Nitrate reductase activity quantitatively predicts the rate of nitrate incorporation under steady state light limitation: A revised assay and characterization of the enzyme in three species of marine phytoplankton

摘要

The enzyme nitrate reductase (NR) has been proposed as an index of nitrate incorporation rates in marine phytoplankton, but it has proven difficult to interpret NR measurements in field settings because many previous NR assays have been poorly optimized and NR activity in phytoplankton has been poorly characterized under steady state conditions. An NR assay was developed for the diatom Thalassiosira pseudonana using an extraction in phosphate buffer with Triton X-100, EDTA, dithiothreitol, polyvinyl pyrrolidone, and bovine serum albumin. NR activity in homogenates was stable for up to 1 h, but filtered samples could be stored for 96 h in liquid nitrogen without significant loss of activity. Addition of FAD to crude extracts of T. pseudonana had no effect, whereas the effect on desalted extracts or crude extracts from other species, varied from decreases in NR activity to over 250% increases. Half-saturation constants (K_m) varied between species; high levels of NADH or nitrate were found to be inhibitory in some cases. These results indicate a wide diversity of forms of NR in marine phytoplankton. Under continuous, light-limited growth, NR activity was quantitatively related to calculated rates of nitrate incorporation (μ_N) in T. pseudonana, Skeletonema costatum, and three other diatom species examined. The relationship differed for 10 other species; NR activity was equal to μ_N in some cases, but higher or lower in others. In dinoflagellates, in particular, NR activity was highly correlated with μ_N, but accounted for < 20% of μ_N in Amphidinium carterae.