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Results for “Centrifugation, Isopycnic”

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At least 19 recordsLinked to original sources
PubMed1984

Advantages of rubidium chloride gradients for isopycnic centrifugation of enzymes.

The isopycnic centrifugation of beta-amylase and the marker enzyme beta-galactosidase was carried out using four salts viz. rubidium chloride, potassium bromide, potassium acetate and lithium bromide. Lithium bromide inactivated both beta-galactosidase and beta-amylase. The high viscosity of potassium acetate gradients necessitated an extremely long centrifugation time. The density profiles obtained with rubidium chloride gradients were sharper and permitted better resolution than potassium bromide gradients. Both enzymes were stable in rubidium chloride gradients, while potassium bromide inactivated beta-galactosidase, even in the presence of 10 mM 2-mercaptoethanol.

Amylases↗
PubMed1984

Isopycnic centrifugation of plant viruses in Nycodenz density gradients.

Isopycnic centrifugation of plant viruses in density gradients prepared from a new non-ionic medium (Nycodenz) was investigated. Particle density of luteo-, tymo-, nepo-, cocksfoot mild mosaic, tobamo-, hordei-like, potex- and potyviruses in buffered solutions of Nycodenz ranged between 1.23 and 1.28 g/ml and did not strictly reflect their nucleic acid and protein composition. Isopycnic centrifugation of several partially purified viruses yielded preparations which were pure by electron microscopy and, when used as immunogens in rabbit or hen, gave antisera with low titers of antibodies against host plant antigens, as required for enzyme immunoassays. A preparation of purified potato leafroll virus contained a single dominant protein corresponding to the viral coat protein. It is concluded that isopycnic centrifugation in Nycodenz density gradients is particularly useful for the purification and analysis of plant viruses which are unstable in cesium chloride solutions, such as the luteoviruses, and preferable to zonal centrifugation in sucrose density gradients for the purification of rod-shaped multipartite viruses or viruses which aggregate.

Centrifugation, Isopycnic↗
PubMed1988

Ultrastructural characterization of isolated human head and neck squamous carcinoma cells. Assessment of isopycnic centrifugation.

In an experiment to evaluate the merit of isopycnic centrifugation as a method of separating cell types in human head and neck squamous cell carcinomas, cells have been isolated from four specimens of these tumors and subjected to isopycnic centrifugation in continuous Percoll gradients. Cell types were identified by electron microscopy. The R- (ribosome-rich), T- (tonofilament-rich), and RT- (intermediate) cell types yielded broad bands overlapping extensively with one another, and partially with the bands of leukocytes. The pattern differed for each tumor studied, so that universal density levels separating given cell types could not be found. Isopycnic centrifugation proves less suitable in analyzing cells dispersed from solid tumors than for cells in suspended culture, blood, effusions, etc., probably because of heterogeneous growth conditions of cells in solid tumors.

Carcinoma, Squamous Cell↗
PubMed1976

Isopycnic centrifugation of thyroid iodoproteins: selectivity of endocytosis.

Isopycnic centrifugation in RbCl was shown to be an effective method both for evaluating the iodine content of thyroglobulin labelled in vivo and for the fractionation of thyroglobulin molecules as a function of their iodine content. Iodination and degradation of thyroid iodoproteins were studied by this method and by zonal centrifugation in sucrose density gradients. Based on these methods it was shown that iodination in vivo is a selective process, 19-S and poorly iodinated thyroglobulin having a higher reactivity toward iodine than 27-S and iodine-rich thyroglobulin. The disappearance of iodoproteins from the thyroid was evaluated by equilibrium, labelling the iodoproteins, blocking iodine incorporation with thiourea derivatives and observing (by sucrose gradient and by RbCl isopycnic centrifugation) at different times the properties of the remaining molecules. Among molecules of different size (19-S and 27-S) and among molecules of the same size (19 S) but different iodine content it was shown that reabsorption from the thyroid gland occurred at the same rate. It was concluded, therefore, that the degradative pathway is essentially a random, non-selective process. Newly iodinated (pulse-labelled) iodoproteins were degraded faster than preexisting molecules. Among the pool of those newly iodinated thyroid proteins, 27-S molecules were reabsorbed faster than 19-S molecules and iodine-rich thyroglobulin molecules were reabsorbed faster than the iodine-poor ones. Since iodination in vivo occurs as repeated pulses of iodine incorporation, it is suggested that this latter phenomenon is a regulatory mechanism which minimizes degradation of molecules which are iodine-poor and have a lower hormonal content.

Animals↗
PubMed1980

Proteoglycan complex and proteoglycan subunit polydispersity. Study by isopycnic centrifugation in cesium sulfate density gradients.

A true isopycnic centrifugation method for the study of the bovine nasal cartilage proteoglycan polydispersity is presented. The use of cesium sulfate as gradient forming salt instead of cesium chloride allowed proteoglycan banding without any sedimentation at the bottom of the centrifuge tube. Apparent buoyant densities of proteoglycan monomer and proteoglycan aggregate were different. The present method provides a useful tool for the study of proteoglycan polydispersity and also allows us to follow the distribution of the link proteins in different proteoglycan extracts.

Amino Acids↗
PubMed1980

A rapid method of isolation and fractionation of proteoglycans using a vertical tube rotor for isopycnic centrifugation.

A comparison was made between a vertical tube rotor and a fixed angle rotor for isopycnic centrifugation of proteoglycans. In the vertical tube rotor, isopycnic gradient was achieved much faster than in the conventional fixed angle rotor. The use of a vertical tube rotor for isopycnic centrifugation shortens the time considerably for the isolation of proteoglycans from various tissues.

Animals↗
PubMed1981

Isopycnic-centrifugation studies in caesium chloride and in caesium sulphate on dermatan sulphate proteoglycans from bovine sclera.

1. Two proteodermatan sulphate species from bovine sclera (fractions PG-I and PG-II) separable by gel chromatography were studied by isopycnic centrifugations in CsCl and Cs(2)SO(4) both in an analytical and a preparative mode. 2. In CsCl, fraction PG-I formed a broad band at a density rho=1.75g/ml whereas fraction PG-II banded sharply at rho=1.64g/ml. However, in Cs(2)SO(4), fraction PG-II banded at rho=1.51g/ml and fraction PG-I at rho=1.40g/ml, a reversal of the relative banding positions of the two species in CsCl. 3. Preparative isopycnic centrifugations in the two caesium salts permitted further subfractionation of fractions PG-I and PG-II. In both CsCl and Cs(2)SO(4) fraction PG-I was split into subfractions that varied greatly in uronate/protein ratios but had very similar uronate composition. In contrast, isopycnic centrifugation of fraction PG-II in Cs(2)SO(4) gave rise to subfractions with similar uronate/protein ratios but markedly different uronate composition (iduronate content, 88-42%). 4. Subfractions of fractions PG-I and PG-II obtained in preparative centrifugations in CsCl or Cs(2)SO(4) were examined in the analytical ultracentrifuge. These subfractions banded at discrete positions in the gradient. Estimations of apparent molecular weight for these subfractions from data from the analytical isopycnic centrifugations gave values that were much higher (around 1x10(6)) than were those obtained previously for their ;monomeric' states (fraction PG-I 160000-410000, and fraction PG-II 70000-130000). 5. In CsCl, fraction PG-I may be subfractionated according to the number of side chains in the molecule. Fraction PG-I increased its net solvation (i.e. lowered its buoyant density) to a larger extent than did fraction PG-II in going from CsCl to Cs(2)SO(4) (i.e. from lower to higher water activity). It is proposed that the presence of large amounts of iduronate in fraction PG-II makes these molecules relatively less solvated in Cs(2)SO(4). Thus the uronate composition may be an important factor in determining the banding position of proteodermatan sulphates in density-gradient centrifugations.

Animals↗
PubMed1983

Characterization of rat lymphocyte cell membranes by analytical isopycnic centrifugation.

Lymph node cell homogenates were fractionated by differential or isopycnic centrifugation and the fractions analyzed for biochemical markers with particular focus on plasma membrane constituents. Markers for the nucleus (DNA), mitochondria (cytochrome oxidase), and lysosomes (acid hydrolases) showed the expected distributions which were different from those of membrane-bound enzymes. 5'-Nucleotidase, alkaline phosphodiesterase, gamma-glutamyltranspeptidase, and cholesterol were membrane-bound and distributed identically after isopycnic centrifugation with peaks at 1.15. The distributions of the enzymes were all shifted to higher densities by digitonin treatment, confirming their association with plasma membrane-derived elements. The distribution of galactosyltransferase (ovalbumin acceptor), largely overlapped those of plasma membrane markers but it was only slightly shifted by digitonin, suggesting its localization in Golgi apparatus. The distribution of mannosyltransferase (dolichyl phosphate acceptor) also overlapped those of plasma membrane and Golgi markers but it was centered at higher density (1.18) and was unaffected by digitonin. It is a useful marker for endoplasmic reticulum. 50% of the activity was in low speed "nuclear" sediments where it was associated with the nuclear membrane. A number of other putative and previously used markers for the endoplasmic reticulum of lymphocytes were shown not to be localized in these membranes. In particular, NADH-cytochrome c reductase was only partly associated with the endoplasmic reticulum (56%) and the remainder of the activity was in mitochondria (44%). The results show the heterogeneity in equilibrium density of plasma membrane vesicles and the considerable overlap of their distribution with those of other cellular membranes; they should provide a basis for the more rational design of preparative procedures for the lymphocyte plasma membrane.

Animals↗
PubMed1984

Isopycnic centrifugation of mammalian metaphase chromosomes in Nycodenz.

Separation of chromosomes on the basis of buoyant density has proved difficult due to their high density and sensitivity to high ionic strength. Nycodenz is a new non-ionic gradient medium which offers significant advantages over previously described media for separation of chromosomes. The purpose of this study was to examine the banding characteristics of isolated mammalian metaphase chromosomes in Nycodenz gradients. The results indicate that chromosomes can be efficiently concentrated and separated from nuclei using Nycodenz gradients. Furthermore, chromosomes density-labeled with bromodeoxyuridine (BrdU) can be separated from unlabeled chromosomes in Nycodenz gradients. Nycodenz does not appear to alter chromosome morphology or protein complement. The introduction of Nycodenz represents a significant new tool for use in chromosome separation and purification.

Animals↗
PubMed1997

Xyloglucan undergoes interpolymeric transglycosylation during binding to the plant cell wall in vivo: evidence from 13C/3H dual labelling and isopycnic centrifugation in caesium trifluoroacetate.

Xyloglucan from the walls of Rosa cells that had been cultured on [12C]- or [13C]-glucose formed bands in caesium trifluoroacetate with mean buoyant densities of 1.575 or 1.616 g/ml respectively. Incubation of a mixture of [13C,3H]xyloglucan and [12C,1H]xyloglucan in the presence of xyloglucan endotransglycosylase (XET) activity caused the mean buoyant density of the radioactive material to decrease, indicating that interpolymeric transglycosylation could be detected in vitro. We used two 13C/3H-dual-labelling protocols to look for interpolymeric transglycosylation in vivo. In protocol A, [13C]glucose-grown Rosa cells were transferred into [12C]glucose medium 6 h after a approximately 2 h pulse of l-[1-3H]arabinose (which radiolabels the xylose residues of xyloglucan). The mean buoyant density of the wall-bound [3H]xyloglucan decreased during the following 7 days in culture. This indicates that, during or after the wall-binding of newly synthesized [12C,1H]xyloglucan, it became covalently attached to previously wall-bound [13C, 3H]xyloglucan. In protocol B, [12C]glycerol- or [12C]glucose-grown Rosa cells were transferred into [13C]glucose medium, 20 or 60 min before a approximately 2 h pulse of [3H]arabinose. The buoyant density of the earliest wall-bound [3H]xyloglucan showed that it had a 12C/13C ratio of approximately 1:1. This indicates that, during (or, implausibly, before) wall-binding, the newly synthesized [13C, 3H]xyloglucan became covalently attached to previously synthesized [12C]xyloglucan. During the following 7 days in culture, the mean buoyant density of the [3H]xyloglucan increased, showing that later-synthesized [13C,1H]xyloglucan can be covalently attached to previously wall-bound [12C,13C,3H]xyloglucan. The only known mechanism by which segments of xyloglucans could become covalently attached to each other in the cell wall is by interpolymeric transglycosylation catalysed by XET. We conclude that XET-catalysed interpolymeric transglycosylation accompanies, and probably causes, the integration of newly secreted xyloglucan into the cell-wall architecture.

Carbon Isotopes↗
PubMed1994

Isolation and partial characterization of Borrelia burgdorferi inner and outer membranes by using isopycnic centrifugation.

In order to characterize the protein composition of the outer membrane of Borrelia burgdorferi, we have isolated inner and outer membranes by using discontinuous sucrose density step gradients. Outer and inner membrane fractions isolated by this method contained less than 1 and 2%, respectively, of the total lactate dehydrogenase activity (soluble marker) in cell lysate. More importantly, the purified outer membranes contained less than 4% contamination by the C subunit of F1/F0 ATPase (inner membrane marker). Very little flagellin protein was present in the outer membrane sample. This indicated that the outer membranes were relatively free of contamination by cytoplasmic, inner membrane or flagellar components. The outer membrane fractions (rho = 1.19 g/cm3) contained 0.15 mg (dry weight) of protein per mg. Inner membrane samples (rho = 1.12 g/cm3) contained 0.60 mg (dry weight) of protein per mg. Freeze-fracture electron microscopy revealed that the outer membrane vesicles contained about 1,700 intramembranous particles per micron 2 while inner membrane densities for inner and outer membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and nonequilibrium pH gel electrophoresis-SDS-PAGE analyses of inner and outer membrane samples revealed several proteins unique to the inner membrane and 20 proteins that localized specifically to the outer membrane. This analysis clearly shows that the inner and outer membranes isolated by this technique are unique structures.

Bacterial Proteins↗
PubMed1980

Isopycnic centrifugation of human, pigeon and guinea pig erythrocytes.

The buoyant densities of human erythrocytes with normal (HbAA) and abnormal heterozygous haemoglobins (HbSS, HbAS, HbSC) as well as erythrocytes from guinea pig and pigeon were determined by centrifugation in discontinuous density gradients of metrizamide. Guinea pig, pigeon and human erythrocytes from normal human adult (HbAA) and from heterozygote HbAS were found to have the same density of 1.1381 g/cm3, while erythrocytes from a sickle-cell patient (HbS) and from a heterozygote HbSC, HbSS and HbSC, which are known to have a higher lipid content than isovolumic normal cells, had a lower density of 1.1261 g/cm3. When centrifugation was carried out under non-equilibrium conditions (rate zonal), several sedimentation bands were obtained from each erythrocyte preparation. Differences in the sedimentation rates were unrelated to the haemoglobin composition of the erythrocytes. It is suggested that the lower density of the erythrocytes associated with sickle-cell disease may have significant consequences in the circulation of such cells and in the clinical sequence of the sickling process.

Anemia, Sickle Cell↗
PubMed1987

[Isolation of periportal and centrolobular hepatocytes by isopycnic centrifugation].

Suspensions of rat hepatocytes were separated by Ficoll discontinuous density gradient into four fractions. About 85% of the total quantity of cells sedimented within the range from 1.044 to 1.126 g/ml. The activity of key enzymes of glycolysis and gluconeogenesis were measured to determine the acinar origin of hepatocyte subpopulations. The activity of the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase, in hepatocytes with the density of 1.044 g/ml was twice as high as in hepatocytes with the density of 1.073 g/ml. In contrast, glycolytic enzyme, hexokinase, was 3 times more active in heavy than in light cells. The results indicate that light and heavy cells correspond to periportal and centrilobular hepatocytes, respectively.

Animals↗