Density gradient centrifugation cell fractionation

The PemB cellular localisation was determined both in E. chrysanthenu and in an E. coli recombinant strain by Western blot of the cell fractions with a PemB-antiserum. No PemB was detected in the culture supernatant and only trace amounts were found in the soluble cell fractions - periplasm and cytoplasm (Figure 2). PemB was found mostly in the total membrane fraction from which it could be completely extracted by Triton X-100/Mg2+ and partially extracted by Sarkosyl (Figure 2). This behaviour is typical of inner membrane proteins, but since some exceptions have been noticed it does not positively indicate the PemB localisation (15). We performed cell membrane fractionation in sucrose density gradient centrifugation both by sedimentation and flotation, using several markers of inner and outer membrane vesicles. PemB was found in the outer membrane vesicles (data not shown). 

The microsome fractions see Fig. 1) that were prepared from mulberry cortical parenchyma cells were fractionated to 24 or 25 fractions using the 15-50% sucrose linear density gradient centrifugation see Fig. 2). Profiles of the marker enzymes and the protein content are described in Fig. 3. In general, the antimycin A-insensitive cytochrome c reductase activity is exhibited at a lower density than are those of the marker enzymes. The fraction that exhibited the highest antimycin A-insensitive cytochrome c reductase activity for each month was used as the ER-enriched fraction. 

Fig. 4. Localization of WAP27 and WAP20 in the crude microsome fractions and the relation with marker-enzyme activities in three organelles (ER, tonoplast, and Golgi). SDS-PAGE of fractionated proteins by isopycnic linear sucrose density gradient centrifugation of microsome fraction of mulberry cortical parenchyma cells was performed using 6-pL samples in each fraction. Immunoblot analysis was performed with anti-WAP27 and anti-WAP20 antibodies. (From ref. [1], with permission from the American Society of Plant Physiologists.)...

PF had been proposed as the terminal complex (23) and associated pores were reported on the outer membrane EF (24). Due to their proximity to the site of cellulose ribbon extrusion from the cell surface, these structures were assumed to be responsible for cellulose synthesis. A model was advanced in which cellulose synthase was localized on the outer membrane, which invoked adhesion sites between the outer and plasma membranes as a mechanism to explain the transfer of uridine-diphosphoryl-glucose (UDPG) from the cytoplasm to the cellulose synthases (25,26). However, when the outer and plasma membranes of Acetobacter were isolated separately by density-gradient centrifugation, the cellulose synthase activity was localized only in the plasma membrane fraction (27). Therefore, the linear structures observed on the Acetobacter outer membrane, while they may be associated in some manner with cellulose biosynthesis, are probably not the cellulose synthase terminal complexes. Since no ultrastructural evidence for adhesion sites between the outer and plasma membranes has been presented, a thorough investigation of the mechanism of / (1-4) glucan chain translocation from the cytoplasmic membrane to the outer membrane in Acetobacter xylinvm is now in order. 

Enrichment of rare cancer cells from peripheral blood samples is an application that typically requires density gradient centrifugation as a first step. Application of this technique addresses two objectives depletion of erythrocytes and depletion of polymorphonuclear cells. It is expected that cancer cells undergo sedimentation with the mononuclear cell fraction because of their similar density. However, some studies have found that cancer cells are also lost in the polymorphonuclear fraction or the erythrocyte fraction (8,9). Optimization of the density gradient sedimentation step is an important issue in such an application, because it will determine the recovery of rare cells from blood and affect the chances of their detection by immunochemical means. 

Equilibrium density-gradient centrifugation, which separates cellular components according to their densities, can further purify cell fractions obtained by differential centrifugation. 

There is clearly no exclusive site of acylation of glycerol phosphate in the plant cell. Density gradient centrifugation of the 270-g supernatant from castor bean endosperm showed the acylation activity mainly in the ER fraction (ca. 90%) and the remainder in the mitochondria (Vick and Beevers, 1977). However, it is clear from the results cited above that acylation activity is present in the chloroplast envelope (Joyard and Douce, 1977) and in the stroma proteins from chloroplasts (Joyard and Douce, 1977). 

The DNA of a cell can be fractionated according to its base composition (see GC content) by density gradient centrifugation in CsQ. The buoyant density, given as g CsCl/cm , is used to characterize the fractions. Mouse liver DNA, for example, consists of a main fraction (buoyant density 1.702) together with Satellite DNA (see) (1.691). The DNA fractions of Euglena have buoyant densities of 1.707 (nuclear DNA), 1.691 (mitochondrial DNA) and 1.686 (chloroplast DNA). 

In vitro methods are performed outside the whole organism. They are essentially test tube methods, employing crude cell homogenates, subcel-lular fractions thereof, or purified enzymes. For methods of cell disruption, subcellular fractionation and enzyme purification, see Proteins Density gradient centrifugation. 

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