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 x 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, dithiothreitol 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 optimal pH for activity from 7.5 to 9.0. The Km for the substrates DL-isocitrate and NADP+ were 13 and 12 microM, 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 turnover number and Mr of the ovarian enzyme were similar to those found for the enzyme from the liver and adrenal gland.
Jennings
G T
GT
Department of Biochemistry, University of Western Australia, Nedlands.
Sadleir
J W
JW
Stevenson
P M
PM
D000818:Animals; D015153:Blotting, Western; D002413:Cations, Divalent; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003338:Corpus Luteum / Q000201:enzymology*; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005990:Glycerol / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007521:Isocitrate Dehydrogenase / Q000302:isolation & purification* / Q000378:metabolism; D007523:Isocitrates / Q000494:pharmacology; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D008970:Molecular Weight; D009249:NADP / Q000494:pharmacology*; D011489:Protein Denaturation; D013552:Swine
D002413:Cations, Divalent; D007523:Isocitrates; D015636:Magnesium Chloride; D009249:NADP; C034219:isocitric acid; D007521:Isocitrate Dehydrogenase; D005990:Glycerol; D004229:Dithiothreitol