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Purification and Characterization of NAD-Isocitrate Dehydrogenase from Chlamydomonas reinhardtii1

José M. Martínez-Rivas2, * and José M. Vega

Instituto de Bioquímica Vegetal y Fotosíntesis, Centro de Investigaciones Isla de la Cartuja, Universidad de Sevilla-Consejo Superior de Investigaciones Científicas, Avenida Américo Vespucio s/n, 41092-Sevilla, Spain

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 Mn2+ protected the enzyme against thermal inactivation or inhibition by specific reagents for arginine or lysine. NADH was a competitive inhibitor (Ki, 0.14 mM) and NADPH was a noncompetitive inhibitor (Ki, 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.

1   This work was supported by research grant no. PB96-1367 from Dirección General de Investigación Científica y Técnica, Spain. J.M.M.-R. was the recipient of a postdoctoral contract from the Ministerio de Educación y Ciencia, Spain.
2   Present address: Institut für Allgemeine Botanik, Universität Hamburg, Ohnhorststrasse 18, 22609-Hamburg, Germany.
*   Corresponding author; e-mail mrivas{at}cica.es; fax 49-40-822-82-254.

Plant Physiol. (1998) 118: 249-255
Copyright Clearance Center:   0032-0889/98/118//07
© 1998 American Society of Plant Physiologists




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