87] Reconstitution of mitochondrial nicotinamide nucleotide transhydrogenase from beef heart with synthetic phospholipids

Abstract

Nicotinamide nucleotide transhydrogenase from beef heart mitochondria has been purified in a reconstitutively active form. Reconstitution of the enzyme is accomplished by incorporating the enzyme into liposomes, which catalyze an uncoupler-sensitive NAD + plus NADPH-dependent uptake of lipophilic anions, for example, tetraphenylboron. The uptake of tetraphenylboron, which has been proposed to reflect the generation of a transmembrane potential, is dependent on the phospholipid composition of the liposomes; maximal uptake is achieved with mitochondrial lecithin. To improve the reproducibility of the reconstitution procedure, synthetic lecithins have been introduced. The most suitable lecithin appears to be dioleoyl-L-α-phosphatidylcholine, which gives at least a two-fold increase in sensitivity. The procedure is reproducible and is the most sensitive assay available for the function of reconstituted transhydrogenase. The reconstitution conditions are applicable to both partially purified transhydrogenase and pure transhydrogenase.