Purification and characterization of oil-bodies (oleosomes) and oil-body boundary proteins (oleosins) from the developing cotyledons of sunflower (Helianthus annuus L.)

Abstract

Oil-bodies, from the immature cotyledons of sunflower (Helianthus annuus L.), were difficult to purify to homogeneity using conventional techniques. The major protein contaminants were albumin and globulin storage proteins. A protocol has been developed, therefore, based upon the stringent washing of the oil-body fraction in 9 M urea, which effectively removed almost all the contaminating protein as judged by SDS/PAGE. The urea-washed oil-bodies were enriched in two major proteins of M(r) 19000 and 20000. These proteins were oleosins as demonstrated by their amino acid compositions and the sequence analysis of peptides produced by CNBr cleavage. Far-UV CD spectra of the oleosins in trifluoroethanol, trifluoroethanol/water mixtures and as mixed micelles in SDS, were typical of alpha-helical proteins with alpha-helical contents of some 55%. The phospholipid content of the urea-washed preparations was less than 0.1% of that required to form a half-unit membrane surrounding the oil-body. The oil-body surface therefore appears to be an unusual and novel structure, covered largely by an oleosin protein coat or pellicle rather than a conventional fluid membrane, half-unit or otherwise.

Neutral Lipid Bodies in Prokaryotes: Recent Insights into Structure, Formation, and Relationship to Eukaryotic Lipid Depots

“…Isolated TAG bodies from R. opacus and R. ruber exhibited a protein pattern strongly resembling the protein pattern of total cell lysates, indicating that a large number of unspecifically bound proteins were copurified (72). This effect is similar to the situation described for oil bodies from plant seeds, which also always contain contaminants of unspecifically bound proteins (95,133). TAG body-associated proteins were divided into three groups, reflecting their binding properties: (i) probably unspecifically bound proteins by ionic interactions, which could be solubilized by salt treatment or mild detergents; (ii) tightly bound proteins which could be solubilized by treatment with chaotropic reagents; and finally, (iii) extremely tightly bound proteins which even resisted treatment with up to 8% sodium dodecyl sulfate or proteolytic digestion, and which could be only solubilized by sodium dodecyl sulfate plus heat treatment.…”

Integral Proteins in Plant Oil Bodies

“…In contrast, the central anchoring domain of oleosins is highly conserved among diverse species, particularly in a relatively hydrophilic proline knot motif at the middle of the sequence [60][61][62][63]. To date, controversial secondary structures of this domain have been proposed or determined in the past two decades, yet the contents of α-helical and β-stranded structures in these proposed models are extremely different [41,[64][65][66][67][68][69][70][71][72]. The controversy among these proposed structures does not seem to be receded unless a convincing three-dimensional structure of oleosin or at least its central hydrophobic domain is resolved.…”

Cloning and Characterization of the Acid Lipase from Castor Beans

“…To identify this enzyme, oil bodies were isolated from the endosperm of castor beans by floatation centrifugation (16) and stripped of peripheral proteins by washing sequentially with 2 M NaCl 2 and then 9 M urea (17). The polypeptides that are associated with the oil body membrane were solubilized in SDS loading buffer and separated by SDS-PAGE (Fig.…”