Abstract
A Percoll density gradient has been devised for the separation of protein bodies from cytoplasmic membrane components of barley endosperm. Protein bodies band isopycnically at a density of 1.13 g.ml−1, whereas stripped and smooth microsomes band at a density of 1.05 g.ml−1 in a sigmoidal Percoll density gradient generated in situ. Sucrose density gradients are shown to be unsuitable for the isolation of immature barley protein bodies owing to the isopycnic banding of smooth microsomes together with immature protein bodies at a density of 1.18 g.ml−1.
The complex ultrastructure of protein bodies observed in situ in barley endosperm is preserved in the isolated protein bodies prepared from Bomi and Carlsberg II barley and their high-lysine mutants Risø 1508 and Risø 56, respectively. The changes in protein body structure observed in these high-lysine mutants are thought to reflect the qualitative and quantitative changes in hordein biosynthesis caused by these gene mutations.
Electrophoretic analysis of the polypeptide composition of the purified protein bodies reveals that all the major hordein polypeptides synthesized in barley endosperm are stored in protein bodies. However, several additional polypeptides, which are not hordein, are specifically associated with protein bodies of each barley genotype.
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Abbreviations
- DTT:
-
dithiothreitol
- HEPES:
-
N-2-hydroxyethylpiperazine-N′-2-ethane sulfonic acid
- HK:
-
HEPES-K acetate buffer (see 2.1.)
- SDS-PAGE:
-
Sodium dodecylsulfate polyacrylamide gel electrophoresis
- Tris:
-
tris(hydroxymethyl)aminomethane
- TCA:
-
trichloracetic acid
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Cameron-Mills, V. The structure and composition of protein bodies purified from barley endosperm by silica sol density gradients. Carlsberg Res. Commun. 45, 557–576 (1980). https://doi.org/10.1007/BF02932923
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DOI: https://doi.org/10.1007/BF02932923