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NAD+-Linked Isocitrate Dehydrogenase: Isolation, Purification, and Characterization of the Protein from Pea Mitochondria
Plant Physiology (1992)
  • Cecilia McIntosh, University of Idaho
  • David J. Oliver, University of Idaho
Abstract
The NAD+-dependent isocitrate dehydrogenase from etiolated pea (Pisum sativum L.) mitochondria was purified more than 200-fold by dye-ligand binding on Matrix Gel Blue A and gel filtration on Superose 6. The enzyme was stabilized during purification by the inclusion of 20% glycerol. In crude matrix extracts, the enzyme activity eluted from Superose 6 with apparent molecular masses of 1400 ± 200, 690 ± 90, and 300 ± 50 kD. During subsequent purification steps the larger molecular mass species disappeared and an additional peak at 94 ± 16 kD was evident. The monomer for the enzyme was tentatively identified at 47 kD by sodium dodecyl-polyacrylamide gel electrophoresis. The NADP+-specific isocitrate dehydrogenase activity from mitochondria eluted from Superose 6 at 80 ± 10 kD. About half of the NAD+ and NADP+-specific enzymes remained bound to the mitochondrial membranes and was not removed by washing. The NAD+-dependent isocitrate dehydrogenase showed sigmodial kinetics in response to isocitrate (S0.5 = 0.3 mm). When the enzyme was aged at 4°C or frozen, the isocitrate response showed less allosterism, but this was partially reversed by the addition of citrate to the reaction medium. The NAD+ isocitrate dehydrogenase showed standard Michaelis-Menten kinetics toward NAD+ (Km = 0.2 mm). NADH was a competitive inhibitor (Ki = 0.2 mm) and, unexpectedly, NADPH was a noncompetitive inhibitor (Ki = 0.3 mm). The regulation by NADPH may provide a mechanism for coordination of pyridine nucleotide pools in the mitochondria.
Keywords
  • characterization,
  • isocitrate dehydrogenase,
  • isolation,
  • NAD-linked,
  • pea mitochondria,
  • pisum sativum,
  • protein,
  • purification
Publication Date
September 1, 1992
DOI
10.1104/pp.100.1.69
Citation Information
Cecilia McIntosh and David J. Oliver. "NAD+-Linked Isocitrate Dehydrogenase: Isolation, Purification, and Characterization of the Protein from Pea Mitochondria" Plant Physiology Vol. 100 Iss. 1 (1992) p. 69 - 75 ISSN: 1532-2548
Available at: http://works.bepress.com/cecilia-mcintosh/32/