RESOLUTION AND PURIFICATION OF AN ALDEHYDE-GENERATING AND AN ALCOHOL-GENERATING FATTY ACYL-COA REDUCTASE FROM PEA LEAVES (PISUM-SATIVUM L)

Higher plant tissues produce both wax esters generated from fatty alco hols and hydrocarbons generated from fatty aldehydes. If two different reductases are responsible for the synthesis of aldehydes and alcohol s, both types of reductases may be present in such tissues. To test fo r this possibility, pea leaves, known to produce both types of wax com ponents, were examined. Subcellular fractionation showed that acyl-CoA reductase activities were localized mainly in the microsomal fraction . Fatty aldehyde formation was rectilinear for 30 min and subsequently decreased, whereas fatty alcohol formation remained linear for 2 h, T he two activities in the microsomes were differently affected by pH; a lcohol formation was optimal between pH 5 and pH 6, whereas aldehyde f ormation was optimal at around pH 7.5. With solubilized microsomes, pr otein concentration dependence of alcohol formation showed a sigmoidal pattern, possibly suggesting inhibition by hexadecanoyl-CoA at low pr otein concentrations. Bovine serum albumin (BSA) enhanced alcohol form ation. In contrast, the aldehyde generation showed a typical protein c oncentration dependence, and BSA severely inhibited aldehyde generatio n. Phosphatidylcholine showed over twofold stimulation for alcohol for mation, whereas aldehyde formation was only slightly stimulated. All o f this biochemical evidence suggested the presence of two different re ductases. Confirming this hypothesis, an aldehyde-generating and an al cohol-generating reductase were resolved from the solubilized microsom al proteins using flue A agarose, gel filtration, and hexadecanoyl-CoA affinity chromatography. SDS-PAGE of the purified proteins showed tha t the alcohol-generating enzyme was a 58-kDa protein and the aldehyde- forming one was a 28-kDa protein. Ii is proposed that two different el ongating systems are functionally coupled to the alcohol-genand aldehy de-generating reductases, which in turn are coupled to the transacylas e to produce wax esters and to the decarbonylase to produce hydrocarbo ns, respectively. (C) 1997 Academic Press.