posterior pole of UV-sterilized embryos, did not develop into germ cells. Furthermore, the F-3 fraction was unable to induce pole cells when injected into the anterior region of the egg. These results can be explained by assuming that (i) pole cell formation and germ cell determination are controlled by different factors, (ii) pole cell formation requires at least two factors, which are normally localized in the posterior-pole cytoplasm, one of which is sensitive and one resistant to the UV dosage we used, and (iii) the subcellular fraction we obtained contains the UV-sensitive factor but not the UV-resistant factor.

Journal Article↗
PubMed1984

Breakdown of proteins from mouse liver subcellular fractions. Effect of nutritional changes.

The rapid restoration of liver protein mass observed in protein-depleted mice when they are fed with an adequate diet is quantitatively explained by a large decrease in the average rate of breakdown of total liver proteins. This study was performed in order to know whether this inhibition of breakdown affects in the same way all the protein constituents of the tissue, or only affects a group of these proteins belonging to a particular subcellular fraction. Subcellular fractions were obtained by differential centrifugation. The relative rates of breakdown of their proteins were estimated by the conservation of radioactivity in these proteins previously labelled by the administration of NaH14CO3 to mice. The results obtained indicated: 1) a general decrease in the rate of breakdown of proteins of subcellular fractions from re-fed livers compared with livers of protein-depleted mice; 2) a decrease of breakdown of proteins from cytosol in re-fed mice which is higher as lower is the molecular weight of the proteins subunits.

Animals↗
PubMed1991

Structure-metabolism relationships in the hydrolysis of nicotinate esters by rat liver and brain subcellular fractions.

Rat liver and brain subcellular esterase activities toward nicotinic acid esters were studied, under varying conditions, such as pH, organic solvents, protein concentration, duration of incubation, and substrate concentration. Esterases in each subcellular fraction displayed activities that obey Michaelis-Menten kinetics, although subcellular fractions are heterogeneous. The Km values were of the same magnitude, and the Vmax values were lower in microsomes than in cytosol of the liver. Brain activities normalized to protein concentration, were much lower than liver activities, aromatic nicotinates being the best substrates in both tissues. Myelin and brain mitochondria of nerve-ending and neuroglial origin display esterase activity toward phenyl nicotinate. In contrast to brain esterases, liver esterases appear homogeneous, and esterase activities in both tissues react differently to changes in pH. Qualitative and quantitative structure-metabolism relationships are not suggestive of tissue-specific ester hydrolysis.

Animals↗
PubMed1999

Subcellular fractionation of human neutrophils on Percoll density gradients.

Subcellular fractionation has been an important tool in the investigation of neutrophil structural organization including granule heterogeneity, composition and mobilization. The resolution of organelles obtained by subcellular fractionation was improved considerably after the introduction of nitrogen cavitation as an efficient but gentle means of disrupting neutrophils and with Percoll as a density medium. This paper describes in detail the methodology of subcellular fractionation of nitrogen cavitated neutrophils on one-, two-, and three-layer Percoll density gradients. Appropriate marker proteins are presented for neutrophil organelles including azurophil, specific and gelatinase granules, in addition to secretory vesicles and plasma membranes. The dynamics of granule and secretory vesicle exocytosis is demonstrated by subcellular fractionation of resting and activated human neutrophils. Finally, the paper describes the applications of subcellular fractionation in the investigation of the localization of neutrophil constituents, in protein purification schemes and in the study of translocation of cytosolic proteins to isolated neutrophil organelles.

Biological Transport↗
PubMed1986

Distribution of alkylglycerone-phosphate synthase in subcellular fractions of rat liver.

Subcellular fractions of rat liver were isolated by density-gradient centrifugation on a linear Metrizamide gradient and were assayed for marker enzymes of peroxisomes, lysosomes, microsomes and mitochondria. Alkylglycerone-phosphate synthase catalysing the formation of the ether bond in glycerolipids was also determined along the gradient. The enzyme was found to be enriched in the peroxisomal and the microsomal fractions thus, displaying a bimodal distribution pattern. Two reaction-products each, alkylglycerone phosphate and alkylglycerone were obtained in the enzymic assays performed, the ratio of which was clearly dependent upon the fraction employed. Alkylglycerone phosphate was mainly synthesized by the 'peroxisomal synthase', whereas an inverse proportion was observed assaying the microsomal counterpart. Furthermore, comparing the mean specific activities of both the enzymes the microsomal one was shown to be roughly twice as active in metabolizing 1-O-palmitoylglycerone 3-phosphate, simultaneously displaying a somewhat different sensitivity to NaF. These findings provide a first line of evidence, that two separate synthases, one in microsomes and another one in peroxisomes might be engaged in the biosynthesis of 1-O-alkyl-glycerolipids in rat liver.

Alkyl and Aryl Transferases↗
PubMed1994

Specific binding sites for progesterone in subcellular fractions of the porcine corpus luteum.

Subcellular fractionation of porcine corpus luteum (CL) homogenates on continuous sucrose gradients has previously demonstrated that most of the endogenous progesterone of the CL was associated with a unique particulate fraction. Exogenous radiolabelled steroids were also sequestered with some specificity by this fraction. We now report that this particulate fraction is capable of binding high levels of exogenous 3H-labelled progesterone (and pregnenolone) in vitro, but only in the presence of the saponin, digitonin. Binding was dependent on the pH, temperature and duration of incubation, and showed specificity and high affinity for progesterone (Kd, 79 nM). Androgens, oestrogens and pregnenolone competed for porcine luteal [3H]progesterone binding sites, but only at much higher concentrations, whereas cholesterol, a number of progesterone receptor agonist and antagonist analogues and inhibitors of 3 beta-hydroxysteroid dehydrogenase and C17-hydroxylase/C17,20-lyase did not compete. Analysis of profiles for a number of luteal cell-surface membrane and intracellular organelle markers confirmed previous studies showing the association of an NADH-cytochrome C reductase with this fraction. Moreover, the content of endogenous progesterone associated with particulate subcellular fractions isolated from porcine granulosa cell (GC) and CL homogenates at different stages of the luteal phase and early pregnancy waxed and waned with the stage of the luteal phase (and the secretory activity of the CL). Binding of [3H]progesterone in vitro equilibrated at the same buoyant density as endogenous progesterone: levels of both were highest during the mid-luteal phase and during early pregnancy, lower in early and late luteal CL, and undetectable in corpora albicantia. In contrast, relaxin secretory granules were readily resolved from progesterone binding sites. We propose that these particulate progesterone binding sites may be involved in the sequestration and/or packaging of newly-synthesized steroid for secretion by the luteal cell, or may mediate actions of progesterone within the luteal cell.

Animals↗
PubMed1999

Subcellular fractionation, electromigration analysis and mapping of organelles.

Subcellular fractionation has provided the means required to analyze the composition and properties of purified cellular elements. In particular, subcellular fractionation has helped to define membrane boundaries and became necessary for the development of cell-free assays that reconstitute complicated cellular processes. Although cell fractionation techniques have improved over the last decades the purification of organelles to homogeneity is still a barely accessible goal in cell biology. In this article, we will first briefly review the basic principles of subcellular fractionation, and the establishment of different organelle fractions by density centrifugation, using tissue culture cells as a paradigm. Then we will discuss some of the intrinsic problems and will compare gradient purification of cellular extracts with electromigration analysis. Finally, we will describe alternative approaches, such as immunoisolation and flow cytometry to purify organelles from tissue culture cells.

Cell Fractionation↗
PubMedDate not supplied

Extraction of corticosterone from cell homogenates and subcellular fractions of the rat adrenal cortex. III. ACTH-induced temporal subcellular redistributions of steroid precursors to corticosterone.

Cholesterol, pregnenolone, progesterone, 11-deoxycorticosterone (11-DOC) and corticosterone were quantitated in subcellular fractions isolated from in vivo adrenocorticotropin (ACTH)-stimulated rat adrenal zona fasciculata/reticularis. Six adrenal subcellular fractions separated by discontinuous sucrose gradient centrifugation (lipid, 0.125 M sucrose, cytosolic, microsomal, mitochondrial and nuclear) were extracted with alkaline ether/ethanol and assayed by high pressure liquid chromatography (HPLC). Lipid fractions contained the major cholesterol stores, while most pregnenolone and progesterone was found in lipid, microsomal and mitochondrial fractions. The 0.125 M sucrose and cytosol fractions together contained approximately 75% of the total 11-DOC and corticosterone. The five steroids were only present in small amounts in organelle fractions containing steroidogenic enzymes. Homogenate and lipid fraction cholesterol decreased between 10 and 15 min and again 30 min after ACTH injection. In the homogenate, lipid, microsomal and mitochondrial fractions, pregnenolone and progesterone were increased after ACTH injection; peak pregnenolone and progesterone concentrations were often measured in adrenal gland sucrose, cytosolic, microsomal and mitochondrial fractions 15 to 20 min after rats were injected with ACTH. Although ACTH increased 11-DOC and corticosterone in all but the mitochondrial and nuclear fractions, the sucrose, cytosolic and microsomal 11-DOC, and cytosolic corticosterone increased most dramatically. In many fractions, peak 11-DOC and corticosterone concentrations were most often observed between the 10 and 15 min periods and again at 30 min.

Adrenal Cortex↗
PubMed1987

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 microM), 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 micrograms/mg protein and inhibition at higher concentrations. Addition of cholesterol (50-250 micrograms/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.

Animals↗
PubMed1989

Subcellular fractionation of tissue culture cells.

Subcellular fractionation has two major steps, (1) the homogenization of the cells and (2) the subsequent separation of the organelles. The homogenization step is discussed with reference to the problems encountered using tissue culture cells. Promising techniques for the isolation of specific compartments are illustrated using the isolation of the endosomal compartment as the example.

Animals↗
PubMed1975

Effects of freezing and storage on subcellular fractionation of guinea pig and human brain.

Subcellular fractionations were carried out on guinea pig and human brains. Distributions of protein marker enzymes, and galactolipids were examined with guinea pig cerebral cortex that was (Group I) homogenized immediately; (Group II) stored 3 to 5 days at -70 degrees C prior to homogenization; (Group III) stored 3 to 6 months; (Group IV) homogenized after 3 h at R.T. and 16 to 18 h at 4 degrees C and then stored at -70 degrees C for 7 to 9 months. Human frontal lobe obtained at autopsy was fractionated immediately (Group V) or stored at -70 degrees C for 5 to 8 months prior to fractionation (Group VI). Protein recoveries in myelin, microsomal, synaptosomal, and supernatant fractions were decreased in brains that were not frozen for several hours prior to storage (Groups IV-VI). SDH and MAO recoveries in the nuclear and free mitochondrial fractions were increased in these groups. AChE, a membrane marker, was also increased in the free mitochondrial fractions in Groups IV-VI, suggesting increased contamination of mitochondria by synaptosomal membrane fragments. Arylsulfatase, a lysosomal enzyme, was decreased in the free mitochondrial fraction with freezing, but the distributions in tissues not frozen for several hours showed only an increase in the nuclear fraction and a decrease in the microsomal fraction. Freezing brought about an increase in supernatant LDH and a decrease in this enzyme in the free mitochondrial fractions. Total galactolipid contents in synaptosomal and free mitochondrial fractions were increased by freezing and storage. Though some redistribution of enzymes takes place, meaningful subcellular fractions can be obtained after storage of fresh and postmortem brain tissues.

Acetylcholinesterase↗
PubMed1999

Dose response of ethanol ingestion on antioxidant defense system in rat brain subcellular fractions.

This study investigated the response of the antioxidant defense system in brain subcellular fractions after oral graded doses of ethanol to rat. Four groups of male Fischer-344 rats were orally administered saline, ethanol 2 g, 4 g, and 6 g/kg, respectively, and sacrificed 1 hour post treatment. Brain cytosol, synaptosomes, microsomes and mitochondria were separated by density gradient differential centrifugation and assayed for antioxidant system. A significant and dose-dependent-decrease in superoxide dismutase (SOD) activity was observed in all brain subcellular fractions. Catalase (CAT) activity was significantly decreased in brain mitochondria (67% and 80% of control) at higher doses of ethanol; whereas, CAT activity was significantly increased in cytosol, synaptosomes and microsomes. Glutathione peroxidase (GSH-Px) activity was significantly increased in all brain subcellular fractions except in cytosol at higher dose of ethanol. Malondialdehyde (MDA) content was significantly increased in all brain subcellular fractions showing dose response of ethanol-induced oxidative stress. The increase in MDA levels in the brain synaptosomes and microsomes were higher at 6 g dose of ethanol (155% and 163% of control) when compared to mitochondria and cytosol. Glutathione (GSH) levels were significantly increased in brain cytosol and microsomes at higher dose of ethanol (164% and 159% of control); whereas, the GSH concentration was significantly decreased in brain synaptosomes and mitochondria. The antioxidant enzyme (AOE) activity ratios (GSH-Px/SOD and GSH-Px + CAT/SOD) were dose dependently increased in all brain subcellular fractions, particularly in synaptosomes. The GSH/GSSG ratio was dose dependently increased in brain microsomes. The perturbations in the antioxidant defense system and enhanced lipid peroxidation following graded doses of ethanol ingestion indicate a dose-dependent-oxidative 2133stress response in brain subcellular compartments of rats.

Animals↗
PubMed1984

Extraction of corticosterone from cell homogenates and subcellular fractions of the rat adrenal cortex. II. ACTH-induced changes in subcellular corticosterone.

Zona fasciculata-reticularis subcellular structures were implicated in corticosterone transport and secretion by noting changes in subcellular corticosterone during a 30-min period following ACTH stimulation. Six decapsulated adrenal homogenate subcellular fractions separated by gradient centrifugation were characterized cytochemically and morphologically. Predominant components in each of six fractions were: floating lipid droplets, 0.125 M sucrose (no organelles), cytosol (0.25 M sucrose supernatant with 0.25-1.2 micron electron dense granules), microsomes (interface between 0.5 M and 1.1 M sucrose layers), mitochondria (boundary between 1.1 M and 2.2 M sucrose layers) and nuclei (centrifuge pellet). Whole glands and most subcellular fractions showed peak corticosterone levels 10 to 15, and 30 min after stimulation. Sucrose and cytosolic fractions contained about 75% of the total corticosterone, responded to stimulation most significantly, and were rich in protein. In these two fractions only cytosol contained structures; these consisted of 0.15-1.2 micron electron dense granules.

Adrenal Cortex↗
PubMed1992

Prostaglandin profile and synthetic capacity of the colon: comparison of tissue sources and subcellular fractions.

Although there has been intense interest in the physiology and pathophysiology of prostaglandins (PGs) synthesized in the colon, little is known about the PG profile and synthetic capacity of different tissue sources and subcellular fractions as enzyme sources. Subcellular fractions prepared from the mucosa and muscle layer of rat colon were incubated with or without exogenous arachidonic acid ([3H]20:4n-6) for 30 min. In experiments with exogenous [3H]20:4n-6, the prostaglandin synthetic capacity of the colonic muscle layer was significantly higher than that of the mucosa. Among the subcellular fractions, microsomes had the highest PG synthetic capacity in both mucosa and muscle. The major PG product was PGI2 and PGD2 in the mucosal microsomes and PGI2 and PGE2 in the muscularis microsomes. However, production of PGI2 in the mucosa and PGE2 in the muscle was significantly reduced in the fractions containing both cytosol and microsome, resulting in an alteration of the PG profile. Substrate availability (exogenous vs endogenous supply) appears to influence the PG profile of the colon. In the colonic mucosa with exogenous [3H]20:4n-6, the production of PGI2 was 5 times higher than that of PGE2, whereas the production of PGE2 was twice higher than that of PGI2 in experiments with endogenous 20:4n-6. These observations indicate: 1) different PG profile and synthetic capacity of tissue sources and subcellular fractions; 2) alteration of PG profile due to the variation of 20:4n-6 availability. Thus, the outcome of experiments on the physiological role of PG in the colon may be determined, in part, by the tissue source and subcellular fraction selected for analysis. The present study also suggests that the variation of substrate availability in physiological and pathophysiological processes may affect the PG profile of the colon.

4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyraz↗
PubMed1985

Macrophage activation by Tetrahymena pyriformis. I. Active subcellular fractions of Tetrahymena.

Experiments were carried out to determine what subcellular fractions of Tetrahymena pyriformis could, after inoculation into mice, activate macrophages to kill Toxoplasma gondii in vitro. Peritoneal macrophages from mice inoculated intraperitoneally with cilia, pellicles, mitochondria, and microsomes exhibited strong toxoplasmacidal activity and had an enhanced capacity to release hydrogen peroxide (H2O2) by stimulation of a membrane-active agent as compared with resident macrophages. In contrast, macrophages from mice inoculated with macronuclei and postmicrosomal supernatant showed no toxoplasmacidal activity and a low level of H2O2 release. Similar dose response was observed on the active subcellular fractions with regard to the degree of macrophage activation. Treatment of the active subcellular fractions with heating and trypsin markedly reduced their activity.

Animals↗