Peroxisomal NADP-dependent isocitrate dehydrogenase. Characterization and activity regulation during natural senescence

The peroxisomal localization and characterization of NADP-dependent isocitr ate dehydrogenase (perlCDH) in young and senescent pea (Pisum sativum) leav es was studied by subcellular fractionation, kinetic analysis, immunoblotti ng, and immunoelectron microscopy. The subunit molecular mass for perlCDH d etermined by immunoblotting was 46 kD. By isoelectric focusing (IEF) of the peroxisomal matrix fraction, the NADP-ICDH activity was resolved into four isoforms, perICDH-1 to perICDH-4, with isoelectric points (pls) of 6.0, 5. 6, 5.4, and 5.2, respectively. The kinetic properties of the NADP-ICDH in p eroxisomes from young and senescent pea leaves were analyzed. The maximum i nitial velocity was the same in peroxisomes from young and senescent leaves , while the Michaelis constant value in senescent leaf peroxisomes was Ii-f old lower than in young leaf peroxisomes. The protein levels of NADP-ICDH i n peroxisomes were not altered during senescence. The kinetic behavior of t his enzyme suggests a possible fine control of enzymatic activity by modula tion of its Michaelis constant during the natural senescence of pea leaves. After embedding, electron microscopy immunogold labeling of NADP-ICDH conf irmed that this enzyme was localized in the peroxisomal matrix. Peroxisomal NADP-ICDH represents an alternative dehydrogenase in these cell organelles and may be the main system for the reduction of NADP to NADPH for its re-u tilization in the peroxisomal metabolism.