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Journal of Histochemistry and Cytochemistry, Vol. 49, 1123-1132, September 2001, Copyright © 2001, The Histochemical Society, Inc.

ARTICLE

Localization of Cytosolic NADP-dependent Isocitrate Dehydrogenase in the Peroxisomes of Rat Liver Cells: Biochemical and Immunocytochemical Studies

Takashi Yoshiharaa, Tatsuhiko Hamamotob, Ryo Munakatab, Ryosuke Tajirib, Mariko Ohsumia, and Sadaki Yokotab
a Department of Bioscience, Faculty of Science and Engineering, Teikyo University of Science and Technology, Yamanashi, Japan
b Biological Laboratory, Yamanashi Medical University, Yamanashi, Japan

Correspondence to: Sadaki Yokota, Biological Laboratory, Yamanashi Medical University, Tamaho-cho, Yamanashi, 409-3898, Japan.

Two types of NADP-dependent isocitrate dehydrogenases (ICDs) have been reported: mitochondrial (ICD1) and cytosolic (ICD2). The C-terminal amino acid sequence of ICD2 has a tripeptide peroxisome targeting signal 1 sequence (PTS1). After differential centrifugation of the postnuclear fraction of rat liver homogenate, approximately 75% of ICD activity was found in the cytosolic fraction. To elucidate the true localization of ICD2 in rat hepatocytes, we analyzed the distribution of ICD activity and immunoreactivity in fractions isolated by Nycodenz gradient centrifugation and immunocytochemical localization of ICD2 antigenic sites in the cells. On Nycodenz gradient centrifugation of the light mitochondrial fraction, ICD2 activity was distributed in the fractions in which activity of catalase, a peroxisomal marker, was also detected, but a low level of activity was also detected in the fractions containing activity for succinate cytochrome C reductase (a mitochondrial marker) and acid phosphatase (a lysosomal marker). We have purified ICD2 from rat liver homogenate and raised a specific antibody to the enzyme. On SDS-PAGE, a single band with a molecular mass of 47 kD was observed, and on immunoblotting analysis of rat liver homogenate a single signal was detected. Double staining of catalase and ICD2 in rat liver revealed co-localization of both enzymes in the same cytoplasmic granules. Immunoelectron microscopy revealed gold particles with antigenic sites of ICD2 present mainly in peroxisomes. The results clearly indicated that ICD2 is a peroxisomal enzyme in rat hepatocytes. ICD2 has been regarded as a cytosolic enzyme, probably because the enzyme easily leaks out of peroxisomes during homogenization. (J Histochem Cytochem 49:1123–1131, 2001)

Key Words: cytosolic NADP-dependent, isocitrate dehydrogenase, peroxisomes, Nycodenz gradient, centrifugation, immunoelectron microscopy


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