Separation of hepatocytes from suspensions of mouse liver cells using programmed gradient sedimentation in gradients of ficoll in tissue sulture medium

Liver cells were obtained in suspension using a solution of lysozyme in Joklik's modification of minimum essential medium. Hepatocytes were separated in 74.2 ± 12.9% purity from other liver cells having different densities using isopycnic centrifugation, in 97.1 ± 1.9% purity from other liver cells having different diameters using velocity or rate-zonal centrifugation. A previously reported computer integration of the differential sedimentation equation was employed in determining the gradient design and the speed and duration of centrifugation which would permit purification of hepatocytes from other liver cells. More than 98% of the hepatocytes separated by velocity sedimentation excluded trypan blue. Velocity sedimentation is superior to isopycnic centrifugation for the separation of hepatocytes from liver cell suspensions because it gives more highly purified hepatocytes and because it requires lower centrifugal forces for shorter periods of time.     

Separation of epithelial cells from suspensions of cells from the hamster parotid gland in an isokinetic density gradient of Ficoll in tissue culture medium.

Epithelial cells were separated from suspensions of hamster parotid cells by velocity sedimentation in an isokinetic gradient and by isopycnic sedimentation. Epithelial cells were 48.1 ± 18.0% of the cells in the starting sample suspensions of cells from the disaggregated hamster parotid glands. The purest gradient fractions following velocity sedimentation in a previously described isokinetic gradient contained 98.8 ± 1.8% epithelial cells. The purest fractions obtained from isopycnic sedimentation contained 99.9 ± 0.2% epithelial cells. Purification of parotid epithelial cells by velocity sedimentation in the isokinetic gradient seems preferable to purification using isopycnic centrifugation because a larger proportion of the epithelial cells are obtained in the zone of the gradient which contains highly purified epithelial cells and because velocity sedimentation requires lower centrifugal forces for a shorter period of time.     

Characterization of mycoplasmatales virus DNA

The DNA of the group L1 $\text{Mycoplasmatales}$ virus, MVL51, was analyzed using alkaline sucrose velocity sedimentation, neutral and alkaline CsCl isopycnic sedimentation, and treatment of the DNA with nucleases. These treatments show that the viral chromosome is a covalently linked single-stranded DNA circle of molecular weight 2×10⁶ daltons.     

Physical Properties of Lactic Dehydrogenase-Elevating Virus and Its Ribonucleic Acid

Infectious lactic dehydrogenase-elevating virus propagated in primary cultures of mouse peritoneal macrophages in the presence of ³H-uridine and isolated by isopycnic centrifugation was found to have a density of 1.12 g/cm³. Ribonucleic acid extracted from the virus by treatment with sodium dodecyl sulfate was single stranded with a sedimentation coefficient of approximately 48S.     

Role of lysine in the replication of reovirus: I. Synthesis of complete and empty virions

Lysine is essential for the replication of infectious reovirus. Omission of lysine from the extracellular medium not only permitted the continued synthesis of structural viral proteins and viral double-stranded ribonucleic acid (RNA), but also caused an enhanced formation of viral structures which were separable by isopycnic sedimentation of CsCl into a top band consisting of empty particles with a buoyant density of 1.29 g/cm³ and essentially free of viral RNA, and two lower bands which were difficult to resolve and had an average buoyant density of 1.37 g/cm³. The lower bands contained most of the viral nucleic acid. The above effects were reversed when lysine was restored early after infection. In contrast, a single band with a buoyant density of 1.38 g/cm³ was obtained from lysine-plus infected cells.     

Highly purified plasma membranes from rat hepatocytes following rate-isopycnic zonal centrifugation

Plasma membranes from liver parenchymal cells were isolated by rate-isopycnic zonal centrifugation. A method is described for the Beckman size 15 zonal rotor. It involved preparation from a perfused liver of a parenchymal cell-enriched homogenate in isoosmotic sucrose. The nuclear fraction containing membranes was recovered by centrifugation. The resuspended pellet was applied on the gradient of the zonal rotor. The isolated membranes had the same isopycnic banding density as 37% sucrose (w/w). The specific activity of 5′-nucleotidase, a widely used plasma membrane marker, was 105 μmoles·(mg protein)^?1·h^?1 being enriched by a factor of 50 as compared with parenchymal cell homogenate. The plasma membrane fraction was free of the mitochondrial and lysosomal enzymes, succinate dehydrogenase and acid phosphatase. No DNA and 10 μg RNA per mg plasma membrane protein were found. The purity of the membranes and their morphological appearance were controlled by electron microscopy. The preparation consisting of large membrane sheets showed a considerable purification away from other cellular components. A comparison with similar methods indicates that plasma membranes of a higher degree of purity can be obtained from parenchymal cells.     

Use of trypsin for rapid and efficient purification of murine sarcoma and leukemia virus

A method is described for purification of MSV-MuLV from culture supernatant of chronically infected 78A₁ rat embryo cell line. This method involves direct polyethylene glycol-NaCl precipitation of the low speed supernatant of culture fluid followed by digestion of the pellet with trypsin. This procedure efficiently disrupts large aggregates which normally entrap most of the virus. Highly purified virus can be obtained in very good yield by a combination of sedimentation velocity and isopycnic centrifugation : yields up to 100 A₂₈₀ units (17 mg of protein) of purified virus per liter of culture fluid can be observed. This procedure appears well suited for large scale isolation of virion associated enzymatic activities.     

10. Purification of Viruses by Cenlrifugation in Gradients of Inert Substances

ABSTRACT

By using a reorienting gradient centrifuge rotor cut from a block of Nylon and fitted with eight septae, it was possible to separate the components of the haemolymph of the mollusc Turbo sarmaticus into three fractions in a sucrose gradient held in the bowl of the rotor. The fractions were (108 and 98)S, 44S and 16-22S. The success of the experiment was due to the large differences in the sedimentation coefficients of the components. When the rotor was applied to the natural mixture of the five viruses of the caterpillars of Nudaurelia cytheria only the main component could be isolated in a pure state. The viruses were separated by isopycnic centrifugation in “self formed” caesium chloride gradients, using a Beckman Model E analytical centrifuge in which a separation cell fitted with a centerpiece with two perforated partitions was used.

Centrifugation in gradients of inert substances is useful for the separation of components in a mixture¹. There are two principles involved in this type of separation. One, termed reorienting gradient centrifugation (reograd) relies on the differences in masses or, better still on the sedimentation coefficients of the different components in the mixture and the second, termed isopycnic centrifugation², on the densities or specific gravities of the different entities.     

Sedimentation properties of chitosomes fromMucor rouxii

Summary This study was undertaken to assess the distribution and localization of chitin synthetase in a fungal cell and to evaluate the sedimentation behavior of chitosomes (microvesicular containers of chitin synthetase). Chitosomes were isolated from cell-free extracts of yeast cells ofMucor rouxii by rate-zonal and isopycnic sedimentation in sucrose density gradients. Because of their small size and low density, chitosomes were effectively separated from other subcellular particles. Rate-zonal sedimentation was a suitable final step for isolating chitosomes as long as ribosomes had been eliminated by enzymic digestion. By isopycnic centrifugation, chitosomes could be separated directly from a crude cell-free extract; they cosedimented with a sharp symmetrical peak of chitin synthetase at a buoyant density of d=1.14–1.15g/cm³; the only significant contaminants were particles of fatty acid synthetase complex. From such sedimentations, we estimated that 80–85% of the chitin synthetase activity in the cell-free extract was associated with chitosomes; the rest was found in two smaller peaks sedimenting at d=1.19–1.20 and d=1.21–1.22 (5–10%), and in the cell wall fraction (5–10%). By consecutive rate-zonal and isopycnic sedimentations, chitosome preparations with relatively few contaminating particles were obtained. Potassium/sodium phosphate buffer (pH 6.5)+MgCl₂ was the most effective isolation medium for chitosomes. Other buffers such as TRIS-MES+MgCl₂ led to massive aggregation of chitosomes and a change in sedimentation properties. This tendency of chitosomes to aggregate could explain why most of the chitin synthetase activity of a fungus is sometimes found associated with other subcellular structures,e.g., plasma membrane.     

Release of chemical mediators from partially purified human lung mast cells.

Human lung mast cells dispersed by enzymatic digestion of human lung fragments were concentrated to greater than 50% purity by sedimentation in isopycnic and velocity gradients. The dispersed lung mast cells had a characteristic ultrasturctural appearance including granules with a scroll or reticular structural appearance including granules with a scroll or reticular structure surrounded by perigranular membranes. Histamine and preformed eosinophilotactic activity sedimented with mast cells on isopycnic gradients, and mast cells and these mediators were separated from the bulk of the other lung cells after velocity gradient sedimentation. The histamine content of isolated lung mast cells was calculated to range from 1.0 to 5.5 pg/cell. The quantity of SRS-A generated with anti-IgE or specific antigen was relatively limited but confined to the mast cell-rich fractions and associated with release of histamine and eosinophilotactic activity.     

Concentration and purification of two small RNA viruses: mycophage PS-1 and bacteriophage phi6

Two small RNA viruses, mycophage PS-1, and bacteriophage φ6 were concentrated and purified by the sequential use of continuous flow zonal centrifugation, high speed continuous flow sedimentation, and combined rate zonal centrifugation and isopycnic banding. The continuous flow centrifugations were done using the K-II and K-XI rotors while the BXXIX rotor was used for batch rate zonal centrifugation and isopycnic banding. The deseribed procedures are useful for the concentration and purification of virus from large volumes of culture fluid.     

Purification of coated vesicles by agarose gel electrophoresis

We have applied agarose gel electrophoresis as a novel step in the purification of clathrin-coated vesicles. Preparations of coated vesicles obtained by sedimentation velocity and isopycnic centrifugation are resolved into two distinct fractions upon electrophoresis. The slower migrating fraction contains smooth vesicles, whereas the faster contains only coated vesicles and empty clathrin coats. The faster mobility of the coated vesicles is primarily caused by the acidic nature of clathrin. Coated vesicles from three different cell types have different mobilities. In each case, however, all of the major polypeptides previously attributed to coated vesicles comigrate with the now homogeneous particles, even though a powerful ATPase activity is completely removed.     

Fractionation of rat fibroblasts in a zonal rotor by means of a viscosity barrier.

Cell fractionation procedures involving differential sedimentation followed by resuspension of pellets and isopycnic centrifugation are very difficult to apply to the small amounts of material available from tissue culture cells. We have explored the possibility of successive differential and isopycnic sedimentation in a zonal rotor using a short viscosity barrier for the differential sedimentation. The marker enzymes used were cytochrome oxidase, acid phosphatase, catalase, and 5′-nucleotidase. The results of these procedures are compared to the results of one-step isopycnic separations in gradients of sucrose and Stractan. The Stractan gradient was much more effective than the sucrose gradient in separating the marker enzymes from the proteins of a postnuclear supernatant, but neither type of gradient could significantly purify the marker enzymes one from another. A two-step procedure using a viscosity barrier was effective in separating particles carrying catalase from the other marker enzymes assayed and from most of the protein. A three-step procedure resulted in similar purification of mitochondria. Modification of barrier composition and centrifugation times would probably result in further improvement of separations according to individual requirements for yield, purification, and freedom from specific contamination by other subcellular particles.     

Separation of large quantities of chinese hamster chromosomes by velocity sedimentation at unit gravity followed by flow sorting (FACS II)

Separation of large quantities of isolated metaphase chromosomes of Chinese hamster cells was performed by velocity sedimentation at unit gravity in a specially designed sedimentation chamber. This simple and easy technique results in chromosome fractions of relatively high purity as determined by flow cytometry and microscopy. Up to 10¹⁰ chromosomes can be processed depending upon the size of the sedimentation device, and enrichments up to 10 times of individual chromosomes were achieved. In addition, further chromosome purification was performed by fluorescence activated flow sorting using fractions, pre-enriched at unit gravity. The flow sorted chromosomal fractions were pure according to flow cytometric analyses. The combination of l g sedimentation and flow-sorting opens the possibility for preparative chromosome sorting by reducing the flow sorting time considerably.     

STUDIES OF NATIVE GLYCOGEN ISOLATED FROM SYNCHRONIZED TETRAHYMENA PYRIFORMIS (HSM)

Native glycogen was isolated from Tetrahymena pyriformis (HSM) by isopycnic centrifugation in cesium chloride density gradients. A density of 1.62 to 1.65 was isopycnic for glycogen. Most of the banded glycogen existed as 35 to 40 mµ particles which had a sedimentation coefficient of 214. These particles were composed of aggregates of 2 to 3 mµ spherical particles. Extraction of glycogen with hot alkali reduced the sedimentation coefficient of native glycogen from 214 to 64.7 and the particle diameter from approximately 40 to 20 mµ and smaller. Cell division was synchronized by a repetitive 12-hour temperature cycle, and glycogen was measured at several times during the cell cycle. The temperature cycle consisted of 9.5 hours at 12°C and 2.5 hours at 27°C. Approximately 90 per cent of the cells divided during the last 1.5 hours of the warm period. The carbohydrate/protein ratio of cells at the end of the cold period was 0.27 and was reduced slightly during the warm period. Glucose was incorporated into glycogen during both periods, although the rate of incorporation was greater during the warm period. No preferential incorporation on the basis of particle size was noted. Incorporation was measured in both native glycogen and KOH-extracted glycogen. Tetrahymena glycogen is compared with rat liver glycogen previously isolated by similar procedures, and the significance of using combined rate-zonal and isopycnic centrifugation for isolating native glycogen is discussed.     

Effect of rhesus or vervet cytomegalovirus infection of DNA synthesis in untreated and 5-iodo-2''-deoxyuridine-arrested cells.

Infection of permissive cells with either rhesus or vervet cytomegalovirus resulted in suppression of cellular DNA synthesis, only viral DNA was resolved by isopycnic centrifugation after 24 h postinoculation. Infection of 5-iodo-2'-deoxyuridine (IUdR)-arrested cells with either of the simian cytomegaloviruses, however, induced synthesis of cellular DNA of normal density; synthesis of cellular DNA substituted with IUdR as evidenced by resolution of a heavy DNA peak after isopycnic centrifugation was not observed. Stimulation of DNA synthesis in IUdR-arrested cells was not observed with virus inactivated with UV light.     

Purification of eukaryotic ribosomes by isopycnic centrifugation in sucrose

A simple and inexpensive procedure for the isolation and purification of ribosomes from eukaryotes is described. The method avoids pelleting of ribosomes at high centrifugal forces and involves isopyenic centrifugation of the post-mitochrondrial supernatant in sucrose, precipitation of ribosomes with 10% polyethylene glycol, and zonal sucrose gradient centrifugation. The ribosomes obtained in this way are very pure and thus especially suited for the measurements of physical properties. The isopycnic centrifugation can also be used for the purification of other macromolecules and is only limited by a maximum density of sucrose of 1.40 g/cm³ obtained at the bottom of the centrifugation tubes.     

Studies on ethylene binding by cell-free preparations from cotyledons of Phaseolus vulgaris L.: subcellular localization

Abstract. Studies on the subcellular location of ethylene binding activity from developing cotyledons of Phaseolus vulgaris L. are described. Binding activity has been shown to be predominantly membrane bound. When separated by rate-zonal centrifugation more than 70% of this activity was of low sedimentation rate. The slowly sedimenting band of activity was further fractionated into three bands by isopycnic density gradient centrifugation. The three bands occur at sucrose densities of 1.125 g cm⁻³, 1.155 g cm⁻³ and 1.175 g cm⁻³, corresponding to the distribution of putative marker enzymes for the cell endomembrane system and to protein body membranes. Further circumstantial evidence was obtained by electron microscopy and sucrose step gradient centrifugation.     

Ultracentrifugation as a probe of conformation of unhydroxylated procollagen and collagen

Unhydroxylated and hydroxylated procollagen and collagen were compared by zone velocity and isopycnic centrifugation. The sedimentation coefficient of unhydroxylated procollagen(3.7 S) was slightly less than that of hydroxylated procollagen (3.9 S) and the sedimentation coefficient of unhydroxylated collagen (2.9 S) was slightly less than hydroxylated collagen (3.2 S). These differences could be accounted for largely by the slight increase in molecular weight and density of the hydroxylated molecules. The results indicate that the unhydroxylated molecules are in a triple helical conformation composed of three chains rather than analogous helical structures formed by the back-folding of individual chains. We conclude, therefore, that previous experiments demonstrating the decreased thermal stability of unhydroxylated collagen relative to hydroxylated collagen have measured the denaturation of true triple helices.