Cytoplasmic extract centrifugation

Cytoplasmic extracts have been made from immature oocytes, either unactivated or artificially activated (Longo et aL, 1994). Ovaries are dissected from ripe females and incubated in seawater to release germinal vesicle (GV)-stage oocytes. Oocytes are washed three times in seawater by suspension and spinning in a hand centrifuge. 

Fig. 12. Informosomes from loach embryo extracts labeled for 3 hours with uridine. Dotted lines, radioactivity solid lines, optical density. A. Sucrose density gradient centrifugation of cytoplasmic extract (22,000 rpm for 18 hours). B. Recentrifugation of the material of 73 to 62S peak in CsCI density gradient. C. The same but of 45 to 36S zone. (From Ovchinnikov et al. 1960a. Molec. Biot. (U.S.S.RJ, 3 448 3.)...

Fig. 13. Nucleoprotein complexes containing virus-specific RNA in the cytoplasm of HeLa cells infected with vaccina virus. A. Sucrose density gradient centrifugation of cytoplasmic extract, after 30-minute labeling with uridine. Centrifugation 18.5 hours at 22,000 rpm. B. Recentrifugation of RNP isolated from SOS zone sucrose gradient in CsCI density gradient. Centrifugation in preformed CsCI density gradient in SW-39 rotor at 37,000 rpm, 20 hours. (From Belitsina et al. 1968. Molec. BioL (U.S.S.R.), 2 727-735.)...

Viral ERA synthesis in the picornavirus group is associated with membrane structirres (25, 26) and can be isolated in the form of a replication complex by centrifugation of the cytoplasmic extracts at 20,000 g (27). The phosphorylase activity was shown to be associated with the replication complexes (Table 7)> result expected, as dsRRA structures were in the P-20 fraction. 

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). 

In this method the cells are lysed by incubation in a hypotonic solution, which leaves the nuclei intact. The cell debris and nuclei are pelleted by centrifugation leaving the cytoplasmic RNA free from DNA in the supernatant. The RNA is released from the polysomes by incubation with proteinase K and the protein extracted into phenol/chloroform. The RNA is then precipitated from the aqueous phase using ethanol. 

FIGURE 1-3 The universal features of living cells. All cells have a nucleus or nucleoid, a plasma membrane, and cytoplasm. The cytosol is defined as that portion of the cytoplasm that remains in the supernatant after centrifugation of a cell extract at 150,000 g for 1 hour. 

Centrifuge the lysate at 13,000 X gfor 30 min and collect the supernatant (containing GST and other cytoplasmic proteins). This clarified cell extract will be used in the GST purification experiment. 

Following such treatment, the cultured cells in monolayer are washed with phosphate-buffered saline and extracted in 4 ml 0.5% Triton X-100 in saline/EDTA (100 mM NaCl, 10 mM EDTA, pH 8.0) for 2 min at room temperature. This releases most of the cytoplasmic material whilst the nuclei remain attached to the culture dish. 0.5% sodium dodecyl sulphate and 40 //g/ml pancreatic RNAse (preincubated at 80°C for 10 min, to inactivate DNAse) in saline/EDTA (above) is then added and the mixture incubated for 20 min at 37°C. One volume of chloroform/isoamyl alcohol (20 5, v/v) is then added and the phases mixed gently. The aqueous phase is separated by centrifugation and extracted again with chloroform/isoamyl alcohol. DNA is precipitated from the aqueous phase with 2 vol 95% ethanol and resuspended in 0.01 M Tris, pH 7.5. Alkaline sucrose density gradients (5-20%) are prepared in 0.1 M NaCl, 0.1 M NaOH with a final volume of 4.1 ml. Samples of DNA (max 3 fig) are layered on the top of these gradients and spun at 32 000 rpm at 20°C in a SW.50.1 Beckman rotor for 120 min. Fractions are collected and the [3H]DNA precipitated... 

Ascorbic acid within the cells was localized in the cytoplasm (G3). More recent studies in rat liver and ox adrenals by the method of differential centrifugation have shown that most of the ascorbic acid was in the soluble and microsomal fractions (G2). The sum of ascorbic acid in the different fractions exceeded that of the whole tissue analyses by 40 %, suggesting to these workers the existence of bound ascorbic acid not liberated by extraction of the whole tissue. The fact of some localization in microsomes deserves confirmation. 

When cell extracts were prepared using a French press, centrifuged at 30 000 xg, and the supernatant further centrifuged at 150 000 xg for 3 h, most of the activity remained in the soluble fraction, establishing that the enzyme separated from the membrane. A 24-fold purified preparation of the enzyme was obtained. The MW of the enzyme was reported to be M,. 340 000. " For optimal activity, the enzyme required Mn, washed membranes or an extract of phospholipids, and an unidentified heat stable factor of MW <10 000. The reaction was strongly stimulated by dithiothreitol and methanol. Since the substrate of the enzyme 3-octaprenyl-4-hydroxybenzoate (49) is membrane bound and the enzyme is stimulated by phospholipid, it has been suggested that the enzyme normally functions in association with the cytoplasmic membrane m A reaction... 

Fig. 12. Comparison of mitochondria-bound and free cytoplasmic polysomes. Polysomes were prepared from isolated mitochondria by extracting with 2% Triton X-100. Free cytoplasmic polysomes were obtained without the use of detergent. Where indicated, polysomes preparations were made in 1% sodium deoxycholate before centrifugation for 40 min at 227,861gmax in a Beckman SW50-1 rotor through a 10-50% sucrose gradient. The position of the 80 S monosomes is indicated by the arrows. (From Kellems et alJ )...

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