LIPID BODY LIPOXYGENASE CHARACTERIZED BY PROTEIN FRAGMENTATION, CDNA SEQUENCE AND VERY EARLY EXPRESSION OF THE ENZYME DURING GERMINATION OFCUCUMBER SEEDS

Lipid bodies are cellular compartments containing triacylglycerols. Th ey are encompassed by a phospholipid monolayer and decorated with char acteristic proteins. In plants, lipid bodies are synthesized during se ed formation but acquire new proteins during seed germination. In germ inating cucumber (Cu-cumis sativus) seeds, the set of newly synthesize d proteins appealing in the lipid bodies at the early stage of triacyl glycerol mobilization comprises a special form of lipoxygenase. We iso lated the lipid body lipoxygenase and characterized fragments prepared by limited proteolysis and cleavage with cyanogen bromide. A very ear ly expression of lipid body lipoxygenase was found by studying the rat e of ne novo synthesis of lipoxygenase forms during germination. This allowed a clear distinction of this enzyme from other lipoxygenase iso forms. Hence, for determining the molecular structure of lipid body li poxygenase we analyzed a cDNA prepared from mRNA of cotyledons at day 1 of germination. From the cDNA sequence, oligonucleotides were derive d that specifically detected lipid body lipoxygenase mRNA on nothern b lots. The very early expression of lipid body lipoxygenase was corrobo rated by this approach. Good agreement was observed between the amino acid sequence deduced fi om the cDNA sequence and the peptide structur es analyzed biochemically. In particular, the cleavage products: of cy anogen bromide treatment indicated that we had isolated the lipid body lipoxygenase cDNA. The sequence data show a lipoxygenase form charact erized by a molecular mass of 99 655 Da, which is significantly higher than the molecular masses of the cytosolic forms. Compared to the cyt osolic forms that exhibit a molecular mass of 95 kDa, the lipid body f orm has an N-terminal extension of 34 amino acid residues. No evidence for a cotranslational or post-translational proteolytic processing wa s obtained by the size comparison of the in vitro-translated lipoxygen ase and the lipid body form.