Immunological investigation of lipases in germinating oilseed rape, Brassica napus
1989
Murphy, D.J. | Cummins, I. | Kang, A.S.
An anti-lipase antibody has been used to investigate the lipases of cotyledons from germinating seedlings of oilseed rape, Brassica napus L, var Mikado. Immunological and enzymic assays revealed that lipases were confined to germinating seeds and were absent from other tissues, such as developing seeds, leaves, roots and flowers. The antibody totally inhibited microsomal lipase activity at a ratio of 12 micrograms IgG protein: 10 micrograms microsomal protein. It was shown by immunoblotting of microsomal, oil-body and total cotyledon proteins separated by gel electrophoresis that, in each case, the antibody specifically bound to a single polypeptide. This polypeptide had an electrophoretic mobility consistent with a molecular weight of 56 kDa. The appearance and subsequent decline of the 56 kDa polypeptide during the first 10 days of germination closely followed that of the lipase activity, both in microsomal protein and total cotyledon protein fractions. An ELISA for the lipase demonstrated that the amount of immunoreactive microsomal lipase present at each stage of germination was proportional to its enzymic activity. The enzymic and immunological activities of the microsomal lipase each exhibited bouyant densities of 1.087 g ml-1 on sucrose density gradients. Lipases were present in only very small amounts in dry seeds and were synthesized de novo after 2-3 days of germination.
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