10.2.3—
Isolation and Purification

Ribosomes and polysomes are normally isolated from plants and purified by sedimentation through sucrose cushions as originally described by Wettstein

Figure 10.2
Electron micrographs of developing seeds of  Vicia faba.
(a) 25 days after fertilization. Ribosomes free in the cytoplasm. Very little ER present.

et al. (1963). In a typical procedure, the material is homogenized in 0.25 M sucrose containing 200 mM -Tris-HCl, pH 8.5 at 2ºC, 500 mM -KCl and 15 mM -MgC1₂ , with a Willems Polytron for 3 seconds at a speed setting of 8. The homo-

Figure10.2
(b) 55 days after fertilization, showing most of the polysomes bound to ER and
storage protein being laid down. C = Cytoplasm, P = Protein body, CW = Cell Wall,
RER = Endoplasm reticulum with polysomes attached, N = Nucleus, V = Vacuole.
From Boulter et al. (Qual. Plant.  XXIII, 239–50, 1973).

genate is filtered through Miracloth and Triton X-100 is added to a final concentration of 2% (v/v). The filtrate is centrifuged at 10⁴ × g for 10 min at 2°C and the ribosomes and polysomes are recovered from the supernatant by layering over a 3 ml cushion of 0.1 M sucrose in 50 mM -Tris-HCI, pH 8.5, 50 mM -KCl

and 10 mM -MgCl₂ , followed by centrifugation for at least 44 x 10⁷ g-min, as described by Leaver & Dyer (1974) (Fig. 10.3). The inclusion of the detergent

Figure 10.3
Sucrose density profiles of ribosomes and polysomes from  Pisum
sativum.  Ribosomes recovered after 6 h. centrifugation through
1 M -sucrose cushion. E₂₅₄  = absorbance at 254 nm
(From Leaver & Dyer  Biochem. J. 144,  165–7, 1974.)

Triton X-100 is necessary since plant materials contain a proportion of polysomes which are membrane-bound and the addition of Triton X-100 solubilizes the endoplasmic reticulum, so releasing them. The method gives a preparation of both polysomes and ribosomes and the latter can be removed by centrifugation techniques or, alternatively, the whole preparation can be converted to ribosomes by exposure of the plants to nitrogen gas for at least 1 hour prior to extraction. Since different plants and even different tissues from the same plant contain different amounts of membrane-bound to free polysomes and different free polysomes to free monoribosome ratios, isolation conditions may need to be varied for optimum results with different experimental materials. The situation is further complicated by the fact that plant mitochondrial ribosomes, although not functionally of the 80s type, sediment at 80s (Leaver & Harmy, 1973), and can contaminate cytoplasmic preparations to varying extents, depending on the experimental material. Damage to ribosomes, both structural and by the removal of associated proteins, can occur during isolation and preparation. The extent of this damage can be assessed, to some extent, by extracting the RNA and fractionating it by polyacrylamide gel electrophoresis (see Leaver & Key, 1970).