Localization of mitochondrial NADP-isocitrate dehydrogenase in rat heart. A biochemical and immunocytochemical study

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It is known that there are three different types of NADP-isocitrate dehydrogenases in mammalian tissues. One forms a heteropolymer and distributes in mitochondria (MT), and the other form a homodimer and localize in the MT (ICD1) or in the cytosol (ICD2). In previous study, we have shown that in rat liver and kidney ICD2 is localized exclusively in peroxisomes (PO). The result is consistent with the fact that ICD2 has a peroxisomal targeting signal 1 (PTS1) sequence (AKL) at the C-terminus. Since it is difficult to isolate PO by differential centrifugation, some of the enzymes that were regarded as mitochondrial localization have been shown to be present in the PO. In present study, we investigated the subcellular localization of ICD1 using biochemical and immunocytochemical methods. Rabbit antibody to peptide consisting of 14 amino acids at the C-terminus of mouse ICD1 was prepared. Immunoblot analysis showed that the antibody reacts with 51 kDa and 44 kDa bands and that ICD1 was expressed highly in heart and kidney of rat. Immunofluorescence staining visualized a typical mitochondrial pattern in rat heart. Immunoelectron microscopy showed that signals for ICD1 were present exclusively in MT. Double labeling using anti-catalase antibody revealed that PO is negative for ICD1. The results clearly indicate that ICD1 is localized only in the MT.