Regulation of NAD- and NADP-dependent isocitrate dehydrogenases by reduction levels of pyridine nucleotides in mitochondria and cytosol of pea leaves

Abstract

Regulation of NAD- and NADP-dependent isocitrate dehydrogenases (NAD-ICDH, EC 1.1.1.41, and NADP-ICDH, EC 1.1.1.42) by the level of reduced and oxidized pyridine nucleotides has been investigated in pea (Pisum sativum L.) leaves. The affinities of mitochondrial and cytosolic ICDH enzymes to substrates and inhibitors were determined on partially purified preparations in forward and reverse directions. From the kinetic data, it follows that NADP(+)- and NAD(+)-dependent isocitrate dehydrogenases in mitochondria represent a system strongly responding to the intramitochondrial NADPH and NADH levels. The NADPH, NADP(+), NADH and NAD(+) concentrations were determined by subcellular fractionation of pea leaf protoplasts using membrane filtration in mitochondria and cytosol in darkness and in the light under saturating and limiting CO(2) conditions. The cytosolic NADPH/NADP ratio was about 1 and almost constant both in darkness and in the light. In mitochondria, the NADPH/NADP ratio was low in darkness (0.2) and increased in the light, reaching 3 in limiting CO(2) conditions compared to 1 in saturating CO(2). At high reduction levels of NADP and NAD observed at limiting CO(2) in the light, i.e. when photorespiratory glycine is the main mitochondrial substrate, isocitrate oxidation in mitochondria will be suppressed and citrate will be transported to the cytosol ('citrate valve'), where the cytosolic NADP-ICDH supplies 2-oxoglutarate for the photorespiratory ammonia refixation.

Peroxisomal Malate Dehydrogenase Is Not Essential for Photorespiration in Arabidopsis But Its Absence Causes an Increase in the Stoichiometry of Photorespiratory CO2 Release

“…This may suggest that the altered consumption of reductant (NADH) in the peroxisomes due to the lack of PMDH caused the NADH/NAD ratio in the mitochondria to increase. An increase in mitochondrial NADH/NAD has been demonstrated to inhibit the NAD-dependent isocitrate dehydrogenase (Igamberdiev and Gardestrom, 2003) and may explain the relatively large increase in isocitrate in the pmdh1pmdh2 plants. The increase in 2-oxoglutarate could potentially be attributed to an alteration in the Glu requirement for recycling nitrogen through the GS/GOGAT-catalyzed reactions.…”

In Vivo Respiratory Metabolism of Illuminated Leaves

“…In addition, the mitochondrial matrix in the light is supposed to be reduced because of the photorespiratory Gly decarboxylation that lead to a high NAD(P)H/NAD(P) 1 ratio. Some enzymes of the Krebs cycle are inhibited by the high NADH/NAD 1 ratios (for review, see Siedow and Day, 2000), and it has been recently found that isocitrate dehydrogenase from pea is inhibited by high NADPH/NADP 1 ratios, a feature that occurs in illuminated mitochondria (Igamberdiev and Gardeströ m, 2003).…”

“…Enzymes of the Krebs cycle are also assumed to be inhibited in the light because of a high mitochondrial NADH level due to photorespiratory Gly decarboxylation (Atkin et al, 2000). Additionally, it has been shown that the mitochondrial isocitrate dehydrogenase is inhibited by the high NADPH/NADP ratios that occur in the light (Igamberdiev and Gardeströ m, 2003).Although all these enzymatic data suggest that the respiratory pathway is down-regulated in the light regarding both glycolysis and the Krebs cycle, respiratory metabolic fluxes in vivo in leaves are not well known. Some labeling experiments with carbon isotopes ( 13 C or 14 C) have already been done to disentangle respiratory metabolic fluxes in vivo in the light and in the dark, but surprisingly, studies that have focused on labeling of the resulting respired CO 2 are scarce.…”

“…Enzymes of the Krebs cycle are also assumed to be inhibited in the light because of a high mitochondrial NADH level due to photorespiratory Gly decarboxylation (Atkin et al, 2000). Additionally, it has been shown that the mitochondrial isocitrate dehydrogenase is inhibited by the high NADPH/NADP ratios that occur in the light (Igamberdiev and Gardeströ m, 2003).…”

Respiratory metabolism of illuminated leaves depends on CO 2 and O 2 conditions

“…This acceptance is based on several strong lines of evidence, ranging from gas-exchange to molecular studies (for a review, see ref. 4): (i) the inhibition is thought to cause the light-enhanced dark respiration (5); (ii) the pyruvate dehydrogenase (PDH) is down-regulated in the light (6, 7); (iii) the metabolic flux through the TCA cycle in the light is reduced in both extracted mitochondria (8) and intact leaves (9,10); (iv) mitochondria experience high ATP/ADP and NADH/NAD ϩ ratios in the light that inhibit NAD-dependent isocitrate dehydrogenase (11); and (v) carbohydrate molecules such as sucrose (Suc) and glucose (Glc) are prevented from entering glycolysis (9), because of a modification of phosphofructokinase activity by the allosteric effector fructose (Fru)-2,6-bisphosphate (12). Nevertheless, not all leaf cells are photosynthetic (e.g., most epidermal cells, phloem, and xylem) so that some ''heterotrophic'' background respiration in the light is expected, but its contribution is minor.…”