NAD(P)(-DEPENDENT ISOCITRATE DEHYDROGENASES IN MITOCHONDRIA PURIFIED FROM PICEA-ABIES SEEDLINGS())

Isocitrate dehydrogenase (IDH) activities were measured in mitochondri a isolated from aerial parts of 21-day-old spruce (Picea abies L. Kars t.) seedlings. Mitochondria were purified by two methods, involving co ntinuous and discontinuous Percoll gradients. Whatever the method of p urification, the mitochondrial outer membrane was about 69% intact, an d the mitochondria contained very low cytosolic, chloroplastic and per oxisomal contaminations. Nevertheless, as judged by the recovery of fu marase activity, purification on a continuous 28% Percoll gradient gav e the best yield in mitochondria, which exhibited a high degree of inn er membrane intactness (91%). The purified mitochondria oxidized succi nate and malate with good respiratory control and ADP/O ratios. The hi ghest oxidation rate was obtained with succinate as substrate, and mal ate oxidation was improved (+ 60%) by addition of exogenous NAD(+). Ex periments using standard respiratory chain inhibitors indicated that, in spruce mitochondria, the alternative pathway was present. Both NAD( +)-isocitrate dehydrogenase (EC 1.1.1.41) and NADP(+)-isocitrate dehyd rogenase (EC 1.1.1.42) were present in the mitochondrial matrix fracti on, and NAD(+)-IDH activity was about 2-fold higher than NADP(+)-IDH a ctivity. The NAD(+)-IDH showed sigmoidal kinetics in response to isoci trate and standard Michaelis-Menten kinetics for NAD(+) and Mg2+. The NADP(+)-IDH, in contrast, displayed lower K-m values. For NAD(+)-IDH t he pH optimum was at 7.4, whereas NADP(+)-IDH exhibited a broad pH opt imum between 8.3 and 9. In addition, NAD(+)-IDH was more thermolabile. Adenine nucleotides and 2-oxoglutarate were found to inhibit NAD(P)()-IDH activities only at high concentrations.