Abstract
Castor bean endosperm contains a well known acid lipase activity that is associated with the oil body membrane. In order to identify this enzyme, proteomic analysis was performed on purified oil bodies. A approximately 60-kDa protein was identified (RcOBL1), which shares homology with a lipase from the filamentous fungus Rhizomucor miehei. RcOBL1 contains features that are characteristic of an alpha/beta-hydrolase, such as a putative catalytic triad (SDH) and a conserved pentapeptide (GXSXG) surrounding the nucleophilic serine residue. RcOBL1 was expressed heterologously in Escherichia coli and shown to hydrolyze triolein at an acid pH (optima approximately 4.5). RcOBL1 can hydrolyze a range of triacylglycerols but is not active on phospholipids. The activity is sensitive to the serine reagent diethyl p-nitrophenyl phosphate, indicating that RcOBL1 is a serine esterase. Antibodies raised against RcOBL1 were used to show that the protein is restricted to the endosperm where it is associated with the surface of oil bodies. This is the first evidence for the molecular identity of an oil body-associated lipase from plants. Sequence comparisons reveal that families of OBL1-like proteins are present in many species, and it is likely that they play an important role in regulating lipolysis.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY360218, AY360219, AY360220, and AY360221
Antibodies raised against RcOBL1 were used to show that the protein is restricted to the endosperm where it is associated with the surface of oil bodies
Sequence comparisons reveal that families of Oil Body Lipase 1 (OBL1)-like proteins are present in many species, and it is likely that they play an important role in regulating lipolysis
Summary
Castor bean endosperm contains a well known acid lipase activity that is associated with the oil body membrane. The activities are only detectable upon germination and increase concomitantly with the disappearance of TAG (3) These lipase activities are usually membrane-associated and can be found in the oil body, glyoxysome, or microsomal fractions of seed extracts, depending upon the species (3). The enzyme is unusual in that it is extremely active in mature seeds (prior to germination) and has a low pH optima of ϳ4.2 It is associated with the oil body membrane (12), and its catalytic properties are quite well defined (13, 14) and broadly similar to lipases from mammals and fungi. Altaf et al (15) raised an antibody against a major oil body membrane protein of similar molecular weight and showed that it could immunoprecipitate acid lipase activity. The castor acid lipase exhibits homology with several fungal lipases, and orthologs are present in other plant species, defining a new family of putative oil body-associated lipases
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