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Purification of plant peroxisomes in iso‐osmotic metrizamide
Author(s) -
Turley Rickie B.,
Trelease Richard N.
Publication year - 1990
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1111/j.1399-3054.1990.tb02119.x
Subject(s) - endosperm , metrizamide , peroxisome , biology , spinach , organelle , chlorophyll , biochemistry , cotyledon , botany , myelography , neuroscience , gene , spinal cord
Methods were developed for isolating highly‐purified peroxisomes under iso‐osmotic conditions from 3 plant parts, namely cotyledons of cotton ( Gossypium hirsutum L.) seedlings, endosperm of castor bean ( Ricinus communis L.) seedlings and leaves of mature spinach ( Spinaca oleracea L.) plants. Purification was achieved by sedimentation of the organelles into metrizamide gradients centrifuged in a vertical rotor (VTi 50). Gradients consisted of an upper transition layer (1:1 mixture of homogenizing medium and 0.25 M metrizamide), a linear 0.25–0.76 M metrizamide gradient and a 0.76 M metrizamide pad. Peroxisomes from all 3 plant parts were recovered in a major band at a density ranging from 1.24 to 1.27 g cm −3 , which is a density range similar to that for peroxisomes isolated in sucrose gradients. The percent of the total gradient cytochrome c oxidase (mitochondria marker) activity recovered in peroxisome fractions ranged from 1.5% in endosperm to 2.8% in leaves, while a plastid marker (chlorophyll or galactosyl transferase activity) ranged from undetectable in leaf peroxisome fractions to 3.6% in endosperm peroxisome fractions. Intactness of the peroxisomes was judged to be 69%, 89% and 78% for the cotyledon, endosperm and leaf peroxisomes, respectively. Isolated peroxisomes were stable for at least 5 h in metrizamide medium. Microscopic (bright‐field and transmission electron microscopy) assessments verified that the peroxisomes were morphologically intact and fractions were essentially free of contaminating organelles. Metrizamide is an excellent iso‐osmotic medium for purifying peroxisomes from these plant organs and tissue.