NADP+ -dependent isocitrate dehydrogenase activity in marine plankton

Abstract

NADP+-isocitrate dehydrogenase (NADP-IDH), as one of the most active intracellular CO2-producing enzymes, was measured in marine plankton by adapting an enzyme assay to the 0.7–50 μm, 50–200 μm, and 200–2000 μm size fraction of the Canary Islands coastal plankton community. The variability of NADP-IDH activity in relation to pH, temperature, dilution of the sample, centrifugation or substrates was measured. In our hands, the maximum NADP-IDH activity (Vmax) in marine plankton samples was attained in 0.1 M phosphate buffer at pH 8.2 (pKa1 = 7.6 and pKa2 = 8.8), by adding 6 mM MgCl2, 0.3 mM NADP+ and 2 mM DL-trisodium isocitrate. The optimum temperature in these subtropic mesozooplankton samples was 28 °C, with an Arrhenius energy of activation (Ea) of 20.4 kcal mol−1 (85.4 J mol−1), and an Arrhenius collision frecuency factor (A) of 2.9·1011 mol NADPH s−1 (kg of protein)−1. The apparent Michaelis-Menten Km values for the substrates in crude homogenates at pH 8.5 and 18 °C were 271 ± 63 μM for isocitrate and 18 ± 3 μM for NADP+. This enzyme, in addition to its high CO2-producing activity, is also important in regulating other CO2-producing enzymes. Thus, it can be used to calculate: (1) respiration in marine samples; (2) carbon flux in the water column; (3) metabolic adaptations to environmental changes; (4) roles of the plankton components of the food chain; and (5) the reactive oxygen species (ROS) scavenging capacity of the marine plankton resources.