The fractionation of nuclei from mammalian cells by zonal centrifugation.
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ABSTRACT:
SUBMITTER: Johnston IR
PROVIDER: S-EPMC1186761 | biostudies-other | 1968 Aug
REPOSITORIES: biostudies-other
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Project description:1. Plasma membranes were isolated from crude nuclear sediments from mouse and rat liver by a rate-dependent centrifugation through a sucrose density gradient contained in the ;A' type zonal rotor. 2. The membranes were further purified by isopycnic centrifugation, and characterized enzymically, chemically and morphologically. 3. When the plasma-membrane fraction of sucrose density 1.17g/cm(3) was dispersed in a tight-fitting homogenizer, two subfractions of densities 1.12 and 1.18 were obtained by isopycnic centrifugation. 4. The light subfraction contained 5'-nucleotidase, nucleoside diphosphatase, leucine naphthylamidase and Mg(2+)-stimulated adenosine triphosphatase activities at higher specific activities than unfractionated membranes. The heavy subfraction was deficient in the above enzymes but contained higher Na(+)+K(+)-stimulated adenosine triphosphatase activity. 5. The light subfraction contained twice as much phospholipid and cholesterol, and three times as much N-acetylneuraminic acid relative to unit protein weight as the heavy subfraction. Polyacrylamide-gel electrophoresis indicated differences in protein composition. 6. Electron microscopy showed the light subfraction to be vesicular. The heavy subfraction contained membrane strips with junctional complexes in addition to vesicles.
| S-EPMC1185506 | biostudies-other
Project description:1. The nuclei of the cells of the whole rat brain have been fractionated in a B-XIV zonal rotor with a discontinuous gradient of sucrose. Five fractions were obtained. Zone (I) contained neuronal nuclei (70%) and astrocytic nuclei (23%). Zone (II) contained astrocytic nuclei (81%) and neuronal nuclei (15%). Zone (III) contained astrocytic nuclei (84%) and oligodendrocytic nuclei (15%). Zone (IV) contained oligodendrocytic nuclei (92%) and zone (V) contained only oligodendrocytic nuclei. 2. The content of DNA, RNA and protein per nucleus was determined for each zone. Although the amount of DNA per nucleus is constant (7pg) the RNA varies from 4.5 to 2.5pg/nucleus and the protein from 38 to 17.6pg/nucleus. The neuronal nuclei have the greatest amounts of protein. The oligodendrocytic nuclei have the least content of RNA and protein. 3. The effects of pH, ionic strength, and Mg(2+) and Mn(2+) concentration on the activity of the nuclear system for synthesis in vitro of RNA have been investigated for unfractionated nuclei. From these studies a standard set of conditions for the assay of nuclear RNA polymerase has been established. 4. The activity of the RNA polymerase in each of the zonal fractions has been determined in the presence and in the absence of alpha-amanitin. Zone (II) is the most active, followed by zone (I). The nuclei of zones (IV) and (V) have comparable activity, which is 40% of that of zone (II). 5. The extent of incorporation of each of the four labelled nucleoside triphosphates by the nuclei from each zone has been measured. These values have been used to calculate the base composition of the RNA synthesized in vitro in each class of nucleus. 6. The effect of changes in the condition of assay of RNA polymerase in the different classes of nuclei has been investigated. Significant differences in the response to concentrations of metal ions and ammonium sulphate have been observed. 7. Homopolymer formation in each zone of brain nuclei has been determined. The extent of formation of the four homopolymers roughly parallels the RNA polymerase activity.
| S-EPMC1174273 | biostudies-other
Project description:1. Homogenates were prepared from sphaeroplasts of anaerobically grown, glucoserepressed Saccharomyces carlsbergensis, and the distributions of marker enzymes investigated after zonal centrifugation on sucrose gradients containing 2mm-MgCl(2). 2. These homogenates contained no detectable cytochrome c oxidase, succinate-cytochrome c oxidoreductase, succinate-ferricyanide oxidoreductase, l(+)-lactate-cytochrome c oxidoreductase or catalase. Cytochromes a+a(3) and c were not detected. 3. Zonal centrifugation of whole homogenates indicated complex density distributions of the sedimentable portions of NADH- and NADPH-cytochrome c oxidoreductases, adenosine triphosphatases (ATPases), adenosine pyrophosphatase (ADPase), pyrophosphatase and acid p-nitrophenyl phosphatase. Several different ATPases were distinguished on the basis of their sensitivities to oligomycin and ouabain. 4. Differential centrifugation of whole homogenates at 10(5)g-min left 80-90% of the protein, dithionite-reducible cytochrome b, acid hydrolases and pyrophosphatase in a supernatant (S(1)) together with 65 and 56% of the NADH- and NADPH-cytochrome c oxidoreductases respectively, 25% of the ATPases and 71% of the adenosine monophosphatase. 5. Further analysis of supernatant S(1) revealed the presence of a class of small particles containing NADPH-cytochrome c oxidoreductases and ATPases. 6. At least four different populations of large particles were distinguished. 7. Electron microscopy indicated that one of these corresponded to ;promitochondria' as described by other workers.
| S-EPMC1178768 | biostudies-other
Project description:Most previously reported methods for purifying DNA-origami nanostructures rely on agarose-gel electrophoresis (AGE) for separation. Although AGE is routinely used to yield 0.1-1 µg purified DNA nanostructures, obtaining >100 µg of purified DNA-origami structure through AGE is typically laborious because of the post-electrophoresis extraction, desalting and concentration steps. Here, we present a readily scalable purification approach utilizing rate-zonal centrifugation, which provides comparable separation resolution as AGE. The DNA nanostructures remain in aqueous solution throughout the purification process. Therefore, the desired products are easily recovered with consistently high yield (40-80%) and without contaminants such as residual agarose gel or DNA intercalating dyes. Seven distinct three-dimensional DNA-origami constructs were purified at the scale of 0.1-100 µg (final yield) per centrifuge tube, showing the versatility of this method. Given the commercially available equipment for gradient mixing and fraction collection, this method should be amenable to automation and further scale up for preparation of larger amounts (e.g. milligram quantities) of DNA nanostructures.
| S-EPMC3553994 | biostudies-literature
Project description:1. The effects of the inclusion of thioacetamide in the diet on the properties of rat liver nuclei were studied both in adolescent rats, in which the parenchymal cells contain diploid nuclei, and in young adult rats, with a high proportion of tetraploid nuclei. 2. These investigations included a survey of the sedimentation properties of the nuclei, the nuclear volumes, content of DNA, RNA and protein, the incorporation in vivo of [(3)H]thymidine into DNA and [(14)C]orotate into RNA, and measurements of the activity of RNA polymerase and ribonuclease. These studies were conducted on nuclei fractionated by zonal centrifugation. 3. In both groups of animals, exposure to thioacetamide produced large numbers of nuclei that were abnormal in their chemical composition and enzymic activity. The changes were complex as regards both the types of nuclei that were affected and in their variation with time. 4. In adolescent rats two waves of synthesis of DNA and RNA were observed, one at 3 days and the other after 2 weeks of treatment. The first decline in the incorporations into both DNA and RNA coincided with a decrease in the pool sizes of some of the precursors. The activity of RNA polymerase was not substantially altered. A marked increase in the content of protein was observed before the first wave of synthesis. The normal progressive increase in tetraploid nuclei was prevented. 5. In young adult rats two waves of DNA synthesis were detected. Each was preceded by a large increase in the amount of protein per nucleus but was not accompanied by increased RNA synthesis. After 4 weeks of treatment, the diploid stromal nuclei appeared mainly unaffected and large numbers of tetraploid nuclei with a greatly increased quantity of protein were observed.
| S-EPMC1177576 | biostudies-other
Project description:1. Lettrée cells were grown intraperitoneally in MF-1 mice. 2. Cells that were loaded with glycerol were swollen in 0.1 M-sucrose and disrupted by Dounce homogenization. 3. Early-passage Lettrée cells were more easily disrupted than late-passage cells by this method, and the former produced larger fragments of plasma membrane. 4. The membranes were fractionated initially in sucrose gradients (on the basis of sedimentation rate) in a BXIV zonal rotor. 5. Fractions from this gradient were further resolved in isopycnic sucrose gradients. 6. Plasma-membrane and endoplasmic-reticulum fractions were recovered in good yield and high purity.
| S-EPMC1161245 | biostudies-other
Project description:1. Homogenates were prepared from sphaeroplasts of aerobically grown glucose-de-repressed Saccharomyces carlsbergensis and the distributions of marker enzymes were investigated after differential centrifugation. Cytochrome c oxidase and cytochrome c were sedimented almost completely at 10(5)g-min, and this fraction also contained 37% of the catalase, 27% of the acid p-nitrophenyl phosphatase, 53 and 54% respectively of the NADH- and NADPH-cytochrome c oxidoreductases. 2. Zonal centrifugation indicated complex density distributions of the sedimentable portions of these enzymes and of adenosine triphosphatases and suggested the presence of two mitochondrial populations, as well as a bimodal distribution of peroxisomes and heterogeneity of the acid p-nitrophenyl phosphatase-containing particles. 3. Several different adenosine triphosphatases were distinguished in a post-mitochondrial supernatant that contained no mitochondrial fragments; these enzymes varied in their sensitivities to oligomycin and ouabain and their distributions were different from those of pyrophosphatase, adenosine phosphatase and adenosine pyrophosphatase. 4. The distribution of NADPH-cytochrome c oxidoreductase demonstrated that it cannot be used in S. carlsbergensis as a specific marker enzyme for the microsomal fraction. Glucose 6-phosphatase, inosine pyrophosphatase, cytochrome P-450 and five other enzymes frequently assigned to microsomal fractions of mammalian origin were not detected in yeast under these growth conditions.
| S-EPMC1178386 | biostudies-other
Project description:BackgroundCentrifugation is commonly used as a first step to enrich biomarkers from blood. Biomarkers are separated on the basis of density and/or diameter. However, the centrifugation protocol affects the yield and purity of biomarkers, for example, isolation of platelets results in co-isolation with extracellular vesicles (EVs).ObjectiveTo assess the ability of rate zonal centrifugation (RZC) to separate platelets from co-isolated EVs.MethodsUsing a linear Optiprep gradient, RZC was able to separate a mixture of beads with different diameters but similar density. Next, RZC was applied to samples containing both platelets and platelet-derived EVs (n = 3). After RZC, all fractions were collected and stained with anti-CD61-Alexa 488 to measure the concentrations of platelets and platelet-derived EVs by flow cytometry.ResultsWe confirm that RZC separates polystyrene beads with diameters of 140 nm, 380 nm and 1,000 nm. Next, we show that the majority of platelets occur in fractions 8-19, whereas the majority of platelet-derived EVs are detectable in fractions 1-7. Furthermore, each fraction contains a different diameter range of platelets, which suggests that separation is indeed diameter based.ConclusionRZC can partially separate platelets from EVs.
| S-EPMC7443435 | biostudies-literature
Project description:1. The subcellular particles of horse and rat thyroids were fractionated in a B XIV zonal rotor on a non-linear gradient of Ficoll after labelling with radioactive iodine in vitro (horse) or in vivo (rat). In the horse, the resulting fractions were analysed for radioactive iodine, protein and enzymes representative of certain subcellular particles. In the rat, iodine turnover and thyrotrophin stimulation were studied. 2. The population of iodinated particles could be subdivided into three main classes, characterized by differences in beta-galactosidase and acid phosphatase content and position in the gradient. The presence of a fourth class of particles is suggested. 3. It is concluded that iodinated particles isolated from the thyroid are essentially secondary lysosomes. Their heterogeneity is established with respect to their position in the gradient, their content of acid hydrolases and their iodine turnover. 4. The iodine pools of these secondary lysosomes are increased by thyrotrophin without any change in their number. 5. Their functional significance is discussed. 6. The distribution of mitochondria as judged by succinate dehydrogenase was also studied. The succinate dehydrogenase was spread throughout the gradient with a maximum of activity (40%) in the upper layer of the gradient. Separation of mitochondria from lysosomes by this method was not successful.
| S-EPMC1166207 | biostudies-other
Project description:1. Radioactive orotic acid, uridine and adenosine were administered to rats by intracisternal injection. The effects of the size of the dose, the specific radioactivity and time on the incorporation into the RNA of unfractionated nuclei of brain tissue were examined to establish appropriate conditions for studies of the relative activities in vivo of the various sorts of brain nuclei fractionated by zonal centrifugation. Uridine is incorporated more efficiently than either orotic acid or adenosine. 2. With [(3)H]uridine as precursor the astrocytes in zone (II) contain the highest radioactivity except at the beginning of the experiment when the neuronal nuclei of zone (I) are more highly labelled. This fraction utilizes [(14)C]orotic acid more readily than the nuclei of zone (II). The astrocytic nuclei of zone (III) show a general resemblance to those of zone (II). Considerable differences between the incorporations into the two types of oligodendrocyte nuclei in zones (IV) and (V) are observed. 3. The relative synthetic activities of the major types of brain nuclei in vitro and in vivo are discussed.
| S-EPMC1177656 | biostudies-other