Subcellular Location of NADP⁺-Isocitrate Dehydrogenase in Pisum sativum Leaves

Abstract

The subcellular location of NADP(+)-isocitrate dehydrogenase was investigated by preparing protoplasts from leaves of pea seedlings. Washed protoplasts were gently lysed and the whole lysate separated on sucrose gradients by a rate-zonal centrifugation. Organelles were located by marker enzymes and chlorophyll analysis. Most of the NADP(+)-isocitrate dehydrogenase was in the soluble fraction. About 10% of the NADP(+)-isocitrate dehydrogenase was present in the chloroplasts as a partially latent enzyme. Less than 1% of the activity was found associated with the peroxisome fraction. NADP(+)-isocitrate dehydrogenase was partially characterized from highly purified chloroplasts isolated from shoot homogenates. The enzyme exhibited apparent K(m) values of 11 micromolar (NADP(+)), 35 micromolar (isocitrate), 78 micromolar (Mn(2+)), 0.3 millimolar (Mg(2+)) and showed optimum activity at pH 8 to 8.5 with Mn(2+) and 8.8 to 9.2 with Mg(2+). The NADP(+)-isocitrate dehydrogenase activity previously claimed in the peroxisomes by other workers is probably due to isolation procedures and/or nonspecific association. The NADP(+)-isocitrate dehydrogenase activity in the chloroplasts might help supply alpha-ketoglutarate for glutamate synthase action.