Abstract
Isocitrate lyase (EC 4.1.3.1) was purified from the pollen of Pinus densiflora Sieb. et Zucc. to apparent homogeneity as judged by SDS-PAGE. The molecular weight of the enzyme was 200, 000 by glycerol density gradient analysis and gel filtration on Sepharose 6B, and the subunit molecular weight was 65, 000 by SDS-PAGE. The enzyme was maximumally active at pH 7.6 in the presence of 3 mM MgCl2 and 0.5 mM EDTA. The enzyme was completely inactivated by heating at 50°C for 5 min, but no activity was lost by the same treatment in the presence-of 3 mM Mg2+ and 0.5 mM EDTA. The enzyme was inhibited by PCMB, and the inhibition was reversed by the addition of an equimolar amount of cysteine. ATP, ADP, 3-phosphoglycerate, phosphoenolpyruvate, tartrate, maleate, oxalate, and itaconate inhibited the enzyme. The Km value for DL-isocitrate was 6.6 × 10-4 M. Itaconate was an uncompetitive inhibitor with respect to isocitrate, and the Ki value for itaconate was 2.8 × 10-6 M.
