Studies on membrane-associated and soluble enoyl reductases from oil seed rape (Brassica napus)

Abstract

Three forms of enoyl reductase have been identified m Brassica napus seeds, one soluble, which is involved in the synthesis of fatty acids up to CI8, and two membrane- associated forms, which elongate VLCFA's. NADH-utilising membrane-associated and soluble enoyl reductases have been differentiated by their pH profiles and the feet that they are immunologically distinct. Despite their differing properties, both enzymes reduce C20:2-CoA[t2, c11] with NADH and so adequate controls are essential to remove contaminating soluble enoyl-ACP reductase from microsomes when studying the membrane-associated enzyme. The importance of these controls and extensive washing procedures to remove such contamination have not previously been stressed m the literature. In addition to the NADH membrane-associated enoyl reductase, a NADPH- utilising form also exists and these two enzymes have been differentiated by their thermal stabilities. Several methods were attempted to solubilise the membrane-associated enoyl reductases. Initial observations indicating their solubilisation by LDAO were later proved to be due to contaminating soluble enoyl-ACP reductase, again highlighting the importance of appropriate controls. Solubilisation of both forms was achieved using guanidinium thiocyanate, which is a inhibitor of soluble enoyl-ACP reductase. The solubilised enzymes were unusually stable at high temperature but were shown to be proteinaceous and to have molecular weights of greater than 30 kDa. Attempts to obtain a cDNA encoding a membrane-associated enoyl reductase using a probe from a soluble enoyl-ACP reductase cDNA were unsuccessful. However, a cDNA encoding the soluble enzyme was isolated which showed high homology in the open reading frame to, but substantial differences m the 5' and 3' non-translated regions from, the previously isolated cDNA, and so represents a differently regulated enoyl-ACP reductase.