Purification and Characterization of NAD-Isocitrate Dehydrogenase from Chlamydomonas reinhardtii1

Abstract

NAD-isocitrate dehydrogenase (NAD-IDH) from the eukaryotic microalga Chlamydomonas reinhardtii was purified to electrophoretic homogeneity by successive chromatography steps on Phenyl-Sepharose, Blue-Sepharose, diethylaminoethyl-Sephacel, and Sephacryl S-300 (all Pharmacia Biotech). The 320-kD enzyme was found to be an octamer composed of 45-kD subunits. The presence of isocitrate plus Mn 2؉ protected the enzyme against thermal inactivation or inhibition by specific reagents for arginine or lysine. NADH was a competitive inhibitor (K i , 0.14 mM) and NADPH was a noncompetitive inhibitor (K i , 0.42 mM) with respect to NAD ؉ . Citrate and adenine nucleotides at concentrations less than 1 mM had no effect on the activity, but 10 mM citrate, ATP, or ADP had an inhibitory effect. In addition, NAD-IDH was inhibited by inorganic monovalent anions, but L-amino acids and intermediates of glycolysis and the tricarboxylic acid cycle had no significant effect. These data support the idea that NAD-IDH from photosynthetic organisms may be a key regulatory enzyme within the tricarboxylic acid cycle.

A unique homodimeric NAD + ‐linked isocitrate dehydrogenase from the smallest autotrophic eukaryote Ostreococcus tauri

“…With respect to NAD + , NADH was a competitive inhibitor ( K i = 0.14 ± 0.01 mM) of OtIDH ( Fig. 7), as was also observed for C. reinhardtii NAD‐IDH, and NADPH had no effect on OtIDH activity (data not shown), although it was a noncompetitive inhibitor of C. reinhardtii NAD‐IDH (2). In addition, 1 mM citrate had almost no effect on OtIDH activity (Table 4 ) , but it significantly inhibited the activity of C. reinhardtii NAD‐IDH (2).…”

“…I socitrate dehydrogenase (IDH) is a key enzyme in the Krebs cycle that catalyzes the oxidative decarboxylation of isocitrate to yield α‐ketoglutarate coupled with the production of NAD(P)H (1). Eukaryotes usually possess a NAD + ‐dependent IDH (NAD‐IDH, EC 1.1.1.41) that is strictly mitochondrial and several types of NADP + ‐dependent IDH (NADP‐IDH, EC 1.1.1.42) isoforms that are distributed in the mitochondrial matrix, cell cytosol, and peroxisome (2–5). Mitochondrial NAD‐IDHs are believed to be heteromeric in solution, such as the hetero‐octameric yeast NAD‐IDH, composed of 4 heterodimers of regulatory IDH1 and catalytic IDH2 subunits (6), and the heterotetrameric human NAD‐IDH, with 3 types of subunits present in the ratio of 2α:1β:1γ (7–9), whereas most prokaryotic NAD‐IDHs are dimeric, such as NAD‐IDH from Acidithiobacillus thiooxidans (10) and Streptococcus mutans (11).…”

“…Nuclear genome analysis of O. tauri has revealed several unique features, including its heterogeneity with 2 structurally different chromosomes and the intense degree of genome compaction (19). Interestingly, the O. tauri genome contains only one icd gene, whereas another unicellular green algal species, Chlamydomonas reinhardtii , possesses at least 3 icd genes encoding a putative NADP‐IDH and 2 putative subunits of NAD‐IDH, an octamer composed of 45 kDa subunits (2). C. reinhardtii belongs to the genus Chlamydomonas (Chlorophyceae), and O. tauri is, A. member of the genus Ostreococcus (Prasinophyceae).…”

Metabolic Flux-Based Modularity using Shortest Retroactive distances

“…Oxaloacetate is a limiting substrate for citrate synthase, and also a competitive inhibitor of oxidative phosphorylation [24]. Reactions R34 (pyruvate dehydrogenase/citrate synthase), R36 (isocitrate dehydrogenase) and R41 (malate dehydrogenase) are steps in the TCA cycle regulated by ATP, which could explain the robustness of interactions between reaction pairs [R34, R36] and [R34, R41] [23,25,26]. …”

Mitochondrial Ca2+ concentrations in live cells: quantification methods and discrepancies

“…NAD‐ICDH is a hetero‐octamer of 2× (2α,β,γ) subunits which catalyzed the oxidation of isocitrate to 2‐ketoglutarate and CO 2 . This enzyme, located exclusively in the mitochondria in mammalian cells, is a key regulatory enzyme in the TCA cycle.…”