Purification, properties and enhanced expression under nitrogen starvationof the NADP(+)-isocitrate dehydrogenase from the cyanobacterium Phormidiumlaminosum

Nitrogen starvation enhances up to 8-fold the cellular level of the NADP(+) -dependent isocitrate dehydrogenase activity (isocitrate:NADP(+) oxidoreduc tase (decarboxylating), IDH, EC 1.1.1.42) in the thermophilic filamentous n on-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). T he native enzyme showed a pI of 5.9 and it was a dimer of 107 kDa consistin g of two identical subunits of 53 kDa. The activity required the presence o f a divalent metal cation as an essential activator, Mn2+ or Mg2+ being the most effective. The optimum temperature for activity was 55 degrees 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 pK(E1), PKE2 and pK(ES1) Of enzyme ionisatio n groups were 6.0, 8.9 and 6.3, respectively. K-m values of 22, 50 and 24 m u M were calculated for D,L-isocitrate, NADP and Mn2+, respectively, in the Mn2+-dependent reaction and 70, 32 and 159 mu M for D,L-isocitrate, NADP a nd Mg2+, respectively, in the Mg2+-dependent reaction. The decarboxylating activity was inhibited by ATP, ADP and by its reaction products 2-oxoglutar ate and NADPH(2). Polyclonal antibodies raised against the pure IDH were us ed to assess the presence of the enzyme in cells subjected to nitrogen star vation. (C) 1999 Elsevier Science B.V. All rights reserved.