PURIFICATION AND PROPERTIES OF NADP+-DEPENDENT ISOCITRATE DEHYDROGENASE FROM THE CORPUS-LUTEUM

Cytoplasmic NADP+-dependent isocitrate dehydrogenase (isocitrate: NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42) was purified 290-fold from the 15 000 × g supernatant fraction of porcine corpora lutea. The major purification step was by anion-exchange chromatography with an FPLC mono P column. Enzyme lability was overcome by including Mg2+, dl-isocitrate, dithiotheritol and glycerol in the elution buffers. The molecular weight of the denatured enzyme was found to be 48 000 by SDS-polyacrylamide gel electrophoresis. The Stokes' radius was estimated to be 3.7 nm by gel filtration and the isoelectric point was 4.8 as determined by chromatofocusing. The purified enzyme had a specific activity of 57.8 units/mg and a broad optima pH for activity from 7.5 to 9.0. The Km for the substrates dl-isocitrate and NADP2+ were 13 and 12 μM, respectively. Polyclonal antibodies were raised against the purified enzyme. Protein (Western) blotting showed an immunological similarity between the cytoplasmic enzyme of the ovary, liver, adrenal gland and heart. A difference was demonstrated between the ovarian enzyme and the heart mitochondrial enzyme. The substrate turnouver number and Mr of the ovarian enzyme were similar to those found for the enzyme from the liver and adrenal gland. © 1990.