Purification, properties and enhanced expression under nitrogen starvation of the NADP +-isocitrate dehydrogenase from the cyanobacterium Phormidium laminosum

• Published in: Biochimica et biophysica acta 1999-04, Vol.1431 (1), p.87-96
• Main Authors: Pardo, Miguel A, Llama, Marı́a J, Serra, Juan L
• Format: Article
• Language: English
• Subjects: Cyanobacteria - enzymology; Cyanobacterium; Hydrogen-Ion Concentration; Isocitrate dehydrogenase; Isocitrate Dehydrogenase - biosynthesis; Isocitrate Dehydrogenase - chemistry; Isocitrate Dehydrogenase - isolation & purification; Isoelectric Point; Molecular Weight; Nitrates; Nitrogen; Nitrogen starvation; Phormidium laminosum; Polyclonal antibody; Temperature
• ISSN: 0167-4838; 0006-3002; 1879-2588
• DOI: 10.1016/S0167-4838(99)00052-7

Nitrogen starvation enhances up to 8-fold the cellular level of the NADP +-dependent isocitrate dehydrogenase activity (isocitrate:NADP + oxidoreductase (decarboxylating), IDH, EC 1.1.1.42) in the thermophilic filamentous non-N 2-fixing cyanobacterium Phormidium laminosum. The enzyme was purified 650-fold to electrophoretic homogeneity from nitrogen-starved cells with an activity yield of 25% and a specific activity of 500 U (mg protein) −1. The native enzyme showed a p I of 5.9 and it was a dimer of 107 kDa consisting of two identical subunits of 53 kDa. The activity required the presence of a divalent metal cation as an essential activator, Mn 2+ or Mg 2+ being the most effective. The optimum temperature for activity was 55°C and the E a for catalysis was 39.7 kJ mol −1. An optimum pH for activity of 8.5 was found and the calculated p K E1, p K E2 and p K ES1 of enzyme ionisation groups were 6.0, 8.9 and 6.3, respectively. K m values of 22, 50 and 24 μM were calculated for d, l-isocitrate, NADP and Mn 2+, respectively, in the Mn 2+-dependent reaction and 70, 32 and 159 μM for d, l-isocitrate, NADP and Mg 2+, respectively, in the Mg 2+-dependent reaction. The decarboxylating activity was inhibited by ATP, ADP and by its reaction products 2-oxoglutarate and NADPH 2. Polyclonal antibodies raised against the pure IDH were used to assess the presence of the enzyme in cells subjected to nitrogen starvation.