Summary
A strategy for the rapid purification of proteins from glyoxysomes of castor bean (Ricinus communis cv. Hale) is described. The first step was to separate the proteins in the mixture on the basis of hydrophobicity by reversed phase high performance liquid chromatography using a gradient of increasing acetonitrile concentration. Individual protein peaks were collected and fractionated according to molecular mass by preparative polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purified polypeptides were used to produce monospecific, polyclonal antibodies. One of these, an anti-catalase antibody, has been employed to assess the subcellular distribution of catalase in endosperm of maturing seeds, dry seeds and seedlings. During seed maturation 45% of the catalase activity was associated with structures sedimenting at high isopycnic densities (1.21 g/cm3). However, in dry seeds, only 6% or less of the catalase activity was associated with these dense particles. In 4-day seedlings 80% of catalase activity was associated with glyoxysomes (1.24 g/cm3). A novel catalase 59 kDa subunit was found in the cytosol of 4-day seedlings and in isolated organelles from maturing and dry seed.
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Abbreviations
- AN:
-
acetonitrile
- CBBR:
-
Coomassie brilliant blue R-250
- HPLC:
-
high performance liquid chromatography
- SDS:
-
sodium dodecylsulfate
- PAGE:
-
polyacrylamide gel electrophoresis
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González, E., Harley, S.M. & Brush, M.D. Purification of glyoxysomal polypeptides. Protoplasma 156, 130–138 (1990). https://doi.org/10.1007/BF01560651
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DOI: https://doi.org/10.1007/BF01560651