Sucrose density gradient centrifugation washing

Chloroplast ATP synthase is a well-defined complex which may be solubilized fi om thylakoid membranes by treatment with octylglucoside and cholate and purified by ammonium sulphate fi actionation and sucrose density gradient centrifugation [140]. The complex is composed of two assemblies of polypeptides CFj, a peripheral membrane complex, which may be washed fi-om thylakoid membranes with EDTA and which shows latent ATPase activity, and CFg, the intrinsic membrane sector, which translocates protons across the thylakoid membrane. 

Eleven-day-old dark-grown maize plants were used. Sixty-seven grams of leaf tissue was used for each preparation. Each set was permitted to absorb 0.4 mC T-phosphate. Preparation and sucrose density gradient centrifugation were as described for Twice washed 1000 g pellet, Fig. 6. 

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... 

Fig. 5. RNA of plastid fraction (washed 1000g pellet) isolated from leaves maintained in darkness or illuminated for 3 hours after absorption of P-phosphate. RNA was extracted and centrifuged as described in legend for Fig. 3, but fractions here were collected manually and optical densities at 260 m/ were determined after dilution. (The two heavier RNA s, when obtained from maize chloro-plasts purified on a sucrose density gradient, are approximately 22 S and 17 S. The apparent base composition of these RNA s is approximately 25% adenine, 32% guanine, 20% uracil, and 23% cytosine these values are based on the distribution of radioactivity, after paper electrophoresis, among AMP, GMP, UMP, and CMP obtained by alkaline hydrolysis of plastid RNA s prepared from sucrose gradient purified plastids of maize leaves supplied P-phosphate.)...

Fig. 8. The effect of illumination of etiolated maize leaves for 45 minutes on the specific activity [cpm ( P)/O.D.2m] profile obtained by centrifuging an extract of twice-washed plastids in a 10-34% sucrose density gradient (upper figure). The lower figure shows the absorption profile for identification of the position of plastid ribosomes (see Fig. 6).

Fig. 16. Effect of ribosome binding on mitochondrial density. Mitochondria, washed with 2 mM EDTA, were incubated for 15 min at 30°C with 0.46, 0.92, 1.84, 3.68, and 5.52 Azeo units of [ H]-labeled cytochrome ribosomes, B through D, respectively. The incubation mixtures were chilled to 0 C and layered on 40-70% linear-sucrose gradients and centrifuged for 1 h at 243,000gmax in a Beckman SW50-1 rotor. The gradients were fractionated and assayed for cytochrome oxidase activity ( — ), radioactivity (A—A), and density (O-—O). Panel A shows the density of mitochondria without added ribosomes.

Fig. 17. Effect of various conditions on mitochondrial density. Mitochondria were sedimented to equilibrium on linear sucrose gradients. After fractionation, the gradients were analyzed for cytochrome oxidase activity and density. (A) Mitochondria from growing cells. (B) Mitochrondria from cells starved for 1 h. (C) Mitochondria from growing cells washed with 2 mM EDTA. (D) Same as (C), except mitochondria were incubated with PH]-labeled ribosomes before centrifugation. (E) Mitochondria from growing cells. Culture was pretreated with 200 Mg/ml cycloheximide prior to harvesting. (F) Mitochondria from growing cells treated with 200 Mg/ml cycloheximide, then starved for 1 h.

Fig. 5. Isopycnic banding of membranes. An exponentially growing culture of B42 was filtered, washed, and resuspended in medium with PHJglycerol ( ) or [ CJleucine with no glycerol (O). Membranes were prepared after 1 h of growth. A mixture of the two membrane preparations was layered onto a linear sucrose gradient and centrifuged at 25,000 rpm for 2 h —---, density (g/ml). ...

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