Peroxisomal NADP-Dependent Isocitrate Dehydrogenase. Characterization and Activity Regulation during Natural Senescence

Abstract

The peroxisomal localization and characterization of NADP-dependent isocitrate dehydrogenase (perICDH) in young and senescent pea (Pisum sativum) leaves was studied by subcellular fractionation, kinetic analysis, immunoblotting, and immunoelectron microscopy. The subunit molecular mass for perICDH determined 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 (pIs) of 6.0, 5.6, 5.4, and 5.2, respectively. The kinetic properties of the NADP-ICDH in peroxisomes from young and senescent pea leaves were analyzed. The maximum initial velocity was the same in peroxisomes from young and senescent leaves, while the Michaelis constant value in senescent leaf peroxisomes was 11-fold lower than in young leaf peroxisomes. The protein levels of NADP-ICDH in peroxisomes were not altered during senescence. The kinetic behavior of this enzyme suggests a possible fine control of enzymatic activity by modulation of its Michaelis constant during the natural senescence of pea leaves. After embedding, electron microscopy immunogold labeling of NADP-ICDH confirmed 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-utilization in the peroxisomal metabolism.

Figure 1

Purification of peroxisomes from pea leaves. Cell organelles were purified from 50-d-old pea leaves by differential and Suc density-gradient centrifugations, as described by López-Huertas and co-workers (1995). Gradient fractions of 1.5 mL were eluted with a gradient fractionator and assayed for specific marker enzymes to localize cell organelles in the gradient: fumarase for mitochondria and catalase for peroxisomes. Catalase and fumarase activities are expressed in μmol min⁻¹ mL⁻¹ and NADP-ICDH activity in milliunits mL⁻¹. Proteins were expressed as mg mL⁻¹ and density as g cm⁻³. Top: ●, Proteins; ○, density. Middle: ●, Catalase; ○, fumarase. Bottom: NADP-IDH.

Figure 2

Western-blot analysis of peroxisomes from senescent pea leaves. Samples were subjected to SDS-PAGE and then transferred to polyvinylidene difluoride membranes and incubated with a polyclonal antibody against pea NADP-ICDH (1/4,000 dilution). Lane a, Crude extract of pea leaves (50 μg of protein); lane b, matrices of pea leaf peroxisomes (45 μg of protein). Molecular mass standards are indicated on the left in kD.

Figure 3

Effect of 2R,3S-isocitrate concentration on the NADP-ICDH activity of leaf peroxisomes from young (●) and senescent (○) pea leaves. The bottom panel shows the Lineweaver-Burk plot of the kinetic data. mU, Milliunits.

Figure 4

Western-blot analysis of NADP-ICDH protein in peroxisomes from young and senescent pea leaves. The western-blot conditions were identical to those described in Figure 2. The upper panel shows the quantification of NADP-ICDH levels by scanning densitometry at 560 nm expressed as arbitrary absorbance units. The lower panel shows an immunoblot indicating the NADP-ICDH protein levels.

Figure 5

Densitograms of NADP-ICDH isoforms in peroxisomes from young (Y) and senescent (S) pea leaves. Samples of peroxisomal matrices (200 μg of protein) were subjected to IEF in a pH gradient of 3.5 to 7.0. NADP-ICDH isoforms were identified by activity staining, and gels were scanned at 560 nm. 1, ICDH-1; 2, ICDH-2; 3, ICDH-3; 4, ICDH-4.

Figure 6

EM immunocytochemical localization of NADP-ICDH in pea leaves. The electron micrographs are representative of thin sections of pea leaves. Cell sections were probed with preimmune serum (dilution 1:500) (A). Immunogold labeling with anti-pea NADP-ICDH (dilution 1:500) was carried out in young (B) and senescent (C and D) pea leaves. Arrows indicate 15-nm gold particles. CH, Chloroplast; CW, cell wall; M, mitochondrion; P, peroxisome; CY, cytosol. Bars = 0.5 μm.