Intracellular location of oleate desaturase and associated constituents in developing sunflower (Helianthus annuus) seeds

The location of sunflower seed oleate desaturase (ODS; EC 1.3.1.35) activity and its putative component cytochrome b has been investigated in fractions obtained by isopycnic centrifugation of seed homogenates prepared in the absence and in the presence of added Mg2+ ions. Electron microscopy showed that the addition of mM Mg2+ resulted in maximal retention of ribosome association with the separated microsomes, whilst omission of this ion from the homogenisation medium yielded preparations containing denuded vesicles. Both ODS activity and cytochrome b were present in the fractions containing smooth vesicles which co-sedimented with the ER marker enzyme NCR, in fractions of density 1.09 g ml(-1) in the latter and with vesicles having a slightly less than maximal population of associated ribosomes, sedimenting at a density of 1.18 g ml(-1), in the former preparations. Whilst the enzymes LPCAT and CPT, which may be concerned in the formation of the phosphatidylcholine substrate of ODS, co-sedimented with the desaturase enzyme in the smooth microsomal preparations of the homogenates prepared in the absence of Mg2+ addition of this ion resulted in preparations showing a polydisperse distribution of both enzymes and the marker NCR in which negligible CPT activity and 38% of the LPCAT activity was present in the fractions containing ODS activity.